Updated on 2024/04/18

写真a

 
IMAMURA Koreyoshi
 
Organization
Faculty of Environmental, Life, Natural Science and Technology Professor
Position
Professor
External link

Degree

  • Doctor of Engineering ( 1999.6   Kyoto University )

  • Master of Engineering ( Kyoto University )

Research Interests

  • material physical chemistry

  • Biochemical engineering

  • Drying technology

  • Food Technology

  • Chemical Engineering

  • 食品工学

  • 化学工学

Research Areas

  • Manufacturing Technology (Mechanical Engineering, Electrical and Electronic Engineering, Chemical Engineering) / Biofunction and bioprocess engineering  / protein, hydrophobic drug, enzyme reaction

  • Life Science / Pharmaceutical analytical chemistry and physicochemistry  / amorphous solid dispersion, glass transition

  • Life Science / Food sciences  / food engineering

  • Manufacturing Technology (Mechanical Engineering, Electrical and Electronic Engineering, Chemical Engineering) / Transport phenomena and unit operations  / drying, amorphous sugar, solid dispersion

Education

  • Kyoto University   工学研究科   化学工学

    - 1997

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    Country: Japan

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  • Kyoto University    

    - 1997

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  • Kyoto University    

    - 1992

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  • Kyoto University   工学部   化学工学科

    - 1992

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    Country: Japan

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Research History

  • Okayama University   Faculty of Environmental, Life, Natural Science and Technology   Professor   PhD

    2023.4

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  • Okayama University   Faculty of Natural Science and Technology   Professor   PhD

    2021.4 - 2023.3

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    Country:Japan

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  • Okayama University   Graduate School of Natural Science and Technology   Professor

    2012.6 - 2021.3

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  • Okayama University   Graduate School of Natural Science and Technology   Associate Professor

    2005.4 - 2012.5

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  • Okayama University   Faculty of Engineering   associate professor

    2003.12 - 2005.3

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  • - Cleaning of solid surfaces using radical

    1998

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  • Okayama University   Faculty of Engineering   assistant professor

    1997.4 - 2003.11

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  • - Adsorption of protein, peptide, &

    1997

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  • - Stabilization of protein by sugar

    1995

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  • oxidizing reaction

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  • amino acidly auto solid surfaces

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Professional Memberships

Committee Memberships

  • Japan Society for Food Engineering   director  

    2017.7   

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    Committee type:Academic society

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  • 日本生物工学会   西日本支部会計  

    1999   

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    Committee type:Academic society

    日本生物工学会

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  • 日本食品工学会   理事  

    1906.1   

 

Papers

  • Flavor retention characteristics of amorphous solid dispersion of flavors, prepared by vacuum-foam- and spray-drying under different conditions Reviewed

    Yuna Nitta, Haruna Sato, Rina Yamamoto, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Drying Technology   1 - 11   2023.11

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    Authorship:Last author, Corresponding author   Publishing type:Research paper (scientific journal)   Publisher:Informa UK Limited  

    DOI: 10.1080/07373937.2023.2285413

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  • Water sorption and glass-to-rubber transition of amorphous sugar matrices, vacuum foam- and spray-dried from alcohols

    Koji Takeda, Shinta Miyazaki, Takashi Okamoto, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    JOURNAL OF FOOD ENGINEERING   349   2023.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCI LTD  

    The objective of this study is to understand the impact of preparation conditions on the physicochemical characteristics of amorphous sugar. Amorphous sugar (alpha-maltose, isomaltulose, trehalose) matrices were pre -pared from alcohol (methanol, ethanol) solutions, and their water sorption behavior and glass transition tem-peratures, Tg, were compared with those of aqueous freeze-dried amorphous sugar. Vacuum-foam-and spray -drying procedures were employed as drying methods. Water sorption for amorphous sugars, dried by different methods, was generally significant in the following order: vacuum-foam-drying < spray-drying <= freeze-drying. In the same order, the Tg values for the thoroughly dried amorphous sugars increased, and water sorption reduced the differences in Tg for given amounts of sorbed water. The solvent type in vacuum-foam-drying slightly affected the water sorption and the dependence of Tg on the sorbed water amount. The obtained findings sug-gested that physicochemical characteristics of amorphous sugar may relate to the sugar molecule conformations in solvent and dried state.

    DOI: 10.1016/j.jfoodeng.2023.111483

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  • Evaluation of interaction forces between hydrophobic surfaces in surfactant solutions by atomic force microscopy

    Shota Kage, Koreyoshi Imamura, Naoyuki Ishida

    Colloid and Polymer Science   301 ( 7 )   775 - 782   2023.7

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    Publishing type:Research paper (scientific journal)  

    The interaction forces between silica surfaces with various degrees of hydrophobicity were measured using colloidal probe atomic force microscopy to clarify the relationship between the hydrophobicity of the surfaces and hydrophobic attraction in detail. The interaction forces between silica surfaces pre-hydrophobised with octadecyltrichlorosilane were measured in aqueous solutions with three different (cationic, anionic, and nonionic) surfactants. Hydrophobic attraction was observed between surfaces from 10 to 15 nm without surfactant, and the range of this attraction decreased when the surface hydrophobicity decreased with increasing surfactant concentration. The hydrophobic attraction disappeared when the surface contact angle was below 80°–90° in the presence of all surfactants used in this study. The range of the hydrophobic attraction was found to depend strongly on the contact angle and not on the surfactant type and concentration. The adhesion force was also highly dependent on the contact angle and comparable to the theoretical capillary force. The possible origin of this force is discussed with reference to the obtained results. Graphical Abstract: [Figure not available: see fulltext.].

    DOI: 10.1007/s00396-023-05118-w

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  • Evaluation of Hydrophobic Attraction between Polystyrene Layer and Silanated Silica Surface by Atomic Force Microscopy

    Yuta Okuda, Koreyoshi Imamura, Naoyuki Ishida

    Chemistry Letters   52 ( 12 )   878 - 882   2023

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    In this study, hydrophobic surfaces were prepared by depositing polystyrene (PS) on a glass substrate using three different methods to measure the interaction forces against a hydrophobized particle by atomic force microscopy. We found that smooth surfaces prepared by spin coating and living radical polymerization generated hydrophobic attraction, whereas uneven surfaces obtained by free radical polymerization produced only steric repulsion, even though they had sufficient hydrophobicity. Based on these results, the origin of the hydrophobic attraction was suggested.

    DOI: 10.1246/cl.230363

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  • Kinetics of Enzymatic Reactions at the Solid/Liquid Interface in Nanofluidic Channels

    Koki Yamamoto, Kyojiro Morikawa, Hiroyuki Imanaka, Koreyoshi Imamura, Takehiko Kitamori

    Analytical Chemistry   2022.10

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    Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

    DOI: 10.1021/acs.analchem.2c02878

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  • Extraordinary high preservation of the dispersion state of Au nanoparticles during freeze-thawing and freeze-drying with gum arabic

    Miki Kadowaki, Tsutashi Matsuura, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Colloids and Surfaces A: Physicochemical and Engineering Aspects   639   2022.4

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    A series of edible and non-toxic substances were screened for their ability to prevent Au nanoparticles (AuNPs) from undergoing aggregation during both freeze-thawing and freeze-drying at as low an additive concentration as possible. Gum arabic was found to be an ideal choice. The addition of 20 µg/mL of gum arabic greatly inhibited the aggregation of AuNPs during freeze-thawing and -drying, whereas polyvinylpyrrolidone was the next best among the tested substances, with approximately 20% aggregation during freeze-drying at a concentration of less than 200 µg/mL. The zeta potential value of the AuNPs indicated the absence of the specific binding of gum arabic to the AuNPs surface. Surface enhanced infrared spectroscopic (SEIRAS) analyses were conducted for the gum arabic solution, frozen and freeze-dried on the Au particles. The results indicated that, in the frozen state, gum arabic molecules were concentrated in remaining unfrozen liquid on the Au coating without any specific orientation toward the Au surface. In contrast, SEIRAS spectra of freeze-dried gum arabic indicated that carboxylic groups in gum arabic molecules preferentially became closer to the Au surface. Considering these findings, during freeze-thawing, we conclude that the high thickening property of gum arabic gum plays an important role in minimizing the mobility and thus the aggregation of AuNPs in the freeze-concentrated regions between frozen ice crystals. The freeze-drying of a gum arabic solution produces a rigid amorphous matrix that has an affinity for the Au surface. The AuNPs were individually separated and immobilized in the freeze-dried matrix of the gum arabic, resulting in the extraordinary inhibition of their aggregation.

    DOI: 10.1016/j.colsurfa.2022.128392

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  • Comparison of improvements of aqueous dissolution of structurally analogous hydrophobic drugs by amorphous solid dispersion

    Takashi Okamoto, Kayoko Yamamoto, Takanari Sekitoh, Akiho Fujioka, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Colloids and Surfaces A: Physicochemical and Engineering Aspects   632   127744 - 127744   2022.1

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    DOI: 10.1016/j.colsurfa.2021.127744

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  • Induction of foaming in vacuum drying by needle stimulation and the impact of solution viscosity, vapor pressure, and the type of solute and solvent

    Akiho Fujioka, Rina Yamamoto, Olivier Tramis, Hiroaki Ishida, Tsutomu Ono, Naoyuki Ishida, Koreyoshi Imamura

    Drying Technology   1 - 13   2021.12

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    Publishing type:Research paper (scientific journal)   Publisher:Informa UK Limited  

    DOI: 10.1080/07373937.2021.2017965

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  • Foaming characteristics of sugar- and polyvinylpyrrolidone-alcohol solutions during vacuum foam drying: A rheological approach

    Olivier Tramis, Akiho Fujioka, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Colloids and Surfaces A: Physicochemical and Engineering Aspects   627   127174 - 127174   2021.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    Vacuum foam drying offers great perspectives to formulate solid-state encapsulated active drugs. Taking into account the specific need of pharmaceutical formulations to keep drug molecules active and dispersed, we show in this paper that vacuum drying of pharmaceutical formulations can be substantially improved by using original rheological approaches. Typically, our formulations totally dried-up in less than 5 min, a delay much shorter than the long hours of the traditional vacuum drying process. Steady-shear rheology was used to evaluate the solution's specific viscosity against the solutions' concentrations, in order to correlate the dilution regime to the foamabiltiy. This rheological approach indicated that in miscible solutions, spontaneous foaming occurred in the semi-dilute entangled regime, near the transition to the concentrated regime; for partially miscible solutions, it occurred in solutions at a concentration near to the percolation concentration. The proposed methodology is versatile, and should provide a simple way to assess the foamability of pharmaceutical formulations.

    DOI: 10.1016/j.colsurfa.2021.127174

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  • Inhibiting Au nanoparticle aggregation in freeze-thawing by presence of various additives

    Miki Kadowaki, Hidetaka Yokota, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Advanced Powder Technology   32 ( 10 )   3517 - 3524   2021.10

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    DOI: 10.1016/j.apt.2021.08.002

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  • Direct measurements of interaction forces of bovine serum albumin and lysozyme with stainless steel by atomic force microscopy Reviewed

    Hikaru Kawaguchi, Hiroyuki Imanaka, Koreyoshi Imamura, Naoyuki Ishida

    Colloids and Surfaces A: Physicochemical and Engineering Aspects   627   127137 - 127137   2021.10

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    DOI: 10.1016/j.colsurfa.2021.127137

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  • Sole-amorphous-sugar-based solid dispersion of curcumin and the influence of formulation composition and heat treatment on the dissolution of curcumin

    Takanari Sekitoh, Takashi Okamoto, Akiho Fujioka, Olivier Tramis, Koji Takeda, Tsutashi Matsuura, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Drying Technology   39 ( 14 )   2065 - 2074   2021.9

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    Publishing type:Research paper (scientific journal)   Publisher:Informa UK Limited  

    An amorphous solid dispersion technique, in which hydrophobic drug molecules are dispersed in an amorphous matrix comprised solely of a sugar, was applied to curcumin that has excellent physiological functions. The sole-amorphous-sugar-based solid dispersion (SAS-SD) of curcumin was prepared by vacuum foam drying from methanol, using various disaccharides as the drug carrier-forming component. Aqueous dissolution of curcumin from the SAS-SD showed “spring-and-parachute” shape profile and was markedly increased when a sugar (α-maltose or trehalose) was added in a composition range ≥100 g-sugar/g-curcumin. Further dissolution was achieved by heating the SAS-SD at close to and above the melting points of curcumin.

    DOI: 10.1080/07373937.2020.1752711

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  • Crystallization characteristics of amorphous trehalose dried from alcohol Reviewed

    Takanari Sekitoh, Takashi Okamoto, Akiho Fujioka, Tomohiko Yoshioka, Shinji Terui, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Journal of Food Engineering   292   110325 - 110325   2021.3

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    File: P0TreCry-pub.pdf

    DOI: 10.1016/j.jfoodeng.2020.110325

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  • Direct measurement of interaction force between hydrophilic silica surfaces in triblock copolymer solutions with salt by atomic force microscopy

    Takumi Deguchi, Takato Nakahara, Koreyoshi Imamura, Naoyuki Ishida

    Advanced Powder Technology   32 ( 1 )   30 - 36   2021.1

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    DOI: 10.1016/j.apt.2020.11.011

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  • Spontaneous foaming during vacuum drying of polyvinylpyrrolidone- and sugar-alcohol mixtures and enhancement of water-dissolution of water insoluble drug Reviewed

    Olivier Tramis, Akiho Fujioka, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Drying Technology   1 - 11   2020.9

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    DOI: 10.1080/07373937.2020.1822863

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  • A suitable and effective stepwise oxidative refolding procedure for highly‐cationic tetrameric avidin in nucleic acid free conditions International journal

    Shuichiro Kimura, Koreyoshi Imamura, Junichiro Futami

    Biotechnology Progress   36 ( 5 )   e3031   2020.9

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Wiley  

    Optimized conditions are needed to refold recombinant proteins from bacterial inclusion bodies into their biologically active conformations. In this study, we found two crucial requirements for efficient refolding of cationic tetrameric chicken avidin. The first step is to eliminate nucleic acid contaminants from the bacterial inclusion body. The electrostatic interactions between the remaining nucleic acids and proteins strongly enhanced protein aggregation during the refolding process. The cysteine specific reversible S-cationization procedure was successfully employed for large-scale preparation of nucleic acid free denatured protein without purification tag system. The second step is the intramolecular disulfide formation prior to refolding in dialysis removing denaturant. Disulfide intact monomeric avidin showed efficient formation of biologically active tetrameric conformation during the refolding process. Using this optimized refolding procedure, highly cationic avidin derivative designed as an intracellular delivery carrier of biotinylated protein was successfully prepared.

    DOI: 10.1002/btpr.3031

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    Other Link: https://onlinelibrary.wiley.com/doi/full-xml/10.1002/btpr.3031

  • The Use of a Combination of a Sugar and Surfactant to Stabilize Au Nanoparticle Dispersion against Aggregation during Freeze-Drying Reviewed

    Hidetaka Yokota, Miki Kadowaki, Tsutashi Matsuura, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Langmuir   36 ( 24 )   6698 - 6705   2020.6

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    DOI: 10.1021/acs.langmuir.0c00695

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  • Immobilization of surface non-affinitive protein onto a metal surface by an external electric field Reviewed

    Olivier Tramis, Ryosuke Iizuka, Hajime Nakao, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Journal of Bioscience and Bioengineering   129 ( 3 )   348 - 353   2020.3

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    DOI: 10.1016/j.jbiosc.2019.09.008

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  • Picoliter enzyme reactor on a nanofluidic device exceeding the bulk reaction rate Invited Reviewed

    Koki Yamamoto, Kyojiro Morikawa, Hiroyuki Imanaka, Koreyoshi Imamura, Takehiko Kitamori

    The Analyst   145 ( 17 )   5801 - 5807   2020

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    Publishing type:Research paper (scientific journal)   Publisher:Royal Society of Chemistry (RSC)  

    <p>A picoliter enzyme reactor using a trypsin immobilized nanochannel realized 25 times faster reaction than the bulk reaction.</p>

    DOI: 10.1039/d0an00998a

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  • Controlling the drying process in vacuum foam drying under low vacuum conditions by inducing foaming by needle stimulation of the solution Reviewed

    Fumihiro Hidaka, Tomo Satoh, Akiho Fujioka, Koji Takeda, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Drying Technology   37 ( 12 )   1520 - 1527   2019.9

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)  

    Vacuum foam drying is a promising drying technique but an extremely high vacuum is needed to achieve “foaming.” The findings reported herein show that, when a solution is partially vacuum-dried to 0.05–2 g-solvent/g-dry matter (initial drying) and the solution is then punctured with a steel needle (needle stimulation), vacuum drying resumes as a result of the solution foaming, even under conditions of an insufficient vacuum (ca. 1,000 Pa) where foaming is minimal. Methanol, ethanol, and isopropanol were used as solvents, and sugar and different molecular weight polyvinylpyrrolidone were employed as solutes. The results indicate that needle puncturing introduces minute bubbles, which then triggers foaming.

    DOI: 10.1080/07373937.2018.1517363

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  • Physical Stability of an Amorphous Sugar Matrix Dried From Methanol as an Amorphous Solid Dispersion Carrier and the Influence of Heat Treatment Reviewed

    Koji Takeda, Takanari Sekitoh, Akiho Fujioka,, Kayoko Yamamoto, Takashi Okamoto, Tsutashi Matsuura, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Journal of Pharmaceutical Sciences   108 ( 6 )   2056 - 2062   2019.6

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    Authorship:Corresponding author   Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE INC  

    An amorphous sugar matrix, after drying from an organic solvent, was investigated for use as a method for dispersing hydrophobic drugs (solid dispersion). However, the amorphous sugar, originally contained in the organic solvent, had a significantly low glass transition temperature (T-g), thus rendering it physically unstable. In this study, we examined the physicochemical properties of a sugar in a dried matrix and in an organic solvent, using alpha-maltose and methanol as a representative sugar and organic solvent. The apparent molar volume of alpha-maltose was similar to 30% smaller in methanol than in water. The methanol-originated amorphous alpha-maltose exhibited a much greater degree of hydrogen bonding than the water-originated one. Considering these findings, we conclude that the alpha-maltose maintained its compact conformation in the dried state and consequently caused the markedly low T-g. Second, it was found that heating under appropriate conditions resulted in an increase in the T-g of the methanol-originated amorphous alpha-maltose as well as a decrease in the level of hydrogen bonding. The aqueous dissolution of 2 model hydrophobic drugs (indomethacin and ibuprofen) from the solid dispersion was also improved as the result of the heat treatment, whereas, to the contrary, the dissolution of another model drug (curcumin) was lowered. (c) 2019 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.xphs.2019.01.008

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  • Adsorption characteristics of various proteins on a metal surface in the presence of an external electric potential Reviewed

    Ei Ei Htwe, Yuhi Nakama, Yuko Yamamoto, Hiroshi Tanaka, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    Colloids and Surfaces B: Biointerfaces   166   262 - 268   2018.6

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    The effect of the properties of a protein on its adsorption to a metal surface in the presence of external electric potential was investigated. Protein adsorption processes at different surface potentials were measured for fifteen types of proteins using an in-situ ellipsometry. The tested proteins were classified into three groups, based on the amount of protein that was adsorbed as a function of the surface potential: In First group of proteins, an increasing trend for the amount adsorbed with a more positive surface potential was found
    The amount adsorbed of α-chymotrypsinogen A and ribonuclease A (Second group) were roughly constant and independent of the applied surface electric potentials
    In Third group, the amount adsorbed decreased with increasing surface potential. This protein classification was correlated with the isoelectric points of the proteins (First group: ≤9.3
    Second group: 9.3–10
    Third group: &gt
    10). Increasing the pH positively and negatively shifted the surface potentials, allowing ß-lactoglobulin (First group) and lysozyme (Third) to become adsorbed, respectively. The surface potential range for protein adsorption was also markedly shifted depending on the metal substrate type. These findings were interpreted based on the electrostatic interactions among the protein, surface hydroxyl groups, and the applied external electric field.

    DOI: 10.1016/j.colsurfb.2018.03.035

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  • Hydrophobic Attraction Measured between Asymmetric Hydrophobic Surfaces Reviewed

    Naoyuki Ishida, Kohei Matsuo, Koreyoshi Imamura, Vincent S. J. Craig

    Langmuir   34 ( 12 )   3588 - 3596   2018.3

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society  

    The interaction forces between silica surfaces modified to different degrees of hydrophobicity were measured using colloidal probe atomic force microscopy (AFM). A highly hydrophobic silica particle was prepared with octadecyltrichlorosilane (OTS), and the interaction forces were measured against silica substrates modified to produce surfaces of varying hydrophobicity. The interaction forces between the highly hydrophobic particle and a completely hydrophilic silicon wafer surface fitted well to the DLVO theory, indicating that no additional (non-DLVO) forces act between the surfaces. When the silicon wafer surface was treated to produce a contact angle of water on surface of 40°, an additional attractive force that is longer ranged than the van der Waals force was observed between the surfaces. The range and magnitude of the attractive force increase with the contact angle of water on the substrate. Beyond the effect on the contact angle, the hydrocarbon chain length and the terminal groups of hydrophobic layer on the substrate only have a minor effect on the magnitude of the force, even when the substrate is terminated with polar carboxyl groups, provided the hydrophobicity of the other surface is high.

    DOI: 10.1021/acs.langmuir.7b04246

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  • Influence of an external electric field on removal of protein fouling on a stainless steel surface by proteolytic enzymes Reviewed

    Ei Ei Htwe, Yuhi Nakama, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    COLLOIDS AND SURFACES B-BIOINTERFACES   159   118 - 124   2017.11

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:ELSEVIER SCIENCE BV  

    Enzymatic cleaning is a potentially useful method for removing proteinaceous fouling from solid surfaces under mild conditions. Herein, the influence of an external electric field on the enzymatic cleaning of a metal surface fouled with a protein was investigated. The model fouling protein (BSA; lysozyme) was prepared on a stainless steel (St) surface, and the resulting surface subjected to enzymatic cleaning with an electric potential being applied to the St plate. Trypsin, alpha-chymotrypsin, and thermolysin were used as model proteases. The amounts of protein remaining on the plate before and during the cleaning process were measured by means of a reflection absorption technique using Fourier transform infrared spectroscopy. In the case for BSA fouling, the cleaning efficiency of the protease tended to increase at more negative applied potentials. Whereas, there was an optimum applied potential for removing the lysozyme fouling. Atomic force microscopy analyses indicated that applying an adequate range of electric potential enhanced the enzymatic removal of protein fouling inside scratches on the St plate surface. These findings suggest the existence of two modes of electrostatic interactions for the external electric field, one with protease molecules and the other with digested fragments of the fouling protein. (C) 2017 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.colsurfb.2017.07.074

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  • Characteristics of proteinaceous additives in stabilizing enzymes during freeze-thawing and -drying Reviewed

    Takanori Shimizu, Tamayo Korehisa, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   81 ( 4 )   687 - 697   2017.4

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:TAYLOR & FRANCIS LTD  

    Protein-stabilizing characteristics of sixteen proteins during freeze-thawing and freeze-drying were investigated. Five enzymes, each with different instabilities against freezing and dehydration, were employed as the protein to be stabilized. Proteinaceous additives generally resulted in greater enzyme stabilization during freeze-thawing than sugars while the degree of stabilization for basic lysozyme and protamine were inferior to that of neutral and acidic proteins. Freeze-drying-induced inactivation of enzyme was also reduced by the presence of a proteinaceous additive, the extent of which was lower than that for a sugar. In both freeze thawing and freeze drying, the enzymes stabilization by the proteinaceous additive increased with increasing additive concentration. The enhancement of enzyme inactivation caused by pH change was also reduced in the presence of proteinaceous additives. The combined use of a sugar such as sucrose and dextran tended to increase the stabilizing effect of the proteinaceous additive.

    DOI: 10.1080/09168451.2016.1274637

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  • Surfactant-Free Solid Dispersions of Hydrophobic Drugs in an Amorphous Sugar Matrix Dried from an Organic Solvent Reviewed

    Koji Takeda, Yuto Gotoda, Daichi Hirota, Fumihiro Hidaka, Tomo Sato, Tsutashi Matsuura, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    MOLECULAR PHARMACEUTICS   14 ( 3 )   791 - 798   2017.3

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:AMER CHEMICAL SOC  

    The technique for homogeneously dispersing hydrophobic drugs in a water-soluble solid matrix (solid dispersion) is a subject that has been extensively investigated in the pharmaceutical industry. Herein, a novel technique for dispersing a solid, without the need to use a surfactant, is reported. A freeze-dried amorphous sugar sample was dissolved in an organic solvent, which contained a soluble model hydrophobic component. The suspension of the sugar and the model hydrophobic component was vacuum foam dried to give a solid powder. Four types of sugars and methanol were used as representative sugars and the organic medium. Four model drugs (indomethacin, ibuprofen, gliclazide, and nifedipine) were employed. Differential scanning calorimetry analyses indicated that the sugar and model drug (100:1) did not undergo segregation during the drying process. The dissolution of the hydrophobic drugs in water from the solid dispersion was then evaluated, and the results indicated that the C-max and AUC(0-60) (min) of the hydrophobic drug in water were increased when the surfactant-free solid dispersion was used. Palatinose and/or alpha-maltose were superior to the other tested carbohydrates in increasing C-max and AUC(0-60 min), for all tested model drugs, and the model drug with a lower water solubility tended to exhibit a greater extent of over-dissolution.

    DOI: 10.1021/acs.molpharmaceut.6b01048

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  • Adsorption of lysozyme on base metal surfaces in the presence of an external electric potential Reviewed

    Ei Ei Htwe, Yuhi Nakama, Hiroshi Tanaka, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    COLLOIDS AND SURFACES B-BIOINTERFACES   147   9 - 16   2016.11

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    The impact of external electric potential on the adsorption of a protein to base metal surfaces was examined. Hen egg white lysozyme (LSZ) and six types of base metal plates (stainless steel SUS316L (St), Ti, Ta, Zr, Cr, or Ni) were used as the protein and adsorption surface, respectively. LSZ was allowed to adsorb on the surface under different conditions (surface potential, pH, electrolyte type and concentration, surface material), which was monitored using an ellipsometer. LSZ adsorption was minimized in the potential range above a certain threshold and, in the surface potential range below the threshold, decreasing the surface potential increased the amount of protein adsorbed. The threshold potential for LSZ adsorption was shifted toward a positive value with increasing pH and was lower for Ta and Zr than for the others. A divalent anion salt (K2SO4) as an electrolyte exhibited the adsorption of LSZ in the positive potential range while a monovalent salt (KCI) did not. A comprehensive consideration of the obtained results suggests that two modes of interactions, namely the electric force by an external electric field and electrostatic interactions with ionized surface hydroxyl groups, act on the LSZ molecules and determine the extent of suppression of LSZ adsorption, All these findings appear to support the view that a base metal surface can be controlled for the affinity to a protein by manipulating the surface electric potential as has been reported on some electrode materials. (C) 2016 Elsevier B.V. All rights reserved.

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  • Molecular design of proteinaceous cushion for sensitive biomolecular interaction detection system Reviewed

    Hiroyuki Imanaka, Koki Dare, Koreyoshi Imamura

    NEW BIOTECHNOLOGY   33   S72 - S72   2016.7

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  • Surfactant-free solid dispersion of fat-soluble flavour in an amorphous sugar matrix Reviewed

    Tomo Satoh, Fumihiro Hidaka, Kento Miyake, Natsuki Yoshiyama, Koji Takeda, Tsutashi Matsuura, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    FOOD CHEMISTRY   197   1136 - 1142   2016.4

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    A solid dispersion technique to homogeneously disperse hydrophobic ingredients in a water-soluble solid without using surfactant was examined as follows: first, freeze-dried amorphous sugar was dissolved in an organic medium that contained a soluble model hydrophobic component. Second, the mixed solution of sugar and the model hydrophobic component was vacuum dried into a solid (solid dispersion). Methanol and six fat-soluble flavours, including cinnamaldehyde, were used as organic media and model hydrophobic components. The retention of flavours in the solid dispersion during drying and storage under vacuum was evaluated. The amorphised disaccharides dissolved in methanol up to 100 mg/mL, even temporarily (20 s to 10 days) and could be solidified without any evidence of crystallisation and segregation from flavour. The solid dispersion, prepared using a-maltose usually showed 65-95% flavour retention during drying (and storage for cinnamaldehyde), whereas &gt;= 50% of the flavour was lost when the flavour was O/W emulsified with a surfactant and then freeze-dried with sugar. (C) 2015 Elsevier Ltd. All rights reserved.

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  • The use of a proteinaceous "cushion" with a polystyrene-binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface Reviewed

    Hiroyuki Imanaka, Daisuke Yamadzumi, Keisuke Yanagita, Naoyuki Ishida, Kazuhiro Nakanishi, Koreyoshi Imamura

    BIOTECHNOLOGY PROGRESS   32 ( 2 )   527 - 534   2016.3

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    In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a cushion between the PS-affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS-affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527-534, 2016

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  • Improvement of Biomolecular Interaction Detection Sensitivity through Molecular Design of Cushion Protein

    Imanaka Hiroyuki, Date Koki, Matoba Haruka, Ishida Naoyuki, Imamura Koreyoshi

    Abstract of annual meeting of the Surface Science of Japan   36   37 - 37   2016

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  • Effect of surface hydrophobicity on short-range hydrophobic attraction between silanated silica surfaces Reviewed

    Yuhei Soga, Hiroyuki Imanaka, Koreyoshi Imamura, Naoyuki Ishida

    ADVANCED POWDER TECHNOLOGY   26 ( 6 )   1729 - 1733   2015.11

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    The interaction forces between silanated silica surfaces without nanobubbles were measured using colloidal probe atomic force microscopy (AFM). To obtain hydrophobic surfaces without nanobubbles, an aqueous solution was introduced between the surfaces following an exchange process involving several solvents. In the obtained approaching force curves, an attractive force was observed from a distance of 10-20 nm, which is an additional attractive force stronger than typical van der Waals attractions. When the surface hydrophobicity decreased, the range of this attraction decreased slightly; the attraction disappeared when the surface contact angle was below 90 degrees. In contrast, measurements in the water-ethanol mixtures revealed that the attraction persisted even when the contact angle was well below 90 degrees. The possible origin of this force was discussed on the basis of the obtained results. (C) 2015 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan.

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  • Inhibitory effects of additives and heat treatment on the crystallization of freeze-dried sugar Reviewed

    Kohshi Kinugawa, Mitsunori Kinuhata, Ryo Kagotani, Hiroyuki Imanaka, Naoyuki Ishida, Mizuki Kitamatsu, Kazuhiro Nakanishi, Koreyoshi Imamura

    JOURNAL OF FOOD ENGINEERING   155   37 - 44   2015.6

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    An amorphous matrix of a sugar is frequently used as a bulk-forming and stabilizing agent in the food industry but tends to crystallize as the result of water uptake and increase in temperature. Additives and methods used to inhibit the crystallization of amorphous sugar (sucrose) were screened in this study. Freeze-dried amorphous sucrose containing 0.5-5 wt% of additive, including salts, different types of sugars, and polymers, the crystallization temperature (T-cry) and isothermal crystallization characteristics were examined. Certain types of salts markedly increased the Tcry and prolonged the induction period for crystal nucleation. The use of 1 wt% MgCl2 was particularly effective in inhibiting sugar crystallization. The heat treatment of crystalline sucrose under appropriate conditions was also found to result in diminished sucrose crystallization. MALDI-TOF mass spectra of the heat-treated sucrose suggested that sucrose derivatives containing multiple pyranose groups were formed, which would closely relate to the crystallization inhibition. Finally, the protein stabilizing effects of the matrices were evaluated. The results indicated that both the addition of additives and the heat treatment resulted in an improvement of the protein stabilizing effect of amorphous sugar matrix, compared to that of sucrose alone. (C) 2015 Elsevier Ltd. All rights reserved.

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  • Influence of sugar surfactant structure on the encapsulation of oil droplets in an amorphous sugar matrix during freeze-drying Reviewed

    Shota Nakayama, Yoshifumi Kimura, Sayuri Miki, Jun Oshitani, Takashi Kobayashi, Shuji Adachi, Tsutashi Matsuura, Hiroyuki Imanaka, Naoyuki Ishida, Hiroko Tada, Kazuhiro Nakanishi, Koreyoshi Imamura

    FOOD RESEARCH INTERNATIONAL   70   143 - 149   2015.4

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    The encapsulation of O/W emulsion droplets in a freeze-dried amorphous sugar matrix was investigated, focusing on the impact of the molecular structure of the emulsifying surfactant. O/W emulsions, containing various surfactants, were freeze-dried in the presence of a sugar. Thirty types of surfactants, including eighteen different sugar surfactants and ten types of commercially available sugar ester mixtures, were used. Linoleic acid methyl ester and trehalose were used as the oil phase and sugar. The amounts of oil droplets encapsulated in freeze-dried amorphous sugar matrix were analyzed by Fourier transform Infrared spectroscopy. Sugar surfactants were generally superior to the other classes of surfactants for oil droplet encapsulation during freeze-drying, and there was the optimum alkyl chain length of the sugar surfactant. Sugar esters generally exhibited greater oil encapsulation than sugar ethers. Larger sugar head group appeared to result in better encapsulation in the case of sugar esters, but the opposite tendency was found for sugar ethers. A limited combination of sugar surfactants (15% sucrose mono- and 85% di-stearate) resulted in the maximum oil droplet encapsulation efficiency although these surfactants are individually quite poor in the encapsulation and other tested combinations did not improve the encapsulation efficiency relative to their individual effectiveness. (C) 2015 Elsevier Ltd. All rights reserved.

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  • Effect of Lipid Amount on Surfactant-free Solid Lipid Nanoparticle Formation by Hot Homogenization Reviewed

    Jun Oshitani, Natsumi Murakami, Mikio Yoshida, Naoyuki Ishida, Koreyoshi Imamura, Hideki Ichikawa

    CHEMISTRY LETTERS   43 ( 7 )   1011 - 1013   2014.7

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    The yield and size distribution of surfactant-free solid lipid nanoparticles (SF-SLNs) formed by hot homogenization were investigated by changing the amount of lipid (palmitic acid). The yield was 100% and monodispersed same-sized particles of 120-200 nm were formed at the lipid amount &lt;= 2.8 x 10(-4) g-lipid (g-water)(-1). The yield decreased owing to phase separation, and the size increased "step-likely" to be 200 350 nm while keeping the monodispersity when the lipid amount was just above &lt;= 2.8 x 10(-4) g-lipid (g-water)(-1). The size and the lipid amount continued to increase gradually at the lipid amount &gt;2.8 x 10(-4) g-lipid (g-water)(-1). The results indicate that submicron-sized solid lipid particles can be formed without surfactants and that the decrease of yield and the step-like increase of particle size occur at the same lipid amount. The total surface area of the SF-SLNs was estimated using the experimental data. It is suggested that the total surface area and the lipid amount are correlated to each other.

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  • Characteristics of Sugar Surfactants in Stabilizing Proteins During Freeze-Thawing and Freeze-Drying Reviewed

    Koreyoshi Imamura, Katsuyuki Murai, Tamayo Korehisa, Noriyuki Shimizu, Ryo Yamahira, Tsutashi Matsuura, Hiroko Tada, Hiroyuki Imanaka, Naoyuki Ishida, Kazuhiro Nakanishi

    JOURNAL OF PHARMACEUTICAL SCIENCES   103 ( 6 )   1628 - 1637   2014.6

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    Sugar surfactants with different alkyl chain lengths and sugar head groups were compared for their protein-stabilizing effect during freeze-thawing and freeze-drying. Six enzymes, different in terms of tolerance against inactivation because of freeze-thawing and freeze-drying, were used as model proteins. The enzyme activities that remained after freeze-thawing and freeze-drying in the presence of a sugar surfactant were measured for different types and concentrations of sugar surfactants. Sugar surfactants stabilized all of the tested enzymes both during freeze-thawing and freeze-drying, and a one or two order higher amount of added sugar surfactant was required for achieving protein stabilization during freeze-drying than for the cryoprotection. The comprehensive comparison showed that the C10-C12 esters of sucrose or trehalose were the most effective through the freeze-drying process: the remaining enzyme activities after freeze-thawing and freeze-drying increased at the sugar ester concentrations of 1-10 and 10-100 M, respectively, and increased to a greater extent than for the other surfactants at higher concentrations. Results also indicate that, when a decent amount of sugar was also added, the protein-stabilizing effect of a small amount of sugar ester through the freeze-drying process could be enhanced. (c) 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci

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  • Stabilization of Protein by Sugar Surfactant

    IMAMURA Koreyoshi

    Oleoscience   14 ( 11 )   487 - 493   2014

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    <p>Protein is one of the substances that are indispensable in the food and pharmaceutical industries and, however, is apt to lose its native conformation and consequently bioactivity under nonphysiological conditions. Hence, to date, the methodologies to protect proteins against denaturation have been investigated and various substances including sugars, amino acids, and certain types of polymers have been known to serve to maintain the protein conformation under different conditions. On the other hand, the sugar surfactants that are frequently used as food additives have been found to stabilize protein in various situations. Herein, the effectiveness of sugar surfactant as a protein stabilizing agent against denaturation in aqueous solution, freezing step, and freeze-drying are demonstrated as well as the denaturation pathways of proteins. Particularly, this review focuses on the differences among the protein stabilizing effects of various sugar surfactants in freezing and freeze-drying. The impacts on the protein stabilizing characteristics by alkyl chain length, sugar head group structure, and ester/ether bondings of sugar surfactant are discussed as well as the possible stabilizing mechanism.</p>

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  • Effect of Surface Hydrophobicity on Short-Range Hydrophobic Attraction between Silanated Silica Surfaces

    Soga Yuuhei, Imanaka Hiroyuki, Imamura Koreyoshi, Ishida Naoyuki

    Journal of the Society of Powder Technology, Japan   51 ( 5 )   343 - 348   2014

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    The interaction forces between silanated silica surfaces with no nanobubbles were measured using colloidal probe atomic force microscopy (AFM). To obtain hydrophobic surfaces without nanobubbles, an aqueous solution was introduced between the surfaces following an exchange process involving several solvents. In the obtained approachingforce curves, an attractive force was observed from a distance of 10~20 nm, which is an additional attractive force stronger than typical van der Waals attractions. When the surface hydrophobicity decreased, the range of this attraction decreased slightly ; the attraction disappeared when the surface contact angle was below 90°。. In contrast, measurements in the water-ethanol mixtures revealed that the attraction persisted even when the contact angle was well below 90°。. The possible origin of this force was discussed on the basis of the obtained results.

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  • 2P078 Screening of a novel gold affinity peptide and its application on protein immobilization(01D. Protein: Function,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

    Shigemori Yojiro, Yoshida Kaori, Imamura Koreyoshi, Takahashi Yuichiro, Imanaka Hiroyuki

    Seibutsu Butsuri   54 ( 1 )   S207   2014

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  • 2P085 Design of highly sensitive peptide-protein interaction detection system adopting CS complex formation as the model(01F. Protein: Engineering,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

    Matsushita Runa, Ishida Naoyuki, Imamura Koreyoshi, Imanaka Hiroyuki

    Seibutsu Butsuri   54 ( 1 )   S209   2014

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  • Improving the physical stability of freeze-dried amorphous sugar matrices by compression at several hundreds MPa Reviewed

    Ryo Kagotani, Kohshi Kinugawa, Mayo Nomura, Hiroyuki Imanaka, Naoyuki Ishida, Koreyoshi Imamura

    JOURNAL OF PHARMACEUTICAL SCIENCES   102 ( 7 )   2187 - 2197   2013.7

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    Amorphous matrices, composed of sugars, are markedly plasticized by moisture uptake, which results in physical instability. Our previous studies, in the compression pressure range 443 MPa, indicated that when a matrix is compressed, the amount of sorbed water at given relative humidities (RHs) decreases, whereas the glass transition temperature (Tg) remains constant. Herein, the effect of higher compression pressures than those used previously was explored to investigate the feasibility of using compression to improve the physical stability of amorphous sugar matrix against water uptake and subsequent collapse. Amorphous sugar samples were prepared by freeze-drying and then compressed at 0-665 MPa, followed by rehumidification at given RHs. The physical stability of the amorphous sugar sample was evaluated by measuring Tg and crystallization temperature (Tcry). The amounts of sorbed water, different in the interaction state, were determined using an FTIR technique. It was found that the compression at pressures of 443 MPa decreased the amount of sorbed water, which is a major factor in plasticization and crystallization, and thus markedly increased the Tg and Tcry relative to that for the uncompressed sample. Hence, the compression at several hundreds MPa appears to be feasible for improving the physical stability of amorphous sugar matrix. (c) 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2187-2197, 2013

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  • Improvement on the freeze-thaw stability of corn starch gel by the polysaccharide from leaves of Corchorus olitorius L. Reviewed

    Eiji Yamazaki, Toru Sago, Yoshiaki Kasubuchi, Kazuhito Imamura, Toshio Matsuoka, Osamu Kurita, Hironobu Nambu, Yasuki Matsumura

    Carbohydrate Polymers   94 ( 1 )   555 - 560   2013.4

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    Effect of the polysaccharide from leaves of Corchorus olitorius L. (PLC) on the freeze-thaw (FT) stability of corn starch gel was studied. PLC was incorporated into the starch gel at 0.7% and total solid was adjusted to 6.0%. The syneresis was measured by the centrifugal-filtration method and, as a result, addition of PLC reduced effectively the syneresis of the starch gel even after 5 FT cycles, which was less than one third that of the normal starch gel. The rheological changes of the starch/PLC gel during the FT treatments were evaluated while the gel remained on the rheometer plate. The starch/PLC gel had less significant changes in the rheological parameters during the FT cycles than starch/guar gum or xanthan gum gel systems. SEM images showed that PLC stabilized the gel matrix surrounding pores, which would contribute to both a lower syneresis production and a higher stability in the rheological behavior at FT. © 2013 Elsevier Ltd. All rights reserved.

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  • Characteristics of amorphous matrices composed of different types of sugars in encapsulating emulsion oil droplets during freeze-drying Reviewed

    Koreyoshi Imamura, Yoshifumi Kimura, Shota Nakayama, Miki Sayuri, Seiji Ogawa, Tatsuya Hoshino, Jun Oshitani, Takashi Kobayashi, Shuji Adachi, Tsutashi Matsuura, Hiroyuki Imanaka, Naoyuki Ishida, Kazuhiro Nakanishi

    FOOD RESEARCH INTERNATIONAL   51 ( 1 )   201 - 207   2013.4

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    The encapsulation of emulsion oil droplets by amorphous sugar matrices, formed by freeze-drying, was investigated, with a focus on the influence of the type of sugar. An oil-in-water emulsion, comprised of linoleic acid methyl ester (LME) and sucrose monolaurate (SML) as an oil phase and surfactant, respectively, were freeze-dried in the presence of different types of sugars. LME-droplet encapsulation during and after freeze-drying were evaluated by FTIR analysis. The loss of LME largely occurred in the early stage of freeze-drying. The size distribution of the encapsulated LME droplets remained unchanged before and after freeze-drying in most cases. The encapsulated fractions of LME droplets could be correlated with the glass transition temperature of the sugars in the fully hydrated state (T-g*), and the existence of an optimum T-g* value for the sugar matrix was predicted. The encapsulation ability of an amorphous sugar matrix was maximized when mono- and polysaccharide were combined so as to give a value for T-g* of approximately -50 degrees C, although, individually, mono- and polysaccharides were quite poor for oil droplet encapsulation. These findings suggest that the structural flexibility of the amorphous sugar matrix is a major determinant in oil droplet encapsulation by an amorphous sugar matrix during freeze-drying. (C) 2012 Elsevier Ltd. All rights reserved.

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  • On the preparation of indoxyl red from indican and some new characteristics Reviewed

    Jingyuan Song, Mizuki Kitamatsu, Koreyoshi Imamura, Hitoshi Ohmori, Kouji Watanabe, Kazuhiro Nakanishi

    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS   23 ( 3 )   627 - 629   2013.2

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    An indole compound with a strong purple-red color was produced by boiling a solution of indican under acidic conditions and purified by chromatographies on DEAE-650S Toyopearl TSK-gel and silica-gel columns. The purple-red compound purified was identified as indoxyl red, on the basis of FAB Mass, C-13 NMR, H-1 NMR, UV-visible spectra, and IR spectra. Although indoxyl red was first synthesized by Seidel(9) 70 years ago, very little information has been available on its characteristics. We repot here that the compound was purple-red colored at acidic pH and green at pH 13, and showed antiproliferative and cytotoxic activities to the mouse B cell lymphoma cell line NSF202. (c) 2012 Elsevier Ltd. All rights reserved.

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  • The Discoidin Domain of Bacillus circulans beta-Galactosidase Plays an Essential Role in Repressing Galactooligosaccharide Production Reviewed

    Jingyuan Song, Hiroyuki Imanaka, Koreyoshi Imamura, Masashi Minoda, Shotaro Yamaguchi, Kazuhiro Nakanishi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   77 ( 1 )   73 - 79   2013.1

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    The recently cloned beta-galactosidase from Bacillus circulans ATCC 31382, designated BgaD, contains a multiple domain architecture including a F5/8 type C domain or a discoidin (DS) domain in the C-terminal peptide region. Here we report that the DS domain plays an essential role in repressing the production of galactooligosaccharides (GOSs). We prepared deletion mutants and point-mutated forms of rBgaD-A (deletion of the BgaD signal peptide) to compare their reaction behaviors. The yields of GOSs for all of the point-mutated forms as well as the deletion mutants of rBgaD-As increased as compared to rBgaD-A. In particular, W1540A mutant BgaD-A (rBgaD-A_W1540A) produced much more GOSs than rBgaD-A. Surface plasmon resonance experiments indicated that both the wildtype and the W1540A mutant DS domains showed high affinity for galactosyllactose. rBgaD-A, which has a wild-type DS domain, showed high hydrolytic activity toward galactosyllactose, while the hydrolytic activities of rBgaD-D, without a DS domain, and rBgaD-A_W1540A, with a mutant DS domain were extremely low. The findings obtained in this study indicate that the wild-type DS domain of rBgaD-A has a function that aids galactosyllactose molecules to be properly oriented within the active site, so that they can be hydrolyzed efficiently to produce galactose/glucose by inhibiting the accumulation of GOSs.

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  • Impact of compression, physical aging, and freezing rate on the crystallization characteristics of an amorphous sugar matrix Reviewed

    Koreyoshi Imamura, Kohshi Kinugawa, Ryo Kagotani, Mayo Nomura, Kazuhiro Nakanishi

    JOURNAL OF FOOD ENGINEERING   112 ( 4 )   313 - 318   2012.10

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    Amorphous matrices made up of sugar molecules, are frequently used in food and pharmaceutical industries. A drawback to their use is that they are susceptible to collapse, as a result of water uptake and an increase in temperature and subsequently crystallize. Herein, the crystallization characteristics of amorphous sugar (sucrose and alpha-lactose) preparations were analyzed, with the purpose of obtaining knowledge that could lead to the prediction of how long the amorphous state is retained under various conditions. The impact of compression, physical aging and freezing rate on the induction period (t(ind)) for crystallization were examined. Freeze-dried sugar samples were compressed at 74 or 443 MPa (5 min) and then rehumidified at specified RHs. Some freeze-dried sucrose samples were physically aged, and alternatively freeze-drying was conducted under different conditions. The isothermal crystallization of the prepared samples at different temperatures (T), the glass transition and the crystallization temperature (T-cry) were measured, using differential scanning calorimetry. The compression markedly decreased the t(ind), while significantly lowered the hygroscopicity. Physical aging and slower-freezing also shortened the t(ind). The t(ind) was found to be correlated exclusively with (T-cry-T), regardless of rehumidification, compression, sugar type, physical aging and freezing rate in the freeze-drying process. (C) 2012 Elsevier Ltd. All rights reserved.

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  • Heterogeneity of the state and functionality of water molecules sorbed in an amorphous sugar matrix Reviewed

    Koreyoshi Imamura, Ryo Kagotani, Mayo Nomura, Kohshi Kinugawa, Kazuhiro Nakanishi

    CARBOHYDRATE RESEARCH   351   108 - 113   2012.4

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    An amorphous matrix, comprised of sugar molecules, is frequently used in the pharmaceutical industry. An amorphous sugar matrix exhibits high hygroscopicity, and it has been established that the sorbed water lowers the glass transition temperature T-g of the amorphous sugar matrix. It is naturally expected that the random allocation and configuration of sugar molecules would result in heterogeneity of states for sorbed water. However, most analyses of the behavior of water, when sorbed to an amorphous sugar matrix, have implicitly assumed that all of the sorbed water molecules are in a single state. In this study, the states of water molecules sorbed in an amorphous sugar matrix were analyzed by Fourier-transform IR spectroscopy and a Fourier self-deconvolution technique. When sorbed water molecules were classified into five states, according to the extent to which they are restricted, three of the states resulted in a lowering of T-g of an amorphous sugar matrix, while the other two were independent of the plasticization of the matrix. This finding provides an explanation for the paradoxical fact that compression at several hundreds of MPa significantly decreases the equilibrium water content at a given RH, while the T-g remains unchanged. (C) 2011 Elsevier Ltd. All rights reserved.

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  • Rod-shaped Hydroxyapatite Nanoparticle Formation by Maturation with AOT Reviewed

    Jun Oshitani, Nobuhide Kataoka, Mikio Yoshida, Kuniaki Gotoh, Koreyoshi Imamura

    CHEMISTRY LETTERS   40 ( 10 )   1085 - 1086   2011.10

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    Amorphous calcium phosphate nanoparticles were synthesized by mixing aqueous solutions of Ca(NO3)(2) and (NH4)(2)-HPO4. Then the particles were matured with sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) at various temperatures. Rod-shaped hydroxyapatite (HAp) nanoparticles were formed at maturation temperature &gt;= 40 degrees C. These results indicate that addition of AOT nor during the particles synthesis but during the maturation at more equal critical temperature is a key to form the rod-shaped HAp nanoparticles using AOT.

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  • Cloning and Expression of a beta-Galactosidase Gene of Bacillus circulans Reviewed

    Jingyuan Song, Hiroyuki Imanaka, Koreyoshi Imamura, Masashi Minoda, Toru Katase, Yukiko Hoshi, Shotaro Yamaguchi, Kazuhiro Nakanishi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   75 ( 6 )   1194 - 1197   2011.6

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    A gene of beta-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta (R). Using the cloned gene, recombinant beta-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.

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  • Rod-shaped Hydroxyapatite Nanoparticle Formation Using AOT and Water without Oil Reviewed

    Jun Oshitani, Nobuhide Kataoka, Mikio Yoshida, Kuniaki Gotoh, Koreyoshi Imamura, Hidekazu Tanaka

    CHEMISTRY LETTERS   40 ( 4 )   400 - 401   2011.4

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    We synthesized rod-shaped hydroxyapatite (HAp) nanoparticles using sodium bis(2-ethylhexyl) sulfosuccinate (AOT) as an additive in water without an oil. The length of the particles increases from 40 to 100 nm with increasing the concentration ratio AOT/Ca while maintaining a width of 12 nm. These results indicate that an oil phase is not necessarily needed to form the rod-shaped HAp nanoparticles using AOT.

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  • Influence of compression on water sorption, glass transition, and enthalpy relaxation behavior of freeze-dried amorphous sugar matrices Reviewed

    Koreyoshi Imamura, Ryo Kagotani, Mayo Nomura, Kazuhiro Tanaka, Kohshi Kinugawa, Kazuhiro Nakanishi

    INTERNATIONAL JOURNAL OF PHARMACEUTICS   408 ( 1-2 )   76 - 83   2011.4

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    An amorphous matrix comprised of sugar molecules are frequently used in the pharmaceutical industry. The compression of the amorphous sugar matrix improves the handling. Herein, the influence of compression on the water sorption of an amorphous sugar matrix was investigated. Amorphous sugar samples were prepared by freeze-drying, using several types of sugars, and compressed at 0-443 MPa. The compressed amorphous sugar samples as well as uncompressed samples were rehumidified at given RHs, and the equilibrium water content and glass transition temperature (T-g) were then measured. Compression resulted in a decrease in the equilibrium water content of the matrix, the magnitude of which was more significant for smaller sized sugars. Diffusivity of water vapor in the sample was also decreased to one-hundredth by the compression. The T-g value for a given RH remained unchanged, irrespective of the compression. Accordingly, the decrease in T-g with increasing water content increased as the result of compression. The structural relaxation of the amorphous sugar matrices were also examined and found to be accelerated to the level of a non-porous amorphous sugar matrix as the result of the compression. The findings indicate that pores contained in freeze-dried sugar samples interfere with the propagation of structural relaxation. (C) 2011 Elsevier B.V. All rights reserved.

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  • Causes of the Production of Multiple Forms of beta-Galactosidase by Bacillus circulans Reviewed

    Jingyuan Song, Kiriko Abe, Hiroyuki Imanaka, Koreyoshi Imamura, Masashi Minoda, Shotaro Yamaguchi, Kazuhiro Nakanishi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   75 ( 2 )   268 - 278   2011.2

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    The presence of multiple types of beta-galactosidases in a commercial enzyme preparation from Bacillus circulans ATCC 31382 and differences in their transgalactosylation activity were investigated. Four beta-galactosidases, beta-Gal-A, beta-Gal-B, beta-Gal-C, and beta-Gal-D, which were immunologically homologous, were isolated and characterized. The N-terminal amino acid sequences of all of the enzymes were identical and biochemical characteristics were similar, except for galactooligosaccharide production. beta-Gal-B, beta-Gal-C, and beta-Gal-D produced mainly tri- and tetra saccharides at maximum yields of 20-30 and 9-12%, while beta-Gal-A produced trisaccharide with 7% with 5% lactose as substrate. The Lineweaver-Burk plots for all of the enzymes, except for beta-Gal-A, showed biphasic behavior. beta-Gal-A was truncated to yield multiple beta-galactosidases by treatment with protease isolated from the culture broth of B. circulans. Treatment of beta-Gal-A with trypsin yielded an active 91-kDa protein composed of 21-kDa and 70-kDa proteins with characteristics similar to those for beta-Gal-D.

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  • Multiple States of Sorbed Water Molecules in Amorphous Sugar Matrix andtheir Different Impacts on Physical Properties of the Matrix Reviewed

    Koreyoshi Imamura

    japan journal of food engineering   12 ( 1 )   1 - 9   2011

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    Amorphous materials, comprised of sugar molecules, exhibit high hygroscopicity and the sorbed water exerts a major influence on physical stability of the matrix. To date, the water sorption behavior of amorphous sugars has been extensively investigated. However, most studies of the behavior of water, when sorbed to an amorphous sugar matrix, have implicitly assumed that all of the sorbed water molecules are in a single state: It is naturally expected that the random allocation and configuration of sugar molecules would result in heterogeneity of states for the sorbed water. Recently, we demonstrated the heterogeneity of state and functionality of water sorbed in amorphous sugar matrices by combining a Fourier transform IR spectroscopy and Fourier self-deconvolution technique
    The sorbed water molecules were classified into five states, and the three of them mainly served to lower the glass transition temperature of an amorphous sugar matrix while the other two appeared to be independent of physical properties of the matrix. © 2011, Japan Society for Food Engineering. All rights reserved.

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  • Water Sorption, Glass Transition, and Protein-Stabilizing Behavior of an Amorphous Sucrose Matrix Combined With Various Materials Reviewed

    Koreyoshi Imamura, Toru Yokoyama, Atsushi Fukushima, Mitsunori Kinuhata, Kazuhiro Nakanishi

    JOURNAL OF PHARMACEUTICAL SCIENCES   99 ( 11 )   4669 - 4677   2010.11

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    The effects of various additives on the physical properties of an amorphous sugar matrix were compared. Amorphous, sugar-additive mixtures were prepared by freeze-drying and then rehumidified at given RHs. Sucrose and eighteen types of substances were used as the sugar and the additive, respectively, and water sorption, glass-to-rubber transition, and protein stabilization during freeze-drying for the various sucrose-additive mixtures were examined. The additives were categorized into two groups according to their effects on T(g) and water sorption. Presence of polysaccharides, cyclodextrins, and polymers (large-sized additives) resulted in a decrease in equilibrium water content from the ideal value calculated from individual water contents for sucrose and additive, and in contrast, low MW substances containing ionizable groups (small-ionized additives) resulted in an increase. The increase in T(g) by the addition of large-sized additives was significant at the additive contents &gt;50 wt.% whereas the T(g) was markedly increased in the lower additive content by the addition of small-ionized additives. The addition of small-ionized additives enhanced the decrease in T(g) with increasing water content. The protein stabilizing effect was decreased with increasing additive content in the cases of the both groups of the additives. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:4669-4677, 2010

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  • Development of a highly efficient indigo dyeing method using indican with an immobilized beta-glucosidase from Aspergillus niger Reviewed

    Jingyuan Song, Hiroyuki Imanaka, Koreyoshi Imamura, Kouichi Kajitani, Kazuhiro Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   110 ( 3 )   281 - 287   2010.9

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    A highly efficient method for dyeing textiles with indigo is described. In this method, the substrate, indican is first hydrolyzed at an acidic pH of 3 using an immobilized beta-glucosidase to produce indoxyl, under which conditions indigo formation is substantially repressed. The textile sample is then dipped in the prepared indoxyl solution and the textile is finally exposed to ammonia vapor for a short time, resulting in rapid indigo dyeing. As an enzyme, we selected a beta-glucosidase from Aspergillus niger, which shows a high hydrolytic activity towards indican and was thermally stable at temperatures up to 50-60 degrees C, in an acidic pH region. The A. niger beta-glucosidase, when immobilized on Chitopearl BCW-3001 by treatment with glutaraldehyde, showed an optimum reaction pH similar to that of the free enzyme with a slightly higher thermal stability. The kinetics for the hydrolysis of indican at pH 3, using the purified free and immobilized enzymes was found to follow Michaelis-Menten type kinetics with weak competitive inhibition by glucose. Using the immobilized enzyme, we successfully carried out repeated-batch and continuous hydrolyses of indican at pH 3 when nitrogen gas was continuously supplied to the substrate solution. Various types of model textiles were dyed using the proposed method although the color yield varied, depending on the type of textile used. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

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  • Influences of properties of protein and adsorption surface on removal kinetics of protein adsorbed on metal surface by H2O2-electrolysis treatment Reviewed

    Koreyoshi Imamura, Manabu Oshita, Masumi Iwai, Toshiki Kuroda, Ippei Watanabe, Takaharu Sakiyama, Kazuhiro Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   345 ( 2 )   474 - 480   2010.5

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    "H2O2-electrolysis" treatment is an alternative method for removing proteinaceous materials that are adsorbed to metal surfaces. The method is based on the generation of hydroxyl radicals by electrolysis of hydrogen peroxide and the subsequent decomposition of organic substances adhering to the metal surface. We herein investigated the influence of some parameters on the kinetics of protein removal by H2O2-electrolysis. These parameters included the properties of proteins and the type of metal surface. Sixteen types of proteins and nine types of metal surfaces were used. The removal of adsorbed protein from a metal surface during the treatment was monitored by ellipsometry. Apparent first-order rate constants for removal, k(cl), for various adsorption and treatment conditions were determined. The k(cl) value varied markedly with the type of protein and was also influenced by the pH used in the adsorption. The isoelectric point (pI) of protein used was found to be a major factor. The amount of adsorbed protein removed by a unit amount of (OH)-O-center dot was larger for a metal surface with a lower pI. The impact of the properties of the protein and metal surface on the removal kinetics are discussed, focusing on relationships with the adsorption characteristics of the protein. (C) 2010 Elsevier Inc. All rights reserved.

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  • Impacts of Compression on Crystallization Behavior of Freeze-Dried Amorphous Sucrose Reviewed

    Koreyoshi Imamura, Mayo Nomura, Kazuhiro Tanaka, Nobuhide Kataoka, Jun Oshitani, Hiroyuki Imanaka, Kazuhiro Nakanishi

    JOURNAL OF PHARMACEUTICAL SCIENCES   99 ( 3 )   1452 - 1463   2010.3

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    An amorphous matrix comprised of sugar molecules is used as excipient and stabilizing agent for labile ingredients in the pharmaceutical industry. The amorphous sugar matrix is often compressed into a tablet form to reduce the volume and improve handling. Herein, the effect of compression on the crystallization behavior of an amorphous sucrose matrix was investigated. Amorphous sucrose samples were prepared by freeze-drying and compressed under different conditions, followed by analyses by differential scanning calorimetry, isothermal crystallization tests, X-ray powder diffractometry, Fourier transform infrared spectroscopy (FTIR), and gas pycnometry. The compressed sample had a lower crystallization temperature and a shorter induction period for isothermal crystallization, indicating that compression facilitates the formation of the critical nucleus of a sucrose crystal. Based on FTIR and molecular dynamics simulation results, the conformational distortion of sucrose molecules due to the compression appears to contribute to the increase in the free energy of the system, which leads to the facilitation of critical nucleus formation. An isothermal crystallization test indicated an increase in the growth rate of sucrose crystals by the compression. This can be attributed to the transformation of the microstructure from porous to nonporous, as the result of compression. (C) 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1452-1463, 2010

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  • Cultivation characteristics and gene expression profiles of Aspergillus oryzae by membrane-surface liquid culture, shaking-flask culture, and agar-plate culture Reviewed

    Hiroyuki Imanaka, Soukichi Tanaka, Bin Feng, Koreyoshi Imamura, Kazuhiro Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   109 ( 3 )   267 - 273   2010.3

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    We cultivated a filamentous fungus, Aspergillus oryzae IAM 2706 by three different cultivation methods, i.e., shaking-flask culture (SFC), agar-plate culture (APC), and membrane-surface liquid culture (MSLC), to elucidate the differences of its behaviors by different cultivation methods under the same media, by measuring the growth, secretion of proteases and alpha-amylase, secreted protein level, and gene transcriptional profile by the DNA microarray analysis. The protease activities detected by MSLC and APC were much higher than that by SFC, using both modified Czapek-Dox (mCD) and dextrin-peptone-yeast extract (DPY) media. The alpha-amylase activity was detected in MSLC and APC in a much larger extent than that in SFC when DPY medium was used. On the basis of SDS-PAGE analyses and N-terminal amino acid sequences, 6 proteins were identified in the supernatants of the culture broths using DPY medium, among which oryzin (alkaline protease) and alpha-amylase were detected at a much higher extent for APC and MSLC than those for SFC while only oryzin was detected in mCD medium, in accordance with the activity measurements. A microarray analysis for the fungi cultivated by SFC, APC, and MSLC using mCD medium was carried out to elucidate the differences in the gene transcriptional profile by the cultivation methods. The gene transcriptional profile obtained for the MSLC sample showed a similar tendency to the APC sample while it was quite different from that for the SFC sample. Most of the genes specifically transcribed in the MSLC sample versus those in the SFC sample with a 10-fold up-regulation or higher were unknown or predicted proteins. However, transcription of oryzin gene was only slightly up-regulated in the MSLC sample and that of alpha-amylase gene, slightly down-regulated. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

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  • 低温度領域における糖類アモルファスマトリクスのタンパク質安定化作用と糖分子の束縛状態

    今村 維克, 横山 徹, 大山 健一, 絹畠 光倫, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2010   584 - 584   2010

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  • 各種糖類アモルファスマトリクスに収着した水の状態とガラス転移温度との関係

    籠谷 亮, 衣川 耕史, 野村 真世, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2010   583 - 583   2010

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  • Streptmyces mobaraensis由来epsilon-Lysine acylaseによるN-epsilon-Lauroyl-L-Lysineの効率的合成

    内田 達也, 田口 友造, 今中 洋行, 今村 維克, 是石 真友子, 中西 一弘

    化学工学会 研究発表講演要旨集   2010   1003 - 1003   2010

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  • クッションタンパク質を用いた機能的ペプチド固定化法の検討

    國方 俊暢, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2010   997 - 997   2010

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  • Development of peptide immobilization method using PS-tagged cushion protein Reviewed

    Hiroyuki Imanaka, Daisuke Yamazumi, Toshinobu Kunikata, Koreyoshi Imamura, Kazuhiro Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   108   S161 - S161   2009.11

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  • Formation characteristics of calcium phosphate deposits on a metal surface by H2O2-electrolysis reaction under various conditions Reviewed

    Kazuaki Kanamoto, Koreyoshi Imamura, Nobuhide Kataoka, Jun Oshitani, Hiroyuki Imanaka, Kazuhiro Nakanishi

    COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS   350 ( 1-3 )   79 - 86   2009.10

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    The cathodic electrolysis of H2O2 (H2O2 + e(-) -&gt; OH- + (OH)-O-center dot) on a metal surface in the presence of calcium and phosphate ions results in the formation of calcium phosphate deposits oil the metal surface. In this Study, the deposits formed under various treatment conditions (pHs, concentrations and ratios of calcium/phosphate ions, and so on) were characterized by scanning electron spectroscopy (SEM). and X-ray diffractometry. The exclusive formation of hydroxyapatite, HAP, was observed under comparatively narrow conditions (pH 3-4, [Ca+]/[PO43-] = 25 mM/15 mM), which is clearly different from the reported conditions for the deposition of HAP on titanium substrates. HAP was deposited in the form of a layer. comprised of morphologically amorphous HAP flakes that were less than 20 nm thick. SEM and FTIR analyses of the deposit at different stages of H2O2-electrolysis revealed that a few dozen nanometer-sized spheres of amorphous calcium phosphate were formed in the first step and then fused with each other to form ribbon-like flakes of HAP or broken glass-like brushite, depending on the pH. The pH for HAP formation oil a stainless steel surface was markedly lower than that used for titanium, and the observed process by which amorphous calcium phosphate is converted to HAP was markedly different from that for the electrochemical deposition (electrolysis of water) of HAP on a titanium substrate. (C) 2009 Elsevier B.V. All rights reserved.

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  • Temperature Scanning FTIR Analysis of Secondary Structures of Proteins Embedded in Amorphous Sugar Matrix Reviewed

    Koreyoshi Imamura, Ken-Ichi Ohyama, Toru Yokoyama, Yoshinobu Maruyama, Nakanishi Kazuhiro

    JOURNAL OF PHARMACEUTICAL SCIENCES   98 ( 9 )   3088 - 3098   2009.9

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    Heat-induced changes in secondary structures of five proteins (bovine serum albumin, BSA; human serum albumin, HSA; myoglobin; ribonuclease A, RNase A; and, beta-lactoglobulin, beta-Lg) in an amorphous sugar matrix were analyzed by temperature-scanning Fourier transform infrared spectroscopy to elucidate the mechanism of heat-induced conformational change of solid-phase proteins. Three sugars, trehalose, maltose, and dextran (MW 6000), were used. Loss of alpha-helices due to increasing temperature was observed for BSA, HSA, and myoglobin, which are rich in a-helices. RNase A showed a marked decrease in predominant secondary structural components (beta-sheet) with increasing temperature. However, no noticeable changes in the content of secondary structures, except for a slight loss of alpha-helices, were observed for beta-Lg, which is also beta-sheet-rich. These heat-induced conformational changes were significant at temperatures above the glass transition temperature. The heat-induced conformational change in BSA dried with sugar appeared time-independent and was clearly different from that due to dehydration and from the thermal conformational change for a solution of BSA. In particular, differences in secondary structural components that increased due to loss of alpha-helices were noted. (c) 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3088-3098, 2009

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  • Purification, Characterization, Molecular Cloning, and Expression of a New Aminoacylase from Streptomyces mobaraensis That Can Hydrolyze N-(Middle/Long)-chain-fatty-acyl-L-amino Acids as Well as N-Short-chain-acyl-L-amino acids Reviewed

    Mayuko Koreishi, Yasuyuki Nakatani, Manami Ooi, Hiroyuki Imanaka, Koreyoshi Imamura, Kazuhiro Nakanishi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   73 ( 9 )   1940 - 1947   2009.9

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    We report here on the purification, characterization, molecular cloning, and expression of a new aminoacylase, initially isolated from the supernatant of Streptomyces mobaraensis (Sm-AA). Purified wild-type Sm-AA was found to be a monomeric protein with a molecular mass of 55kDa. The cloned gene of Sm-AA contained an ORF of 1,383 bp, encoding a polypeptide of 460 amino acids. A BLAST search revealed that Sm-AA belongs to the peptidase M20 family, with identities to a hypothetical protein from Streptomyces pristinaespiralis, a putative peptidase from Streptomyces averinitilis, peptidase M20 from Frankia sp., succinyl-diaminopimelate desuccinylase from Hemophilus influenzae, and aminoacylase-1 from porcine kidney at 89, 88, 67, 29, and 25% respectively. The Sm-AA gene was subcloned into an expression vector, pSH19, and was expressed in Streptomyces lividans TK24. The amount of the recombinant Sm-AA expressed in the S. lividans cells was approximately 42-fold higher than that of Sm-AA found in the supernatant of S. mobaraensis. Sm-AA showed high hydrolytic activity towards various N-acetyl-L-amino acids and N-(middle/long)-chain-fatty-acyl-L-amino acids, with a preference for the acyl derivatives of L-Met, L-Ala, L-Cys, etc. with an optimum pH and temperature for reaction of about 7.5 and 50 degrees C (at pH 7.5).

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  • Efficient N epsilon-lauroyl-L-lysine production by recombinant epsilon-lysine acylase from Streptomyces mobaraensis Reviewed

    Mayuko Koreishi, Ryoko Kawasaki, Hiroyuki Imanaka, Koreyoshi Imamura, Yasuaki Takakura, Kazuhiro Nakanishi

    JOURNAL OF BIOTECHNOLOGY   141 ( 3-4 )   160 - 165   2009.5

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    epsilon-Lysine acylase from Streptomyces mobaraensis (Sm-ELA), which specifically catalyzes hydrolysis of the epsilon-amide bond in various N epsilon-acyl-L-lysines, was cloned and sequenced. The Sm-ELA gene consists of a 1617-bp open reading frame that encodes a 538-amino acid protein with a molecular mass of 55,816 Da. An NCBI protein-protein BLAST search revealed that the enzyme belongs to the YtcJ-like metal-dependent amidohydrolase family, which is further characterized as the metallo-dependent hydrolase superfamily. The Sm-ELA gene was ligated into a pUC702 vector for expression in Streptomyces lividans TK24. Expression of recombinant Sm-ELA in S. lividans was approximately 300-fold higher than that in wild-type S. mobaraensis. The recombinant Sm-ELAs from the cell-free extract and Culture supernatant were purified to homogeneity. The specific activities of the purified Sm-ELAs were 2500-2800 U/mg, which were similar to that obtained for the wild-type Sm-ELA. Using the cell-free extract of the recombinant S. lividans cells, N epsilon-lauroyl-L-lysine was synthesized from 500 mM L-lysine hydrochloride and 50, 100, or 250 mM lauric acid in an aqueous buffer Solution at 37 degrees C. The yields were close to 100% after 6 and 9 1) of reaction for 50 and 100 mM lauric acid, respectively. and 90% after 24 h for 250 mM lauric acid. (C) 2009 Elsevier B.V. All rights reserved.

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  • PS-tag連結タンパク質を利用したバイオ分子間相互作用解析

    今中 洋行, 上崎 英範, 國方 俊暢, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2009   934 - 934   2009

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  • 糖類アモルファス材料の水分収着特性とタンパク質安定化作用に及ぼす高圧処理の影響

    今村 維克, 野村 真世, 木村 佳文, 伊久 珠代, 絹畠 光倫, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2009   779 - 779   2009

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  • クッションタンパク質を利用したバイオ分子固定化技術の開発と応用

    麻那古 秀友, 山隅 大輔, 大塚 隆尚, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2009   1060 - 1060   2009

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  • オートトランスポーターを用いたバイオ分子表層提示系

    今中 洋行, 植田 久子, 山下 麻衣, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2009   606 - 606   2009

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  • 糖類アモルファスマトリクスにおける糖-高分子間相互作用の形成特性

    今村 維克, 横山 徹, 大山 健一, 野村 真世, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2009   608 - 608   2009

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  • Recent advances in controlled immobilization of proteins onto the surface of the solid substrate and its possible application to proteomics Reviewed

    Kazuhiro Nakanishi, Takaharu Sakiyama, Yoichi Kumada, Koreyoshi Imamura, Hiroyuki Imanaka

    Current Proteomics   5 ( 3 )   161 - 175   2008.10

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    Proteome analysis plays a key role in the elucidation of the functions and applications for numerous proteins. For proteome analyses, various microplate- and microarray-based techniques have been developed by a number of researchers. Their intent was to immobilize proteins on the surface of a solid substrate in a site-directed manner while retaining structure and native biological function. In this review, we focus on recent advances in immobilization methodology for proteins/enzymes on a surface, including those using the affinity peptides screened by random peptide library systems. We also discuss applications of the affinity peptide-mediated immobilization method in fields related to proteome analysis, particularly our recent work concerning immunoassay and protein-protein interaction analysis. ©2008 Bentham Science Publishers Ltd.

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  • Adsorption Characteristics of Various Proteins to a Titanium Surface Reviewed

    Koreyoshi Imamura, Miyuki Shimomura, Shungo Nagai, Megumi Akamatsu, Kazuhiro Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   106 ( 3 )   273 - 278   2008.9

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    Adsorption characteristics of 18 proteins, with different sizes and isoelectric points, to a titanium oxide surface were studied. The adsorption isotherms were categorized based on protein type and pH: type 1, irreversible adsorption; type 2, Langmuir-type reversible adsorption; and type 3, reversible and irreversible adsorption. Most of the proteins tested were irreversibly adsorbed in the pH range of 3-8, whereas most adsorbed reversibly at pH 8.5-9.4. Protamine, with a pI value of 12, adsorbed reversibly in the pH range of 3-9. pH values that gave maximal sums of irreversibly and reversibly adsorbed proteins were in the pH range of 3-8 and tended to increase slightly with the pI value of the corresponding protein. pH values that gave maximal quantities of irreversibly adsorbed protein ranged between 4-6 and were nearly independent of pI.

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  • True density analysis of a freeze-dried amorphous sugar matrix Reviewed

    Koreyoshi Imamura, Yoshinobu Maruyama, Kazuhiro Tanaka, Tohru Yokoyama, Hiroyuki Imanaka, Kazuhiro Nakanishi

    JOURNAL OF PHARMACEUTICAL SCIENCES   97 ( 7 )   2789 - 2797   2008.7

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    True density of an amorphous matrix represents the state of molecular packing in the matrix, which is closely related to the physical/chemical properties of the material. Dry gas pycnometry is one possible technique for measuring the true density of an amorphous sugar matrix prepared by freeze-drying. We herein report on the influence of conditions used for pycnometry on the measured density value and propose a protocol for obtaining the true density. The technique is sufficiently accurate to permit values for matrices comprised of different types of sugar to be compared. Using the protocol, the true densities of several amorphous sugar samples containing different types of sugar, freeze-drying conditions (temperature and sugar concentration at the time of freezing of an aqueous sugar solution), pretreatment (compaction and grind) were determined and the results were compared. A model for simulating an amorphous matrix of sugar (trehalose) was constructed using molecular dynamics/mechanics calculations, and the true density of the simulated sugar matrix was found to agree with the value experimentally determined using the proposed protocol. The relationship among the true density, the states of intermolecular interactions, and strain of sugar molecules in the matrix are discussed using the simulated amorphous sugar matrix. (C) 2007 Wiley-Liss, Inc.

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  • Characteristics of hydrogen bond formation between sugar and polymer in freeze-dried mixtures under different rehumidification conditions and its impact on the glass transition temperature Reviewed

    Koreyoshi Imamura, Yoko Asano, Yoshinobu Maruyama, Tohru Yokoyama, Mayo Nomura, Seiji Ogawa, Kazuhiro Nakanishi

    JOURNAL OF PHARMACEUTICAL SCIENCES   97 ( 3 )   1301 - 1312   2008.3

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    The characteristics of hydrogen bond formation between trehalose and polyvinylpyrrolidone (PVP) in amorphous mixtures at different hydration states were quantitatively investigated. Amorphous trehalose-PVP mixtures were prepared by freeze-drying and equilibrated at different relative humidities (RH). Infrared (IR) spectra of the trehalose-PVP mixtures were obtained by Fourier transform IR spectroscopy,(FTIR) and the IRband corresponding to C=O groups of PVP was deconvolved into the component bands responsible for C=O groups that were free and restricted by hydrogen bonds, to estimate the degree of the trehalose-PVP interactions. The FTIR analysis indicated that approximately 80% of the C=O groups of PVP formed hydrogen bonds with trehalose in the presence of more than 3 g of trehalose per gramme of PVP, independent of the RH. IR analysis of the O-H stretching vibration of the sugar demonstrated that the presence of PVP lead to an increase in the free hydroxyl groups of trehalose that did not form hydrogen bonds at RH 0%. On the other hand, the water sorption behavior of the trehalose-PVP mixtures suggested that rehumidification diminished the effect of PVP on increasing the free OH groups. Thus a peculiar relationship may exist between T-g, RH and the composition of the mixture: The presence of PVP increased T-g at RHs 0 and above 23% but decreased T-g at 11%. (C) 2007 Wiley-Liss, Inc.

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  • Temperature scanning FTIR analysis of interactions between sugar and polymer additive in amorphous sugar-polymer mixtures Reviewed

    Koreyoshi Imamura, Ken-Ichi Ohyama, Toru Yokoyama, Yoshinobu Maruyama, Hiroyuki Imanaka, Kazuhiro Nakanishi

    JOURNAL OF PHARMACEUTICAL SCIENCES   97 ( 1 )   519 - 528   2008.1

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    The impact of a polymer additive (polyvinylpyrrolidone, PVP) on hydrogen bonding in amorphous sugar matrices as well as on the glass transition temperature, T-g, were examined by temperature scanning Fourier transform infrared spectroscopy (TS-FTIR). An amorphous sugar matrix containing PVP was prepared by air-drying an aqueous solution of a sugar-PVP mixture. The hydrogen bonds in the sugar-PVP mixture (sugar-PVP and sugar-sugar hydrogen bonds) were analyzed from the IR peak positions corresponding to the stretching vibration of C=O groups of PVP and O-H groups of the sugar and the temperature dependence of the peak position of the O-H stretching vibration band. The addition of PVP to amorphous mono and disaccharides significantly lowered the extent of hydrogen bond formation while interactions between sugars and the PVP tended to prevent the disruption of hydrogen bonds due to increasing temperature, the magnitude of which was larger for larger oligomers. The T-g value for the amorphous sugar was increased by the addition of PVP in many cases. As the size of sugar molecule became larger, the relative magnitude of the increased T-g by PVP to the difference between the T-g values for sugar alone and PVP alone became larger and then reached a certain level; it was slight in the case of glucose. Collectively, these results demonstrate that the magnitude of the impact of PVP on an amorphous sugar matrix strongly vary and are dependent on the types of sugar. (C) 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:519-528, 2008.

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  • Fourier self-deconvolution analysis of hydrogen bonding states of polyvinylpyrrolidone in an amorphous sugar matrix below and above the glass transition temperature Reviewed

    Koreyoshi Imamura, Ken-ichi Ohyama, Kazuko Tani, Toru Yokoyama, Yoshinobu Maruyama, Hiroyuki Imanaka, Kazuhiro Nakanishi

    SPECTROSCOPY LETTERS   41 ( 6 )   305 - 312   2008

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    In an amorphous mixture of sugar and polyvinylpyrrolidone (PVP), PVP carbonyl groups form hydrogen bonds with sugar hydroxyl groups, thereby improving the physical stability of the amorphous matrix against a glass-to-rubber transition. Herein, Fourier self-deconvolved IR bands due to the C=O stretching vibration of PVP in sugar-PVP mixtures were analyzed. The C=O groups in sugar-PVP mixtures generally had four vibrational states, corresponding with free and hydrogen-bonded C=O in three different modes. Changes in these vibrational states induced by increasing the temperature were compared among various sugar-PVP mixtures. Formation and thermal disruption characteristics of different modes of sugar-PVP hydrogen bondings are discussed.

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  • Factors Affecting Performance of Cleaning Technique for Metal Surfaces Based on Electrolysi of Hydrogen Peroxide, H2O2-electrolysis

    IMAMURA Koreyoshi

    NIHON SHOKUHIN KOUGAKUKAISHI (Japan Journal of Food Engineering)   9 ( 4 )   229 - 238   2008

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    We have developed a novel technique for removing organic soilings from metal surfaces using hydroxy radicals (•OH) . In the cleaning system, a metal surface fouled with organic soilings is made to contact with an aqueous solution containing hydrogen peroxide and supporting electrolyte. A slightly negative potential is then applied into the metal. The •OHs, which are generated by the electrolysis of hydrogen peroxide (H2O2+e-> •OH+OH-) on the metal surface, effectively attack and subsequently remove the adsorbed organic soils. In this study, the removal behavior of model organic soilings (protein) during the H2O2-electrolysis cleaning using different types and concentrations of supporting electrolyte was investigated. The presence of ammonium compounds and potassium phosphate led to marked increase in the removal rate. The influences of the types of metal substrate and adsorbed material on the removal behavior were also investigated, which suggested that the adsorption state of organic soiling on a metal surface strongly affect the removal characteristics. Furthermore, the removal rates in the presence of various organic coexisting materials were investigated. It was found that the removal rate constant was only slightly lowered in many cases.

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  • 高圧処理における糖類アモルファスマトリクスの物理特性変化

    今村 維克, 野村 真世, 横山 徹, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2008   339 - 339   2008

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    DOI: 10.11491/scej.2008.0.339.0

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  • 膜面液体培養法を用いた麹菌の培養特性及び遺伝子発現

    田中 創吉, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2008   378 - 378   2008

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  • 親和性ペプチドを利用したポリスチレンおよびガラス表面への蛋白質固定化とその利用

    今中 洋行, 上崎 英範, 石村 遼太, 熊田 陽一, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2008   345 - 345   2008

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  • クッションタンパク質を利用したバイオ分子固定化技術の開発

    山隅 大輔, 柳田 圭介, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2008   418 - 418   2008

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  • 糖類アモルファスマトリクスの包括安定化作用および物理的諸特性に及ぼす高圧処理の影響

    今村 維克, 野村 真世, 田中 一宏, 木村 佳文, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2008   175 - 175   2008

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  • ヒト由来転写因子とDNAとの相互作用解析システムの検討

    今中 洋行, 前川 真光, 今村 維克, 近藤 英作, 中西 一弘

    化学工学会 研究発表講演要旨集   2008   955 - 955   2008

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    DOI: 10.11491/scej.2008f.0.955.0

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  • TPR domain of Ser/Thr phosphatase of Aspergillus oryzae shows no auto-inhibitory effect on the dephosphorylation activity Reviewed

    Bin Feng, Chun-Hui Zhao, Soukichi Tanaka, Hiroyuki Imanaka, Koreyoshi Imamura, Kazuhiro Nakanishi

    INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES   41 ( 3 )   281 - 285   2007.8

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    A Ser/Thr phosphatase gene cloned from Aspergillus oryzae, aoppt, revealed that the tetratricopeptide repeat (TPR) and catalytic domains of the full-length AoPPT are located at the N- and C-terminal regions, respectively, similar to those of human Ser/Thr phosphatase 5 (PP5) and yeast Ppt1. Four different regions of AoPPT, namely, a full-length polypeptide, the catalytic domain, the catalytic domain plus C-terminal 15 amino-acid residues and the TPR domain were expressed in Escherichia coli and their roles in dephosphorylation activity were examined, using p-nitrophenyl phosphate as the substrate. The full-length AoPPT showed the highest dephosphorylation activity while the catalytic domain had the lowest activity. The activity of the catalytic domain was not inhibited by the presence of the TPR domain and arachidonic acid did not increase the activity of the full-length enzyme. These findings suggest that the integrity of the entire enzyme would be necessary for its full activity to be expressed. (c) 2007 Elsevier B.V. All rights reserved.

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  • Enzymatic synthesis of beta-lactam antibiotics and N-fatty-acylated amino compounds by the acyl-transfer reaction catalyzed by penicillin V acylase from Streptomyces mobaraensis Reviewed

    Mayuko Koreishi, Kazuha Tani, Yuuichi Ise, Hiroyuki Imanaka, Koreyoshi Imamura, Kazuhiro Nakanishi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   71 ( 6 )   1582 - 1586   2007.6

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    Penicillin V acylase from Streptomyces mobaraensis (Sm-PVA) showed high acyl-transfer activity in reactions using methyl esters of carboxylic acid (acyl donor) and amino compounds (nucleophile), to produce the corresponding amides. Moreover, Sm-PVA had broad substrate specificity, as indicated by the fact that it catalyzed the efficient synthesis of fl-lactam antibiotics, capsaicin derivatives, and N-fatty-acyl-amino acid/N-fatty-acyl-peptide derivatives.

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  • Cloning and characterization of penicillin V acylase from Streptomyces mobaraensis Reviewed

    Demin Zhang, Mayuko Koreishi, Hiroyuki Imanaka, Koreyoshi Imamura, Kazuhiro Nakanishi

    JOURNAL OF BIOTECHNOLOGY   128 ( 4 )   788 - 800   2007.3

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    We report on the molecular cloning and characterization of penicillin V acylase (PVA) from an actinomycete, Streptomyces mobaraensis (Sm-PVA), which was originally isolated as an acylase that efficiently hydrolyzes the amide bond of various N-fatty-acyl-L-amino acids and N-fatty-acyl-peptides as well as capsaicin (8-methyl-N-vanillyl-6-nonenamide). In addition, the purified Sm-PVA hydrolyzed penicillin V with the highest activity (k(cat)) among the PVAs so far reported, penicillin G, and 2-nitro-5-phenoxyacetamide benzoic acid. The BLAST search revealed that the Sm-PVA precursor is composed of a polypeptide that is characteristic of enzymes belonging to the beta-lactam acylase family with four distinct segments; a signal sequence (43 amino acids), an a subunit (173 amino acids), a linker peptide (28 amino acids), and a beta subunit (570 amino acids). The mature, active Sm-PVA is a heterodimeric protein with alpha and beta subunits, in contrast to PVAs isolated from Bacillus sphaericus and B. subtilis, which have a homotetrameric structure. The amino acid sequence of Sm-PVA showed identities to PVA from S. lavendulae, N-acylhomoserine lactone-degrading acylase from Streptomyces sp., cyclic lipopeptide acylase from Streptomyces sp., and aculeacin A acylase from Actinoplanes utahensis with 68, 67, 67, and 41% identities, respectively. (c) 2007 Elsevier B.V. All rights reserved.

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  • Protein-protein interaction analysis using an affinity peptide tag and hydrophilic polystyrene plate Reviewed

    Yoichi Kumada, Chunhui Zhao, Ryota Ishimura, Hiroyuki Imanaka, Koreyoshi Imamura, Kazuhiro Nakanishi

    JOURNAL OF BIOTECHNOLOGY   128 ( 2 )   354 - 361   2007.2

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    A sandwich ELISA method using peptide tags showing a specific affinity to a hydrophilic polystyrene surface (PS-tags), PS 19 composed of RAFIASRRIKRP and KPS19R10 of KRAFIASRRIRRP and a hydrophilic polystyrene (phi-PS) plate was used to analyze protein-protein interactions. An Escherichia coli cysteine synthase complex, in which serine acetyltransferase (SAT) interacts with O-acetylserine sulfhydrylase-A (OASS) was used as a model system. When the interaction was detected by the conventional sandwich ELISA method using a hydrophobic polystyrene (pho-PS) plate, for the exclusive use of ELISA, the signal intensity was barely detectable due to conformational change of the ligand protein, OASS in the adsorbed state. On the contrary, when OASS, genetically fused with PS19 (OASS-PS19) or chemically conjugated with KPS19R10 (OASS-KPS19R10), was immobilized on the phi-PS plate, a high signal intensity was detected. Furthermore, by applying the two-step sandwich ELISA, in which OASS-PS19 or OASS-KPS19R10 formed a complex with SAT in the blocking solution before immobilization on the phi-PS plate, the signal intensity was further increased with a much shorter operational time, because SAT in the blocking solution formed a complex with OASS-PS19 or OASS-KPS19R10 without any steric hindrance. (c) 2006 Elsevier B.V. All rights reserved.

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  • Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface Reviewed

    Yoichi Kumada, Shigeo Katoh, Hiroyuki Imartaka, Koreyoshi Imamura, Kazuhiro Nakanishi

    JOURNAL OF BIOTECHNOLOGY   127 ( 2 )   288 - 299   2007.1

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    Glutathione S-transferase genetically fused with an affinity peptide tag, PS 19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS 19R10, in which (10)Lys in PS 19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS 19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immumosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein. (c) 2006 Elsevier B.V. All rights reserved.

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  • Adsorption characteristics of oligopeptides composed of acidic and basic amino acids on titanium surface Reviewed

    Koreyoshi Imamura, Yuuki Kawasaki, Takeshi Nagayasu, Takaharu Sakiyama, Kazuhiro Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   103 ( 1 )   7 - 12   2007.1

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    The adsorption characteristics of octapeptides, containing different numbers of aspartic acid, lysine, and alanine residues (i.e., D(4)K(0)A(4), D(4)K(1)A(3), D(4)K(3)A(1), D(4)K(4)A(0), and D(0)K(4)A(4)) on the surface of titanium (Ti) particles were investigated in the pH range of 3.0-8.8 at 30 degrees C. The adsorption isotherms for octapeptides having four plural aspartic acid residues with or without lysine residues showed two distinct adsorption modes, i.e., irreversible and reversible modes, at pHs 3.0-6.5; at pH 7.0 or higher, the adsorption mode was reversible. Increasing the number of lysine residues at a fixed number of aspartic acid residues (i.e., 4) decreased the amount of peptides adsorbed in both modes. D(4)K(4)A(0) adsorbed irreversibly at pHs 3.0-6.5, due to the fact that negatively charged carboxyl groups directly interact with a positively charged Ti surface, whereas positively charged amino groups of lysine residues are directed in an opposite direction toward the solution side, as predicted by molecular mechanics/dynamics calculations.

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  • オートトランスポーターを利用した異種タンパク質の大腸菌細胞表層提示系の構築

    今中 洋行, 山下 麻衣, 盛永 鈴香, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2007   417 - 417   2007

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  • PS-tagを用いたタンパク質間相互作用の検出

    熊田 陽一, Zhao Chunhui, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2007   427 - 427   2007

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  • 糖-高分子複合アモルファスマトリクスにおける分子間相互作用と物理的安定性

    今村 維克, 浅野 容子, 大山 健一, 丸山 佳伸, 崎山 高明, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2007   368 - 368   2007

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  • 過酸化水素-電気分解における金属表面でのハイドロキシアパタイト相の形成とその特性評価

    金本 和明, 今村 維克, 片岡 信秀, 押谷 潤, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2007   126 - 126   2007

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  • オートトランスポーターを用いた大腸菌細胞表層提示系に及ぼすシグナルペプチドの影響

    今中 洋行, 辰本 渉, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2007   842 - 842   2007

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  • 大腸菌由来Cysteine synthaseの複合体形成特性

    今中 洋行, 森賀 雄大, 趙 春暉, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2007   841 - 841   2007

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  • 過酸化水素-電気分解により生成したハイドロキシアパタイトの特性評価および骨芽様細胞の増殖・分化特性

    今村 維克, 金本 和明, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2007   990 - 990   2007

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  • H2O2-電気分解による金属表面および水溶液中の有機物質の分解・除去

    今村 維克, 黒田 俊樹, 小崎 正晴, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2007   702 - 702   2007

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  • Identification of genes from Aspergillus oryzae that are preferentially expressed in membrane-surface liquid culture Reviewed

    Bin Feng, Masakazu Morita, Hiroyuki Imanaka, Koreyoshi Imamura, Kazuhlro Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   102 ( 5 )   470 - 473   2006.11

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    We identified 22 genes from Aspergillus oryzae that are preferentially expressed in membrane-surface liquid culture (MSLC), among which Ser/Thr protein kinase (aopk1) and phosphatase (aoppt) genes were cloned. We also revealed that aopk1 encodes a protein with an N-terminal sequence 150 amino acid residues longer than that predicted from the registered sequence in GenBank.

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  • Effects of the supporting electrolyte on the kinetics of the removal of proteins adsorbed on a stainless steel surface by H2O2-electrolysis Reviewed

    Koreyoshi Imamura, Ippei Watanabe, Takaharu Sakiyama, Kazuhiro Nakanishi

    LANGMUIR   22 ( 16 )   7035 - 7040   2006.8

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    The effects of different types of supporting electrolytes on the removal of beta-lactoglobulin (beta-Lg) after being adsorbed to a stainless steel surface by a H2O2-electrolysis treatment was investigated. In this process, hydroxyl radicals (center dot OH), generated by the electrolysis of hydrogen peroxide, decompose the substances adhering to the surface. The removal of the adsorbed protein from the stainless steel surface during the treatment was monitored in situ by ellipsometry. The apparent first-order removal rate constants, k(cl), for 17 types of supporting electrolytes were determined, as well as the current corresponding to the rate of generation of center dot OH. The kcl and generated current values for LiCl, NaCl, KCl, KNO3, K2SO4, CH3COOK, and K2CO3 were all similar. Ca2+ and Mg2+ strongly suppressed the removal of the adsorbed protein. The presence of ammonium compounds led to an increase in kcl and current values. In H2O2-electrolysis in the presence of potassium phosphate, the removal was extremely rapid, and an apparent increase in the thickness of the adsorbed layer was observed. The mechanisms responsible for the peculiar effects of calcium, magnesium, phosphate, and ammonium compounds were investigated by means of a Fourier transform infrared (FTIR) spectroscopic analysis, as well as by the characteristics of the removal under different treatment conditions.

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  • Temperature scanning FTIR analysis of hydrogen bonding states of various saccharides in amorphous matrixes below and above their glass transition temperatures Reviewed

    Koreyoshi Imamura, Keisuke Sakaura, Ken-ichi Ohyama, Atsushi Fukushima, Hiroyuki Imanaka, Takaharu Sakiyama, Kazuhiro Nakanishi

    JOURNAL OF PHYSICAL CHEMISTRY B   110 ( 31 )   15094 - 15099   2006.8

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    Temperature scanning Fourier transform infrared, TS-FTIR, spectroscopy of various amorphous sugar matrixes was conducted to investigate the relationship between the glass transition temperature, T-g, of an amorphous sugar matrix and the nature of the hydrogen bonds in the matrix. An amorphous sugar matrix was prepared by air-drying an aqueous solution of sugar, and the degree of formation of hydrogen bonds in the matrix was evaluated at different temperatures using the peak positions of the IR band corresponding to the O-H stretching vibration at around 3400 cm(-1). The T-g value increased with increasing peak position of the O-H stretching vibration at T-g and were correlated reasonably well with the magnitude of the peak shift by the temperature increase (from 25 degrees C) to the T-g value. This demonstrates that the amorphous sugar matrix, in which the segments are fixed by fewer hydrogen bonds, has a higher thermal resistance. The glycosidic linkage largely contributes to the restriction of the segments, pyranose ring, rather than a hydrogen bond. As the degree of polymerization of pyranose rings increases, the degree of hydrogen bond formation needed to hold the matrix in a fixed position decreases. However, the magnitude of the restriction of pyranose rings by a glycosidic linkage changes depending on the type: the restrictions imposed by alpha-1,1 and -1,6 glycosidic linkages are the tightest and most flexible of all of the types of glycosidic linkages, respectively.

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  • Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli Reviewed

    C Zhao, Y Kumada, H Imanaka, K Imamura, K Nakanishi

    PROTEIN EXPRESSION AND PURIFICATION   47 ( 2 )   607 - 613   2006.6

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    O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis Of L-Cysteine from O-acetyl- L-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-13 was characterized and its properties compared with OASS-A. OASS-13 did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis. (c) 2006 Elsevier Inc. All rights reserved.

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  • Protease susceptibility of beta-lactoglobulin adsorbed on stainless steel surface as evidence of contribution of its specific segment to adsorption Reviewed

    Takaharu Sakiyama, Atsuko Aya, Mari Embutsu, Koreyoshi Imamura, Kazuhiro Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   101 ( 5 )   434 - 439   2006.5

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    beta-Lactoglobulin (beta-Lg) is a major constituent of fouling deposits in the dairy industry. To determine the interaction between beta-Lg and stainless steel surfaces, beta-Lg irreversibly adsorbed on stainless steel particles was subjected to lysyl endopeptidase treatment and the course of fragmentation was compared with that observed for beta-Lg in solution. The results showed a distinct difference between the courses of fragmentation: a fragment (residues 102-135) was liberated readily from beta-Lg in solution but scarcely from beta-Lg irreversibly adsorbed on stainless steel particles. This result strongly suggests that residues 102-135 include a segment primarily responsible for the interaction of beta-Lg with stainless steel surfaces. This supports our previous results [Sakiyama et al., J. Biosci. Bioeng., 88, 536-541 (1999)] that showed that residues 125-135 of beta-Lg have a strong affinity toward stainless steel surfaces and probably a major contribution to the adsorption of beta-Lg.

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  • Screening and characterization of affinity peptide tags specific to polystyrene supports for the orientated immobilization of proteins Reviewed

    Y Kumada, Y Tokunaga, H Imanaka, K Imamura, T Sakiyama, S Katoh, K Nakanishi

    BIOTECHNOLOGY PROGRESS   22 ( 2 )   401 - 405   2006.3

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    Dodecapeptides that exhibit a high affinity specific to a polystyrene surface (PS-tags) were screened using an Escherichia coli random peptide display library system, and the compounds were used as a peptide tag for the site-specific immobilization of proteins. The various PS-tags obtained after 10 rounds of biopanning selection were mainly composed of basic and aliphatic amino acid residues, most of which were arranged in close proximity to one another. Mutant-type glutathione S-transferases (GSTs) fused with the selected PS-tags. PS19 (RAFIASRRIKRP) and PS23 (AGLRLKKAAIHR) at their C-terminus, GST-PS19 and GST-PS23, when adsorbed on the PS latex beads had a higher affinity than the wild-type GST, and the specific remaining activity of the immobilized mutant-type GSTs was approximately 10 times higher than that of the wild-type GST. The signal intensity detected for GST-PS19 and GST-PS23 adsorbed on hydrophilic and hydrophobic PS surfaces using an anti-peptide antibody specific for the N-terminus peptide of GST was much higher than that for the wild-type GST. These findings indicate that the mutant-type GSTs fused with the selected peptide tags, PS19 and PS23, could be site-specifically immobilized on the surface of polystyrene with their N-terminal regions directed toward the solution. Thus, the selected peptide tags would be useful for protein immobilization in the construction of enzyme-linked immunosorbent assay (ELISA) systems and protein-based biochips.

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  • On the interaction site of serine acetyltransferase in the cysteine synthase complex from Escherichia coli Reviewed

    CH Zhao, Y Moriga, B Feng, Y Kumada, H Imanaka, K Imamura, K Nakanishi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   341 ( 4 )   911 - 916   2006.3

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    Cysteine synthase from Escherichia coli is a bienzyme complex comprised of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase A. The site of interaction of a SAT molecule was investigated by gel chromatography and surface plasmon technique using various mutant-type SATs, to better understand the mechanism involved in complex formation. The C-terminus of SAT, Ile 273, along with Glu 268 and Asp 271, was found to be essential for complex formation. The effects of O-acetyl-L-serine and sulfide on the affinity for the complex formation were also studied using a surface plasmon technique. (c) 2006 Elsevier Inc. All rights reserved.

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  • A novel acylase from Streptomyces mobaraensis that efficiently catalyzes hydrolysis/synthesis of capsaicins as well as N-Acyl-L-amino acids and N-acyl-peptides Reviewed

    M Koreishi, DM Zhang, H Imanaka, K Imamura, S Adachi, R Matsuno, K Nakanishi

    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY   54 ( 1 )   72 - 78   2006.1

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    A novel enzyme that catalyzes efficient hydrolysis of capsaicin (8-methyl-N-vanillyl-6-nonenamide) was isolated from the culture broth of Streptomyces mobaraensis. The enzyme consisted of two dissimilar subunits with molecular masses of 61 and 19 kDa. The enzyme was activated and stabilized in the presence of Co2+. It showed a pH optimum of about 8 and was stable at temperatures of up to 55 degrees C for 1 h at pH 7.8. The specific activity of the enzyme for the hydrolysis of capsaicin was 10(2)-10(4) times higher than those for the enzymes reported to date. In an aqueous/n-hexane biphasic system, capsaicin analogues such as octanoyl, decanoyl, and lauroyl vanillylamides were synthesized from the corresponding fatty acids and vanillylamine at yields of 50% or greater. In addition, the enzyme catalyzed the deacylation of N-lauroyl-L-amino acids and N-lauroyl-L-dipeptides and the efficient synthesis of N alpha-lauroyl-L-lysine, N epsilon-lauroyl-L-lysine, and various N-lauroyl-peptides in aqueous solution in both the absence and the presence of glycerol.

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  • 各種糖分子からなるアモルファスマトリクスの物理的諸特性

    今村 維克, 丸山 佳伸, 横山 徹, 大山 健一, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2006   413 - 413   2006

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    DOI: 10.11491/scej.2006f.0.413.0

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  • 金属表面へのタンパク質付着配向制御

    柳田 圭介, 中泉 雅人, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2006   1002 - 1002   2006

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  • 放線菌 Streptomyces mobaraensis 由来アシラーゼによる転移反応とその応用

    是石 真友子, 谷 和葉, 伊勢 雄一, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2006   949 - 949   2006

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  • 高度な物理的安定性とタンパク質安定化作用を兼ね備えた糖類アモルファスマトリクスの創製

    横山 徹, 丸山 佳伸, 大山 健一, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2006   995 - 995   2006

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  • ポリスチレン親和性ペプチドを利用したタンパク質間相互作用の分析

    熊田 陽一, 石村 遼太, 上崎 英範, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2006   996 - 996   2006

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  • Purification and characterization of a novel aminoacylase from Streptomyces mobaraensis Reviewed

    M Koreishi, F Asayama, H Imanaka, K Imamura, M Kadota, T Tsuno, K Nakanishi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   69 ( 10 )   1914 - 1922   2005.10

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    A novel aminoacylase was purified to homogeneity from culture broth of Streptomyces mobaraensis, as evidenced by SDS-polyacrylamide gel electrophoresis (PAGE). The enzyme was a monomer with an approximate molecular mass of 100 kDa. The purified enzyme was inhibited by the presence of 1,10-phenanthroline and activated by the addition of Co2+. It was stable at temperatures of up to 60 degrees C for 1 h at pH 7.2. It showed broad substrate specificity to N-acetylated L-amino acids. It catalyzed the hydrolysis of the amide bonds of various N-acetylated L-amino acids, except for N epsilon-acetyl-L-lysine and N-acetyl-L-proline. Hydrolysis of N-acetyl-L-methionine and N-acetyl-L-histidine followed Michaelis-Menten kinetics with K-m values of 1.3 +/- 0.1 mm and 2.7 +/- 0.1 mm respectively. The enzyme also catalyzed the deacetylation of 7-aminocephalosporanic acid (7-ACA) and cephalosporin C. Moreover, feruloyl-amino acids and L-lysine derivatives of ferulic acid derivatives were synthesized in an aqueous buffer using the enzyme.

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  • A novel epsilon-lysine acylase from Streptomyces mobaraensis for synthesis of N epsilon-acyl-L-lysines Reviewed

    M Koreishi, R Kawasaki, H Imanaka, K Imamura, K Nakanishi

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   82 ( 9 )   631 - 637   2005.9

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    A novel epsilon-lysine acylase (N-6-acyl-L-lysine amido-hydrolase; EC 3.5.1.17) was isolated from Streptomyces mobaraensis and purified to homogeneity by SDS-PAGE from the culture broth. The purified enzyme was monomeric, with a molecular mass of approximately 60 kDa. The enzyme was inactivated by the presence of 1,10-phenanthroline and activated in the presence of Co2+ and Zn2+. The enzyme showed a pH optimum of 8.0 and was stable at temperatures of up to 50 degrees C for 1 h at pH 8.0. The enzyme specifically catalyzed the hydrolysis of the amide bond of various N epsilon-acyl-L-lysines. Furthermore, the enzyme efficiently catalyzed the synthesis of NF-acyl-L-lysines with fatty and aromatic acyl groups in an aqueous buffer. in the syntheses of N epsilon-decanoyl-L-lysine, N epsilon-lauroyl-L-lysine, and N epsilon-myristoyl-L-lysine, the product precipitated and the yield was 90% or higher using 10 mM FA and 0.5 M L-lysine as the substrate.

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  • Adsorption characteristics of various organic substances on the surfaces of tantalum, titanium, and zirconium Reviewed

    T Nagayasu, K Imamura, K Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   286 ( 2 )   462 - 470   2005.6

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    Adsorption characteristics of carboxylic acids, amines, an octapeptide composed of four L-alanine and four L-aspartic acid residues (Peptide-A(4)D(4)), and beta-lactoglobulin (beta-Lg) on tantalum (Ta), titanium (Ti), and zirconium (Zr) particles were examined at 30 degrees C and in some case, were compared with their adsorption onto SUS316L stainless steel particles (S6L). The adsorption isotherms on the Ta, Ti, and Zr particles could usually be expressed either by a Langmuir-type equation for reversible adsorption or by a modified Langmuir-type adsorption equation including terms for both reversible and irreversible adsorption. The adsorption equilibrium of benzoic acid, benzylamine, and m-xylylenediamine on all the metal surfaces followed a Langmuir-type equation, while those of phthalic acid, mellitic acid, and Peptide-A(4)D(4) could be fitted to the modified Langmuir-type adsorption equation. The adsorption characteristics of different adsorbates on the different surfaces were discussed particularly with reference to the pH dependencies of the q(irrev), q(rev), and K values and the electrostatic properties of the oxidized surface of the metal particles. Fourier transform infrared spectroscopic analyses using a reflection/absorption technique (RA-IR) indicated that phthalic acid and mellitic acid are adsorbed in similar adsorption states irrespective of the type of metal. beta-Lg was adsorbed onto the surfaces principally in an irreversible manner. The desorption behavior of beta-Lg from Ta, Ti, and S6L surfaces was examined, in order to evaluate the extent of interaction between beta-Lg and the metals. (c) 2005 Elsevier Inc. All rights reserved.

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  • Enzymatic Synthesis of Non-proteinaceous Amino Acids Catalyzed by Cysteine Synthase Coupled with Acetyl-CoA Regeneration

    Zhao Chunhui, Imanaka Hiroyuki, Imamura Koreyoshi, Nakanishi Kazuhiro

    2005   271 - 271   2005

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  • 金属表面に吸着した各種タンパク質のH2O2-電気分解処理における脱離特性と脱離促進因子

    今村 維克, 尾下 学, 岩井 真澄, 金本 知明, 今中 洋行, 中西 一弘

    化学工学会 研究発表講演要旨集   2005   294 - 294   2005

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  • 放線菌 Streptomyces mobaraensis 由来新規アシラーゼの精製・特性解析および合成反応

    是石 真友子, 川崎 涼子, 今中 洋行, 今村 維克, 中西 一弘, 丹尾 式希

    化学工学会 研究発表講演要旨集   2005   270 - 270   2005

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  • 膜面液体培養法を用いた遺伝子組換え麹カビによるNuclease S1生産

    森田 正和, 安達 昇, 奥村 敦, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2005   242 - 242   2005

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  • 温度走査フーリエ変換赤外分光分析による糖-高分子複合アモルファスマトリクスにおける分子間相互作用の解析

    大山 健一, 谷 加寿子, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2005   293 - 293   2005

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  • 異なる金属表面への種々の有機物質の吸着挙動の比較

    長安 武司, 今中 洋行, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2005   292 - 292   2005

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  • ポリスチレン親和性ペプチド融合タンパク質の吸着特性とその応用

    徳永 安秀, 熊田 陽一, 今石 大輔, 今中 洋行, 今村 維克, 崎山 高明, 中西 一弘

    化学工学会 研究発表講演要旨集   2005f   311 - 311   2005

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  • Characteristics of alpha-glucosidase production from recombinant Aspergillus oryzae by membrane-surface liquid culture in comparison with various cultivation methods Reviewed

    M Morita, H Shimamura, N Ishida, K Imamura, T Sakiyama, K Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   98 ( 3 )   200 - 206   2004.9

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    alpha-Glucosidase was produced using recombinant Aspergillus oryzae by membrane-surface liquid culture (MSLC), a method previously developed by the authors and the results compared with other methods, including shaking flask culture (SFC), agar-plate culture (APC), culture on urethane sponge supports (USC), and liquid surface culture (LSC) to determine possible reasons for the advantageous features of MSLC. When yeast extract was used as a nitrogen source, the amount of enzyme produced by MSLC was 5 or more times higher than those for SFC and LSC, but similar to that using APC. Enzyme production in USC was slightly lower than in MSLC and APC. Cell growth was similar irrespective of the cultivation method used. When NaNO3, a typical inorganic nitrogen source was used, enzyme production in all the cultures was lower than that using yeast extract. However, even using NaNO3, the amount of the enzyme produced by MSLC was 8 to 20 times higher than those by SFC, APC, USC, and LSC. Although Cell growth using NaNO3 was similar to that for yeast extract in MSLC, it was markedly decreased in SFC, APC, and LSC. The reason for the difference in enzyme productivity for various cultivation methods using yeast extract and NaNO3 as a nitrogen source is discussed, on the basis of the experimental findings. The role of the oxygen transfer effect and gene expression levels in enzyme production were also examined.

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  • Production of nonproteinaceous amino acids using recombinant Escherichia coli cells expressing cysteine synthase and related enzymes with or without the secretion of O-acetyl-L-serine Reviewed

    CH Zhao, K Ohno, K Sogoh, K Imamura, T Sakiyama, K Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   97 ( 5 )   322 - 328   2004.5

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    beta-(Pyrazol-1-yl)-L-alanine (beta-PA), a model nonproteinaceous amino acid, was specifically synthesized by two methods using recombinant Escherichia coli cells that express cysteine synthase, comprising serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS-A) and related enzymes from E. coli. In the first method (method A), recombinant cells that express wildtype SAT, OASS-A, acetate kinase (AK), and phosphotransacetylase (PTA) showed the highest beta-PA production. beta-PA was produced at 140 mM from 200 MM L-serine and 200 mM pyrazole under optimum conditions. Using the cells expressing SATDeltaC20 (truncated SAT), OASS-A, AK, and PTA, beta-PA was produced at a level of only 80 mM, whereas O-acetyl-serine (OAS) was found to be secreted into the broth. Under optimum conditions, OAS accumulated at levels of around 105 mM from 300 mM L-serine. Thus, in the second method (method B), the secreted OAS was used as the substrate for the syntheses of beta-PA and beta-(triazol-1-yl)-L-alanine (beta-TA). The OAS that accumulated in the broth was efficiently converted to beta-PA and beta-TA at levels of around 90 mM from 105 mM OAS using free OASS-A. In both methods A and B, the addition of glucose was essential for the efficient production of beta-PA and OAS, respectively.

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  • Factors Affecting Performance of Cleaning Technique for Metal Surfaces Based on Electrolysis of Hydrogen Peroxide, H2O2-electrolysis Reviewed

    Koreyoshi Imamura

    nihon shokuhin kougakukaishi (japan journal of food engineering)   9 ( 4 )   229 - 238   2004

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    We have developed a novel technique for removing organic soilings from metal surfaces using hydroxy radicals (·OH). In the cleaning system, a metal surface fouled with organic soilings is made to contact with an aqueous solution containing hydrogen peroxide and supporting electrolyte. A slightly negative potential is then applied into the metal. The ·OHs, which are generated by the electrolysis of hydrogen peroxide (H2O2+e--ē·OH+OH”) on the metal surface, effectively attack and subsequently remove the adsorbed organic soils. In this study, the removal behavior of model organic soilings (protein) during the H2O2-electrolysis cleaning using different types and concentrations of supporting electrolyte was investigated. The presence of ammonium compounds and potassium phosphate led to marked increase in the removal rate. The influences of the types of metal substrate and adsorbed material on the removal behavior were also investigated, which suggested that the adsorption state of organic soiling on a metal surface strongly affect the removal characteristics. Furthermore, the removal rates in the presence of various organic coexisting materials were investigated. It was found that the removal rate constant was only slightly lowered in many cases. © 2008, Japan Society for Food Engineering. All rights reserved.

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  • Production of Non-Proteinaceous Amino Acids using Recombinant E. coli Cells

    Nakanishi Kazuhiro, Zhao Chunhui, Imamura Koreyoshi, Ohno Katsuhiro

    Asian Pacific Confederation of Chemical Engineering congress program and abstracts   2004   815 - 815   2004

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    β-(Pyrazol-1-yl)-L-alanine (β-pyrazole-L-Ala; β-PA), a model non-proteinaceous amino acid was synthesized using recombinant Escherichia coli cells that express cysteine synthase and enzymes that regenerate acetyl-CoA. In Method A, β-PA was synthesized from L-Ser and pyrazole using recombinant cells that express wild-type serine acetyltransferase (SAT), O-acetylserine sulfhydrylase-A (OASS-A), acetate kinase (AK), and phosphotransacetylase (PTA). Under the optimized condition, β-PA was produced at 140 mM from 200 mM L-Ser and 200 mM pyrazole. On the other hand, in Method B, O-acetyl-L-serine (OAS) was secreted into the broth, using the cells that express SATΔC20 (truncated SAT), OASS-A, AK, and PTA. Under optimized conditions OAS accumulated in the broth at levels of around 100 mM. The secreted OAS was efficiently converted to β-PA at levels of around 90 mM from 105 mM OAS using free OASS-A. In both Methods A and B, the addition of glucose was essential for the efficient production of β-PA and OAS, respectively.

    DOI: 10.11491/apcche.2004.0.815.0

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  • Removal Behavior of Protein Adsorbed on Metal Surface by H2O2-Electrolysis Treatment

    Imamura Koreyoshi, Watanabe Ippei, Aramoto Yumi, Sakiyama Takaharu, Nakanishi Kazuhiro

    Asian Pacific Confederation of Chemical Engineering congress program and abstracts   2004   353 - 353   2004

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    A novel method for generating hydroxyl radicals, ·OH, was developed, by applying slightly negative electric potentials (-0.2 ∼ -0.8 V vs Ag/AgCl) to the surface of a metal in contact with a hydrogen peroxide solution containing a supporting electrolyte. Namely, ·OH radicals are generated at the surface by the electrolysis of hydrogen peroxide according to the equation, H2O2 + e- -> ·OH + OH-. In this study, the kinetics of the removal of a model proteinaceous soiling (ß-lactoglobulin, ß-Lg) adsorbed to a stainless steel surface by the H2O2-electrolysis treatment were investigated. The rate of removal of the adsorbed ß-Lg from the stainless steel surface during the treatment was monitored in situ by ellipsometry, and the dependencies of the removal rate on H2O2 concentration, the electric potential applied to the surface, and the concentration and type of supporting electrolyte were examined.

    DOI: 10.11491/apcche.2004.0.353.0

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  • Effects of water activity and lipid addition on secondary structure of zein in powder systems Reviewed

    Y Mizutani, Y Matsumura, K Imamura, K Nakanishi, T Mori

    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY   51 ( 1 )   229 - 235   2003.1

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    The effects of water activity (A(w)) and lipid addition on the secondary structure of powdery zein were investigated using Fourier transform infrared spectroscopy. Two fatty acid esters, i.e., the linolenic and eicosapentaenoic acid ethyl esters (LAE and EPE), were mixed with the zein powder. The powders were stored in the "dry" state (with silica gel) and the "humid" state (A(w) = 0.9). The powdery zein without the lipids was shown to have a high content of the intermolecular hydrogen-bonded beta-sheet in the "dry" state, indicating the presence of protein aggregates. An increase in AW induced a decrease in this beta-sheet, concomitant with increases in the alpha-helix and beta-turn structures. The addition of LAE caused decreases in the alpha-helix and intermolecular hydrogen-bonded beta-sheet of zein when the powder was stored in the "humid" state, suggesting the strong interaction of LAE and zein molecules. However, LAE did not affect the secondary structure of zein in the "dry" state. The addition of EPE hardly influenced the secondary structure of zein, irrespective of A(w). These results are discussed in relation to the antioxidative activity of zein in the powder system, which had studied previously.

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  • ステンレス表面上の耐熱性アミノペプチダーゼの付着特性と活性発現

    長安 武司, 山越 憲吾, 吉岡 千里, 松本 訓智, 大西 宏輔, 今村 維克, 崎山 高明, 中西 一弘

    化学工学会 研究発表講演要旨集   2003   682 - 682   2003

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    DOI: 10.11491/scej.2003f.0.682.0

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  • 温度走査FTIRによる各種糖類アモルファスマトリクスにおける相互作用の解析

    今村 維克, 坂浦 啓介, 福島 厚志, 大山 健一, 谷 加寿子, 崎山 高明, 中西 一弘

    化学工学会 研究発表講演要旨集   2003   688 - 688   2003

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    DOI: 10.11491/scej.2003f.0.688.0

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  • Regulation of the Excretion of O-Acetyl-L-serine from Recombinant Escherichia coli Cells and Its Application to Synthesis of L-Cysteine and Non-protein Amino Acids

    ZHAO Chunhui, OHNO Katsuhiro, SOGOH Kohji, IMAMURA Koreyoshi, SAKIYAMA Takaharu, NAKANISHI Kazuhiro

    化学工学会 研究発表講演要旨集   2003   685 - 685   2003

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    DOI: 10.11491/scej.2003f.0.685.0

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  • 膜面液体培養法(MSLC)と振盪フラスコ培養(SFC)を用いたα-glucosidase生産性の差についての考察

    森田 正和, 今村 維克, 崎山 高明, 中西 一弘

    化学工学会 研究発表講演要旨集   2003   681 - 681   2003

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    DOI: 10.11491/scej.2003f.0.681.0

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  • 天然由来の高分子電解質を素材とした複合ゲルの調製とその膨潤特性

    崎山 高明, 今村 維克, 中西 一弘

    化学工学会 研究発表講演要旨集   2003   995 - 995   2003

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    DOI: 10.11491/scej.2003f.0.995.0

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  • Isolation of a Novel Acylase from Streptomyces mobaraensis for Synthesis of Useful Amide Compounds

    Zhang Demin, Koreishi Mayuko, Imamura Koreyoshi, Sakiyama Takaharu, NAKANISHI Kazuhiro

    化学工学会 研究発表講演要旨集   2003   136 - 136   2003

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    DOI: 10.11491/scej.2003f.0.136.0

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  • Enzymatic synthesis of N-acyl-L-amino acids in a glycerol-water system using acylase I from pig kidney Reviewed

    E Wada, M Handa, K Imamura, T Sakiyama, S Adachi, R Matsuno, K Nakanishi

    JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY   79 ( 1 )   41 - 46   2002.1

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    N-Medium- and long-chain acyl-L-amino acids were enzymatically synthesized from the corresponding L-amino acids and fatty acids using a reverse hydrolysis. Enzymes that are suitable for the synthetic reaction of N-acyl-L-amino acids were screened on the basis of hydrolytic activity toward N-lauroyl-L-glutamic acid as an indicator. Acylase I from pig kidney (EC 3.5.1.14) showed the highest N-acyl-L-amino acid hydrolytic activity among 57 commercially available enzymes tested. Acylase I effectively catalyzed the synthesis of N-lauroyl-L-amino acids except for N-lauroyl-L-proline and N-lauroyl-L-tyrosine in a glycerol-water system. Under the optimized reaction conditions, N-lauroyl-L-arginine and N-lauroyl-L-glutamic acid were obtained in conversions of 82 and 44%, respectively. The equilibrium constants calculated from the conversion obtained were 5.6, 15.4, 18.0, and 39.4 for the syntheses of N-lauroyl-L-glutamic acid, N.-lauroyl-L-lysine, N-lauroyl-L-glutamine, and N-lauroyl-L-methionine, respectively. N-Acyl-L-arginines with myristic acid and palmitic acid as the fatty acid were also synthesized using acylase I.

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  • Enzymatic modification of beta-lactoglobulin with N-fatty-acyl-dipeptide by transglutaminase from Streptomyces mobaraense Reviewed

    E Wada, H Masuda, K Imamura, T Sakiyama, S Adachi, R Matsuno, K Nakanishi

    BIOTECHNOLOGY LETTERS   23 ( 17 )   1367 - 1372   2001.9

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    beta -Lactoglobulin was enzymatically acylated with N-lauroyl-L-glutaminyl-glycine and N-lauroyl-L-glutamyl-L-lysine using transglutaminase from Streptomyces mobaraense. The modification of the protein with N-fatty-acyl-dipeptide was confirmed by SDS-PAGE, gel chromatography, HPLC, amino acid analysis, and TOF-MS. The degrees of the protein modification with N-lauroyl-L-glutaminyl-glycine and N-lauroyl-L-glutamyl-L-lysine were estimated to be 2-4 and 1.5 residues per molecule, respectively.

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  • Role of sucrose-LDH hydrogen bond for thermal stabilizing effect of sucrose on freeze-dried LDH Reviewed

    T Suzuki, K Imamura, H Fujimoto, M Okazaki

    DRYING TECHNOLOGY   17 ( 7-8 )   1429 - 1439   1999

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    Relationship between thermal stabilizing effect of sugar on freeze-dried protein and sugar-protein hydrogen bond is studied. Sucrose and lactate dehydrogenase (LDH) are used as models for sugar and protein. Samples of freeze-dried LDH involved in sucrose of different crystallinity were prepared, and measurement of X-ray diffractometry (XRD) and Fourier-Transform Infrared (FT-IR) spectroscopy was done. It is found that when sucrose is amorphous, the degree of hydrogen bond formation is high and LDH is stabilized; when sucrose is crystalline, hydrogen bond is less formed and LDH is inactivated. These results indicate that the stabilizing effect of sugar is closely related to sugar-protein hydrogen bond. It is also found that there is an optimum sucrose content for the thermal stabilizing effect. This is because amorphous structure of sucrose is stabilized and protected from crystallization by LDH. Thus we can deduce that sugars and proteins work together to keep the activities of proteins.

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  • Relation between thermal stabilizing effect of sucrose on LDH and sucrose-LDH hydrogen bond Reviewed

    T Suzuki, K Imamura, H Fujimoto, M Okazaki

    JOURNAL OF CHEMICAL ENGINEERING OF JAPAN   31 ( 4 )   565 - 570   1998.8

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    The thermal stabilizing effect of sugar on freeze-dried protein is studied, in particular on the relationship between the thermal stabilizing effect and the degree of sugar-protein hydrogen bond formation. Sucrose and lactate dehydrogenase (LDH) are used as models for sugar and protein. Several freeze-dried samples of LDH involved in sucrose were prepared; they differed in the degree of crystallinity of sucrose, By X-ray diffractometry (XRD) and Fourier transform infrared (FT-IR) spectroscopy, it is found that when sucrose is amorphous in the samples, the degree of hydrogen bond formation is high and LDH is stabilized remarkably. In contrast, when sucrose is crystalline, the degree of hydrogen bond formation is low and LDH is inactivated. These results indicate that the stabilizing effect of sugar is closely related to the sugar-protein hydrogen bond. The influence of sucrose content on the thermal stabilizing effect is also investigated. It is found that there is an optimum sucrose content for the thermal stabilizing effect. This is because, by the presence of LDH, the amorphous structure of sucrose is stabilized and protected from crystallization that causes loss of sucrose-LDH hydrogen bonds. Thus, we can deduce that sugars and proteins work together to maintain protein activities.

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  • Thermal stabilization of freeze-dried enzymes by sugars Reviewed

    T Suzuki, K Imamura, K Yamamoto, T Satoh, M Okazaki

    JOURNAL OF CHEMICAL ENGINEERING OF JAPAN   30 ( 4 )   609 - 613   1997.8

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    The thermal stabilization effect of sugars on freeze-dried proteins is studied, particularly the relation between stabilizing effect and the degree of sugar crystallinity. Three kinds of enzymes, alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and malate dehydrogenase (MDH), were used as model proteins. Aqueous solutions of enzymes with sugars were freeze-dried and stored in dry air at 65 degrees C. The stabilities of freeze-dried enzymes improve remarkably by addition of trehalose or raffinose. By measurement of X-ray diffractometry, the sugars are found to form fully amorphous matrix in freeze-dried samples. Furthermore, sucrose stabilizes enzymes to a great extent when it is amorphous in samples, though it shows Little stabilizing effect when it is crystalline. These results indicate that the stabilizing effect of sugars closely relates to the amorphous matrix formed by the sugars.

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  • Stabilizing effect on freeze-dried proteins by amorphous matrices of sugars Reviewed

    T Suzuki, K Imamura, K Yamamoto, M Okazaki

    DRYING '96, VOL B   1261 - 1266   1996

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  • A New Method for Measurement of Powder Characteristics Based on Reentrainment Phenomena Reviewed

    Hiroaki Masuda, Shuji Matsusaka, Koreyoshi Imamura

    KONA Powder and Particle Journal   12   133 - 143   1994

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    A new method has been developed to measure and evaluate the operational powder characteristics for fine particle processing based on the reentrainment phenomena. Experiments on particle reentrainment were carried out using an accelerated air flow for 21 different test powders. Both the reentrainment fluxes, which were measured by an electrostatic method, and the average air velocities were automatically sampled by a computer. The sampled data were processed to obtain the reentrainment profiles as a function of the average air velocity. The cumulative reentrainment efficiencies were also obtained and represented as a function of the wall shear stress. It was found that fine particles having tendencies to form large aggregates were reentrained mainly in the early stage of the measurement when the flow velocity was as low as 5 m·s−1. Also, the mass ratio of the reentrained large aggregates to the total particles was used to obtain information on the particle-particle (cohesion) or particle-wall interactions (adhesion). On the other hand, the cumulative reentrainment efficiency-curves must be applied to determine the operational conditions controlling the amount of adhered particles in various aerosol processes. © 1994, Hosokawa Powder Technology Foundation. All rights reserved.

    DOI: 10.14356/kona.1994021

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  • A New Method for the Measurement of Powder Characteristics Based on the Reentrainment Phenomena.

    MASUDA Hiroaki, MATSUSAKA Shuji, IMAMURA Koreyoshi

    Journal of the Research Association of Powder Technology, Japan   29 ( 12 )   897 - 905   1992

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    A new method has been developed to measure and evaluate the operational powder characteristics for fine particle processing based on the reentrainment phenomena. Experiments on particle reentrainment were carried out by use of an accelerated air flow for 21 different test powders. Both the reentrainment fluxes, which were measured by an electrostatic method, and the average air velocities were automatically sampled in a computer. The sampled data were processed to give the reentrainment profiles as a function of the average air velocity. The cumulative reentrainment efficiencies were also obtained and represented by a function of the wall shear stress.<br>It was found that fine particles having tendencies to form large aggregates were reentrained mainly in the early stage of the measurement where the flow velocity was as low as 5m·s-1. Also, the mass ratio of the reentrained large aggregates to the total particles was well applied to obtain the information on the particle-particle or particle-wall interactions. On the other hand, the cumulative reentrainment efficiency-curves would be applied to determine the operational conditions controlling the amount of adhered particles in various aerosol processes.

    DOI: 10.4164/sptj.29.897

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  • パッキング相互作用に着目したタンパク質結晶化法

    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 今村維克, 玉田太郎

    日本結晶学会年会講演要旨集   2019   2019

  • パッキングのコントロールにより,難結晶性蛋白質の結晶化を促進する結晶化法

    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 今村維克, 玉田太郎

    日本細胞生物学会大会(Web)   71st   2019

  • 難結晶性蛋白質のパッキングをコントロールする結晶化法の開発と評価

    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 今村維克, 玉田太郎

    日本結晶学会年会講演要旨集   2018   2018

  • タンパク質のパッキングをコントロールする疎水性残基導入

    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 今村維克, 玉田太郎

    日本結晶学会年会講演要旨集   2017   107   2017.11

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  • 疎水性残基導入が難結晶性タンパク質の結晶化に与える影響

    小坂恵, 山田秀徳, 二見淳一郎, 多田宏子, 今村維克, 玉田太郎

    日本生化学会大会(Web)   90th   ROMBUNNO.3P‐0100 (WEB ONLY)   2017

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  • Sugar surfactantの応用 : 油滴包括およびタンパク質安定化

    今村 維克, 石田 尚之, 今中 洋行

    日本食品工学会誌 = Japan journal of food engineering   17 ( 3 )   A - 7-11   2016.9

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  • Surfactant-Free Solid Dispersion of Fat-Soluble Flavor in an Amorphous Sugar Matrix, Based on Over-Dissolution of Sugar in Organic Media

    ( 28 )   67 - 76   2015

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  • 2P-064 Screening of a novel Pd affinity peptide tag and characterization of tag conjugated protein immobilization

    Matoba Haruka, Ishida Naoyuki, Imamura Koreyoshi, Imanaka Hiroyuki

    67   190 - 190   2015

  • 3P-195 Influences of sequence at the 5'-terminal region and media components on Escherichia coli recombinant expression system

    Abo Hirotaka, Hara Hirofumi, Umetsu Mitsuo, Ishida Naoyuki, Imamura Koreyoshi, Imanaka Hiroyuki

    67   319 - 319   2015

  • 2P-020 Influence of codon bias and media components on the Escherichia coli recombinant protein expression system and transcriptional profile of E. coli

    Abo Hirotaka, Miki Syunya, Hara Hirofumi, Yamazoe Atsushi, Hosoyama Akira, Tsuchikane Keiko, Imamura Koreyoshi, Imanaka Hiroyuki

    66   111 - 111   2014

  • Utilization of Hydrophilic Polystyrene Affinity Peptide for Functional Biomolecular Immobilization and Effective Site Specific Peptide Binder Screening

    H. Imanaka, R. Matsushita, T. Takimoto, T. Kunikata, K. Imamura, K. Nakanishi

    BIOPOLYMERS   100 ( 3 )   270 - 270   2013.5

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  • 3P-180 Screening of novel gold affinity peptide and its application on protein immobilization

    Shigemori Yojiro, Ishida Naoyuki, Imamura Koreyoshi, Takahashi Yuichiro, Imanaka Hiroyuki

    65   233 - 233   2013

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  • 2P-076 The influence of orientation and density of immobilized ligand molecule on biomolecular interaction

    Matsushita Runa, Ishida Naoyuki, Imamura Koreyoshi, Imanaka Hiroyuki

    65   123 - 123   2013

  • 4Cp02 Screening of NFkB(p50) affinity peptides by adopting functional biomoleculer immobilization

    Takimoto Takayuki, Miyahara Tetsuya, Imamura Koreyosi, Imanaka Hiroyuki, Kondou Eisaku, Nakanishi Kazuhiro

    64   195 - 195   2012

  • 4Cp03 Examination of the site specific peptide drug screening method transcription factor, FOXP3, as the target

    Kawasaki Kazuki, Matsumoto Kohei, Imanaka Hiroyuki, Imamura Koreyoshi, Kondou Eisaku

    64   195 - 195   2012

  • 11p04 Investigation of overexpression of ε-Lysine acylase from Streptomyces mobaraensis in Escherichia coli as a host

    Imanaka Hiroyuki, Taguchi Yuzo, Uchida Tatsuya, Kumon Kazuki, Kuroki Kentaro, Imamura Koreyoshi, Nakanishi Kazuhiro

    63   58 - 58   2011

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  • 2Ep03 Screening of NFkB(p50)inhibitory peptides using affinity tag mediated functional protein immobilization

    Takimoto Takayuki, Miyahara Tetsuya, Imanaka Hiroyuki, Imamura Koreyoshi, Kondo Eisaku, Nakanishi Kazuhiro

    63   145 - 145   2011

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  • Development of a highly efficient indigo dyeing method using indican with an immobilized beta-glucosidase from Aspergillus niger

    Jingyuan Song, Hiroyuki Imanaka, Koreyoshi Imamura, Kouichi Kajitani, Kazuhiro Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   110 ( 3 )   281 - 287   2010.9

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    A highly efficient method for dyeing textiles with indigo is described. In this method, the substrate, indican is first hydrolyzed at an acidic pH of 3 using an immobilized beta-glucosidase to produce indoxyl, under which conditions indigo formation is substantially repressed. The textile sample is then dipped in the prepared indoxyl solution and the textile is finally exposed to ammonia vapor for a short time, resulting in rapid indigo dyeing. As an enzyme, we selected a beta-glucosidase from Aspergillus niger, which shows a high hydrolytic activity towards indican and was thermally stable at temperatures up to 50-60 degrees C, in an acidic pH region. The A. niger beta-glucosidase, when immobilized on Chitopearl BCW-3001 by treatment with glutaraldehyde, showed an optimum reaction pH similar to that of the free enzyme with a slightly higher thermal stability. The kinetics for the hydrolysis of indican at pH 3, using the purified free and immobilized enzymes was found to follow Michaelis-Menten type kinetics with weak competitive inhibition by glucose. Using the immobilized enzyme, we successfully carried out repeated-batch and continuous hydrolyses of indican at pH 3 when nitrogen gas was continuously supplied to the substrate solution. Various types of model textiles were dyed using the proposed method although the color yield varied, depending on the type of textile used. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2010.03.010

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  • Temperature-Scanning Fourier Transform IR Spectroscopic Analysis of Amorphous Sugar Matrices : Changes in Interaction States with Temperature

    IMAMURA Koreyoshi

    Calorimetry and thermal analysis.   37 ( 4 )   154 - 161   2010.8

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  • Effects of the Cultivation Method on the Characteristics or Gene Expression Profiles of Aspergillus oryzae Using mCD or DPY Media

    Imanaka Hiroyuki, Tanaka Soukichi, Feng Bin, Imamura Koreyoshi, Nakanishi Kazuhiro

    Memoirs of the Faculty of Engineering, Okayama University   44   73 - 83   2010.1

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  • 3P-1085 Identification of a key amino acid residue for the formation of cysteine synthase complex using a highly functional peptide immobilization

    SHIMIZU Yuki, IMANAKA Hiroyuki, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    22   79 - 79   2010

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  • 2P-1103 Influence of amino acid sequence on its translocation to E. coli cell surface by autotransporter

    IMANAKA Hiroyuki, UEDA Hisako, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    22   34 - 34   2010

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  • 3P-1055 Multiformity of β-Galactosidase Produced from Bacillus circulans :

    SONG Jingyuan, IMANAKA Hiroyuki, IMAMURA Koreyoshi, MINODA Masashi, YAMAGUCHI Shoutaro, NAKANISHI Kazuhiro

    22   71 - 71   2010

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  • MN-P23 Development of peptide immobilization method using PS-tagged cushion protein(Section X Micro/Nano Technology for Analysis and Cell Manipulation)

    Imanaka Hiroyuki, Yamazumi Daisuke, Kunikata Toshinobu, Imamura Koreyoshi, Nakanishi Kazuhiro

    Journal of bioscience and bioengineering   108 ( 1 )   S161   2009.11

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  • Efficient Ethanol Production from Wheat Bran by Enzymatic Saccharification Using Commercially Available Enzyme Products and Fermentation Using Bakers' Yeast

    KOREISHI Mayuko, IMANAKA Hiroyuki, IMAMURA Koreyoshi, KARIYAMA Masahiro, NAKANISHI Kazuhiro

    Seibutsu-kougaku Kaishi   87 ( 5 )   216 - 223   2009.5

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    We aimed to produce ethanol efficiently by enzymatic saccharification coupled with ethanol fermentation using wheat bran as a biomass. For this purpose, we examined pretreatment conditions of biomass and a combination of commercially available enzyme products for saccharification. When a 3.3% (w/v) wheat bran suspension was pretreated using subcritical water at 140℃ with a holding time of 0h, followed by saccharification at 45℃ using a mixture of commercially available Meiselase (composed of cellulase, cellobiase/β-glucosidase, and xylanase) and Novozyme^[○!R] 188 (composed of cellobiase/β-glucosidase, α-amylase, and glucoamylase) at a ratio of 4 to 1 by weight, glucose was released at 10.6g/l, which corresponded to 90% of the value calculated on the basis of the total amount of glucose-based saccharides contained in the wheat bran. On the other hand, from a pretreated 33% (w/v) wheat bran suspension, 85g/l glucose was produced, which corresponded to only 72% of the theoretical value, owing to the inhibitory effect of glucose on β-glucosidase activity. However, when fermentation was carried out at 30℃ following 24-h saccharification of the pretreated wheat bran using a mixture of Meiselase and Novozyme^[○!R] 188, the inhibitory effect of glucose was considered to be reduced and as a result, after 21-h fermentation, ethanol was produced at 5.2% (w/v), which corresponded to 88% of the theoretical maximum value.

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  • 2Ea05 Investigation of peptide surface display system by autotransporter

    UEDA Hisako, YAMASHITA Mai, IMANAKA Hiroyuki, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    21   59 - 59   2009

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  • 2Ip05 Investigation of Peptide-Protein Interaction Detection System by Cushion Protein

    IMANAKA Hiroyuki, KUNIKATA Toshinobu, YAMANOUCHI Mai, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    21   126 - 126   2009

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  • 2Da01 Construction of expression systems of a novel aminoacylase from Streptomyces mobaraensis

    OOI Manami, NAKATANI Yasuyuki, KOREISHI Mayuko, IMANAKA Hiroyuki, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    21   44 - 44   2009

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  • 3Ga07 Development of rapid and sensitive detection system of human FOXP3-DNA interaction

    MAEKAWA Masami, IMANAKA Hiroyuki, IMAMURA Koreyoshi, KONDO Eisaku, NAKANISHI Kazuhiro

    20   186 - 186   2008

  • 2Aa03 Purification and Characterization of Recombinant Monoacylglycerol Lipase from Rhodanobacter sp. strain LP7315

    TERAKAWA Shuji, MORI Masashi, IMANAKA Hiroyuki, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    20   59 - 59   2008

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  • 3Da04 Immobilization of Biomolecules by Cushion Protein on Solid Surfaces

    IMANAKA Hiroyuki, YAMAZUMI Daisuke, YANAGITA Keisuke, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    20   126 - 126   2008

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  • Enzymatic modification of proteolytic fragments of food proteins with fattyacid using a novel acylase

    Annual report of study for food under the supporting program by The Skylark Food Science Institute   ( 21 )   109 - 115   2008

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  • Enzymatic synthesis of beta-lactam antibiotics and N-fatty-acylated amino compounds by the acyl-transfer reaction catalyzed by penicillin V acylase from Streptomyces mobaraensis

    Mayuko Koreishi, Kazuha Tani, Yuuichi Ise, Hiroyuki Imanaka, Koreyoshi Imamura, Kazuhiro Nakanishi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   71 ( 6 )   1582 - 1586   2007.6

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    Penicillin V acylase from Streptomyces mobaraensis (Sm-PVA) showed high acyl-transfer activity in reactions using methyl esters of carboxylic acid (acyl donor) and amino compounds (nucleophile), to produce the corresponding amides. Moreover, Sm-PVA had broad substrate specificity, as indicated by the fact that it catalyzed the efficient synthesis of fl-lactam antibiotics, capsaicin derivatives, and N-fatty-acyl-amino acid/N-fatty-acyl-peptide derivatives.

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  • 2F14-4 Production of protease and gene expression analysis of Aspergillus oryzae cultivated by membrane-surface liquid culture and shaking flask culture

    TANAKA Soukichi, FENG Bin, IMANAKA Hiroyuki, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    19   141 - 141   2007

  • 1C11-1 Characterization and cloning of long-chain aminoacylase and shortchain aminoacylase from Streptomyces mobaraensis

    NAKATANI Yasuyuki, KOREISHI Mayuko, IMANAKA Hiroyuki, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    19   79 - 79   2007

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  • 1C09-2 Influence of signal peptide on the construction of cell surface display system by autotransporter

    TATSUMOTO Wataru, IMANAKA Hiroyuki, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    19   77 - 77   2007

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  • 1C11-2 Enzymatic synthesis of functional compounds by the acyl-transfer reaction catalyzed by penicillin V acylase from Streptomyces mobaraensis

    KOREISHI Mayuko, ISE Yuuichi, TANI Kazuha, IMANAKA Hiroyuki, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    19   79 - 79   2007

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  • ポリスチレン親和性ペプチド標識タンパク質を用いる迅速免疫測定

    中西一弘, 熊田陽一, 今中洋行, 今村維克

    日本食品工学会年次大会講演要旨集   8th   2007

  • Stabilization of Proteins during Freeze-drying and Storage by "Sugar Glass"

    IMAMURA Koreyoshi

    81 ( 949 )   908 - 912   2006.11

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  • Protease susceptibility of beta-lactoglobulin adsorbed on stainless steel surface as evidence of contribution of its specific segment to adsorption

    Takaharu Sakiyama, Atsuko Aya, Mari Embutsu, Koreyoshi Imamura, Kazuhiro Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   101 ( 5 )   434 - 439   2006.5

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    beta-Lactoglobulin (beta-Lg) is a major constituent of fouling deposits in the dairy industry. To determine the interaction between beta-Lg and stainless steel surfaces, beta-Lg irreversibly adsorbed on stainless steel particles was subjected to lysyl endopeptidase treatment and the course of fragmentation was compared with that observed for beta-Lg in solution. The results showed a distinct difference between the courses of fragmentation: a fragment (residues 102-135) was liberated readily from beta-Lg in solution but scarcely from beta-Lg irreversibly adsorbed on stainless steel particles. This result strongly suggests that residues 102-135 include a segment primarily responsible for the interaction of beta-Lg with stainless steel surfaces. This supports our previous results [Sakiyama et al., J. Biosci. Bioeng., 88, 536-541 (1999)] that showed that residues 125-135 of beta-Lg have a strong affinity toward stainless steel surfaces and probably a major contribution to the adsorption of beta-Lg.

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  • ポリスチレン表面に親和性を示すペプチドタグを付加した酵素の設計と特性解析

    今石大輔, 熊田陽一, 今中洋行, 今村維克, 崎山高明, 中西一弘

    日本農芸化学会中四国支部講演会講演要旨集   14th (Web)   2006

  • ポリスチレン親和性ペプチド標識タンパク質を用いる迅速免疫測定

    熊田陽一, 今中洋行, 今村維克, 加藤滋雄, 中西一弘

    化学工学会年会研究発表講演要旨集   71st   2006

  • Isolation and Characterization of a Thermostable Monoacylglycerol Lipase from Soil Bacterium

    Hiroyuki Imanaka, Masashi Mori, Koreyoshi Imamura, Takaharu Sakiyama, Kazuhiro Nakanishi

    Proceedings for Asia-Pacific Biochemical Engineering Conference (APBioChEC’05)   2005

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  • 2E11-4 Orientated immobilization of protein by use of affinity peptide specific to polystyrene

    KUMADA Yoichi, IMANAKA Hiroyuki, IMAMURA Koreyoshi, KATOH Shigeo, SAKIYAMA Takaaki, NAKANISHI Kazuhiro

    17   153 - 153   2005

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  • Sugar-Protein Interaction and Stabilization of Protein in Amorphous Sugar Matrix(Papers presented at the Seminar, "Amorphous Science for Food and Pharmaceutical System")

    IMAMURA Koreyoshi

    Cryobiology and cryotechnology   51 ( 1 )   31 - 35   2005

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    Preserving the native conformation of protein during freeze-drying would lead to a greater stability against denaturation during storage. A number of investigators have reported that freeze-drying in the presence of sugar aids in preserving the native conformation of a protein. When a protein solution is freeze-dried in the presence of sugars, amorphous matrices of sugars are formed, which could embed protein molecules. The embedding of protein molecules in the amorphous sugar matrices is generally thought to prevent conformational changes in the protein during dehydration and storage due to interactions between protein and sugar molecules. In this review we describe the methods which are used to study the sugar-protein hydrogen bond. The effects of various sugars on the structural stabilization of protein in the dried state are also reviewed and discussed.

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  • ポリスチレン親和性ペプチド融合タンパク質の吸着特性とその応用

    徳永安秀, 熊田陽一, 今石大輔, 今中洋行, 今村維克, 崎山高明, 中西一弘

    化学工学会秋季大会研究発表講演要旨集(CD-ROM)   37th   2005

  • Effects of carboxyl groups on the adsorption behavior of low-molecular-weight substances on a stainless steel surface

    T Nagayasu, C Yoshioka, K Imamura, K Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   279 ( 2 )   296 - 306   2004.11

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    The adsorption isotherms of various carboxylic acids and several amines on a stainless steel surface were taken as a function of pH and the ionic strength of the solution at 30 degreesC. In particular, the effect of the number of carboxyl groups on the adsorption behavior was investigated. Monocarboxylic acids such as benzoic, acid and n-butyric acid were reversibly adsorbed on the stainless steel particles and showed a Langmuir-type adsorption isotherm, i.e., Q = Kq(m)C/(1 + KC), where Q and C are, respectively, the amount of adsorbate adsorbed and the equilibrium concentration in the bulk solution, q(m), the maximum adsorbed amount, and K is the adsorption equilibrium constant. Carboxylic acids having plural carboxyl groups had much higher affinity to the surface and were adsorbed in both reversible and irreversible modes. The adsorption isotherms for the carboxylic acids having plural carboxyl groups could be expressed by a modified Langmuir-type adsorption isotherm, i.e., Q = q(irrev) + K q(rev)C/(1 + KC), where q(irrev) and q(rev) are, respectively, the maximum amounts adsorbed irreversibly and reversibly. The K and q(irrev) values increased with an increase in the number of carboxyl groups except for isophthalic acid and terephthalic acid. On the basis of the pH dependencies of K, q(m), q(irrev), and q(rev) as well as the surface properties of the stainless steel, both reversible and irreversible adsorptions were considered to occur through the electrostatic interaction between negatively charged carboxyl groups and the positively charged sites on the surface. The dependency of the qirrev value on ionic strength was discussed on the basis of the differences in their adsorbed state with the interaction forces to the surface and repulsive forces among the adsorbed molecules. The adsorption of amine components was quite weak. The RA-IR and molecular dynamics calculation were done to investigate the adsorption states of phthalic acid, trimellitic acid, and mellitic acid. (C) 2004 Elsevier Inc. All rights reserved.

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  • Characteristics of alpha-glucosidase production from recombinant Aspergillus oryzae by membrane-surface liquid culture in comparison with various cultivation methods

    M Morita, H Shimamura, N Ishida, K Imamura, T Sakiyama, K Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   98 ( 3 )   200 - 206   2004.9

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    alpha-Glucosidase was produced using recombinant Aspergillus oryzae by membrane-surface liquid culture (MSLC), a method previously developed by the authors and the results compared with other methods, including shaking flask culture (SFC), agar-plate culture (APC), culture on urethane sponge supports (USC), and liquid surface culture (LSC) to determine possible reasons for the advantageous features of MSLC. When yeast extract was used as a nitrogen source, the amount of enzyme produced by MSLC was 5 or more times higher than those for SFC and LSC, but similar to that using APC. Enzyme production in USC was slightly lower than in MSLC and APC. Cell growth was similar irrespective of the cultivation method used. When NaNO3, a typical inorganic nitrogen source was used, enzyme production in all the cultures was lower than that using yeast extract. However, even using NaNO3, the amount of the enzyme produced by MSLC was 8 to 20 times higher than those by SFC, APC, USC, and LSC. Although Cell growth using NaNO3 was similar to that for yeast extract in MSLC, it was markedly decreased in SFC, APC, and LSC. The reason for the difference in enzyme productivity for various cultivation methods using yeast extract and NaNO3 as a nitrogen source is discussed, on the basis of the experimental findings. The role of the oxygen transfer effect and gene expression levels in enzyme production were also examined.

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  • Use of a novel affinity tag selected with a bacterial random peptide library for improving activity retention of glutathione S-transferase adsorbed on a polystyrene surface

    T Sakiyama, S Ueno, K Imamura, K Nakanishi

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   28 ( 4-6 )   207 - 214   2004.6

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    Aiming at developing a novel affinity tag for site-specific immobilization of functional proteins onto polystyrene (PS) surfaces, Escherichia coli random peptide display library was screened for dodecapeptides exhibiting a high affinity toward PS plate,. The selected peptides were commonly rich in hydrophobic amino acid residues and had two or three basic amino acid residues. Adsorption and desorption experiments for one of the selected peptide named PSI (KGLRGWREMISL) showed that it was well and irreversibly adsorbed onto PS latex particles. To study its performance as an affinity tag, PSI was genetically fused to a model enzyme, glutathione S-transferase (GST), in several manners, and the fusion enzymes were compared to the original GST in terms of the adsorption behavior onto the PS latex particles as well as the specific activities before and after the adsorption. The fusion GSTs in solution showed lower specific activities than the original one, and their adsorption behaviors were also altered. In particular, the fusion of PS 1 to the N-terminal region of GST resulted in severe losses both in the specific activity and in the adsorptive ability. However, two types of GSTs fused with PSI at the C-terminal region were well adsorbed onto the PS latex particles, and their specific activities after the adsorption were significantly higher than the original GST adsorbed on the PS latex particles. The fusion of PSI to the C-terminal region of GST was thus shown to reduce the activity loss upon the adsorption onto the PS latex particles. (C) 2004 Elsevier B.V. All rights reserved.

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  • Production of nonproteinaceous amino acids using recombinant Escherichia coli cells expressing cysteine synthase and related enzymes with or without the secretion of O-acetyl-L-serine

    CH Zhao, K Ohno, K Sogoh, K Imamura, T Sakiyama, K Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   97 ( 5 )   322 - 328   2004.5

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    beta-(Pyrazol-1-yl)-L-alanine (beta-PA), a model nonproteinaceous amino acid, was specifically synthesized by two methods using recombinant Escherichia coli cells that express cysteine synthase, comprising serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS-A) and related enzymes from E. coli. In the first method (method A), recombinant cells that express wildtype SAT, OASS-A, acetate kinase (AK), and phosphotransacetylase (PTA) showed the highest beta-PA production. beta-PA was produced at 140 mM from 200 MM L-serine and 200 mM pyrazole under optimum conditions. Using the cells expressing SATDeltaC20 (truncated SAT), OASS-A, AK, and PTA, beta-PA was produced at a level of only 80 mM, whereas O-acetyl-serine (OAS) was found to be secreted into the broth. Under optimum conditions, OAS accumulated at levels of around 105 mM from 300 mM L-serine. Thus, in the second method (method B), the secreted OAS was used as the substrate for the syntheses of beta-PA and beta-(triazol-1-yl)-L-alanine (beta-TA). The OAS that accumulated in the broth was efficiently converted to beta-PA and beta-TA at levels of around 90 mM from 105 mM OAS using free OASS-A. In both methods A and B, the addition of glucose was essential for the efficient production of beta-PA and OAS, respectively.

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  • Adsorption characteristics of bovine serum albumin and its peptide fragments on a stainless steel surface

    T Sakiyama, J Tomura, K Imamura, K Nakanishi

    COLLOIDS AND SURFACES B-BIOINTERFACES   33 ( 2 )   77 - 84   2004.1

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    Adsorption characteristics of peptide fragments prepared by lysyl endopeptidase treatment of bovine serum albumin (BSA) were studied in comparison with those of BSA itself using stainless steel particles (type 316L) as a substrate surface. BSA was adsorbed onto the stainless steel surface remarkably at acidic pHs but scarcely at alkaline pHs. The peptide fragments were also scarcely adsorbed at alkaline pHs. At acidic pHs, however, the affinity toward the stainless steel surface depended on the type of peptide fragment; several types of the peptide fragments, relatively rich in acidic amino acid residues, such as DLGEEHFK (residues 13-20) and DAIPEDLPPLTADFAEDK (residues 295-312), were considerably adsorbed onto the stainless steel surface at acidic pHs. Similarly to BSA, the adsorption isotherms of those two types of peptide fragments showed very high affinities toward the stainless steel surface at pH 3 resulting in irreversible adsorption. Adsorption experiments for their synthetic analogues showed that the acidic amino acid residues were essential for the adsorption at pH 3. Furthermore FT-IR analysis suggested that the carboxyl groups of these acidic amino acid residues on the stainless steel surface were dissociated and hence had electrostatic interaction with the stainless steel surface. Thus the importance of carboxyl groups in the adsorption of peptide fragments on the stainless steel surface at acidic pHs was indicated. Close similarity of adsorption behaviors at acidic pHs found between BSA and those peptide fragments suggests that the acidic amino acid residues of the corresponding segments of a BSA molecule make a major contribution to the adsorption at acidic pHs. However, when those adsorbed at pH 3 were treated with 0.02 N NaOH, complete removal was observed for the peptide fragments but not for BSA. (C) 2003 Elsevier B.V. All rights reserved.

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  • ランダムペプチドライブラリーから選別した新規アフィニティータグによるポリスチレン表面に吸着したグルタチオンSトランスフェラーゼの活性保持の改善

    J. Mol. Catal. B: Enzymatic   28 (4), pp. 207-214   2004

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  • ステンレス表面に対する牛血清アルブミンとそのペプチド断片の吸着特性

    Colloids Surf. B: Biointerfaces   vol. 33(2), pp. 77-84   2004

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  • システインシンセターゼおよび関連酵素を発現し,O-アセチルセリンを分泌能が異なる組み替え大腸菌を用いた非タンパク性アミノ酸の生産

    J. Biosci. Bioeng.   97(5), pp. 322-328   2004

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  • ステンレス表面に対する低分子物質の吸着におけるカルボキシル基の寄与

    Journal of Colloid and Interface Science   279(2), pp. 289-295   2004

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  • Contribution of acidic amino residues to the adsorption of peptides onto a stainless steel surface

    K Imamura, Y Kawasaki, T Awadzu, T Sakiyama, K Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   267 ( 2 )   294 - 301   2003.11

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    The role of the acidic amino acid residues in the adsorption of peptides/proteins onto stainless steel particles was investigated using a peptide fragment from bovine beta-lactoglobulin, Thr-Pro-Glu-Val-Asp-Asp-Glu-Ala-Leu-Glu-Lys (T5 peptide), which has a high affinity to a stainless steel surface at acidic pHs, and its mutant peptides substituted with different numbers of acidic amino acid residues. The adsorption behavior of the mutant peptides as well as the T5 peptide were studied at pH 3 with respect to concentration and ionic strength dependencies and the reversibility of the adsorption process. The behavior of the peptides was generally characterized as two distinct irreversible adsorption modes, Mode I and Mode II. In Mode I, the amounts adsorbed lay on the ordinate at zero equilibrium concentration in the solution, while in Mode II, the amount adsorbed increased with increased equilibrium concentration. The area occupied by the peptides was predicted by molecular mechanics and molecular dynamics. The state of the peptides, when adsorbed, was investigated using FT-IR analysis. The FT-IR analyses revealed that the side carboxylic groups of the peptides adsorbed on the stainless steel surface were ionized, while they were unionized in the solution at pH 3. Thus, the interactions between the carboxylic groups of the peptide and the stainless steel surface can be considered to be largely electrostatic. The peptide having four acidic amino acid residues took a maximum adsorbed amount, the reason for which is discussed. (C) 2003 Elsevier Inc. All rights reserved.

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  • The kinetics of removal of proteins adsorbed on a stainless steel surface by H2O2-electrolysis and factors affecting its performance

    K Imamura, Watanabe, I, M Imada, T Sakiyama, K Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   265 ( 1 )   49 - 55   2003.9

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    The kinetics of the removal of beta-lactoglobulin (beta-Lg) adsorbed on a stainless steel surface by H2O2-electrolysis treatment, in which hydroxyl radicals (OHs) generated by the electrolysis of hydrogen peroxide decompose the substances adhering on the surface, was investigated. The rate of removal of the adsorbed beta-Lg from the stainless steel surface during the treatment was monitored in situ by ellipsometry. The dependencies of the removal rate on the H2O2 concentration, the electric potential applied to the surface, and the supporting electrolyte concentration were examined and the results were compared with those obtained for the treatment of a titanium surface. Differences in the removal rates of the protein from the stainless steel and titanium surfaces are discussed with respect to differences in the nature of the interaction between the protein and the surface. The atomic compositions of the stainless steel surface before and after treatment were analyzed by Auger electron spectroscopy, and the stainless steel surface was found not to be affected by the H2O2-electrolysis treatment. The influences of various coexisting materials on removal characteristics during the H2O2-electrolysis treatment were also investigated. The difference between the effect of coexisting substances on the decomposition rate for the radical reaction in the H2O2-electrolysis treatment and that for the well-known UV-H2O2 treatment in bulk solution is discussed. (C) 2003 Elsevier Inc. All rights reserved.

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  • 放線菌由来新規アシラーゼによるカプサイシン誘導体の合成とその抗酸化性の評価

    KOREISHI MAYUKO, CHO TOKUMIN, KAWANAMI KAZUKO, IMAMURA YUKIKATSU, SAKIYAMA TAKAAKI, MATSUMURA YASUO, NAKANISHI KAZUHIRO

    日本食品工学会年次大会講演要旨集   4th   117   2003.7

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  • 食品製造プロセスにおける新規洗浄方法の原理とその特性 (特集 食品工場の洗浄・除菌技術)

    今村 維克, 崎山 高明, 中西 一弘

    ジャパンフ-ドサイエンス   42 ( 6 )   27 - 33   2003.6

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  • Effects of types of sugar on the stabilization of protein in the dried state

    K Imamura, T Ogawa, T Sakiyama, K Nakanishi

    JOURNAL OF PHARMACEUTICAL SCIENCES   92 ( 2 )   266 - 274   2003.2

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    The effects of various sugars on the structural stabilization of protein during freeze-drying were investigated. The degree of native structure of protein that was freeze-dried and rehumidified at constant relative humidities (RHs) was evaluated by measurement of the alpha-helix content by Fourier-transform infrared spectroscopy. Bovine serum albumin (BSA) and several types of sugars, including sucrose, trehalose, and dextrans, were used as a model protein and sugars, respectively. The glass transition temperature, T-g for the amorphous sugar samples was measured by differential scanning calorimetry (DSC) to characterize the structural stability of sugars. The dependence of the alpha-helix content (Calpha-helix) of BSA on the sugar content (c(sugar)) could, in most cases, be represented by a Langmuir-type equation: Calpha-helix = K x (C-alpha-helix(max)-C-alpha-jelix(0)) x c(sugar)/(1 + K x c(sugar)) + C-alpha-helix(0), where K is a constant, indicating the ability of amorphous sugar matrix to embed protein, and C-alpha-helix(0) and C-alpha-helix(max) indicate the alpha-helix content in the absence of sugar and saturating levels of sugar, respectively. The preservation effects of the sugars could be characterized by K and C-alpha-helix(max). Both K and C-alpha-helix(max) values tended to be higher with decreasing T-g values for the amorphous sugar, probably because an amorphous sugar matrix with lower T-g values is structurally more flexible. The rehumidification of protein that was freeze-dried in the presence of sugar induced the refolding of protein structure, whereas the protein dried alone did not show any recovery of its native structure. (C) 2003 Wiley-Liss, Inc.

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  • Effects of types of sugar on the stabilization of protein in the dried state

    K Imamura, T Ogawa, T Sakiyama, K Nakanishi

    JOURNAL OF PHARMACEUTICAL SCIENCES   92 ( 2 )   266 - 274   2003.2

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    The effects of various sugars on the structural stabilization of protein during freeze-drying were investigated. The degree of native structure of protein that was freeze-dried and rehumidified at constant relative humidities (RHs) was evaluated by measurement of the alpha-helix content by Fourier-transform infrared spectroscopy. Bovine serum albumin (BSA) and several types of sugars, including sucrose, trehalose, and dextrans, were used as a model protein and sugars, respectively. The glass transition temperature, T-g for the amorphous sugar samples was measured by differential scanning calorimetry (DSC) to characterize the structural stability of sugars. The dependence of the alpha-helix content (Calpha-helix) of BSA on the sugar content (c(sugar)) could, in most cases, be represented by a Langmuir-type equation: Calpha-helix = K x (C-alpha-helix(max)-C-alpha-jelix(0)) x c(sugar)/(1 + K x c(sugar)) + C-alpha-helix(0), where K is a constant, indicating the ability of amorphous sugar matrix to embed protein, and C-alpha-helix(0) and C-alpha-helix(max) indicate the alpha-helix content in the absence of sugar and saturating levels of sugar, respectively. The preservation effects of the sugars could be characterized by K and C-alpha-helix(max). Both K and C-alpha-helix(max) values tended to be higher with decreasing T-g values for the amorphous sugar, probably because an amorphous sugar matrix with lower T-g values is structurally more flexible. The rehumidification of protein that was freeze-dried in the presence of sugar induced the refolding of protein structure, whereas the protein dried alone did not show any recovery of its native structure. (C) 2003 Wiley-Liss, Inc.

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  • ステンレス表面に吸着したタンパク質のH2O2-電気分解による脱離の速度論と処理効果に及ぼす諸因子の影響

    Journal of Colloid and Interface Science   265 ( 1 )   49 - 55   2003

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  • Ionization characteristics of polyelectrolyte complex gels: analysis based on their swelling behaviors

    T. Sakiyama, T. Tsutsui, E. Masuda, K. Imamura, K. Nakanishi

    Macromolecules   36 ( 13 )   5039 - 5042   2003

  • ペプチドのステンレス表面への吸着に対する酸性アミノ酸残基の寄与

    Journal of Colloid and Interface Science   276 ( 2 )   294 - 301   2003

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  • 酵素法による脂質付加食品素材物質の大量合成法の開発と機能性の解明

    中西 一弘, 今村 維克, 安達 修二

    年報   2003   271 - 277   2003

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  • In situ analysis of the removal behavior of protein adsorbed on a titanium surface by H2O2-electrolysis treatment

    K Imamura, T Sakiyama, K Nakanishi

    LANGMUIR   18 ( 21 )   8033 - 8039   2002.10

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    The kinetics of the removal of beta-lactoglobulin (beta-Lg) adsorbed to a titanium surface using a method involving the electrolysis of hydrogen peroxide (H2O2)-electrolysis treatment) was examined. The rate of decrease in the thickness of the layer of beta-Lg adsorbed to the titanium surface during the H2O2-electrolysis treatment was measured in situ by ellipsometry, The rate for this process followed first-order reaction kinetics. Relationships between the rate constant for removal and such factors as the H2O2 concentration, the potential applied to the titanium surface, the supporting electrolyte concentration, and temperature were experimentally studied. The rate of removal of the adsorbed beta-Lg from a stainless steel surface was found to be much slower than that from a titanium surface, indicating that the type of metal also affects the removal characteristics during the H2O2-electrolysis treatment.

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  • In situ analysis of the removal behavior of protein adsorbed on a titanium surface by H2O2-electrolysis treatment

    K Imamura, T Sakiyama, K Nakanishi

    LANGMUIR   18 ( 21 )   8033 - 8039   2002.10

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    The kinetics of the removal of beta-lactoglobulin (beta-Lg) adsorbed to a titanium surface using a method involving the electrolysis of hydrogen peroxide (H2O2)-electrolysis treatment) was examined. The rate of decrease in the thickness of the layer of beta-Lg adsorbed to the titanium surface during the H2O2-electrolysis treatment was measured in situ by ellipsometry, The rate for this process followed first-order reaction kinetics. Relationships between the rate constant for removal and such factors as the H2O2 concentration, the potential applied to the titanium surface, the supporting electrolyte concentration, and temperature were experimentally studied. The rate of removal of the adsorbed beta-Lg from a stainless steel surface was found to be much slower than that from a titanium surface, indicating that the type of metal also affects the removal characteristics during the H2O2-electrolysis treatment.

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  • Water sorption and glass transition behaviors of freeze-dried sucrose-dextran mixtures

    K Imamura, A Fukushima, K Sakaura, T Sugita, T Sakiyama, K Nakanishi

    JOURNAL OF PHARMACEUTICAL SCIENCES   91 ( 10 )   2175 - 2181   2002.10

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    The water sorption and glass transition behaviors of freeze-dried disaccharide-polysaccharide mixtures at various contents were investigated at relative humidities (RHs) of 0, 11, 23, and 33%. Sucrose and three types of dextrans, which differ in molecular weight, were used as model di- and polysaccharides, respectively. The relationship between the dextran and water contents of the sucrose-dextran mixture at different constant RHs indicated that a mixture of sucrose and dextran was lower than that calculated by the Lang and Steinberg mass balance equation. In the RH range of 0-23%, the glass transition temperature, T-g, increased to a considerable extent when the dextran content was equal to or higher than 50%, while the increase in T-g, at dextran contents lower than 50% was small. A marked increase in T-g, was observed at RH 33% for dextran contents of 0-25% as well as in the range above 50%. This suggests that the physical stability of the highly hydrated amorphous disaccharide is effectively strengthened by the addition of a small amount of polysaccharide. These tendencies were similar for the three dextrans of different molecular weights. Furthermore, it was demonstrated that the addition of a small amount of dextran is quite effective in preventing the collapse of amorphous sugar during freeze drying. (C) 2002 Wiley-Liss, Inc.

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  • A Novel Cleaning Method Using Electrolysis of Hydrogen Peroxide

    IMAMURA Koreyoshi, NAKANISHI Kazuhiro

    77 ( 899 )   800 - 805   2002.9

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  • 粉末系におけるゼインの二次構造に及ぼす水分活性と脂質添加の影響

    MIZUTANI YUKIKO, MATSUMURA YASUO, IMAMURA TADAKATSU, NAKANISHI KAZUHIRO, MORI TOMOHIKO

    日本食品科学工学会大会講演集   49th   152   2002.8

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  • Removal of proteinaceous soils using hydroxyl radicals generated by the electrolysis of hydrogen peroxide

    K Imamura, Y Tada, H Tanaka, T Sakiyama, K Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   250 ( 2 )   409 - 414   2002.6

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    We have developed here for the first time a novel method to generate hydroxyl radicals, .OH, by applying slightly negative electric potentials (-0.2 - -0.8 V vs Ag/AgCl) to the surface of a metal (or metal oxide) that is in contact with hydrogen peroxide solution containing a supporting electrolyte. Namely, .OH radicals were generated at the surface by the electrolysis of hydrogen peroxide according to the equation, H2O2 + e(-) --&gt; .OH + OH-. This method was used to clean a stainless steel fouled with a model protein, beta-lactoglobulin. The .OHs generated at the surface were effective in removing beta-lactoglobulin that had been irreversibly adsorbed, by several minutes of treatment at room temperature (22 +/- 2degreesC). The removal rates measured for various concentrations of H2O2, and supporting electrolyte and different potentials were determined exclusively by the electric current. (C) 2002 Elsevier Science (USA).

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  • Removal of proteinaceous soils using hydroxyl radicals generated by the electrolysis of hydrogen peroxide

    K Imamura, Y Tada, H Tanaka, T Sakiyama, K Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   250 ( 2 )   409 - 414   2002.6

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    We have developed here for the first time a novel method to generate hydroxyl radicals, .OH, by applying slightly negative electric potentials (-0.2 - -0.8 V vs Ag/AgCl) to the surface of a metal (or metal oxide) that is in contact with hydrogen peroxide solution containing a supporting electrolyte. Namely, .OH radicals were generated at the surface by the electrolysis of hydrogen peroxide according to the equation, H2O2 + e(-) --&gt; .OH + OH-. This method was used to clean a stainless steel fouled with a model protein, beta-lactoglobulin. The .OHs generated at the surface were effective in removing beta-lactoglobulin that had been irreversibly adsorbed, by several minutes of treatment at room temperature (22 +/- 2degreesC). The removal rates measured for various concentrations of H2O2, and supporting electrolyte and different potentials were determined exclusively by the electric current. (C) 2002 Elsevier Science (USA).

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  • 食品製造プロセスで発生する汚れの新規洗浄方法 (特集:食品製造装置の洗浄殺菌)

    中西 一弘, 崎山 高明, 今村 維克

    食品工業   45 ( 2 )   18 - 24   2002.1

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  • Adsorption behavior of methylene blue and its congeners on a stainless steel surface

    K Imamura, E Ikeda, T Nagayasu, T Sakiyama, K Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   245 ( 1 )   50 - 57   2002.1

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    Methylene blue and its congeners as model dyes were adsorbed onto stainless steel particles at different ionic strengths, pH values, and ethanol contents, and the adsorption mechanism was investigated. A Fourier transform infrared spectroscopy (FTIR) analysis of the dyes adsorbed on the stainless steel plate was carried out to determine the orientations of the adsorbed dyes on stainless steel surface. The adsorption isotherms for all the dyes tested were approximated by a Langmuir equation (Q = K q(m) C/(1 + KC)) in most cases except under strongly basic conditions. From the ionic strength and ethanol content dependencies of the K value in the Langmuir equations, both the electrostatic and hydrophobic interactions were indicated to contribute to the adsorption of the dyes at neutral pH. By comparing the K and q(m) values for the methylene blue congeners and with the aid of the FTIR analyses, it was found that the kind of substituent groups at most positions of the polyheterocycles of methylene blue strongly affects the adsorption behavior, particularly the area occupied by an adsorbed dye molecule, the affinity for the stainless steel surface, and the orientation of the adsorbed dye molecule on the stainless steel surface. (C) 2002 Elsevier Science.

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  • Adsorption behavior of methylene blue and its congeners on a stainless steel surface

    K Imamura, E Ikeda, T Nagayasu, T Sakiyama, K Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   245 ( 1 )   50 - 57   2002.1

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    Methylene blue and its congeners as model dyes were adsorbed onto stainless steel particles at different ionic strengths, pH values, and ethanol contents, and the adsorption mechanism was investigated. A Fourier transform infrared spectroscopy (FTIR) analysis of the dyes adsorbed on the stainless steel plate was carried out to determine the orientations of the adsorbed dyes on stainless steel surface. The adsorption isotherms for all the dyes tested were approximated by a Langmuir equation (Q = K q(m) C/(1 + KC)) in most cases except under strongly basic conditions. From the ionic strength and ethanol content dependencies of the K value in the Langmuir equations, both the electrostatic and hydrophobic interactions were indicated to contribute to the adsorption of the dyes at neutral pH. By comparing the K and q(m) values for the methylene blue congeners and with the aid of the FTIR analyses, it was found that the kind of substituent groups at most positions of the polyheterocycles of methylene blue strongly affects the adsorption behavior, particularly the area occupied by an adsorbed dye molecule, the affinity for the stainless steel surface, and the orientation of the adsorbed dye molecule on the stainless steel surface. (C) 2002 Elsevier Science.

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  • Water sorption and glass transition behaviors of freeze-dried sucrose-dextran mixtures

    Koreyoshi Imamura, Atsushi Fukushima, Keisuke Sakaura, Takuo Sugita, Takaharu Sakiyama, Kazuhiro Nakanishi

    Journal of Pharmaceutical Sciences   91 ( 10 )   2175 - 2181   2002

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    The water sorption and glass transition behaviors of freeze-dried disaccharide-polysaccharide mixtures at various contents were investigated at relative humidities (RHs) of 0, 11, 23, and 33%. Sucrose and three types of dextrans, which differ in molecular weight, were used as model di- and polysaccharides, respectively. The relationship between the dextran and water contents of the sucrose-dextran mixture at different constant RHs indicated that a mixture of sucrose and dextran was lower than that calculated by the Lang and Steinberg mass balance equation. In the RH range of 0-23%, the glass transition temperature, Tg, increased to a considerable extent when the dextran content was equal to or higher than 50%, while the increase in Tg at dextran contents lower than 50% was small. A marked increase in Tg was observed at RH 33% for dextran contents of 0-25% as well as in the range above 50%. This suggests that the physical stability of the highly hydrated amorphous disaccharide is effectively strengthened by the addition of a small amount of polysaccharide. These tendencies were similar for the three dextrans of different molecular weights. Furthermore, it was demonstrated that the addition of a small amount of dextran is quite effective in preventing the collapse of amorphous sugar during freeze drying. © 2002 Wiley-Liss, Inc.

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  • Influences of the Suspension Method of Thermolysin in Organic Solvents on Enzyme Activity

    Keizo Tanaka, Koreyoshi Imamura, Takaharu SAkiyama, Kazuhiro Nakanishi

    Japan Journal of Food Engineering   3 ( 1 )   15 - 23   2002

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    Thermolysin (EC 3. 4. 24. 4) was suspended in organic solvents containing a small amount of water (aqueous buffer solution) by two different methods, and the catalytic activity as well as the conformational changes in the enzyme were compared. In one method (Method 1), dried powder enzyme was directly suspended in organic solvents containing a small amount of water while in the second method (Method 2), the dried enzyme was first dissolved in the buffer solution and then added to the anhydrous organic solvents containing the same final water content as that used in Method 1. In Method 2, the enzymes showed a catalytic activity 2-10 times as high as that in Method 1. The ratio of the activity obtained in Method 2 to that in Method 1 strongly depended on kinds of organic solvents, and its dependency was reasonably explained by the ET(30) values, which represent the extent of polarity of an organic solvent. The Fourier transform infrared spectroscopy (FT-IR) analyses indicated that the enzyme conformation in Method 2 was closer to its native one than that in Method 1. The influences of the suspension method on the enzyme activities of the different forms of the enzyme (as donated, dialyzed, and cross-linked) and the courses of the enzyme activities during incubation in Methods 1 and 2 were also investigated. © 2002, Japan Society for Food Engineering. All rights reserved.

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  • S219 Prospects for micro-bioreactor systems with respect to reactor and biocatalyst designs

    Nakanishi Kazuhiro, Sakiyama Takaharu, Imamura Koreyoshi

    14   21 - 21   2002

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  • 308 Analysis and control of adsorption behavior of enzyme onto a solid surface

    Ohnishi Kousuke, Imamura Koreyoshi, Sakiyama Takaharu, Nakanishi Kazuhiro

    14   28 - 28   2002

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  • 306 Water sorption and glass transition of freeze-dried disaccharidepolymer mixtures

    Fukushima Atsushi, Sakaura Keisuke, Sugita Takuo, Imamura Koreyoshi, Sakiyama Takaharu, Nakanishi kazuhiro

    14   28 - 28   2002

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  • 310 Removal of protein adsorbed on a metal surface by H_2O_<2^->electrolysis treatment

    Imamura Koreyoshi, Watanabe Ippei, Sakiyama Takaharu, Nakanishi Kazuhiro

    14   29 - 29   2002

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  • 309 Basic study on the control of enzyme binging onto polystyrene surfaces

    Ueno Shunsuke, Sakiyama Takaharu, Imamura Koreyoshi, Nakanishi Kazuhiro

    14   29 - 29   2002

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  • 549 Various substrate specificities of a novel acylase from Streptomyces sp.

    Koreishi Mayuko, Zhang Demin, Takayanagi Eiko, Hata Kazuki, Imamura Koreyoshi, Sakiyama Takaharu, Nakanishi Kazuhiro

    14   85 - 85   2002

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  • Evaluation of hydration states of protein in freeze-dried amorphous sugar matrix

    K Imamura, M Iwai, T Ogawa, T Sakiyama, K Nakanishi

    JOURNAL OF PHARMACEUTICAL SCIENCES   90 ( 12 )   1955 - 1963   2001.12

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    A model to analyze the hydration state of protein in freeze-dried amorphous sugar matrix was proposed, based on the assumptions that there is a limit to the amount of protein that a given amount of amorphous sugar could embed and that the freeze-dried sugar-protein mixture is composed of the four components, i.e., sugars with and without hydrogen bonding to proteins and proteins with and without hydrogen bonding to sugar. Bovine serum albumin and three kinds of disaccharides, i.e., sucrose, maltose, and trehalose, were used as samples. Using the analytical equations derived from the model and experimental sugar content dependencies of the water sorption at various relative humidities, the amount of hydration water for bovine serum albumin, and the minimum amount of sugar to embed the protein were determined. On the basis of these results, the degree of interaction between the three sugars and protein was discussed, with respect to their stabilizing effect on the protein. (C) 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association.

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  • Cleaning of a stainless steel surface fouled with protein using a UV-H2O2 technique

    K Imamura, Y Tada, H Tanaka, T Sakiyama, A Tanaka, Y Yamada, K Nakanishi

    JOURNAL OF CHEMICAL ENGINEERING OF JAPAN   34 ( 7 )   869 - 877   2001.7

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    The UV-H2O2 technique, which is commonly known as a method of decomposing organic substances from wastewater, was applied into cleaning of a stainless steel surface fouled with protein. On a stainless steel surface fouled with bovine beta -lactoglobulin (beta -Lg), H2O2 Solution was made to now, and UV rays were irradiated over the flowing liquid. The amounts of beta -Lg adsorbed before and during the UV-H2O2 cleaning were measured by a reflection absorption technique using Fourier transform infrared spectroscopy (RA-IR). The adsorbed amount approximately decreased linearly with time during the UV-H2O2 cleaning. There was an optimum H2O2 concentration for the removal rate. The H2O2 concentration dependency of the removal rate could be explained by considering the formation and disappearance rates of hydroxyl radicals (. OH), the decay of UV illuminance along the depth of the flow, and the rate of reaction between adsorbed beta -Lg and -OH.

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  • Increase in the stability of serine acetyltransferase from Escherichia coli against cold inactivation and proteolysis by forming a bienzyme complex

    K Mino, K Imamura, T Sakiyama, N Eisaki, A Matsuyama, K Nakanishi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   65 ( 4 )   865 - 874   2001.4

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    Cysteine synthetase from Escherichia coli is a bienzyme complex composed of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A, (OASS), The effects of the complex formation on the stability of SAT against cold inactivation and proteolysis were investigated. SAT was reversibly inactivated on cooling to 0 degreesC. Ultracentrifugal analysis showed that SET (a hexamer) was dissociated mostly into two trimers on cooling to 0 degreesC in the absence of OASS, while in the presence of BASS one trimer of the SAT subunits formed a complex with one dimer of BASS subunits. In the presence of BASS, trot only the cold inactivation rate was reduced but also the reactivation rate was increased. Furthermore, SAT became stable against proteolytic attack bg alpha -chymotrypsin and V8 protease by forming the complex with OASS, On the other hand, SAT was degraded by trypsin in the same manner both in the presence and in the absence of OASS. The different tendency in the stability against proteolysis with the different proteases was discussed with respect to the substrate specificity of the proteases and amino acid sequence of the C-terminal region of SAT that interacts with OASS.

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  • On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon

    K Nakanishi, T Sakiyama, K Imamura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   91 ( 3 )   233 - 244   2001.3

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    Adsorption of proteins on solid surfaces and their interaction are major concerns in a number of fields such as biology, medicine, biotechnology and food processing, and play an important role from various points of view. Based on practical viewpoints, information on the conformation of the adsorbed protein as well as adsorption characteristics is essential for a system's performance. Although there are still many problems to be solved, extensive studies in recent years, owing to the development in instrumentation and instrumental techniques, reveal the adsorption behavior of proteins in detail. Here, we stress the importance and interesting aspect of protein adsorption on solid surfaces by reviewing findings that have been obtained in recent years.

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  • 凍結乾燥によって得られた糖類アモリファスマトリクスにおけるタンパク質の水和状態の評価

    Journal of Pharmacentical Sciences   91(12), 1955-1964   2001

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  • UV/H2O2処理を利用したステンレス表面に付着したタンパク質の洗浄

    34 ( 7 )   869 - 877   2001

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  • Enzymatic modification of β-lactoglobulin with N-fatty-acyl-dipeptides by transglutaminase from Streptomyces mobaraense.

    Wada Eiko, Masuda Haruki, Imamura Koreyoshi, Sakiyama Takaharu, Adachi Shuji, Matsuno Ryuichi, Nakanishi Kazuhiro

    13   158 - 158   2001

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  • Purification of capsaicin-hydrolyzing enzyme from Streptomyces sp.

    Koreishi Mayuko, Zhang Demin, Imamura Koreyoshi, Sakiyama Takaharu, Adachi Syuji, Matsuno Ryuichi, Nakanishi Kazuhiro

    13   159 - 159   2001

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  • pH-Responsive swelling of chitosan/chondroitin sulfate complex gels and its application.

    Sakiyama Takaharu, Ikuta Yuji, Imamura Koreyoshi, Nakanishi Kazuhiro

    13   282 - 282   2001

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  • Cleaning of a stainless steel surface fouled with protein by H_2O_2-electrolysis technique

    Imamura Koreyoshi, Tada Yoichiro, Tanaka Hirofumi, Sakiyama Takaharu, Nakanishi Kazuhiro, Tanaka Atsumi, Yamada Yosuke

    13   282 - 282   2001

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  • Cultivation characteristics of rerecombinant Aspergillus oryzae for production of enzyme

    Shimamura Hiroko, Morita Masakazu, Yonashiro Ryota, Imammura Koreyoshi, Sakiyama Takaharu, Ninetoki Toshitaka, Nakanishi Kazuhiro

    13   199 - 199   2001

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  • バイオ・食品製造プロセスにおける汚れの付着と洗浄 (特集 最近の生物工学の展望)

    中西 一弘, 崎山 高明, 今村 維克

    ケミカルエンジニヤリング   45 ( 11 )   854 - 861   2000.11

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  • Adsorption behavior of amino acids on a stainless steel surface

    K Imamura, T Mimura, M Okamoto, T Sakiyama, K Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   229 ( 1 )   237 - 246   2000.9

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    The adsorption behavior of various amino acids on a stainless steel surface was investigated at 30 degrees C and over a pH range of 3-10. Acidic and basic amino acids except histidine adsorbed remarkably at pH 3-4 and 7-10, respectively, and showed Langmuir-type adsorption isotherms. The effects of pH and ionic strength on the adsorption isotherms were investigated to analyze the interactions between amino acids and adsorption sites on the stainless steel. Hydrophobic amino acids and glycine showed only small adsorbed amounts at all pHs tested. For the acidic and basic amino acids, reversibility of the absorption and the influence of the ionic strength on the adsorption behavior were examined. The adsorption isotherms of the derivatives of aspartic acid were also measured in order to examine the contribution of the carboxylic groups of acidic amino acids to the adsorption. Furthermore, a Fourier-transform infrared spectroscopic analysis and semiempirical molecular orbital calculation were carried out to analyze the ionization states and the configuration of the amino acids adsorbed on a stainless steel surface. These investigations suggest that the acidic and basic amino acids adsorb through two electrostatic interactions of two ionized groups in the amino acid with a stainless steel surface, (C) 2000 Academic Press.

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  • Characteristics of serine acetyltransferase from Escherichia coli deleting different lengths of amino acid residues from the C-terminus

    K Mino, K Hiraoka, K Imamura, T Sakiyama, N Eisaki, A Matsuyama, K Nakanishi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   64 ( 9 )   1874 - 1880   2000.9

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    Some properties of serine acetyltransferases (SATs) from Escherichia coli, deleting 10-25 amino acid residues from the C-terminus (SAT Delta C10-Delta C25) were investigated. The specific activity depended only slightly on the length of the C-terminal region deleted. Although the sensitivity of SAT Delta C10 to inhibition by L-cysteine was similar to that for the wild-type SAT, it became less with further increases in the length of the amino acid residues deleted. SAT Delta C10 was inactivated on cooling to 0 degrees C and dissociated into dimers or trimers in the same manner as the wild-type SAT, but Met-256-Ile mutant SAT as web as SAT Delta C14, SAT Delta C20, and SAT Delta C25 were stable. Since SAT Delta C10, SAT Delta C14, and SAT Delta C25 did not form a complex with O-acetyl-serine sulfhydrylase-A (OASS-A) in a way similar to SAT Delta C20. it was indicated that 10 amino acid residues or fewer from the C-terminus of the wild-type SAT are responsible for the complex formation with OASS-A. The C-terminal peptide of the 10 amino acid residues interacted competitively with OASS-A with respect to OAS although its affinity was much lower than that for the wild-type SAT.

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  • Adsorption behavior of amino acids on a stainless steel surface

    K Imamura, T Mimura, M Okamoto, T Sakiyama, K Nakanishi

    JOURNAL OF COLLOID AND INTERFACE SCIENCE   229 ( 1 )   237 - 246   2000.9

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    The adsorption behavior of various amino acids on a stainless steel surface was investigated at 30 degrees C and over a pH range of 3-10. Acidic and basic amino acids except histidine adsorbed remarkably at pH 3-4 and 7-10, respectively, and showed Langmuir-type adsorption isotherms. The effects of pH and ionic strength on the adsorption isotherms were investigated to analyze the interactions between amino acids and adsorption sites on the stainless steel. Hydrophobic amino acids and glycine showed only small adsorbed amounts at all pHs tested. For the acidic and basic amino acids, reversibility of the absorption and the influence of the ionic strength on the adsorption behavior were examined. The adsorption isotherms of the derivatives of aspartic acid were also measured in order to examine the contribution of the carboxylic groups of acidic amino acids to the adsorption. Furthermore, a Fourier-transform infrared spectroscopic analysis and semiempirical molecular orbital calculation were carried out to analyze the ionization states and the configuration of the amino acids adsorbed on a stainless steel surface. These investigations suggest that the acidic and basic amino acids adsorb through two electrostatic interactions of two ionized groups in the amino acid with a stainless steel surface, (C) 2000 Academic Press.

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  • Water sorption and glass transition of amorphous sugars containing BSA

    K Imamura, T Suzuki, T Tatsumichi, S Kirii, M Okazaki

    JOURNAL OF CHEMICAL ENGINEERING OF JAPAN   33 ( 4 )   657 - 660   2000.8

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    Water sorption and glass transition of four amorphous sugars (lactose, maltose, sucrose, and trehalose) containing bovine serum albumin (BSA) are investigated. Freeze-dried sugar-BSA samples equilibrated at several water activities ranging from 0 to 0.43 were prepared. Moisture content and glass transition temperature (T-g) were measured. For the all sugars, it is found that BSA lowers T-g at low water activity, and raises it at high water activity. It Is also found that the difference between T-g of the sugar-BSA samples and that of the corresponding amorphous sugar samples (T-go) depends mainly on T-go.

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  • Kinetics and equilibrium for thermolysin-catalyzed syntheses of dipeptide precursors in aqueous/organic biphasic systems

    M Miyanaga, M Ohmori, K Imamura, T Sakiyama, K Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   90 ( 1 )   43 - 51   2000.7

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    The initial kinetics for the syntheses of N-(benzyloxycarbonyl)-L-alanyl-L-phenylanine methyl ester (ZAPM) and N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (ZDPM) in an aqueous/organic biphasic system, using free thermolysin were elucidated, both experimentally and theoretically. As model organic solvents, ethyl acetate and tert-amyl alcohol were used. The substrate concentration dependencies of the initial rate of syntheses for ZAPM and ZI)PM observed in the biphasic system were well simulated using the overall partition coefficients of the substrates and product taking into consideration the effect of the formation of ion-pair complexes between the acid and amine components of the substrate, the initial rate equations determined in an aqueous buffer saturated with the organic solvent, and the pH dependence of the rate constant. The equilibrium yield for the synthesis of ZDPM was also in good agreement with the calculated result using the overall partition coefficients and equilibrium constant measured in the aqueous buffer.

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  • Kinetic analysis for synthesis of a dipeptide precursor using an immobilized enzyme in water-immiscible organic solvents

    M Miyanaga, K Imamura, T Sakiyama, K Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   90 ( 1 )   112 - 114   2000.7

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    N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the synthetic sweetener aspartame, was synthesized, using thermolysin immobilized onto Amberlite XAD-7, both in ethyl acetate and in tert-amyl alcohol. The initial rates for synthesis of Z-AspPheOMe in the organic solvents were predicted on the basis of a model proposed for an aqueous/organic biphasic reaction and compared with the experimentally observed substrate concentration dependencies. The experimental synthetic rates using the enzyme immobilized at a high enzyme concentration were lower than the calculated ones over a wide range of the substrate concentration. It was suggested as a reason for this discrepancy that the enzyme molecules form compact aggregates and those existing inside the aggregates cannot be utilized for reaction. The experimental results with the enzyme immobilized at a low concentration in ethyl acetate coincided well with the calculated ones. On the other hand, when tert-amyl alcohol was used, the experimental results were different in tendency irrespective of the amount of enzyme loaded, probably due to the fact that a distinct water phase does not exist around the enzyme aggregates inside the support.

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  • Development of novel advanced oxidation system using combined UV/H2O2 technique and kinetic analysis for decomposition of dye solutions

    K Imamura, A Hiramatsu, M Imada, T Sakiyama, A Tanaka, Y Yamada, K Nakanishi

    JOURNAL OF CHEMICAL ENGINEERING OF JAPAN   33 ( 2 )   253 - 261   2000.4

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    A novel advanced oxidation system using a combined UV/H2O2 technique was constructed for application to wastewater treatment. In this system, wastewater containing H2O2 flows along a channel with a flat surface in a thin film, onto which surface UV rays with a wavelength of 253.7 nm are irradiated. As a model wastewater, we used various dye solutions and investigated decoloration rates under various conditions differing in H2O2 concentration, temperature, UV illuminance, and so on. Based on the model, which takes into consideration the formation as well as the disappearance rate of hydroxyl radical (. OH), distribution of UV illuminance along the depth of the flow, and the rate of reaction between dye and . OH, we could simulate the course of dye decomposition. Furthermore, we showed the existence of an optimum H2O2 concentration for decomposition on the basis of this model.

    DOI: 10.1252/jcej.33.253

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  • 膜面液体培養法を用いた海洋植物プランクトンOscillatoria属の培養と特性解明

    中西 一弘, 崎山 高明, 今村 維克

    財団法人ソルト・サイエンス研究財団助成研究報告集   1998 ( 1 )   279 - 286   2000.3

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  • BSAを含有した糖アモルファスの水分収着およびガス転移

    Journal of Chemical Engineering of Japan   33 ( 4 )   657 - 660   2000

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  • 紫外線/H2O2処理を利用した新規促進酸化システムの開発と色素水溶液の分解過程の速度論の解析

    33 ( 2 )   253 - 261   2000

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  • Synthesis of capsaicin analogues by aminoacylase.

    Koreishi Mayuko, Kamakura Kaoru, Imamura Koreyoshi, Sakiyama Takaharu, Adachi Syuji, Matsuno Ryuichi, Nakanishi Kazuhiro

    12   218 - 218   2000

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  • Enzymatic synthesis of N-acylamino acids in cosolvents.

    Wada Eiko, Handa Masato, Imamura Koreyoshi, Sakiyama Takaharu, Adachi Shuji, Matsuno Ryuichi, Nakanishi Kazuhiro

    12   219 - 219   2000

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  • Synthesis of nonproteinous amino acid by cysteine synthetase with acetyl-CoA regeneration system.

    Ohno Katsuhiro, Mino Koshiki, Imamura Koreyoshi, Sakiyama Takaharu, Eisaki Naoki, Matsuyama Asahi, Nakanishi Kazuhiro

    12   218 - 218   2000

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  • Maintenance of protein structure in dry state by amorphous sugar

    Imamura Koreyoshi, Ogawa Tomohiro, Sakiyama Takaharu, Nakanishi Kazuhiro

    12   280 - 280   2000

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  • Increase in the stability of serine acetyltransferase from Escherichia coli against cold inactivation and proteolysis by forming a biezyme complex

    Mino Koshiki, Imamura Koreyoshi, Sakiyama Takaharu, Eisaki Naoki, Matsuyama Asahi, Nakanishi Kazuhiro, RITE

    12   225 - 225   2000

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  • Analysis for partition equilibrium of amino acid derivatives in aqueous/organic biphasic systems

    M Miyanaga, K Imamura, K Tanaka, T Sakiyama, K Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   88 ( 6 )   651 - 658   1999.12

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    The overall partition coefficients of the acid and amine components of amino acid derivatives, the substrates for proteinase-catalyzed synthesis of oligopeptide precursors, in an aqueous/organic biphasic system were studied both experimentally and theoretically. In a single-component system containing either an acid or amine component, the overall partition coefficient was well expressed by a model using the acid dissociation constant and partition coefficients for the non-ionized and ionized forms of the component. The overall partition coefficient in a binary-component system containing both acid and amine components was well simulated by the model when the formation of ion-pair complexes in the organic solvent phase was taken into consideration in addition to the partitioning of the non-ionized and ionized forms of the components. The formation of ion-pair complexes in the organic solvent phase was indicated by an analysis using Fourier transform infrared ray spectroscopy (FT-IR). In addition to the partition equilibrium, the aqueous-phase pH change after partitioning could be predicted by the model.

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  • Adsorption characteristics of tryptic fragments of bovine beta-lactoglobulin on a stainless steel surface

    T Sakiyama, K Tanino, M Urakawa, K Imamura, T Takahashi, T Nagai, K Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   88 ( 5 )   536 - 541   1999.11

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    As a strategy for the analysis of the mode of protein adsorption onto stainless steel surfaces, peptides obtained by tryptic digestion of bovine beta-lactoglobulin were subjected to adsorption experiments after identification of their primary structures. In the presence of I mM KOH, the peptides were scarcely adsorbed onto the surfaces of stainless steel particles from the peptide mixture. The adsorption experiments on isolated peptides showed that the affinities of the peptides for stainless steel surfaces in the presence of 1 mM HNO3 were significantly different from each other. Peptides without any acidic amino acid residues were scarcely adsorbed onto the surface, whereas some peptides with acidic amino acid residues were found to be irreversibly adsorbed onto the surfaces in the acidic pH region. As for the latter peptides, the amount adsorbed on the surface increased with increasing ionic strength. These results indicated that the carboxyl groups on the side chains of the peptides play an important role in the adsorption. Furthermore, the adsorption behavior of beta-lactoglobulin itself was found to be very similar to that of one of the latter peptides.

    DOI: 10.1016/S1389-1723(00)87672-0

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  • Stability of immobilized thermolysin in organic solvents

    M Miyanaga, M Ohmori, K Imamura, T Sakiyama, K Nakanishi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   87 ( 4 )   463 - 472   1999.4

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    Various factors affecting the stability of thermolysin immobilized by cross-linking with glutaraldehyde were elucidated, particularly in the water-immiscible organic solvents such as ethyl acetate and tert-amyl alcohol. The main reason for enzyme inactivation in water-immiscible organic solvents was found to be autolysis in the water phase, which may surround the enzyme immobilized inside the support. By contrast, in water-miscible organic solvents thermal denaturation was the predominant cause of enzyme inactivation. Courses of inactivation were expressed by second-order kinetics in the initial stage, after which inactivation proceeded at a slower rate. The extent of autolysis was found to strongly depend on the kind of organic solvent, the water content, and type of support and these dependencies were explained by the difference in the amount and state of water inside the support. Thermolysin was immobilized onto Amberlite XAD-7 as a compact aggregate inside the support which may increase the stability of the enzyme. Finally, it was shown that the stability of the immobilized enzyme could be correlated with the logP value for water-miscible organic solvents and with the solubility of water for water- immiscible organic solvents.

    DOI: 10.1016/S1389-1723(99)80095-4

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  • 食品製造・バイオプロセスにおける洗浄とタンパク性汚れの付着(<特集>殺菌・洗浄のバイオテクノロジー)

    中西 一弘, 今村 維克, 崎山 高明

    生物工学会誌 : seibutsu-kogaku kaishi   77 ( 6 )   241 - 245   1999

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    Other Link: http://dl.ndl.go.jp/info:ndljp/pid/10513378

  • Characteristics and kinetics of cysteine synthetase from Escherichia coli.

    Mino Koshiki, Imamura Koreyoshi, Sakiyama Takaharu, Eisaki Naoki, Matsuyama Asahi, Nakanishi Kazuhiro, RITE

    11   170 - 170   1999

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  • Adsorption behaviors of protein, peptide, and amino acid onto stainless steel surface

    Imamura Koreyoshi, Sato Masako, Tomura Junji, Okamoto Makoto, Sakiyama Takaharu, Nakanishi Kazuhiro, Takahashi Tokio, Nagai Tadashi

    11   333 - 333   1999

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  • Production of pigment from Monascus pilosus k using membrane surface liquid culture

    Tsushima Youko, Mino Akira, Imamura Koreyoshi, Sakiyama Takaharu, Nakanishi Kazuhiro, Suga Sadaharu, Danou Yasuhumi

    11   220 - 220   1999

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  • Purification and characterization of serine acetyltransferases truncated at C-terminal regions.

    Hiraoka Kenji, Mino Koshiki, Imamura Koreyoshi, Sakiyama Takaharu, Eisaki Naoki, Matsuyama Asahi, Nakanishi Kazuhiro, RITE

    11   167 - 167   1999

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  • Synthesis of β-pyrazole alanine using cysteine synthetase coupled with acetyl-CoA regeneration system.

    Ohno Katsuhiro, Mino Koshiki, Yamanoue Tsuyoshi, Imamura Koreyoshi, Sakiyama Takaharu, Eisaki Naoki, Matsuyama Asahi, Nakanishi Kazuhiro, RITE

    11   184 - 184   1999

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  • Characteristics of enzyme production from recombinant Aspergillus oryzae using membrane surface liquid culture

    Shimamura Hiroko, Ishida Natsuko, Sugita Takuo, Imamura Koreyoshi, Sakiyama Takaharu, Nakanishi Kazuhiro, Ozeki Kenji

    11   220 - 220   1999

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  • Characteristics of production of kojic acid from Aspergillus oryzae NRRL 484 using membrane surface liquid culture

    Sugita Takuo, Segawa Takayuki, Shimamura Hiroko, Imamura Koreyoshi, Sakiyama Takaharu, Nakanishi Kazuhiro

    11   219 - 219   1999

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  • Influence of protein on phase transition of amorphous sugar

    K Imamura, T Suzuki, S Kirii, T Tatsumichi, M Okazaki

    JOURNAL OF CHEMICAL ENGINEERING OF JAPAN   31 ( 3 )   325 - 329   1998.6

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    In order to investigate the influence of protein on the phase transitions of amorphous structure of sugar, we carried out differential scanning calorimetry (DSC) analysis of freeze-dried sucrose containing various contents of water and bovine serum albumin (BSA). Glass transition temperature Tg, crystallization temperature Tcr, and melting temperature Tm were measured. Tg, Tcr, and Tm decreased with increasing water activity. Addition of BSA raised Tg except the case that water activity was low (up to about 0.1). Tcr increased linearly with BSA content, and Tm did not depend on BSA content. The increase in Tg and Tcr with BSA suggests that protein contributes to stabilizing amorphous structure of sugar.

    DOI: 10.1252/jcej.31.325

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  • Fouling and cleaning of stainless steel surface: Adsorption and desorption behavior of bovine serum albumin and gelatin

    T Sakiyama, T Toyomasu, A Nagata, K Imamura, K Nakanishi, T Takahashi, T Nagai

    JOURNAL OF CHEMICAL ENGINEERING OF JAPAN   31 ( 2 )   208 - 213   1998.4

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    Adsorption and desorption behavior of bovine serum albumin (BSA) and gelatin on the surface of stainless steel particles are studied. The amount of BSA adsorbed increases significantly with temperature above 60 degrees C whereas that of gelatin decreases slightly with increasing temperature, Results of adsorption experiments with S-carboxymethylated BSA show that the thermal aggregation of BSA molecules at the surface through intermolecular thiol-disulfide interchange reactions plays an important role in the adsorption of BSA at elevated temperatures. Furthermore, the initial desorption rate constants and residual amounts of the two proteins during caustic and enzymatic cleanings of the fouled particles are compared under various conditions. As a result, a large difference is found in the temperature dependence of the initial desorption rate constant in caustic cleaning, suggesting different modes of adsorption of the proteins.

    DOI: 10.1252/jcej.31.208

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  • Performance of protease as a cleaning agent for stainless steel surfaces fouled with protein

    T Sakiyama, T Toyomasu, A Nagata, K Imamura, T Takahashi, T Nagai, K Nakanishi

    JOURNAL OF FERMENTATION AND BIOENGINEERING   85 ( 3 )   297 - 301   1998

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    Cleaning experiments were performed by feeding protease solutions as cleaning agents into a packed column of stainless steel particles fouled with beta-lactoglobulin or gelatin. By evaluating the initial desorption rate constants (Brst-order rate constants) and residual amounts of protein, the cleaning efficiencies of the proteases were compared. The initial desorption rate constant depended on the kind of protease used and also on the type of protein to be removed. It increased with protease concentration and reached a constant value at a limit concentration. Kinetic analysis of the proteolytic reactions catalyzed by the proteases revealed that protease with a large Vm(ax)/EKm value for the proteolytic reaction resulted in a large value of the initial desorption rate constant in the enzymatic cleaning at a low protease concentration. The K-m value affected the limit concentration of protease yielding a saturation of initial desorption rate constant.

    DOI: 10.1016/S0922-338X(97)85678-4

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  • Development of a cylindrical apparatus for membrane-surface liquid culture and production of kojic acid using Aspergillus oryzae NRRL484

    Y Wakisaka, T Segawa, K Imamura, T Sakiyama, K Nakanishi

    JOURNAL OF FERMENTATION AND BIOENGINEERING   85 ( 5 )   488 - 494   1998

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    To apply membrane-surface liquid culture (MSLC), reported in previous papers (J. Ferment. Bioeng., 80: 35-40, 41-46, 1995) to large-scale production, a novel bioreactor was constructed in which molds were grown on the outer surface of a cylindrical porous membrane facing the air with liquid medium running down along its inner surface. We cultivated Aspergillus oryzae NRRL484 to produce kojic acid as a model secondary metabolite. An SE20 membrane composed of polysulfone with a nominal pore diameter of 0.2 mu m (Fuji Photo Film Co.) was found to be most suitable based on the facts that the mass transfer through the membrane had no appreciable effect on the kojic acid production and the mycelia did not pass through the membrane pores. Kojic acid was produced at higher levels than in shaking flask culture. The highest kojic acid concentration in the batch MSLCs using the cylindrical membrane module with an active surface area of 220 cm(2) was 14, 45, and 60 mg/ml for glucose concentrations in the medium of 5, 10, and 20%, respectively. In the shaking flask cultures using 10 and 20% glucose, the maximum kojic acid concentrations were 24 and 22 mg/ml, respectively. In the MS;LC, the liquid medium was quite clear without any contamination of cells. Furthermore, the mold was highly stable against autolysis, and continuous cultivation with holding tells on the membrane surface could be conducted for over 70 d. When the average feed rate of the medium containing 10% glucose and 0.1% yeast extract was 30 ml/d, the steady-state kojic acid concentration at the outlet of the MSLC apparatus was in the range of 45-50 mg/ml.

    DOI: 10.1016/S0922-338X(98)80067-6

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  • 糖類アモルファス構造の相転移に及ぼすタンパク質の影響

    Journal of Chemical Engineering of Japan   31 ( 3 )   325 - 329   1998

  • 新規に開発したカビの膜面液体培養法を用いた食品加工用酵素の生産に関する基礎研究

    中西 一弘, 崎山 高明, 今村 維克

    食に関する助成研究調査報告書   ( 11 )   51 - 58   1998

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  • Adsorption of amino acids and poptides onto the surface of stainless steel particles.

    Mimura Tomoya, Tanino Kei, Imamura Koreyoshi, Sakiyama Takaharu, Nakanishi Kazuhiro

    9   190 - 190   1997

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  • Cultivation of mold by membrane-surface liquid culture and its scale-up

    Segawa Takayuki, Imamura Koreyoshi, Sakiyama Takaharu, Nakanishi Kazuhiro

    9   96 - 96   1997

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Presentations

  • 金属表面の高分子コーティングによるタンパク質の三次元固定化

    田坂 隆貴, 西川 葵, 今中 洋行, 石田 尚之, 今村 維克

    化学工学会岡山大会2022  2022.12.19 

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    Event date: 2022.12.19

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Awards

  • study award

    2019.8   Japanese Society of Food Engineering   Engineering study on the technique for enbedding and stabilizing biomolecules including food proteins

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  • encouragement award

    2007.8   Japanese Society of Food Engineering   Developmemt and innovation of novel cleaning technique based on electrolysis of hydrogen peroxide

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  • encouragement award

    2002   Society of Chemical Engineering of Japan   Study on advanced cleaning technique utilizing hydroxyl radical

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    Country:Japan

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Research Projects

  • Physico-chemical stabilization of surfactant-free solid dispersion and improvement of aqueous dissolution of water-insoluble drugs

    Grant number:19H02499  2019.04 - 2023.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    今村 維克

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    Grant amount:\16120000 ( Direct expense: \12400000 、 Indirect expense:\3720000 )

    難水溶性薬剤の生体内における溶解性を改善する手法として,水可溶性のキャリアマトリクスに非晶質化した薬剤を分散する技術がある.申請者は,本来は“水と油”の関係にある糖と疎水性薬剤を,両親媒性物質を一切用いることなく,分子レベルで均一に混合できる原理的に新規な固体分散技術(Sole Amorphous Sugar based Amorphous Solid Dispersioin, SAS-SD, “surfactant-free固体分散”から呼称変更)を開発した.このSAS-SD試料において糖は水中で瞬時に溶解するため,疎水性薬剤の対水溶解性を既存の固体分散技術と同等以上に改善することができる.このSAS-SD技術は難水溶性成分の水溶性を高度に改善するが, ガラス転移温度 (Tg) が低く保存安定性に乏しいことが分かっていた.また他の固体分散技術と同様に, 難水溶性成分の溶解時間にも改善の余地が大きかった.そこでいくつかのモデル薬剤と糖を用いてSAS-SDを作成し,そのSAS-SD試料を難水溶性成分の融点以上の温度で熱処理(アニーリング)すると難水溶性成分の対水溶解性, Tgともに改善することが分かった.この難水溶性物質の溶解挙動の変化機構について基礎的な知見を得るためDSC等により固体分散試料の熱特性, 相互作用状態を評価した.その結果,アニーリングすることで糖分子間の相互作用密度が緩和され, 糖分子がより安定な構造をとっているためであることが示唆された.さらに,同じ骨格構造は持つ複数の誘導体(ibuprofen等)をモデル薬剤としてSAS-SD試料を作成し,アニーリング条件と薬剤の対水溶解性,およびモデル薬剤の物理化学的特性の関係について検討した結果,SAS-SDおよびそのアニーリングによる薬剤の対水溶解性の改善度は薬剤の融点を用いて相関できることを見出した.さらにSAS-SDの調製手法・条件の検討を行った.具体的にはSAS-SD試料を真空foam乾燥と噴霧乾燥の二種類の方法で調製し,その物性を比較・検討した.

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  • Induction of Foaming under Low Vacuum by Needle Stimulation of Solution for Controlling Drying Process in Vacuum Foam Drying

    Grant number:15K14206  2015.04 - 2017.03

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Exploratory Research

    Imamura Koreyoshi

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    “Vacuum foam drying” is a promising drying technique that allows quick and effective desolvation without heating. However, “foaming” occurs randomly even under a sufficiently foamable condition and the timing of the foaming during the drying is hard to be controlled; The heterogeneous foaming results in different drying histories and consequently different characteristics of the dried products. On the other hand, we eventually found that, that, when the vacuum drying was aborted at a certain extent of drying and stimulated by sticking the sample solution with a steel needle, the resurgence of the vacuum drying immediately bring about the foaming at 100% probability. In order to establish the technique to uniform the product qualities obtained by “vacuum foam drying,” the impact of the needle stimulation on the solution foaming was herein investigated under different conditions. The mechanism by which the needle stimulation induces the inevitable foaming was also discussed.

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  • Heterogeneity of the state and functionality of water molecules sorbed in an amorphous sugar matrix

    Grant number:23560908  2011 - 2013

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    IMAMURA Koreyoshi

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    Amorphous materials, comprised of sugar molecules, exhibit high hygroscopicity and the sorbed water exerts a major influence on physical stabilities of the matrix. To date, the water sorption behavior of amorphous sugars has been extensively investigated. However, most studies of the behavior of water, when sorbed to an amorphous sugar matrix, have implicitly assumed that all of the sorbed water molecules are in a single state: It is naturally expected that the random allocation and configuration of sugar molecules would result in heterogeneity of states for the sorbed water. Recently, we demonstrated the heterogeneity of state and functionality of water sorbed in amorphous sugar matrices by combining a Fourier transform IR spectroscopy and Fourier self-deconvolution technique; The sorbed water molecules were classified into five states, and the three of them mainly served to lower the glass transition temperature of an amorphous sugar matrix while the other two are independent.

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  • Development of Site-Directed Immobilization Method to ConstructHigh-Efficient Bioreactors

    Grant number:22560771  2010 - 2012

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    NAKANISHI Kazuhiro, IMAMURA Koreyoshi, IMANAKA Hiroyuki

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    In this study, a method to immobilize enzymes/proteins on the solid surface without suffering from denaturation that might occur upon coming in contact with the solid surface was investigated. Conjugating peptide tags that show a high affinity to silica and polystyrene surfaces, toenzymes enabled a strong, nearly irreversible immobilization without appreciable loss of activity.

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  • Extraordinarily High Stabilizing Effect of Amorphous Sugar Matrix at Temperatures below Zero-Mobility Temperature

    Grant number:20560702  2008 - 2010

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    IMAMURA Koreyoshi

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    Grant amount:\4420000 ( Direct expense: \3400000 、 Indirect expense:\1020000 )

    An amorphous matrix, comprised of sugar molecules, is known to stabilize labile substances such as proteins against chemical and physical degradations. On the other hand, this study revealed that a critical temperature, T*, exists, below which the disruption of sugar-sugar hydrogen bonds due to increasing temperature was substantially decreased relative to that at temperatures above the T*. In order to determine whether T* is the boundary temperature for achieving minimum molecular mobility, the relationship between T* with the mobility of sugar molecules in an amorphous matrix was investigated based on the structural relaxation behavior of amorphous sugar matrices at different temperatures. Furthermore, the T* values and the storage-stabilities of a protein for several types of sugars were examined and compared.

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  • 親水性ポリスチレン高親和性ペプチドを用いた革新的蛋白質相互作用解析システムの創製

    Grant number:19656221  2007 - 2008

    日本学術振興会  科学研究費助成事業  萌芽研究

    中西 一弘, 今村 維克, 今中 洋行

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    Grant amount:\3300000 ( Direct expense: \3300000 )

    本年度は、親水性ポリスチレン親和性ペプチドを用いた、1)コアストレプトアビジン(Core-SA)-DNA相互作用解析と2)相互作用に関与する機能性ペプチド部位を連結したクッションタンパク質を用いた相互作用解析を計画したが、両課題共に、初期の予定を達成できた。1)の課題においては、4量体のCore-SAに連結する親和性ペプチド(PSペプチド)の個数を変えて、その固定化特性とオリゴDNAとの相互作用をELISA法で検出した。結果として、4つのPSペプチドを連結したCore-SAは共存する高濃度牛血清アルブミンとの競合に影響されることなく、最も強固に固定化できることを示した。さらに、ビオチン標識オリゴDNAを連結したペプチドタグ連結コアストレプトアビジンは、市販のSA-coated plateよりも高い検出感度を示すことがわかった。一方、2)の課題に関しては,システイン合成酵素を構成するO-acetylserine sulfhydryrase-A(OASS-A)とそのC末端で相互作用することが知られているserine acetyltransferase(SAT)との間の相互作用に着目した。SATのC末端20残基からなるオリゴペプチドを、そのN末端にPSペプチドを連結したクッションタンパク質(RNaseHII)のC末端に連結し、オリゴペプチドのC末端アミノ酸を変化させてOASS-Aとの相互作用をELISA法で検出した結果、C末端アミノ酸がイソロイシンの野生型ペプチドを提示させた場合にのみ、相互作用することがわかった。同様に、Strep-tagIIをクッションタンパク質に提示したが、そのN末端をクッションタンパク質に向けて結合した場合にのみstreo-tactinと強い相互作用を示した。これらの結果から、本研究で検討した、親水性PS特異的親和性ペプチを用いる相互作用解析方法は、極めて高い検出感度と特異性を有することが明らかにされた。

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  • 糖類アモルファスマトリクスのゼロモビリティー温度と不安定物質の半永久的保存

    Grant number:18760594  2006 - 2007

    日本学術振興会  科学研究費助成事業  若手研究(B)

    今村 維克

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    Grant amount:\3500000 ( Direct expense: \3500000 )

    糖によって形されたアモルファスマトリクスは,タンパク質などの不安定物質を包括すると,その劣化を抑制する働きがある.この糖類アモルファスマトリクスによる包括安定化作用は,マトリクスを構成する糖分子の状態と密接な関係があると考えられる.従って,室温以下の低温域においては,極めて高度な保存性が期待できる.本研究では,昇温過程における糖分子の赤外線吸収(IR)スペクトルを連続的に測定できる温度操作フーリエ変換赤外分光分析(TS-FTIR)により,-20℃からTg前後の温度域における糖分子間相互作用(水素結合)の形成状態を解析した.すなわち,糖水酸基の伸縮振動に起因するIR吸収帯のピーク波数を温度に対してプロットした.その結果,ピーク波数は温度上昇に伴い高波数側にシフトしていくが,Tgより数十℃低いある温度(T*)においてもピーク波数の増加勾配が顕著に小さくなることが分かった.これより,糖分子間水素結合が極めて安定に存在する温度域(<T*)が存在することが示唆された.また,その温度域の上限(T*)はsucroseで顕著に低く,糖の分子量とともに高くなる傾向が見られた.
    一方,糖類アモルファスマトリクスの構造エンタルピーの緩和過程を示差走査熱量分析(DSC)により測定し,異なる温度における糖分子のモビリティーを評価・比較した.その結果,上述のT*以下の温度領域では,各糖試料の構造エンタルピーの緩和が認められなかった.さらに,酵素を用いて糖類アモルファスマトリクスで包括したときの失活速度の温度依存性を調べたところ,T*以下では酵素の失活がほとんど生じないことが分かった.以上のことから,T*は糖分子のモビリティーが極端に制限される温度領域の上限値を表しており,この温度以下では極めて高度な包括安定化作用が発現することが明らかとなった.

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  • Molecular mechanism for adsorption and adsorption of soil components formed on the surface of equipment wall during manufacturing and development of an efficient cleaning method

    Grant number:16206076  2004 - 2007

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (A)

    NAKANISHI Kazuhiro, IMAMURA Koreyoshi, IMANAKA Hiroyuki

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    Grant amount:\46800000 ( Direct expense: \36000000 、 Indirect expense:\10800000 )

    In various manufacturing processes, cleaning of the equipment and piping systems used for manufacturing of various products is requisite for maintaining sanitary conditions and/or functions of the equipments. In particular, in the food- and bio-products manufacturing processes, a large amount of energy and detergents are usually consumed in addition to time, since soils or residues of the products strongly interact with the equipment surface. In this study, we studied adsorption mechanisms of various proteins/peptides and low-molecular weight substances as a model soil component on the surfaces of various metal oxides, plastics, and glasses. We clarified the molecular mechanisms for reversible/irreversible adsorptions, states and orientation of the molecules adsorbed, the effects of the pI values of the metal oxide surfaces/solution pHs on the adsorption characteristics, and conformational change of the adsorbed proteins, on the basis of adsorption/desorption experiments, IR spectrum analyses, and molecular calculations. Furthermore, we investigated the effects of various factors such as the kinds of the co-existing salts and their concentrations on the removal rates of the metal oxide surface fouled with proteins, using H2O2-electrolysis cleaning method that was developed by the author's group. Furthermore, we developed a method to immobilize proteins with controlled orientation and structure for various uses in chip-based biotechnology, which was derived in the course of investigation on the adsorption mechanism of proteins.

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  • 高度なタンパク質安定化作用と物理的安定性を兼備した新規糖類アモルファス構造の創製

    Grant number:15760558  2003 - 2004

    日本学術振興会  科学研究費助成事業  若手研究(B)

    今村 維克

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    Grant amount:\3500000 ( Direct expense: \3500000 )

    目的
    高度なタンパク質安定化作用と物理的安定性を併せ持つ新規な糖類アモルファスマトリクスの構築を最終目標として,高度なタンパク質安定化作用を有する少糖と高い物理的安定性を有する多糖を組み合わせた少糖-多糖複合アモルファスの物理特性およびマトリクス内における分子間相互作用について検討した.
    実験方法
    スクロ-スおよびデキストランを様々な割合(0〜100%)で含む水溶液を調製した.一部の試料溶液には塩を0.1〜100mMになるように添加した.試料溶液を液体窒素で急速凍結して室温で1日凍結乾燥した.その後,さらにP_2O_5を入れた真空デシケータ内(37℃)で3日間絶乾処理することにより,少糖-多糖複合アモルファス試料を得た.少糖-多糖複合アモルファス内の分子間相互作用を解析するため,試料を一定相対湿度(RH 11,23,33%)下で平衡化後,含水率,ガラス転移温度(Tg)を測定した.試料の含水率は重量法およびKarl-Fischer法により測定し,ガラス転移温度は示差走査熱分析により測定した.
    結果
    スクロースーデキストラン複合アモルファス試料の水分収着特性を解析した結果,複合試料の平衡含水率はスクロースおよびデキストランそれぞれ単体のときの値から算出される理論値よりも低くなることが分かった.これはスクロースとデキストランが共存することでスクロース-デキストラン分子間に相互作用が形成し,それぞれ単体で存在するときよりも水和サイトが減少したことを意味している.一方,スクロース-デキストラン複合アモルファス試料のガラス転移温度に及ぼす塩の影響を調べた結果,リン酸塩の共存によるガラス転移温度の変化は認められなかったが,塩化ナトリウムや塩化アンモニウムはガラス転移温度を低下させることが分かった.

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  • Adhesion and Removal Mechanisms of Composite Soils Formed on the Equipment Surface During Food Processing

    Grant number:13450319  2001 - 2003

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    NAKANISHI Kazuhiro, IMAMURA Koreyoshi, SAKIYAMA Takaharu

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    Grant amount:\15100000 ( Direct expense: \15100000 )

    In food manufacturing processes, proteinaceous soils derived from foods tend to strongly adhere to the サball surface of equipment/pipes and thus a lot of energy and detergents are usually consumed for cleaning of the equipment. Although it is strongly required to save energy and detergents by optimizing the cleaning conditions, few systematic approaches have been made. In this project, the intended to reveal scientifically the mechanism for adsorption of proteinaceous soils onto the metal surfaces and that for removal. As a model proteinaceous soil, we usually adopted β-lactoglobulin (β-Lg) and sometimes used a composite of the protein and tannic acid. Particles and plates of stainless steel were mostly used as a model surface. The findings obtained in this projects are summarized as follows.
    1. Interaction peptide regions of β-Lg that directly interact with a stainless steel surface was analyzed by the two methods : 1) peptide fragments of β-Lg digested with protease were first in contact with the surface and peptides strongly interacting were identified and 2) β-Lg that had been adsorbed on the surface was subjected to digestion with protease and then the kinds and composition of the remaining peptides were analyzed. As a result, we found that peptide regions containing plural, acidic amino, acids are responsible for direct interaction with the surface at a pH region lower than neutral pH. With increasing the number of acidic amino acids of the peptide, affinity as well as the amount of adsorptiotn increased. Adsorption isotherms could be explained by taking into consideration the both reversible and irreversible adsorption modes.
    2. We developed a novel cleaning method, in which negative electropotential (around -0.2〜-0.8V vs. Ag/AgCl) is added to the metal surface being in contact with dilute H_2O_2 solution to generate hydroxyl radicals and generated radials could quickly decompose proteinaceous soils adsorbed on the surface. The proteinaceous soil strongly adsorbed on a stainless steel surface there found to be removed completely within 2 min at room temperature while the removal rate by caustic cleaning was much slower. The various factors including coexisting salt concentration, potential, and current on the removal kinetics there analyzed in details.
    3. Adsorption mechanism of the composite soil composed of β-Lg and tannic acid was analyzed under different conditions ~ using RAS-IR. The H_2O_2-electrolysis cleaning method was quite effective to remove the soil from the surface. Namely, tannic acid was removed effectively even by caustic cleaning while sufficient removal of β-Lg was possible only by the H_2O_2-electrolysis cleaning method at room temperature.

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  • 高度なタンパク質安定化機能を有する糖類アモルファスマトリクスの創製

    Grant number:12750664  2000 - 2001

    日本学術振興会  科学研究費助成事業  奨励研究(A)

    今村 維克

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    Grant amount:\2200000 ( Direct expense: \2200000 )

    目的 不安定なタンパク質を安定化する手法の一つとして、タンパク質を糖などの安定化剤とともに凍結乾燥することでタンパク質を糖のアモルファスマトリクスに包埋し、保存中の変性、失活を低減する手法がある。このタンパク質安定化剤としてはスクロースなどの低分子量の糖は高い安定化作用を有するものの、吸湿や熱により容易にコラプスするという欠点がある.一方,デキストランなどの多糖類は安定化作用は極めて低いものの,物理的安定性に優れている。従って,高い安定化作用を有する少糖と高い物理的安定性を有する多糖を組み合わせることで,高度な安定化作用と物理的安定性を併せ持つ新規な糖類アモルファスマトリクスを構築出来る可能性がある.この新規な安定化剤である少糖-多糖複合アモルファスの物理的安定性(ガラス転移温度,Tg)およびタンパク質安定化作用について検討した。
    方法 少糖としてsucrose, erythritol,多糖として分子量の異なる2種類のdextran(MW1500, 6000),タンパク質としてlactate dehydrogenase (LDH)を用いた.これらの少糖・多糖を様々な割合(0〜100%)で含む凍結乾燥試料を作成し,凍結乾燥直後のLDHの相対残存活性,一定相対湿度下で平衡化したときのTgを測定した.
    結果 sucrose-dextran, erythritol-dextran試料のTgはいずれの相対湿度においてもdextran含有率が40%以上で顕著に上昇することが分かった。一方,凍結乾燥直後の残存酵素活性は,sucrose crythritol単体の場合はいずれも50%であったのに対し,sucrose-dextran試料では最大70%, erythrito1-dextran試料では最大95%となった.これらのことより少糖-多糖複合アモルファスマトリクスの極めて高い有効性が明らかとなった.

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  • 固体表面に対する配向の精密制御されたタンパク質の創製

    Grant number:12019251  2000

    日本学術振興会  科学研究費助成事業  特定領域研究(A)

    中西 一弘, 今村 維克, 崎山 高明

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    Grant amount:\2000000 ( Direct expense: \2000000 )

    タンパク質(酵素)の固体表面に対する付着は、分析、反応、生体材料設計など生物生体工学の多岐の分野においてみられれ現象であり、それぞれの応用を考える際には付着に対する親和性と配向の制御が重要な鍵となる。そこで、本研究では、タンパク質の固体表面に対する相互作用と直接付着に関与する部位の解明を行うと共に固体表面に対する親和性と配向の制御されたタンパク質の創製を最終目的としている。タンパク質として分子量が小さく、性質や立体構造が明らかにされているウシ膵臓ribonucleaseA(RNaseA)に着目して、固体表面に直接相互作用するタンパク質分子内ペプチド部位の解明と固体表面に対する親和性及び配向の制御された酵素の創製を行うために、変異RNaseAの調製法を確立すると共にその付着挙動を調べた。変異RNaseAとしては、ステンレス表面に対する付着部位と推定されている、^<47>His-Glu-Ser-Leu-Ala-Asp-Val^<57>中のAlaをAspに置換したAla55Asp変異RNaseAとGluとAspのそれぞれあるいは両方をAlaに置換した、Glu55Ala変異RNaseAとAsp55Ala変異RNaseA、及びPS Latexに対し強い付着を示す^<117>Pro-Val-His-Phe-Asp-Ala-Ser-Val^<124>中のPheをAlaに置換したPhe120Ala変異RNaseAを調製した。いずれの場合も変異RNaseA遺伝子をプラスミドpBXRに組み込んだ発現プラスミドを作成し、大腸菌(BL21)内で発現させた。培養大腸菌を回収後に超音波破砕を行い、封入体を回収した。回収した封入体を変性・再生後にイオン交換クロマトグラフィーとゲル濾過により精製した。精製酵素はSDS-PAGE上で均一であることを確認した、精製酵素の収率は10%前後であった。精製酵素のステンレス粒子及びPS Latexに対する付着特性を調べた。予備的検討の結果、予想した通りにAla55Asp変異RNaseAはステンレス粒子に対して野生型RNaseAよりも強い付着力を示したが、Phe120Ala変異RnaseAでは変化がみられなかった。

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  • 固体表面に対する親和性と配向の精密制御されたタンパク質の創製

    Grant number:11132250  1999

    日本学術振興会  科学研究費助成事業  特定領域研究(A)

    中西 一弘, 今村 維克

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    Grant amount:\2000000 ( Direct expense: \2000000 )

    本研究では、タンパク質と固体表面の直接の相互作用に着目して、固体表面に対する親和性と配向の制御されたタンパク質の創製を最終目的として、1)固体表面に直接相互作用するタンパク質分子内ペプチド部位を特定する方法の確立と、2)ペプチド部位の固体表面に対する親和性の強さとそのアミノ酸組成・長さ・二次構造等との関係を明らかにすることである。本年度はモデルタンパク質として、主として分子量が小さく三次元構造が明らかにされている、ウシ膵臓由来リボヌクレアーゼA(RNaseA)を、固体表面としてステンレス粒子とポリスチレンラテックス(Pslatex)を用いて、固体表面に直接相互作用する部位の同定と相互作用に関する知見を得ることを目的とした。RNaseAをカルボキシメチル化した後、トリプシン処理し、得られたペプチド混合液あるいは合成したペプチドを試験液に溶解し、吸着等温線を測定した。主な成果は次の3点に要約される。
    1)RNaseAの各pHにおけるステンレスに対する吸着等温線はいずれもLangmuir型を示したが、高pH領域では、殆ど吸着がみられなかった。Pslatexの場合では吸着等温線はpHに殆ど依存せず、不可逆吸着を示した。
    2)トリプシン処理によって得られたRNaseA由来ペプチドを用いて吸着実験を行ったところ、ステンレスに対しては酸性アミノ酸残基を含むペプチド10が、Pslatexに対しては、疎水性アミノ酸残基を多く含むペプチド9が顕著に吸着していた。3)ペプチド9-1の疎水性アミノ酸残基であるIleをGlyに、ペプチド9-3のPheをGlyに、10-1の酸性アミノ酸残基GluとAspをGlyに置換したペプチドを合成し、吸着特性の変化を調べたところ、いずれのペプチドも吸着力は激減した。これらの結果から、ステンレスに対する吸着には酸性アミノ酸残基が、Pslatexに対する吸着には、疎水性アミノ酸残基による疎水相互作用とベンゼン環同士のπ-π相互作用が深く関与していることが示唆された。

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  • Clarification of Adhesion Mechanisms of Soils onto the Equipment Wall Surfaces and Its Desorption in Food Manufacturing Process

    Grant number:10450287  1998 - 2000

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B).

    NAKANISHI Kazuhiro, IMAMURA Koreyosi, SAKIYAMA Takaharu

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    Grant amount:\14100000 ( Direct expense: \14100000 )

    In this study, we focussed to the following three subjects, i.e., 1) clarification of adsorption characteristics of proteins, peptides, and amino acids, 2) identification of peptide regions in protein molecules that interact with solid surfaces, and 3) cleaning behaviors of the surfaces fouled with proteins using proteolytic enzymes and hydroxyl radicals generated by combined hydrogen peroxide and ultraviolet rays. Adsorption behaviors of β-lactoglobulin, bovine serum albumin, and gelatin onto the stainless steel surface were investigated under different conditions. With increasing the temperature, the adsorbed amount of β-lactoglobulin and bovine serum albumin were increased to form disulfide linkages, whereas that of gelatin decreased. Furthermore, adsorption behaviors of amino acids and peptides were examined in detail, which showed the importance of acidic amino acids in the adsorption behaviors. The adsorption behaviors of peptides obtained by digestion of β-lactoglobulin with trypsin were investigated. It was found that a peptide fragment (^<125>Thr-Pro-Glu-Val-Asp-Asp-Glu-Ala-Leu-Glu-Lys^<136>) showed an irreversible adsorption behavior and this region was thought to be one of the interaction sites in the protein. FT-IR analyses revealed that the acidic amino acids were dissociated on adsorption onto the stainless steel surface. Cleaning behaviors of the stainless steel particles fouled with proteins by enzyme cleaning (proteases) and hydroxyl radical cleaning were carried out under various conditions to compare their performances with caustic cleaning and their cleaning mechanisms were studied.

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  • Development of a Novel Cultivation Method for Molds and Its Characterization

    Grant number:10555291  1998 - 2000

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B).

    NAKANISHI Kazuhiro, NAGASAWA Tooru, IMAMURA Koreyosi, SAKIYAMA Takaharu

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    Grant amount:\13200000 ( Direct expense: \13200000 )

    In this, study we applied a newly-developed membrane-surface liquid culture (abbreviated as MSLC) to production of useful substances using molds. We not only intended to optimize the cultivation conditions but also clarify the reasons for the higher productivity obtained in MSLC in comparison with the results obtained in shaking flask cultures. We studied three fermentation systems, i.e., 1) kojic acid fermentation using Aspergillus oryzae, 2) red pigment production using Monascus pilosus, and 3) production of α-glucosidase using recombinant Aspergillus oryzae under various cultivation conditions, different in factors such as pH of the medium and C-and N-source concentrations. In MSLC, we first examined the effect of kinds of porous membranes on the cultivation behaviors. The most suitable membrane was found to be such a membrane that have a pore diameter of around 0.2μm and hydrophilic property. MSLC gave a higher productivity in the all three cultivation systems examined than those with shaking flask cultures. We examined the effect of oxygen supply in the shaking flask cultures to seek the reasons for the higher productivity in MSLC.The reasons for the higher productivity in MSLC were found not to be in the higher oxygen supply. It was suggested that differences in gene expression, the amount of relating enzyme produced in the cells, and permeability of the product through the cell membrane may affect the higher productivity in MSLC.

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  • 糖-タンパク質間水素結合のタンパク質の安定化に対する寄与とその評価

    Grant number:10750548  1998 - 1999

    日本学術振興会  科学研究費助成事業  奨励研究(A)

    今村 維克

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    Grant amount:\2000000 ( Direct expense: \2000000 )

    タンパク質水溶液に糖を添加し凍結乾燥すると,タンパク質は糖によって形成されたアモルファス構造に包埋され,その結果,タンパク質の安定性が顕著に向上する.この糖によるタンパク質安定化機構についてはこれまで多くの研究がなされているが,依然未解明な点が多く,実際の安定化操作の改善に繋がる知見は乏しい.そこで本研究では糖としてスクロース,マルトース,トレハロース,マルトトリオース,および平均分子量1500,6000のデキストラン,タンパク質として牛血清アルブミン(BSA)を様々な割合で含有する糖-タンパク質凍結乾燥資料を作成し,試料中におけるタンパク質の構造保持の度合いを比較した.タンパク質の構造保持度はフーリエ変換赤外分光分析計(FT-IR)を用いて二次構造を解析することにより評価し,各種糖アモルファスの物理的特性は示差走査熱量計(DSC)を用いて分析した.また,タンパク質安定化作用との密接な関係が注目されている糖-タンパク質間水素結合を水分収着特性から定量し,タンパク質の構造保持度との関係について検討を加えた.その結果,タンパク質の構造保持度をαへリックスの含有率を指標として評価した場合,構造保持の度合いはスクロース>マルトース>トレハロース>マルトトリオース>デキストラン1500>デキストラン6000の順となり,スクロースを用いた場合,糖添加量が1g/g-BSAのとき水溶液中の値とほぼ一致した.各種糖アモルファスのガラス転移温度(Tg)を測定した結果,Tgが低い糖ほどタンパク質の構造保持度が高いことが分かった.これはTgが低い糖アモルファスほど糖分子の構造柔軟性が高く,タンパク質との相互作用が形成され易いためと考えられる.また,糖-タンパク質間水素結合の形成度を評価した結果,糖-タンパク質間水素結合の形成度は構造保持の度合いと大略傾向は一致することが明らかになった.

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  • 固体表面に対する親和性と配向の精密制御された抗体の創製

    Grant number:10145237  1998

    日本学術振興会  科学研究費助成事業  特定領域研究(A)

    中西 一弘, 今村 維克

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    Grant amount:\2000000 ( Direct expense: \2000000 )

    平成10年度は、比較的分子量の小さいRNAseやβ-ラクトグロブリン(β-Lg)をモデルタンパク質として用いて、固体表面に直接接触しているぺプチド部位の特定と相互作用の強さに関して検討を試みた。直接付着に関与するぺプチド部位を特定する方法としては、タンパク質のプロテアーゼ消化により得られるぺプチド類の付着挙動から推定する間接的な方法を用いた。タンパク濃度一定条件下では、等電点近傍のpH5近傍で最大の吸着量を示した。吸着等温線(30℃)は見掛けLangmuir型を示した。酸性領域(pH3〜4)では、イオン強度が高いほど、吸着平衡定数は大きい結果となった。β-Lgをトリプシンで処理することにより14種類のペプチドを単離した。各ペプチドの組成と配列を一次構造とアミノ酸分析により同定した。ペプチド混合溶液あるいは各ペプチド単独の溶液を用いて種々の条件下(PH、温度、イオン強度)で吸着挙動を調べた。β-Lgが高い吸着を示した酸性pH領域では酸性アミノ酸を複数個含むペプチドがステンレス粒子に対する親和性が高いことが判明した。これは、ペプチド分子中の酸性アミノ酸残基がマイナスに誘起されて、ステンレス表面のプラスの電荷と多点結合するためであることが示唆された。同様にトリプシン処理によって得られたカルポキシメチル化RNaseA由来ペプチドを単離(10種類)し、そのステンレス粒子及びラテックスに対する吸着挙動をペプチド混合溶液を用いて検討した。いずれのペプチドもステンレスに対する吸着は、殆どみられなかった。一方、ラテックスに対する吸着は疎水性アミノ酸残基が多く含むペプチドにおいて強いことから、ラテックスに対する吸着には疎水性相互作用が寄与していることが示唆されたが、ペプチドとRNaseAの吸着挙動には、β-Lgのような明確な相関関係はみられなかった。

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  • バイオおよび食品製造プロセスにおける洗浄操作の高効率化によるエミッションの削減

    Grant number:10141228  1998

    日本学術振興会  科学研究費助成事業  特定領域研究(A)

    中西 一弘, 今村 維克

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    Grant amount:\2000000 ( Direct expense: \2000000 )

    本研究の目的は,バイオプロセスにおいて膨大なエミッションを生み出す洗浄操作の合理化ある.平成10年度はOHラジカルの高い酸化力に着目し,(1)固体表面汚れの効率的除去と(2)洗浄排水の低減の可能性を検討した.
    (1) 付着汚れの除去への応用は次のような原理に基づいている.H_2O_2水溶液を汚れの付着した固体表面に薄い液膜として流し,その上部から紫外線を照射すると,H_2O_2の紫外線分解によりOHラジカルが発生し,付着状態および脱着・遊離した汚れ物質を酸化分解する.さらに,光照射により汚れ物質の脱着が促進される.モデルは汚れ物質として脂肪酸を付着させたステンレス表面を用いて,付着汚れのラジカル酸化除去効果および紫外線による脱離促進効果を検証した.脂肪酸の付着・残留量はRAS-FT-IRにより定量した.その詰果,水流だけでは脂肪酸はほとんど脱離しないが,紫外線の照射により顕著に脂肪酸が脱離することが分かった.
    (2) 洗浄排水処理へのラジカル酸化分解の応用については,モデル有機物質として色素を含む着色水溶液のUV-H_2O_2処理における脱色特性を明らかにした.いづれの色素についても脱色反応は一次反応に従い,見かけの一次反応速度定(K)のH_2O_2濃度依存性を求めたところ,H_2O_2濃度に最大値が存在した.これはH_2O_2濃度の増加伴い,OHラジカルの発生速度とともにUVの減衰が顕著になるためであると考えられる.そこで,紫外線照射下のH_2O_2-色素水溶液におけるラジカル反応(H_2O_2の紫外線分解反応,OHラジカルとH_2O_2との反広,OHラジカルと色素との反応)とH_2O_2および色素による紫外線の減衰を考慮したモデルを設定し,kの理論値を計算した.その結果,実験値と良好に一致し,本モデルにより有機物質のラジカル酸化速度に対する条件の最適化が可能である.

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  • Improvement of stability and reaction behavior of enzymes in organic solutions by immobilization onto nano-porous supports

    Grant number:09555256  1997 - 1998

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    IMAMURA Koreyosi, UTAGAWA Takashi, NAKANISHI Kazuhiro

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    Grant amount:\1100000 ( Direct expense: \1100000 )

    In this study, we intended to establish theoretical bases, which are required to realize enzymatic syntheses in organic solvents. We studied the synthetic reaction of aspartame precursor, Z-L-Asp-L-PheOMe, from respective amino acid derivatives (Z-Asp and PheOMe) catalyzed by thermolysin. First, we investigated various factors affecting stability of the immobilized thermolysin and analyzed its mechanism. Autolysis was found to be a main cause for inactivation in water-immiscible organic solvents and the immobilized enzyme tended to be inactivated in organic solvents with lower solubility of water. In particular, the immobilized was stable in tert-amyl alcohol probably because that aqueous phase around the enzyme immobilized is thin and as a result autolysis tended to be repressed. The method to predict the overall partition coefficient of the substrates containing acid and amine components was proposed, by taking into consideration the effect of ion-pair complexes formed between the substrates.
    The synthetic rate with the immobilized enzyme in an organic solvent, containing a small amount of water could be in principle estimated on the basis of the model for the biphasic reaction when the amount of enzyme loaded was not so much. Namely, in the synthetic reactions, the both components of the substrate are partitioned into the water phase inside the support, and then converted to the product by the action of the enzyme, and finally the product is transferred into the bulk organic phase.

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  • Adsorption & desorption of protein, peptide, & amino acids

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    Grant type:Competitive

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  • Application of radical oxidation to waste water treatment & cleaning of Solid Surfaces

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  • Stabilization of protein by sugars

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    Grant type:Competitive

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  • Adsorption & desorption of protein, peptide, & aminoocids

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    Grant type:Competitive

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  • Application of radical oxidation to waste water treatment & cleaning of Sclid Surfaces

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  • Stabilization of protein by sugar

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  • ラジカル酸化反応の排水処理,固体表面の洗浄への応用

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    Grant type:Competitive

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  • 糖によるタンパク質の安定化

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  • タンパク質・ペプチド・アミノ酸の金属壁面への付着・脱離

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