谷氨酸脱羧酶，一种磷酸吡哆醛（PLP）依赖性酶，能专一、不可逆地催化L-谷氨酸脱羧得到γ-氨基丁酸（GABA）。构建了产Lactobacillus brevisWJH3谷氨酸脱羧酶重组大肠杆菌E.coliBL21（DE3）/pET-24a-gad，以此作为菌种进行摇瓶发酵诱导培养，发酵过程中一次性添加0.05mmol/LPLP培养24h，破壁上清酶活达81.7U/mL，是不添加PLP对照酶活的1.8倍。对酶转化L-谷氨酸钠生成GABA反应条件进行了优化，结果表明，在转化体系不添加PLP的情况下，底物谷氨酸钠浓度为250g/L，反应初始pH5.0，温度37℃，加酶量60U/g底物，转速200r/min，在此条件下反应18h，GABA转化率达到100%，为γ-氨基丁酸的工业化生产奠定基础。%Glutamate decarboxylase(GAD),a pyridoxal 5'-phosphate(PLP)-dependent enzyme,irreversibly catalyzes the decarboxylation of L-glutamate to be the valuable food additive γ-aminobutyric acid(GABA). In this study,a recombinant Escherichia coli BL21(DE3)/pET-24a-gad producing Lactobacillus brevis WJH3 GAD was constructed as strain in the flask culturing of fermentation and induction. The activity of GAD produced in the supernatant of culturing for 24 h medium supplemented one-time with 0.05 mmol/L PLP was 81.7 U/mL,and this was 1.8-fold of that without PLP supplementation. Furthermore,the condition for GABA preparation by enzymatic conversion was optimized;under the condition of 250 g/L monosodium glutamate(MSG),pH5.0,37℃,60 U GAD per gram substrate incubated for 18 hours,and rotation rate 200 r/min,100% of the MSG was transformed into GABA. These results establish the utility of PLP supplementation and lay the foundation for large-scale enzymatic production of GABA.
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