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

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)32-38
Number of pages7
JournalJournal of Microbiological Methods
Volume71
Issue number1
DOIs
Publication statusPublished - 2007 Oct 1
Externally publishedYes

Keywords

  • Aldehyde dehydrogenase
  • Biocatalysis
  • Enzyme assay
  • Thermophilic enzyme
  • Unnatural amino acids
  • α-aminotransferase
  • ω-aminotransferase

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

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