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Directed evolution and mutagenesis of glutamate decarboxylase from Lactobacillus brevis Lb85 to broaden the range of its activity toward a near-neutral pH

Paper ID Volume ID Publish Year Pages File Format Full-Text
17014 42632 2014 9 PDF Available
Title
Directed evolution and mutagenesis of glutamate decarboxylase from Lactobacillus brevis Lb85 to broaden the range of its activity toward a near-neutral pH
Abstract

•The active pH range of L. brevis GadB1 was broadened by directed evolution.•Its active range was expanded to near-neutral pH by site-specific mutagenesis.•Enzymatic properties of the wild-type and mutant GadB1 were examined.•GABA biosynthesis was improved by expressing mutant gadB1 in C. glutamicum.•Directed evolution was demonstrated as a useful strategy to improve GAD activity.

Glutamate decarboxylase (GAD) transforms l-glutamate into γ-aminobutyric acid (GABA) with the consumption of a proton. GAD derived from lactic acid bacteria exhibits optimum activity at pH 4.0–5.0 and significantly loses activity at near-neutral pH. To broaden the active range of the GAD GadB1 from Lactobacillus brevis Lb85 toward a near-neutral pH, irrational design using directed evolution and rational design using site-specific mutagenesis were performed. For directed evolution of GadB1, a sensitive high-throughput screening strategy based on a pH indicator was established. One improved mutant, GadB1T17I/D294G/Q346H, was selected from 800 variants after one round of EP-PCR. It exhibited 3.9- and 25.0-fold increase in activity and catalytic efficiency, respectively at pH 6.0. Through site-specific mutagenesis, several improved mutants were obtained, with GadB1E312S being the best one. The combined mutant GadB1T17I/D294G/E312S/Q346H showed even higher catalytic efficiency, 13.1- and 43.2-fold that of wild-type GadB1 at pH 4.6 and 6.0, respectively. The amount of GABA produced in gadB1T17I/D294G/Q346H-, gadB1E312S- and gadB1T17I/D294G/E312S/Q346H-expressing Corynebacterium glutamicum ATCC 13032 from endogenous l-glutamate increased by 9.6%, 20.3% and 63.9%, respectively. These results indicate that these mutations have beneficial effects on expanding the active pH range and on GABA biosynthesis, suggesting these GadB1 variants as potent candidates for GABA production.

Keywords
Glutamate decarboxylase; γ-Aminobutyric acid; Active pH range; Error prone-PCR; Directed evolution; Site-specific mutagenesis
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Directed evolution and mutagenesis of glutamate decarboxylase from Lactobacillus brevis Lb85 to broaden the range of its activity toward a near-neutral pH
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volumes 61–62, July–August 2014, Pages 35–43
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us