Screening,Identification,Fermentation Optimization And Molecular Modification Of Glutamate Decarboxylase Gene Of High GABA-producing Lactic Acid Bacteria

Gamma-aminobutyric acid(GABA)is a non-protein amino acid that widely found in living organisms.Studies have shown that it has many important physiological functions,such as lowering blood pressure,calming and preventing Alzheimer's disease,etc.Therefore,GABA has great application potential in the pharmaceutical,food,health care and other industries.Glutamate decarboxylase(GAD),which has found in many organisms,is the unique enzyme capable of converting glutamate(L-glu)into GABA.With the improvement of the global economy,the demand for GABA is increasing.However,the current domestic production of GABA wild strains is generally low,and at the same time,the optimum pH of GAD is low during fermentation production.Generally between 4.0 and 5.0,when the pH reaches 6.0,the enzyme is inactivated by depolymerization.In the industrial production process,the acid is additionally added at the initial stage of the fermentation to maintain the fermentation environment,which greatly increases the production cost.On the other hand,the acidic conditions exacerbate the corrosion of the equipment materials.Dued to the characteristics of the strains and the enzymatic properties of GAD,its industrial application is limited.Therefore,a strain capable of high-producing GABA is obtained,and a large amount of GAD is obtained on the basis of this.On the foundation of keeping the enzyme activity,the pH adaptation range is broadened,which becomes an urgent demand for industrial production.The main research contents and results are as follows:1.Screening and identification of GABA-producing strains.In this study,four GABA-producing strains were screened out from pickles in Zigong.Four GABA-producing strains were cultured by fermentation respectively.The GABA production of standard strains were 0.736 g/L,0.691 g/L,1.11 g/L and 1.92 g/L.The highest produced strain Liulab84 was identified as Lactobacillus plantarum by morphology,physiology,biochemistry and 16S rDNA molecular biology,that was named Lactobacillus plantarumLC84.2.Optimizing fermentation conditions for GABA-producing by strains.In the fermentation process,through single factor and orthogonal test,the optimum fermentation conditions of Lactobacillus plantarum LC84 were obtained as follows:fermentation temperature 39 C,pH 6.0,inoculume 4%,time 60h.Under these conditions,the content of GABA was 3.83 g/L.The results showed that A3B2C1D3 was the best combination.3.Cloning and expression of glutamate decarboxylase gene.The cloned GAD gene is similar to the base sequence of glutamate decarboxylase gene published on GeneBank.Decarboxylase also differs greatly in amino acid sequence.SDS-PAGE analysis showed that a fusion protein of about 53KDa was expressed.The optimum pH of glutamate decarboxylase was 4.8,the optimum temperature was 40-50?,and the optimum amount of coenzyme PLP was 50 ?m/L.The metal ions added in the study had no obvious promoting effect on the enzyme activity.Cu2+ and Ag+had obvious inhibitory effect on the enzyme,while Ag+almost inhibited the activity of GAD.4.Molecular mutation of glutamate decarboxylase.Dpnl method was used to mutate wild glutamate decarboxylase successfully.Aspartic(Asp)at 294 sites was mutated to glycine(Gly),serine(Ser)at 307 sites to asparagine(Asn),glutamic acid(Glu)at 312 sites to serine(Ser),glutamine(Gln)at 346 sites to histidine(His).After that,the optimum enzyme activity of E312S mutant was about 1.6 times of that of wild type.At pH 6.6,the enzyme activity of E312S mutant was about 5 times higher than that of wild type.In the range of 4.8-5.8,the acid stability of E312S mutant was over 60%.The enzyme activity of Q346H mutant was slightly higher than that of wild type at pH 4.8.At pH 6.6,the enzyme activity of Q346H mutant increased by about 20%.In the range of 4.8-5.8,the enzyme activity of Q346H mutant maintained over 40%.In summary,wild GABA-producing Lactobacillus plantarum was obtained from pickles,and its fermentation conditions were optimized to maximize the performance of GABA production.At the same time,GAD in Lactobacillus plantarum was exogenously expressed by genetic engineering.On this basis,site-directed mutagenesis was carried out,which broadened the reaction pH of the enzyme and provided a basis for the modification of GAD enzymatic properties.It lays a foundation for reducing the cost of production and equipment wastage in the industrial application of GABA production.