来自链霉菌属X-119-6的L-谷氨酸氧化酶的重组表达、生化特性及通过蛋白酶解实现的稳定化
Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp. X-119-6.
作者信息
Arima Jiro, Tamura Takashi, Kusakabe Hitoshi, Ashiuchi Makoto, Yagi Toshiharu, Tanaka Hidehiko, Inagaki Kenji
机构信息
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530.
出版信息
J Biochem. 2003 Dec;134(6):805-12. doi: 10.1093/jb/mvg206.
L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure alpha2beta2gamma2. The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of alpha2beta2gamma2 and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.
来自链霉菌属X-119-6的L-谷氨酸氧化酶（LGOX）是一种150 kDa的蛋白质，具有α2β2γ2六聚体结构。编码LGOX的基因被克隆并在大肠杆菌中进行异源表达。从大肠杆菌转化体中分离出的LGOX具有单链多肽结构。尽管重组LGOX表现出催化活性，但其催化效率低于从链霉菌属X-119-6中分离出的LGOX。与从链霉菌属X-119-6中分离出的LGOX相比，重组LGOX的热稳定性较低，且呈聚集形式。用灰色链霉菌的金属内肽酶（Sgmp）对重组LGOX进行蛋白水解，可提高其在不同pH值下的催化效率。此外，经Sgmp处理的重组LGOX具有α2β2γ2亚基结构，其酶学特性与从链霉菌属X-119-6中分离出的LGOX几乎相同。经Sgmp处理的重组LGOX未检测到重组LGOX中观察到的高分子量物种。这些结果证明，Sgmp的蛋白水解作用参与了重组LGOX的稳定性维持。
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