Shi Feng, Xie Yilong, Jiang Junjun, Wang Nannan, Li Yongfu, Wang Xiaoyuan
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China.
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China.
Enzyme Microb Technol. 2014 Jul-Aug;61-62:35-43. doi: 10.1016/j.enzmictec.2014.04.012. Epub 2014 May 1.
Glutamate decarboxylase (GAD) transforms l-glutamate into γ-aminobutyric acid (GABA) with the consumption of a proton. GAD derived from lactic acid bacteria exhibits optimum activity at pH 4.0-5.0 and significantly loses activity at near-neutral pH. To broaden the active range of the GAD GadB1 from Lactobacillus brevis Lb85 toward a near-neutral pH, irrational design using directed evolution and rational design using site-specific mutagenesis were performed. For directed evolution of GadB1, a sensitive high-throughput screening strategy based on a pH indicator was established. One improved mutant, GadB1(T17I/D294G/Q346H), was selected from 800 variants after one round of EP-PCR. It exhibited 3.9- and 25.0-fold increase in activity and catalytic efficiency, respectively at pH 6.0. Through site-specific mutagenesis, several improved mutants were obtained, with GadB1(E312S) being the best one. The combined mutant GadB1(T17I/D294G/E312S/Q346H) showed even higher catalytic efficiency, 13.1- and 43.2-fold that of wild-type GadB1 at pH 4.6 and 6.0, respectively. The amount of GABA produced in gadB1(T17I/D294G/Q346H)-, gadB1(E312S)- and gadB1(T17I/D294G/E312S/Q346H)-expressing Corynebacterium glutamicum ATCC 13032 from endogenous l-glutamate increased by 9.6%, 20.3% and 63.9%, respectively. These results indicate that these mutations have beneficial effects on expanding the active pH range and on GABA biosynthesis, suggesting these GadB1 variants as potent candidates for GABA production.
谷氨酸脱羧酶（GAD）利用一个质子将L-谷氨酸转化为γ-氨基丁酸（GABA）。来源于乳酸菌的GAD在pH 4.0 - 5.0时表现出最佳活性，在接近中性pH时活性显著丧失。为了将短乳杆菌Lb85的GAD GadB1的活性范围拓宽至接近中性pH，进行了基于定向进化的非理性设计和基于位点特异性诱变的理性设计。对于GadB1的定向进化，建立了一种基于pH指示剂的灵敏高通量筛选策略。经过一轮易错PCR（EP-PCR）后，从800个变体中筛选出一个改进的突变体GadB1(T17I/D294G/Q346H)。它在pH 6.0时活性和催化效率分别提高了3.9倍和25.0倍。通过位点特异性诱变，获得了几个改进的突变体，其中GadB1(E312S)是最佳的一个。组合突变体GadB1(T17I/D294G/E312S/Q346H)表现出更高的催化效率，在pH 4.6和6.0时分别是野生型GadB1的13.1倍和43.2倍。在表达gadB1(T17I/D294G/Q346H)、gadB1(E312S)和gadB1(T17I/D294G/E312S/Q346H)的谷氨酸棒杆菌ATCC 13032中，内源性L-谷氨酸产生的GABA量分别增加了9.6%、20.3%和63.9%。这些结果表明这些突变对扩大活性pH范围和GABA生物合成具有有益作用，表明这些GadB1变体是GABA生产的有力候选者。
