一株重组大肠杆菌/pET-28a-lpgad的构建及其高效生产γ-氨基丁酸转化条件的优化

Construction of a recombinant Escherichia coli BL21/ pET-28a-lpgad and the optimization of transformation conditions for the efficient production of γ-aminobutyric acid

摘要:

为实现微生物法高效率生产γ-氨基丁酸 (GABA)，从一株经多次诱变筛选的具有较高谷氨酸脱羧酶活力植物乳杆菌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，5 L发酵罐水平转化24 h产量可达143.5 g/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5 L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。

Abstract:

In order to enhance γ-aminobutyric acid production from L-glutamate efficiently, we amplified the key enzyme glutamate decarboxylase (GAD) encoding gene lpgad 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 show that the recombinant E. coli BL21 (pET-28a-lpgad) 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 γ-aminobutyric acid transformation in 5 L fermentor under the optimum transformation conditions. Accordingly, the yield of γ-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 γ-aminobutyric acid was improved by 42.5% compared with that under the unoptimized transformation conditions. This paved a way for the γ-aminobutyric acid construction of the industrial applications.