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.

Characterization and immobilization on nickel-chelated Sepharose of a glutamate decarboxylase A from Lactobacillus brevis BH2 and its application for production of GABA

“…The yield of GABA in the present system was relatively higher than that of the immobilized E. coli GAD (91.2%). 15) As proven by the presented data, the prepared Ni-Sepharose particles were applicable for immobilization of GadA with high conversion yield of GABA, suggesting that high binding capacity of the Ni-Sepharose support allowed concentrated enzyme immobilization and a high rate of conversion.…”

“…15,24) The advantage of Ni-Sepharose resin used in this study is its high binding capacity (about 40 mg protein /mL resin ) for enzymes while retaining their activity. The recombinant GadA was immobilized on Ni-Sepharose that consists of 90 μm beads of highly cross-linked agarose and Ni 2+ ions.…”

“…The binding capacity of previously studied supports was limited to 0.24 and 0.05 mg GAD /g dry bead for Eupergit 250 C and calcium alginate, respectively, 9) and 1.91 mg GAD /g wet avicel and 9.23 mg GAD /mL resin (immobilization yield, 92.3%) for Avicel and Ni-Sepharose, respectively. 14,15) Whereas the immobilization yield of GadA on the resin reached 95.8% when the conditions were as follows: coupling time of 1 h at 25°C in working buffer and enzyme loading of 42 mg GadA /g wet resin .…”

“…The reusability of E. coli GAD immobilized onto Ni-Sepharose was limited to 58.1% residual activity after 10 consecutive uses. 15) Thus, the high reusability makes the immobilized GadA very attractive for consecutive conversion of L-glutamate solution.…”

“…9,[13][14][15] Immobilization of GAD on calcium alginate, Eupergit 250 C, and bacterial cellulose membrane was attempted by Lammens et al and Yao et al 9,13) Park et al and Lee et al studied the immobilization and reuse of GAD on the affinity support using the cellulose binding domain or histidine tag. 14,15) However, the immobilized GAD is still limited in E. coli one. LAB have been interested as possible GABA producers due to their commercial potential as a starter for fermented foods.…”