| Glutamate decarboxylase（GAD）acts specifically on theα-carboxyl group of L-glutamic acid or its sodium salt with pyridoxal phosphate（PLP）,making it irreversibly remove a CO2 to obtainγ-aminobutyric acid.GABA,a valuable functional amino acid that functions as an inhibitory neurotransmitter,which is widely used as food additives in the food industry.Enzymatic preparation of GABA has the advantages of high efficiency,no limitation of resources and environment,and is gradually used in industrial production.In this study,GAD derived from Escherichia coli was expressed in food-grade host Bacillus subtilis.The effects of coenzyme precursors pyridoxal（PL）on the activity and enzymatic properties of recombinant GAD enzyme were studied.In addition,the effects of relatively inexpensive coenzyme precursors pyridoxine（PN）on the production and enzymatic properties of GAD were investigated by co-expression of GAD and pyridoxine phosphate oxidase（PNPO）during the fermentation process.The process of synthesizing GABA by whole cells was optimized.Finally,shake-flask and 3-L fermentor experiments was optimized through choosing better strains,which laid the foundation for industrial preparation of food grade GABA.This paper achieves following results:（1）Successfully constructed the recombinant B.subtilis/pHY300PLK-gadB,which contains GAD gene derived from E.coli.The effects of PL concentration on the growth of recombinant bacteria and the expression of GAD was investigated.The results showed that the optimal concentration of PL was 0.1 mmol·L-1,the maximum activity of GAD was 28.28 U·mL-1,which was increased by 1.55 times than GAD（control group）.Then the enzymatic properties of GAD（control group）and GAD+PL were examined.The results showed that the optimum temperature was 55℃and 60℃,respectively,and the optimal pH of both was 4.5,the half-life of 40℃was 70 h and 107 h,respectively.（2）Successfully constructed the recombinant B.subtilis/pHY300PLK-gadB-PdxH,which is co-expression of pyridoxine phosphate oxidase（PNPO）derived from E.coli and GAD.Effects of supplement concentration of coenzyme precursors pyridoxine（PN）on the yield of GAD was studied.The results showed that the optimal concentration of PN was 0.2 mmol·L-1,the maximum activity of GAD was 31.86 U·m L-1,which was 1.59 times higher than GAD-PNPO（control group）.And the enzymatic properties of GAD-PNPO+PN were examined.The results showed that the optimum temperature was 60℃,the optimal pH was 4.5,and the retention rate of enzyme activity was over 95%in the range of pH 4.0-7.0,which was basically consistent with GAD+PL,but the half-life of 40℃was 90 h,17 h lower than GAD+PL.（3）The preparation of GABA by whole cells was investigated.It was found that the optimal amount of GAD（control group）,GAD+PL and GAD-PNPO+PN was 50,40 and 40 U·g-1（glutamic acid）,respectively.When the concentration of 400 g·L-1（glutamic acid）,the GABA yield reached273.62 g·L-1、275.60 g·L-1 and 274.51 g·L-1,respectively.（4）Considering the cost,the recombinant B.subtilis/pHY300PLK-gadB-PdxH was selected for fermentation optimization.The results showed that 25 g·L-1 corn steep powder and 5 g·L-1 soy peptone were the best compound nitrogen source,5 g·L-1 glucose was the best carbon source.When1 mmol·L-1 Ba2+was added,the GAD activity was reaching 29.60 U·mL-1 and the dry weight of bacteria was 5.81 g·L-1.After that,the optimization results in a 3-L fermentor showed that it is the better way to add 3 mmol·L-1 PN in batches at 0 h,24 h and 48 h,respectively.And the GAD activity was reaching of 742 U·mL-1,which was 1.77 times higher than GAD（control group）. |
