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C-terminal truncation of glutamate decarboxylase from Lactobacillus brevis CGMCC 1306 extends its activity toward near-neutral pH

Paper ID Volume ID Publish Year Pages File Format Full-Text
17266 42655 2012 7 PDF Available
Title
C-terminal truncation of glutamate decarboxylase from Lactobacillus brevis CGMCC 1306 extends its activity toward near-neutral pH
Abstract

Glutamate decarboxylase (GAD) from Lactobacillus brevis is a very promising candidate for biosynthesis of GABA and various other bulk chemicals that can be derived from GABA. However, no structure of GAD of this origin has been reported to date, which limits enzyme engineering strategy to improve its properties for better use in production of GABA. Bacterial GAD exhibits an acidic pH optimum and there is often a sharp pH dependence. In the present work, site-directed mutagenesis was performed to delete the C-terminal residues of GAD to generate a mutant, designated as GADΔC, which exhibited extended activity toward near-neutral pH compared to the wild type. Comparison of the UV–visible, fluorescence and Circular Dichroism spectra of the mutant with those of the wild type revealed that the microenvironment of the active site had been changed. Based on the homology model, we speculated that the substrate entrance was probably enlarged in GADΔC. These results provide evidence for the important role of C-terminal region in the pH-dependent regulation of enzyme activity, and the resulting mutant would be useful in a bioreactor for continuous production of GABA.

► We construct the homology model of GAD form Lactobacillus brevis CGMCC 1306. ► We delete fourteen C-terminal amino acid residues of the enzyme. ► Fluorescence and CD spectra of the mutant show different properties from those of WT. ► The mutant exhibits extended activity toward near-neutral pH compared to WT. ► The C-terminal region is essential to the pH-dependent control of enzyme activity.

Keywords
GAD, glutamate decarboxylase; GABA, γ-aminobutyrate; PLP, pyridoxal 5′-phosphate; LAB, lactic acid bacteria; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CD, Circular DichroismGlutamate decarboxylase; Site-directed mutagenesis; Hom
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C-terminal truncation of glutamate decarboxylase from Lactobacillus brevis CGMCC 1306 extends its activity toward near-neutral pH
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 50, Issues 4–5, 5 April 2012, Pages 263–269
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