Journal
ENZYME AND MICROBIAL TECHNOLOGY
Volume 53, Issue 3, Pages 200-205Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2013.04.006
Keywords
Glutamate decarboxylase; Gamma-aminobutyric acid; Buffer-free enzyme reaction; Enzyme engineering
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Funding
- Ministry of Education, Science and Technology through the National Research Foundation of Korea [NRF-2010-0005998, NRF-2010-0026498]
- Ministry of Knowledge Economy through the Korean Evaluation Institute of Industrial Technology [10033199]
- Korea Evaluation Institute of Industrial Technology (KEIT) [10033199] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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Escherichia coli glutamate decarboxylase (GAD) converts glutamate into gamma-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. (C) 2013 Elsevier Inc. All rights reserved.
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