定向突变谷氨酸脱羧酶及其生物合成γ-氨基丁酸的研究

Site-directed Mutagenesis of Glutamate Decarboxylase and its Synthesis of Gamma Aminobutyric Acid

γ-氨基丁酸(GABA)是一种天然存在的非蛋白质氨基酸,它由谷氨酸脱羧酶催化谷氨酸脱羧生成,作为抑制性神经递质具有广泛的应用。天然的谷氨酸脱羧酶只在酸性环境时有活性,这与菌体的生长环境不一致,不利于一步发酵法生产GABA。针对该问题,对来自大肠杆菌AS1.505的谷氨酸脱羧酶(Gad B)进行定向突变,构建了3个表达载体p ET28a(+)-Gad B、p ET28a(+)-Gad Bm1和p ET28a(+)-Gad Bm2,研究了3株重组菌生物转化谷氨酸合成GABA的情况。结果显示,在中性缓冲液中转化72 h,DE3(p ET28a(+)-Gad Bm1)和DE3(p ET28a(+)-Gad Bm2)转化合成GABA分别是对照菌DE3(p ET28a(+)-Gad B)的3.2和4.6倍。在M9培养基中培养72 h,DE3(p ET28a(+)-Gad Bm1)和DE3(p ET28a(+)-Gad Bm2)分别合成4.32 g/L和4.65 g/L GABA,比对照菌分别提高了1.17和1.34倍。实验结果证明,定向突变的Gad B能在中性p H条件下有效催化谷氨酸脱羧合成GABA。

Gamma aminobutyric acid（ GABA） is a non-protein amino acid,synthesized by microorganisms,plants and animals,and widely used as a bioactive component in controlling neurotransmitter signal. Enzyme is only active under acidic environments,which is inconsistent with the condition for bacterium growth. This is not convenient for GABA production by one-step fermentation. According to this problem,this experiment mutated Gad B of E. coli AS1.505 through site-directed mutagenesis; constructed 3 expression vectors： p ET28a（ ＋）-Gad B,p ET28a（ ＋）-Gad Bm1,and p ET28a（ ＋）-Gad Bm2; and studied their status for GABA production. The results showed that after72 h in neutral buffer solution,DE3（ p ET28a（ ＋）-Gad Bm1） and DE3（ p ET28a（ ＋）-Gad Bm2） had produced GABA3.2 and 4.6 times of the control DE3（ p ET28a（ ＋）-Gad B）,respectively. And after 72 h in M9 minimal medium,DE3（ p ET28a（ ＋）-Gad Bm1） and DE3（ p ET28a（ ＋）-Gad Bm2） had produced GABA 4.32 g / L and 4.65 g / L GABA,respectively,which was 1.17 and 1.34 times higher than that of the contrast. The results indicated that site-directed mutated Gad B could break the cooperativity in activity change and yield a mutant Gad Bm2 retained the activity under neutral PH value.