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

DOI： 10.1021/acs.analchem.2c02878

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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.

File： COLSUA_128392_edit_report.pdf

DOI： 10.1016/j.colsurfa.2022.128392

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

File： P0SF-IBPs-draft.pdf

DOI： 10.1016/j.colsurfa.2021.127744

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

File： P0NeedleEx-draft.pdf

DOI： 10.1080/07373937.2021.2017965

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Authorship：Last author   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.

File： P0NeedleTramis2-pub.pdf

DOI： 10.1016/j.colsurfa.2021.127174

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File： P1ParticleEx-draft.pdf

DOI： 10.1016/j.apt.2021.08.002

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

DOI： 10.1016/j.colsurfa.2021.127137

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Authorship：Last author,　Corresponding author   Language：English   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.

File： P0SF-Cur-draft.pdf

DOI： 10.1080/07373937.2020.1752711

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File： P0TreCry-pub.pdf

DOI： 10.1016/j.jfoodeng.2020.110325

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

DOI： 10.1016/j.apt.2020.11.011

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

File： P0NeedleTramisPVP.pdf

DOI： 10.1080/07373937.2020.1822863

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Authorship：Lead author,　Last author,　Corresponding author   Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Food Engineering  

File： 200815食工研究賞原稿.pdf

DOI： 10.11301/jsfe.20579

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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

Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

File： P1Particle-pub.pdf

DOI： 10.1021/acs.langmuir.0c00695

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File： P2Tramis-pub.pdf

DOI： 10.1016/j.jbiosc.2019.09.008

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Language：English   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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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Chemical and Biological Microsystems Society  

The Needs of shotgun proteomics have been increasing in the field of biology and medicine for understanding biological systems. In study, protein sample with pL, which volume was same scale as single cell volume, was successfully digested and separated and detected by integrated device of pL enzyme reactor fL chromatography and UV-POPS detector. This nanofluidic device will greatly contribute to single cell shotgun proteomics.

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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.

File： LDRT_A_1517363draft.pdf

DOI： 10.1080/07373937.2018.1517363

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Authorship：Last author,　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.

File： P0TgOrganic-pub.pdf

DOI： 10.1016/j.xphs.2019.01.008

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：Chemical and Biological Microsystems Society  

We development a nanofluidic enzyme reactor exceeding the limitation of bulk enzyme reaction. The limit concentration of bulk trypsin solution is approximately 0.5 PM because self-digestion occurs over 0.5 PM solution, which complicates analysis of target proteins by digestion trypsin itself. Using extremely high surface/volume ration of nanospace, trypsin concentration, which indicates number of immobilized trypsin molecules on the nanochannel surface for liquid phase volume, exceed to bulk limit of concentration. As a result, 31 times higher concentration and 33 times faster enzyme reaction compared to bulk was realized. This reactor will be powerful tools which overcomes limitation in bulk and microfluidic method.

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

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.

File： pagination_COLSUB_9236.pdf

DOI： 10.1016/j.colsurfb.2018.03.035

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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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Authorship：Last author,　Corresponding author   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.

File： P3Enzyme-pub.pdf

DOI： 10.1016/j.colsurfb.2017.07.074

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Authorship：Last author,　Corresponding author   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.

File： TBBB_A_1274637r1.pdf

DOI： 10.1080/09168451.2016.1274637

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Authorship：Last author,　Corresponding author   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.

File： P0SF-DrugProof.pdf

DOI： 10.1021/acs.molpharmaceut.6b01048

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

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.

File： P2LSZ-pub.pdf

DOI： 10.1016/j.colsurfb.2016.07.042

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DOI： 10.1016/j.nbt.2016.06.966

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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.

File： P0SurfFree-drft.pdf

DOI： 10.1016/j.foodchem.2015.11.097

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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

File： BTPR_2232corected.pdf

DOI： 10.1002/btpr.2232

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Language：Japanese   Publisher：The Surface Science Society of Japan  

DOI： 10.14886/sssj2008.36.0_37

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

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.

DOI： 10.1016/j.apt.2015.10.017

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

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.

File： P0CryInh-proof.pdf

DOI： 10.1016/j.jfoodeng.2015.01.016

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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.

File： P1AOex-pub.pdf

DOI： 10.1016/j.foodres.2015.02.003

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

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.

DOI： 10.1246/cl.140279

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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

File： P1SugSF-pub.pdf

DOI： 10.1002/jps.23988

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Language：Japanese   Publisher：Japan Oil Chemists' Society  

<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>

DOI： 10.5650/oleoscience.14.487

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Other Link： http://search.jamas.or.jp/link/ui/2015030450

Language：English   Publisher：一般社団法人 粉体工学会  

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.

DOI： 10.4164/sptj.51.343

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S207_6

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S209_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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

DOI： 10.1002/jps.23568

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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.

DOI： 10.1016/j.carbpol.2013.01.079

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

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.

DOI： 10.1016/j.foodres.2012.12.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

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.

DOI： 10.1016/j.bmcl.2012.12.006

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

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.

DOI： 10.1271/bbb.120583

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

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.

DOI： 10.1016/j.jfoodeng.2012.05.004

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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.

DOI： 10.1016/j.carres.2011.12.021

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

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.

DOI： 10.1246/cl.2011.1085

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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.

DOI： 10.1271/bbb.110014

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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.

DOI： 10.1246/cl.2011.400

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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.

DOI： 10.1016/j.ijpharm.2011.01.052

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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.

DOI： 10.1271/bbb.100574

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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.

DOI： 10.11301/jsfe.12.1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

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

DOI： 10.1002/jps.22162

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

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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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

"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.

DOI： 10.1016/j.jcis.2010.01.083

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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

DOI： 10.1002/jps.21890

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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.

DOI： 10.1016/j.jbiosc.2009.09.004

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2010f.0.584.0

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DOI： 10.11491/scej.2010f.0.583.0

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DOI： 10.11491/scej.2010f.0.1003.0

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DOI： 10.11491/scej.2010f.0.997.0

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DOI： 10.1016/j.jbiosc.2009.08.436

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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.

DOI： 10.1016/j.colsurfa.2009.09.007

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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

DOI： 10.1002/jps.21568

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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).

DOI： 10.1271/bbb.90081

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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.

DOI： 10.1016/j.jbiotec.2009.03.008

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2009f.0.934.0

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DOI： 10.11491/scej.2009f.0.779.0

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DOI： 10.11491/scej.2009.0.608.0

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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.

DOI： 10.2174/157016408785909622

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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.

DOI： 10.1263/jbb.106.273

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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.

DOI： 10.1002/jps.21202

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

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.

DOI： 10.1002/jps.21066

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

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.

DOI： 10.1002/jps.21075

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

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.

DOI： 10.1080/00387010802370959

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2008.0.418.0

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Publisher：Japan Society for Food Engineering  

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.

DOI： 10.11301/jsfe2000.9.229

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DOI： 10.11491/scej.2008.0.339.0

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DOI： 10.11491/scej.2008f.0.955.0

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

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.

DOI： 10.1016/j.ijbiomac.2007.03.005

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

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.

DOI： 10.1271/bbb.70052

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

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.

DOI： 10.1016/j.jbiotec.2006.12.017

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

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.

DOI： 10.1016/j.jbiotec.2006.09.018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

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.

DOI： 10.1263/jbb.103.7

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

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.

DOI： 10.1016/j.jbiotec.2006.07.011

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DOI： 10.11491/scej.2007f.0.702.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

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.

DOI： 10.1263/jbb.102.470

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

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.

DOI： 10.1021/la053494s

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

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.

DOI： 10.1021/jp057527o

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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.

DOI： 10.1016/j.pep.2006.01.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

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.

DOI： 10.1263/jbb.101.434

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

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.

DOI： 10.1021/bp050331l

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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.

DOI： 10.1016/j.bbrc.2006.01.054

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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.

DOI： 10.1021/jf052102k

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

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.

DOI： 10.1271/bbb.69.1914

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER OIL CHEMISTS SOC A O C S PRESS  

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.

DOI： 10.1007/s11746-005-1121-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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.

DOI： 10.1016/j.jcis.2005.01.023

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DOI： 10.11491/scej.2005.0.294.0

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DOI： 10.11491/scej.2005f.0.311.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

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.

DOI： 10.1016/S1389-1723(04)00266-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

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.

DOI： 10.1016/S1389-1723(04)70213-3

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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.

DOI： 10.11301/jsfe2000.9.229

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Language：English   Publisher：The Society of Chemical Engineers, Japan  

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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Language：English   Publisher：The Society of Chemical Engineers, Japan  

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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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.

DOI： 10.1021/jf0205007

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Language：Japanese   Publisher：公益社団法人 化学工学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER OIL CHEMISTS SOC A O C S PRESS  

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.

DOI： 10.1007/s11746-002-0432-7

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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.

DOI： 10.1023/A:1011699921628

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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.

DOI： 10.1080/07373939908917625

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

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.

DOI： 10.1252/jcej.31.565

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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.

DOI： 10.1252/jcej.30.609

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：DRUKARNIA PAPAJ  

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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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Language：Japanese   Publisher：The Society of Powder Technology, Japan  

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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Language：Japanese   Publisher：日本食品工学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL  

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Other Link： http://search.jamas.or.jp/link/ui/2014247513

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Other Link： http://search.jamas.or.jp/link/ui/2013125671

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Other Link： http://search.jamas.or.jp/link/ui/2013125672

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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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Language：English   Publisher：Faculty of Engineering, Okayama University  

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Language：English   Publisher：The Society for Biotechnology, Japan  

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Language：Japanese   Publisher：日本生物工学会  

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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Language：English   Publisher：TAYLOR & FRANCIS LTD  

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.

DOI： 10.1271/bbb.70052

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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.

DOI： 10.1263/jbb.101.434

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Language：Japanese   Publisher：Japanese Society for Cryobiology and Cryotechnology  

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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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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.

DOI： 10.1016/j.jcis.2004.06.081

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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.

DOI： 10.1263/jbb.98.200

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Language：English   Publisher：ELSEVIER SCIENCE BV  

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.

DOI： 10.1016/j.molcatb.2003.12.019

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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.

DOI： 10.1263/jbb.97.322

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Language：English   Publisher：ELSEVIER SCIENCE BV  

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.

DOI： 10.1016/j.colsurfb.2003.08.010

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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.

DOI： 10.1016/S0021-9797(03)00700-8

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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.

DOI： 10.1016/S0021-9797(03)00329-1

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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.

DOI： 10.1002/jps.10305

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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.

DOI： 10.1002/jps.10305

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DOI： 10.1021/ma021721g

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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.

DOI： 10.1021/la025893i

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Language：English   Publisher：AMER CHEMICAL SOC  

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.

DOI： 10.1021/la025893i

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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.

DOI： 10.1002/jps.10218

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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).

DOI： 10.1006/jcis.2002.8368

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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).

DOI： 10.1006/jcis.2002.8368

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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.

DOI： 10.1006/jcis.2001.7967

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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.

DOI： 10.1006/jcis.2001.7967

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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.

DOI： 10.11301/jsfe2000.3.15

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Language：English   Publisher：John Wiley and Sons Inc.  

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.

DOI： 10.1002/jps.10218

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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.

DOI： 10.1002/jps.1146

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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.

DOI： 10.1252/jcej.34.869

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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.

DOI： 10.1271/bbb.65.865

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

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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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.

DOI： 10.1006/jcis.2000.7016

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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.

DOI： 10.1271/bbb.64.1874

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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.

DOI： 10.1006/jcis.2000.7016

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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.

DOI： 10.1252/jcej.33.657

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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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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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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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Language：Japanese   Publisher：ソルト・サイエンス研究財団  

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Language：English   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

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.

DOI： 10.1016/S1389-1723(00)87095-4

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Language：English   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

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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Language：English   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

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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Language：Japanese   Publisher：日本生物工学会  

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

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