研究者詳細 - 今中　洋行

論文 - 今中　洋行

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

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

Current Proteomics 5 ( 3 ) 161 - 175 2008年10月

記述言語：英語

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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True density analysis of a freeze-dried amorphous sugar matrix 査読

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

JOURNAL OF PHARMACEUTICAL SCIENCES 97 ( 7 ) 2789 - 2797 2008年7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：JOHN WILEY & SONS INC

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

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

JOURNAL OF PHARMACEUTICAL SCIENCES 97 ( 1 ) 519 - 528 2008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：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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クッションタンパク質を利用したバイオ分子固定化技術の開発

山隅大輔, 柳田圭介, 今中洋行, 今村維克, 中西一弘

化学工学会研究発表講演要旨集 2008 418 - 418 2008年

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

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

SPECTROSCOPY LETTERS 41 ( 6 ) 305 - 312 2008年

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 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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高圧処理における糖類アモルファスマトリクスの物理特性変化

今村維克, 野村真世, 横山徹, 今中洋行, 中西一弘

化学工学会研究発表講演要旨集 2008 339 - 339 2008年

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

今中洋行, 上崎英範, 石村遼太, 熊田陽一, 今村維克, 中西一弘

化学工学会研究発表講演要旨集 2008 345 - 345 2008年

膜面液体培養法を用いた麹菌の培養特性及び遺伝子発現

田中創吉, 今中洋行, 今村維克, 中西一弘

化学工学会研究発表講演要旨集 2008 378 - 378 2008年

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

今村維克, 野村真世, 田中一宏, 木村佳文, 今中洋行, 中西一弘

化学工学会研究発表講演要旨集 2008 175 - 175 2008年

ヒト由来転写因子とDNAとの相互作用解析システムの検討

今中洋行, 前川真光, 今村維克, 近藤英作, 中西一弘

化学工学会研究発表講演要旨集 2008 955 - 955 2008年

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

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

INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 41 ( 3 ) 281 - 285 2007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：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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Enzymatic synthesis of beta-lactam antibiotics and N-fatty-acylated amino compounds by the acyl-transfer reaction catalyzed by penicillin V acylase from Streptomyces mobaraensis 査読

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

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 ( 6 ) 1582 - 1586 2007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：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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Cloning and characterization of penicillin V acylase from Streptomyces mobaraensis 査読

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

JOURNAL OF BIOTECHNOLOGY 128 ( 4 ) 788 - 800 2007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：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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Protein-protein interaction analysis using an affinity peptide tag and hydrophilic polystyrene plate 査読

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

JOURNAL OF BIOTECHNOLOGY 128 ( 2 ) 354 - 361 2007年2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：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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Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface 査読

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

JOURNAL OF BIOTECHNOLOGY 127 ( 2 ) 288 - 299 2007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：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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糖-高分子複合アモルファスマトリクスにおける分子間相互作用と物理的安定性

今村維克, 浅野容子, 大山健一, 丸山佳伸, 崎山高明, 今中洋行, 中西一弘

化学工学会研究発表講演要旨集 2007 368 - 368 2007年