关键词: 白丝北里孢菌, L-谷氨酸氧化酶, α-酮戊二酸, 异源表达, 酶学性质

Abstract: 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.

Key words: Kitasatospora setae, L-glutamate oxidase, α-ketoglutaric acid, heterologous expression, enzymatic properties