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Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
17307 42658 2013 6 PDF Available
Title
Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli
Abstract

•Conversion in the glutamate decarboxylase reaction depends on pH.•Dependency of conversion on pH and kinds of buffers was simulated and confirmed.•In the simulation, glutamic acid was shown to act as a pH buffer as well.•Hyper active enzyme was generated by engineering the C-terminus of the enzyme.•Buffer-free reaction was demonstrated at high concentration of the substrate.

Escherichia coli glutamate decarboxylase (GAD) converts glutamate into γ-aminobutyric acid (GABA) through decarboxylation using proton as a co-substrate. Since GAD is active only at acidic conditions even though pH increases as the reaction proceeds, the conventional practice of using this enzyme involved the use of relatively high concentration of buffers, which might complicate the downstream purification steps. Here we show by simulation and experiments that the free acid substrate, glutamic acid, rather than its monosodium salt can act as a substrate and buffer at the same time. This yielded the buffer- and salt-free synthesis of GABA conveniently in a batch mode. Furthermore, we engineered GAD to hyper active ones by extending or reducing the length of the enzyme by just one residue at its C-terminus. Through the buffer-free reaction with engineered GAD, we could synthesize 1 M GABA in 3 h, which can be translated into a space-time yield of 34.3 g/L/h.

Keywords
Glutamate decarboxylase; Gamma-aminobutyric acid; Buffer-free enzyme reaction; Enzyme engineering
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Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 53, Issue 3, 15 August 2013, Pages 200–205
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