Objectives To improve the production of alpha-ketoglutaric acid (alpha-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 degrees C. The apparent K-m and V-max 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 alpha-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 alpha-KG was established in the recombinant Escherichia coli, and it has a potential prospect in industrial application.