In order to enhance y-aminobutyric acid production from L-glutamate efficiently, we amplified the key enzyme glutamate decarboxylase (GAD) encoding gene Ipgad from the strain Lactobacillus plantarum GB 01-21 which was obtained by way of multi-mutagenesis and overexpressed it in E. Coli BL21. Then we purified GAD by Ni-NTA affinity chromatography and characterized the enzyme to optimize the conditions of the whole-cell transformation. The results showed that the recombinant E. Coli BL21 (Pet-28a-/pgafiT) produced 8.53 U/mg GAD, which was increased by 3.24 fold compared with the GAD activity in L. Plantarum. The optimum Ph and temperature of the enzyme were Ph 4.8 and 37 °C, respectively. At the same time, we found that Ca2+ and Mg2+ could increase the activity significantly. Based on this, we investigated y-aminobutyric acid transformation in 5 L fermentor under the optimum transformation conditions. Accordingly, the yield of y-aminobutyric acid was 204.5 g/L at 24 h when the 600 g L-glutamate was added and the mole conversion rate had reached 97.92%. The production of y-aminobutyric acid was improved by 42.5% compared with that under the unoptimized transformation conditions. This paved a way for the y-aminobutyric acid construction of the industrial applications.%为实现微生物法高效率生产γ-氨基丁酸(GABA),从一株经多次谤变筛选的具有较高谷氨酸脱羧酶(GAD)活力植物乳杆菌GB 01-21全基因组DNA中PCR扩增获得GAD酶基因lpgad,构建重组质粒pET-28a-lpgad,在大肠杆菌E.coli BL21 (DE3)中高效诱导表达.并采用Ni柱亲和层析纯化获得重组GAD,并对其酶学性质进行初步研究,为改良转化工艺提高GABA产量提供可靠理论依据.结果显示,重组大肠杆菌中GAD酶活显著提高,可达8.53 U/mg,是植物乳杆菌GB 01-21中GAD酶活的4.24倍.将该重组菌应用于转化L-谷氨酸生产GABA,5L发酵罐水平转化24 h产量可达143.5g/L,摩尔转化率为97.32％,是植物乳杆菌GB 01-21的2.19倍.纯化后酶学性质进行初步研究表明:其最适pH为4.8；最适温度为37℃;Ca2+、Mg2+对其有较强的激活作用,将上述实验结果用于转化条件的优化,最终5L发酵罐上进行转化实验,批次添加底物L-谷氨酸共600 g,转化24 h,GABA累计浓度可达204.5 g/L,摩尔转化率为97.92％,与最初转化条件相比,GABA浓度提高了42.5％,为其工业化应用打下了良好的基础.
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