NDLI: Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries

Content Provider	Springer Nature Link
Author	Heuson, Egon Petit, Jean Louis Debard, Adrien Job, Aurélie Charmantray, Franck de Berardinis, Véronique Gefflaut, Thierry
Copyright Year	2015
Abstract	In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine.
Starting Page	397
Ending Page	408
Page Count	12
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	100
Issue Number	1
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2015-10-09
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Aminotransferase Transaminase Cysteine sulfinic acid High-throughput screening Enzyme library Colorimetric assay Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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