Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp X-119-6
Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp X-119-6
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DOI：
10.1093/jb/mvg206
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发表时间：
2003-12-01
影响因子：
2.7
通讯作者：
Inagaki, K
中科院分区：
文献类型：
--
作者：
Arima, J;Tamura, T;Inagaki, K
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，具有六聚体结构α（2）β（2）γ（2）。克隆了LGOX基因，并在大肠杆菌中异源表达。从大肠杆菌中分离得到LGOX。大肠杆菌Escherichia coli具有单链多肽结构。虽然重组LGOX具有催化活性，但其催化效率不如从链霉菌X-119-6分离的LGOX。与从链霉菌X-119-6分离的LGOX相比，重组LGOX表现出较低的热稳定性，并且是聚集形式。用灰色链霉菌（Streptomycesgriseus，Sgmp）的金属内肽酶（metallocendopeptidase）对重组LGOX进行蛋白酶解，在不同pH条件下提高了其催化效率。此外，经Sgmp处理的重组LGOX具有α（2）β（2）γ（2）亚基结构，其酶学性质与从Streptomycessp. X-119-6分离的LGOX几乎相同。在重组LGOX中观察到的较高分子物质在Sgmp处理的重组LGOX中未检测到。这些结果证明Sgmp的蛋白水解参与了重组LGOX的稳定。
L-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure alpha(2)beta(2)gamma(2). 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 alpha(2)beta(2)gamma(2) 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.