研究者詳細 - 今村　維克

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

Koreyoshi Imamura, Ippei Watanabe, Takaharu Sakiyama, Kazuhiro Nakanishi

LANGMUIR 22 ( 16 ) 7035 - 7040 2006年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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

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

JOURNAL OF PHYSICAL CHEMISTRY B 110 ( 31 ) 15094 - 15099 2006年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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

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

PROTEIN EXPRESSION AND PURIFICATION 47 ( 2 ) 607 - 613 2006年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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

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

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 101 ( 5 ) 434 - 439 2006年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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

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

BIOTECHNOLOGY PROGRESS 22 ( 2 ) 401 - 405 2006年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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

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

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

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 341 ( 4 ) 911 - 916 2006年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

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

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

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

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 54 ( 1 ) 72 - 78 2006年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER CHEMICAL SOC

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

各種糖分子からなるアモルファスマトリクスの物理的諸特性

今村維克, 丸山佳伸, 横山徹, 大山健一, 今中洋行, 中西一弘

化学工学会研究発表講演要旨集 2006 413 - 413 2006年

ポリスチレン親和性ペプチドを利用したタンパク質間相互作用の分析

熊田陽一, 石村遼太, 上崎英範, 今中洋行, 今村維克, 中西一弘

化学工学会研究発表講演要旨集 2006 996 - 996 2006年

金属表面へのタンパク質付着配向制御

柳田圭介, 中泉雅人, 今中洋行, 今村維克, 中西一弘

化学工学会研究発表講演要旨集 2006 1002 - 1002 2006年

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

是石真友子, 谷和葉, 伊勢雄一, 今中洋行, 今村維克, 中西一弘

化学工学会研究発表講演要旨集 2006 949 - 949 2006年

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

横山徹, 丸山佳伸, 大山健一, 今中洋行, 今村維克, 中西一弘

化学工学会研究発表講演要旨集 2006 995 - 995 2006年

Purification and characterization of a novel aminoacylase from Streptomyces mobaraensis 査読

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

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 ( 10 ) 1914 - 1922 2005年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：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