A high-throughput and generic assay method for the determination of substrate specificities of thermophilic α-aminotransferases

Toshiya Sawai, Daisuke Koma, Ryotaro Hara, Kuniki Kino, Shigeaki Harayama*

*この研究の対応する著者

研究成果: Article査読

3 被引用数 (Scopus)

抄録

For the determination of substrate specificities of thermophilic α-aminotransferases (AATs), a novel high-throughput assay method was developed. In this method, a thermophilic ω-aminotransferase (OAT) and a thermophilic aldehyde dehydrogenase (ALDH) are coupled to the AAT reaction. Glutamic acid is used as an amino group donor for the AAT reaction yielding 2-oxoglutalic acid. 2-Oxoglutalic acid produced by the AAT reaction is used as an amino group acceptor in the OAT reaction regenerating glutamic acid. The amino group donor of the OAT reaction is 5-aminopentanoic acid yielding pentanedioic acid semialdehyde which is oxidized by ALDH to pentanedioic acid with concomitant reduction of NADP+ to NADPH. NADPH thus produced then reduces colorless tetrazolium salt into colored formazan. To construct such a reaction system, the genes for a thermophilic AAT, a thermophilic OAT and a thermophilic ALDH were cloned and expressed in Escherichia coli. These enzymes were subsequently purified and used to determine the activities of AAT for the synthesis of unnatural amino acids. This method allowed the clear detection of the AAT activities as it measures the increase in the absorbance on a low background absorbance reading.

本文言語English
ページ(範囲)32-38
ページ数7
ジャーナルJournal of Microbiological Methods
71
1
DOI
出版ステータスPublished - 2007 10月
外部発表はい

ASJC Scopus subject areas

  • 微生物学
  • 分子生物学
  • 微生物学(医療)

フィンガープリント

「A high-throughput and generic assay method for the determination of substrate specificities of thermophilic α-aminotransferases」の研究トピックを掘り下げます。これらがまとまってユニークなフィンガープリントを構成します。

引用スタイル