Objectives: To improve the production of α-ketoglutaric acid (α-KG) from L-glutamate by whole-cell biocatalysis.
Results: A novel and highly active L-glutamate oxidase, SmlGOX, from Streptomyces mobaraensis was overexpressed and purified. The recombinant SmlGOX was approx. 64 kDa by SDS-PAGE. SmlGOX had a maximal activity of 125 ± 2.7 U mg-1 at pH 6.0, 35 oC. The apparent Km and Vmax values of SmlGOX were 9.3 ± 0.5 mM and 159 ± 3 U mg-1, respectively. Subsequently, a co-expression plasmid containing the SmlGOX and KatE genes was constructed to remove H2O2, and the protein levels of SmlGOX were improved by codon optimization. Finally, by optimizing the whole-cell transformation conditions, the production of α-KG reached 77.4 g l-1 with a conversion rate from L-glutamate of 98.5% after 12 h.
Conclusions: An efficient method for the production of α-KG was established in the recombinant Escherichia coli, and it has a potential prospect in industrial application.
Keywords: L-Glutamate oxidase; Streptomyces mobaraensis; Whole-cell biocatalyst; α-Ketoglutaric acid.