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.
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Acknowledgements
This research was supported by the National Natural Science Foundation of China (No. 31500044, 31571029, 31501475), the Natural Science Foundation of Tianjin (15JCYBJC30300, 15JCTPJC55400), the Key Projects in the Tianjin Science and Technology Pillar Program (No. 11ZCZDSY08600), and the “Hundred Talents Program” of the Chinese Academy of Sciences.
Supporting information
Supplementary Table 1—Strains and plasmids used.
Supplementary Table 2—Primers used.
Supplementary Table 3—The substrate specificity of SmLGOX.
Supplementary Fig. 1—Effects of metal ions on the activity of the recombinant SmLGOX. The relative activity was normalized as percentage activity of the control sample (125.2 ± 2.7 U mg−1). The data are presented as mean ± standard deviation (SD) from three independent experiments.
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Qingdai Liu and Xiaoqian Ma have contributed equally to this work.
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10529_2017_2314_MOESM2_ESM.tif
Supplementary Fig. 1 Effects of metal ions on the activity of the recombinant SmLGOX. The relative activity was normalized as percentage activity of the control sample (125.2 ± 2.7 U mg−1). The data are presented as mean ± standard deviation (SD) from three independent experiments. (TIFF 7626 kb)
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Liu, Q., Ma, X., Cheng, H. et al. Co-expression of l-glutamate oxidase and catalase in Escherichia coli to produce α-ketoglutaric acid by whole-cell biocatalyst. Biotechnol Lett 39, 913–919 (2017). https://doi.org/10.1007/s10529-017-2314-5
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DOI: https://doi.org/10.1007/s10529-017-2314-5