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MISC - 今村　維克

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Protease Susceptibility of β-Lactoglobulin Adsorbed on Stainless Steel Surface as Evidence of Contribution of Its Specific Segment to Adsorption

Sakiyama Takaharu, Aya Atsuko, Embutsu Mari, IMAMURA Koreyoshi, NAKANISHI Kazuhiro

Journal of bioscience and bioengineering 101 ( 5 ) 434 - 439 2006年5月

記述言語：英語出版者・発行元：公益社団法人日本生物工学会

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

DOI： 10.1263/jbb.101.434

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ポリスチレン親和性ペプチド標識タンパク質を用いる迅速免疫測定

熊田陽一, 今中洋行, 今村維克, 加藤滋雄, 中西一弘

化学工学会年会研究発表講演要旨集 71st 2006年

ポリスチレン表面に親和性を示すペプチドタグを付加した酵素の設計と特性解析

今石大輔, 熊田陽一, 今中洋行, 今村維克, 崎山高明, 中西一弘

日本農芸化学会中四国支部講演会講演要旨集 14th (Web) 2006年

Isolation and Characterization of a Thermostable Monoacylglycerol Lipase from Soil Bacterium

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

Proceedings for Asia-Pacific Biochemical Engineering Conference (APBioChEC’04) 2005年

2E11-4 ポリスチレン親和性ペプチドを用いるタンパク質吸着の配向制御(生物化学工学,一般講演)

熊田陽一, 今中洋行, 今村維克, 加藤滋雄, 崎山高明, 中西一弘

日本生物工学会大会講演要旨集 17 153 - 153 2005年

糖類アモルファスマトリクスにおける糖-タンパク質間相互作用とタンパク質の安定化

今村維克

低温生物工学会誌 51 ( 1 ) 31 - 35 2005年

記述言語：日本語出版者・発行元：低温生物工学会

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

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ポリスチレン親和性ペプチド融合タンパク質の吸着特性とその応用

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

化学工学会秋季大会研究発表講演要旨集(CD-ROM) 37th 2005年

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

T Nagayasu, C Yoshioka, K Imamura, K Nakanishi

JOURNAL OF COLLOID AND INTERFACE SCIENCE 279 ( 2 ) 296 - 306 2004年11月

記述言語：英語出版者・発行元：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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Characteristics of α-Glucosidase Production from Recombinant Aspergillus oryzae by Membrane-Surface Liquid Culture in Comparison with Various Cultivation Methods

Morita Masakazu, Shimamura Hiroko, Ishida Natsuko, IMAMURA KOREYOSHI, SAKIYAMA TAKAHARU, NAKANISHI KAZUHIRO

Journal of bioscience and bioengineering 98 ( 3 ) 200 - 206 2004年9月

記述言語：英語出版者・発行元：公益社団法人日本生物工学会

α-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 NaNO_3, a typical inorganic nitrogen source was used, enzyme production in all the cultures was lower than that using yeast extract. However, even using NaNO_3, 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 NaNO_3 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 NaNO_3 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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Use of a novel affinity tag selected with a bacterial random peptide library for improving activity retention of glutathione S-transferase adsorbed on a polystyrene surface

T Sakiyama, S Ueno, K Imamura, K Nakanishi

JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 28 ( 4-6 ) 207 - 214 2004年6月

記述言語：英語出版者・発行元：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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Production of nonproteinaceous amino acids using recombinant Escherichia coli cells expressing cysteine synthase and related enzymes with or without the secretion of O-acetyl-L-serine

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

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 97 ( 5 ) 322 - 328 2004年5月

記述言語：英語出版者・発行元：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： 10.1263/jbb.97.322

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

T Sakiyama, J Tomura, K Imamura, K Nakanishi

COLLOIDS AND SURFACES B-BIOINTERFACES 33 ( 2 ) 77 - 84 2004年1月

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

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

ステンレス表面に対する牛血清アルブミンとそのペプチド断片の吸着特性

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

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

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

ステンレス表面に対する低分子物質の吸着におけるカルボキシル基の寄与

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

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

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

JOURNAL OF COLLOID AND INTERFACE SCIENCE 267 ( 2 ) 294 - 301 2003年11月

記述言語：英語出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

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

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

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

JOURNAL OF COLLOID AND INTERFACE SCIENCE 265 ( 1 ) 49 - 55 2003年9月

記述言語：英語出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

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

放線菌由来新規アシラーゼによるカプサイシン誘導体の合成とその抗酸化性の評価

是石真友子, 張徳民, 川波和子, 今村維克, 崎山高明, 松村康生, 中西一弘

日本食品工学会年次大会講演要旨集 4th 117 2003年7月

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

今村維克, 崎山高明, 中西一弘

ジャパンフ-ドサイエンス 42 ( 6 ) 27 - 33 2003年6月

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