Gene Resource Mining Of Glutamate Decarboxylase And Whole-cell Biocatalyst Production Of γ-Aminobutyric Acid

γ-Aminobutyric acid（GABA）is a four-carbon non-protein amino acid and ubiquitous in nature.GABA plays a role in lowering blood pressure,anti-epileptic,anti-depressive,liver-protecting,kidney-reinforcing and other important physiological functions and has been widely used in the field of medicine as an inhibitory neurotransmitter in human.What’s more,GABA was found that can effectively improve the heat stress response of animals and promote animal growth in the animal husbandry industry.Thus,it can be used to promote the green and healthy development of the animal husbandry industry as a new type of feed additive.GABA exists in a variety of plant tissues with very low content,it is expensive to extract it directly from natural organisms.Therefore,the methods of chemical synthesis and biotransformation draw much attention.Compared to biotransformation,chemical synthesis methods have many disadvantages such as corrosive chemicals,harsh reaction conditions and high energy consumption,which severely limit their widly application.However,bioconversion by enzyme showed the advantages of highly conversion efficiency,mild reaction conditions and easily purification.Among them,glutamate decarboxylase（GAD;EC 4.1.1.15）using pyridoxal 5’-phosphate（PLP）as a cofactor to decarboxylate L-glutamic acid into GABA has attracted widespread attention.At present,some problems in the conversion process were existed,such as poor stability of enzymes,low conversion rate and cumbersome separation,large energy consumption in the later stage purification processes.Based on this,the screening of enzyme producing strains,enzymological characteristics analysis and whole-cell transformation production process of GABA was systematically carried out.The method of GAD-activity screening on the plate was established,combined with the re-screening of shake flask fermentation,three strains,named Z1,Z11,and Z20,respectively,showing glutamate decarboxylase activity were obtained from desert soil samples,Ningxia province.After the colony morphology analysis,16S r DNA sequence alignment and phylogenetic tree analysis,the three strains were preliminarily identified as Bacillus sp.,and preserved in the China Agricultural Microbial Species Conservation Center.The whole genome of the three strains was sequenced,each of them containing a GAD gene,they shared the 94.5-97.3%sequence similarity.The three GAD genes were cloned into E.coli expression vectors separately for overexpression heterologously.All three genes successfully achieved heterologous expression concluded by SDS-PAGE electrophoresis and enzyme activity analysis.The enzymological characteristics of the three purified GAD enzymes were determined.The three recombinant enzymes showed the same p H and temperature optimum at 5.0 and40oC.Among the three GAD enzymes,GADZ11showed the highest specific activity of 98 U/mg.The catalytic efficiency of GADZ11 was 2.19 m M^-1·s^-1 with protential in good application prospects.It is difficult for the GAD enzyme to achieve thr effective catalysis due to the low solubility of L-Glu and the low p H of the dissolving solution.As the microbial whole-cell transformation process is less affected by factors such as environment and space,it considered to be an effective method for large-scale production of GABA.In this study,single factor and crossover experiments were used to systematically investigate the six aspects of substrate specificity,substrate concentration,cell concentration,PLP concentration,conversion time course,and cell repetition rate.The best reaction conditions are:substrate L-Glu 1 M,PLP 0.1 m M,cell concentration OD₆₀₀ 20,reaction temperature37oC,rotation speed 120 rpm,reaction time 1 hour.Under this condition,the conversion rate was as high as 97%,which was close to complete conversion.Further preliminary explorations were made on the purification and crystallization process of GABA in the later stage.Through the basic steps of centrifugal filtration,rotary evaporation and drying,high-purity GABA crystals were obtained.The study provided the strong support for the industrialized large-scale production of GABA.