Qingdai Liu1, Xiaoqian Ma1, Haijiao Cheng2,3, Ning Xu2,3, Jun Liu4,5, Yanhe Ma2,3. 1. Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin, 300457, China. 2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. 3. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. 4. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. liu_jun@tib.cas.cn. 5. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. liu_jun@tib.cas.cn.
Abstract
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.
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.