Production Of ?-Aminobutyric Acid Using Whole-cell Biotransformation

Gamma-aminobutyric acid?GABA?is a no-protein amino acid and widely distributed in nature,from plants to animals and microbes.It is well known that GABA acted as a major inhibitor of neurotransmission in controlling neurotransmitter signal,so it played an important role in human body,such as treatment of headaches,cerebral arterial disorders,lowering blood pressure,oxygen supply,and metabolism in the brain.In addition,GABA can be also used to synthesis 2-pyrrolidone and polyamide nylon 4.Based on these versatile advantages,GABA is widely applied both in food and pharmaceutical industry,as well as in industries.Glutamate decarboxylase?GAD?is a pyridoxal 5'-phosphate?PLP?-dependent enzyme was widely exist in Eukaryotes and prokaryotes.The enzyme catalyzes the irreversible of L-glutamate?L-Glu?or sodium glutamate to GABA and CO₂ and it was the rate-limiting enzyme for biotransformation of GABA.Although there are many reports on the production of GABA,simple and efficient,low-cost access to high-quality GABA were rarely reported.The main contents of this study are as follows:1.Construction of recombinant strainsGAD gene fragment was amplified from the E.coli genome by PCR and cloned into vector pET-23a to construct recombinant plasmid pET-23a-GAD.The recombinant plasmid was transformed into Escherichia coli BL21?DE3?to construct the recombinant strain and then cultured in shake flask.The expression of the target protein was detected by SDS-PAGE and Western-Blot.2.Whole cell transformation of L-Glu to produce GABAThe Escherichia coli BL21?DE3?were used to express the GAD.The cells were collected and used for whole cell catalytic reaction.With lower price sodium glutamate as a precursor substrate,after the saturated solution by adding concentrated hydrochloric acid into glutamic acid,and then glutamic acid as the final substrate,and a PLP with a final concentration of 0.5 mM was added to a500 mL reaction system.The reaction was carried out at 38°C without a buffer system.After 48 h reaction,800 g sodium glutamate nearly 100%converted into GABA.3.High cell density cultivationThe recombinant Escherichia coli BL21?DE3?strain was inoculated with 10%fermentor in a 5L fermentor,and the dissolved oxygen in the growth cycle was maintained at 30%to 50%by adjusting the rotational speed and the ventilation.The induction was started at 6 h before the end of the fermentation.The activity of L-Glu could reach 2 000U/mL,which was 20 times higher than that of shake flask culture.4.Crystallization of GABAAfter the substrate has been completely transformed,the reaction mixture was centrifuged and the supernatant was collected.After concentration evaporation at 70?,4 times the volume of ethanol was added,and the mixture was slowly cooled to obtain GABA crystals.5.GABA purity determinationThe purity of GABA was determined by high performance liquid chromatography?HPLC?,the purity of GABA was above 96.1%,which laid the foundation for future large-scale production.