定向突变谷氨酸脱羧酶及其生物合成γ-氨基丁酸的研究

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。

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

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 GadB of E. coli AS1.505 through site-directed mutagenesis; constructed 3 expression vectors: pET28a(+)-GadB, pET28a(+)-GadBm1, and pET28a(+)-GadBm2; and studied their status for GABA production. The results showed that after 72 h in neutral buffer solution, DE3（pET28a (+)-GadBm1）and DE3（pET28a (+)-GadBm2）had produced GABA 3.2 and 4.6 times of the control DE3（pET28a (+)-GadB）, respectively. And after 72 h in M9 minimal medium, DE3（pET28a (+)-GadBm1）and DE3（pET28a (+)-GadBm2）had produced GABA 4.32 g/L and 4.65 g/L GABA, respectively, which was 1.17and 1.34 times higher than that of the contrast. The results indicated that site-directed mutated GadB could break the cooperativity in activity change and yield a mutant GadBm2 retained the activity under neutral PH value.