Document Detail


A high-throughput and generic assay method for the determination of substrate specificities of thermophilic alpha-aminotransferases.
MedLine Citation:
PMID:  17719665     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
For the determination of substrate specificities of thermophilic alpha-aminotransferases (AATs), a novel high-throughput assay method was developed. In this method, a thermophilic omega-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.
Authors:
Toshiya Sawai; Daisuke Koma; Ryotaro Hara; Kuniki Kino; Shigeaki Harayama
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2007-07-21
Journal Detail:
Title:  Journal of microbiological methods     Volume:  71     ISSN:  0167-7012     ISO Abbreviation:  J. Microbiol. Methods     Publication Date:  2007 Oct 
Date Detail:
Created Date:  2007-10-01     Completed Date:  2008-01-11     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  8306883     Medline TA:  J Microbiol Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  32-8     Citation Subset:  IM    
Affiliation:
Laboratory of Applied Biochemistry, Department of Applied Chemistry, School of Science and Engineering, Waseda University, 3-4-1, Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan.
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MeSH Terms
Descriptor/Qualifier:
Amino Acids / chemical synthesis*,  chemistry
Bacteria / enzymology*,  genetics,  growth & development
Biological Assay*
Gene Expression Regulation, Bacterial
Hot Temperature*
Kinetics
Substrate Specificity
Transaminases / metabolism*
Chemical
Reg. No./Substance:
0/Amino Acids; EC 2.6.1.-/Transaminases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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