白丝北里孢菌L-谷氨酸氧化酶的异源表达及酶学性质分析

为实现从L-谷氨酸到α-酮戊二酸（α-ketoglutaric acid，α-KG）的高效生物转化，将来源于白丝北里孢菌（Kitasatospora setae KM-6054）的L-谷氨酸氧化酶（L-glutamate oxidase，LGOX）在大肠杆菌（Escherichia coli）中实现异源表达，并研究其酶学特性。根据LGOX的氨基酸序列和大肠杆菌系统偏好性合成LGOX全基因序列，并通过pET28a（＋）/DE3系统在大肠杆菌中实现了功能表达。诱导剂异丙基硫代半乳糖苷（isopropyl-β-D-thiogalactoside，IPTG）终浓度为0.1?mmol/L，20?℃诱导18?h，重组大肠杆菌粗酶液酶活力可达49.10?U/mL。亲和层析获得酶比活力为45.98?U/mg纯酶，十二烷基硫酸钠-聚丙烯酰氨凝胶电泳条带显示蛋白分子质量大小约为70?kDa。酶学性质研究表明：其最适反应温度和pH值分别为40?℃和6.0；Km值为1.23?mmol/L，Vmax值为76.24?μmol/（min·mg），L-谷氨酸为该酶的最适底物。本研究确定了LGOX在E.?coli?BL21中的异源表达及酶学特性，为生物转化合成α-KG提供了新的参考途径。

Heterologous Expression and Characterization of Recombinant L-Glutamate Oxidase from Kitasatospora setae

In order to achieve efficient bioconversion of L-glutamic acid to alpha ketoglutaric acid (α-KG), the L-glutamate oxidase (LGOX) gene from Kitasatospora setae KM-6054 was expressed in Escherichia coli BL21. According to the known LGOX sequence from K. setae and the codon bias of E. coli, the optimized LGOX gene sequence was synthesized and cloned into the pET28a (+) vector, which was then transferred into E. coli BL21(DE3) to obtain a recombinant LGOX. And enzymatic properties of the recombinant LGOX were also investigated. Results showed that the recombinant LGOX activity could reach 49.10 U/mL after induction at an IPTG concentration of 0.1 mmol/L at 20 ℃ for 18 h. Subsequently, the recombinant LGOX was purified by affinity column chromatography to a specific activity of 45.98 U/mg and its molecular weight was about 70 kDa as determined by SDS-PAGE analysis. The enzymatic properties showed that the optimal reaction temperature and pH value were 40 ℃ and 6.0, respectively. The Michaelis-Menten constant Km was 1.23 mmol/L, and the maximum reaction rate Vmax was 76.24 μmol/(min·mg). L-glutamic acid was the optimal substrate for the LGOX. The heterologous expression and characterization of recombinant L-glutamate oxidase in this study can provide a new way for biosynthesis of α-KG.