TY - JOUR
T1 - Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli
AU - Kang, Taek Jin
AU - Ho, Ngoc Anh Thu
AU - Pack, Seung Pil
PY - 2013/8/15
Y1 - 2013/8/15
N2 - 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.
AB - 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.
KW - Buffer-free enzyme reaction
KW - Enzyme engineering
KW - Gamma-aminobutyric acid
KW - Glutamate decarboxylase
UR - http://www.scopus.com/inward/record.url?scp=84880038125&partnerID=8YFLogxK
U2 - 10.1016/j.enzmictec.2013.04.006
DO - 10.1016/j.enzmictec.2013.04.006
M3 - Article
C2 - 23830463
AN - SCOPUS:84880038125
SN - 0141-0229
VL - 53
SP - 200
EP - 205
JO - Enzyme and Microbial Technology
JF - Enzyme and Microbial Technology
IS - 3
ER -