title: Gamma-aminobutyric acid production using immobilized glutamate decarboxylase followed by downstream processing with cation exchange chromatography.
abstract: We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7% monomer, 29.2% dimer and 2.3% tetramer with a 97.6% soluble form of the total GAD. The harvested GAD was immobilized to metal affinity gel with an immobilization yield of 92%. Based on an investigation of specific enzyme activity and reaction characteristics, glutamic acid (GA) was chosen over monosodium glutamate (MSG) as a substrate for immobilized GAD, resulting in conversion of 2.17 M GABA in a 1 L reactor within 100 min. The immobilized enzymes retained 58.1% of their initial activities after ten consecutive uses. By using cation exchange chromatography followed by enzymatic conversion, GABA was separated from the residual substrate and leached GAD. As a consequence, the glutamic acid was mostly removed with no detectable GAD, while 91.2% of GABA was yielded in the purification step.
authors: University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 305-350, Korea. eglee@kribb.re.kr.
authors: Seungwoon
authors: S
authors: Lee
authors: Jungoh
authors: J
authors: Ahn
authors: Yeon-Gu
authors: YG
authors: Kim
authors: Joon-Ki
authors: JK
authors: Jung
authors: Hongweon
authors: H
authors: Lee
authors: Eun Gyo
authors: EG
authors: Lee
mesh_terms: D002412:Cations; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012970:Sodium Glutamate / Q000378:metabolism; D013379:Substrate Specificity; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis* / Q000302:isolation & purification*
chemical_list: D002412:Cations; D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; D012970:Sodium Glutamate