title: Buffer-free production of gamma-aminobutyric acid using an engineered glutamate decarboxylase from Escherichia coli.
abstract: 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 1M GABA in 3h, which can be translated into a space-time yield of 34.3g/L/h.
authors.affiliation: Department of Chemical and Biochemical Engineering, Dongguk University-Seoul, Seoul, 100-715, Republic of Korea. taekkang@dongguk.edu
authors.forename: Taek Jin
authors.initials: TJ
authors.lastname: Kang
authors.forename: Ngoc Anh Thu
authors.initials: NA
authors.lastname: Ho
authors.forename: Seung Pil
authors.initials: SP
authors.lastname: Pack
mesh_terms: D002021:Buffers; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012970:Sodium Glutamate / Q000378:metabolism; D014867:Water; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis*
chemical_list: D002021:Buffers; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014867:Water; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; D012970:Sodium Glutamate
keywords: Buffer-free enzyme reaction; Enzyme engineering; Gamma-aminobutyric acid; Glutamate decarboxylase