MISC

基本情報

氏名 古賀 雄一
氏名(カナ) コガ ユウイチ
氏名(英語) Koga Yuuichi
所属 工学部 応用化学科
職名 教授
researchmap研究者コード 5000076449
researchmap機関 岡山理科大学

題名

Modifying the chain-length selectivity of the lipase from Burkholderia cepacia KWI-56 through in vitro combinatorial mutagenesis in the substrate-binding site

単著・共著の別

 

著者

J Yang
Y Koga
H Nakano
T Yamane

概要

The mature lipase of Burkholderia cepacia KWI-56 was synthesized in an enzymatically active form using an in vitro Escherichia coli S30 coupled transcription/translation system by expressing the mature lipase gene (rlip) in the presence of its specific activator. To investigate the substrate specificity of the lipase comprehensively, a large number of mutant lipases were constructed and analyzed in a high throughput manner by combining overlapping PCR and in vitro protein synthesis. In this paper, Phe119 and Leu167, which are located in the acyl portion of the substrate-binding pocket of the lipase of B. cepacia KWI-56, were substituted with six hydrophobic amino acid residues by the in vitro combinatorial mutagenesis. The wild-type and 35 mutant genes amplified by PCR were directly used as templates for the in vitro transcription/translation. The acyl chain-length selectivity of the in vitro expressed lipases against p-nitrophenyl butyrate,p-nitrophenyl caprylate and p-nitrophenyl palmitate, was compared by their relative hydrolysis rates. Two mutant lipases, L167V and F119A/ L167M, which showed a significant shift in substrate selectivity were further expressed in vivo and refolded in vitro. It was found that L167V raised its preference for the short-chain ester, whereas F119A/L167M improved its selectivity for the long-chain ester.

発表雑誌等の名称

PROTEIN ENGINEERING

出版者

OXFORD UNIV PRESS

15

2

開始ページ

147

終了ページ

152

発行又は発表の年月

2002-02

査読の有無

無し

依頼の有無

無し

記述言語

英語

掲載種別

 

ISSN

 

ID:DOI

 

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JGlobalID

 

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