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.
Highlights
Gamma-aminobutyric acid (GABA) is a non-proteinaceous amino acid widely used in the food and pharmaceutical industry, where it serves as an inhibitor of neurotransmission with hypotensive and diuretic effects [1,2,3,4], as well as in the chemical industry, where it can be employed as a precursor for biodegradable polymers as an intermediate to pyrrolidone to synthesize polymer Nylon 4 [5,6]
We have successfully developed a process for GABA production applicable on a large scale
We achieved more than 2 M GABA production in a 1 L scale conversion, and most of the residual substrate and proteins were removed in the downstream part of the process
Summary
Gamma-aminobutyric acid (GABA) is a non-proteinaceous amino acid widely used in the food and pharmaceutical industry, where it serves as an inhibitor of neurotransmission with hypotensive and diuretic effects [1,2,3,4], as well as in the chemical industry, where it can be employed as a precursor for biodegradable polymers as an intermediate to pyrrolidone to synthesize polymer Nylon 4 [5,6]. Recent advances of fermentative production of GABA using fungal strains, such as Monascus, have achieved up to 13.5 g/L of GABA production yield, which resulted in a lower production yield than Lactobacillus strains [15,16,17,18]. GAD on a metal affinity resin and further purification by cation exchange chromatography. The aim of this process is to convert more than 2 molar concentration of GABA from glutamate and to remove impurities to achieve a purity of higher than 99%, potentially providing an alternative method for GABA production for biotechnological and pharmaceutical uses
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