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Cloning and characterization of two distinct water-forming NADH oxidases from Lactobacillus pentosus for the regeneration of NAD

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Abstract

Two uncharacterized nicotinamide adenine dinucleotide (NADH) oxidases (named as LpNox1, LpNox2) from Lactobacillus pentosus ATCC 8041 were cloned and overexpressed in Escherichia coli BL21 (DE3). The sequence analysis revealed that the two enzymes are water-forming Noxs with 64 % and 52 % identity to LbNox from Lactobacillus brevis DSM 20054. The optimal pH and temperature of the purified LpNox1 and LpNox2 were 7.0 and 8.0 and 35 and 40 °C, respectively, with K M of 99.0 μM (LpNox1) and 27.6 μM (LpNox2), and yielding catalytic efficiency k cat/K M of 1.0 and 0.2 μM−1 s−1, respectively. Heat inactivation studies revealed that the two enzymes are relatively instable. The application of LpNox1 for the regeneration of NAD+ was demonstrated by coupling with a glycerol dehydrogenase-catalyzed oxidation of glycerol to 1,3-dihydroxyacetone. The characteristics of the LpNox1 could prove to be of interest in industrial application such as NAD+ regeneration in dehydrogenase-catalyzed oxidations.

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Acknowledgments

This work was financially supported by the Qualified Personnel Foundation of Taiyuan University of Technology (Grant No. tyut-rc201484a) and the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (STIP) (Grant No. 2015132).

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  1. Department of Biological and Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Taiyuan University of Technology, No.79 West Yingze Street, Taiyuan, Shanxi, 030024, People’s Republic of China

    Jian-Dong Zhang, Zhi-Mei Cui, Xiao-Jun Fan, Hua-Lei Wu & Hong-Hong Chang

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  1. Jian-Dong Zhang
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  2. Zhi-Mei Cui
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Correspondence to Jian-Dong Zhang or Xiao-Jun Fan.

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Zhang, JD., Cui, ZM., Fan, XJ. et al. Cloning and characterization of two distinct water-forming NADH oxidases from Lactobacillus pentosus for the regeneration of NAD. Bioprocess Biosyst Eng 39, 603–611 (2016). https://doi.org/10.1007/s00449-016-1542-8

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  • DOI: https://doi.org/10.1007/s00449-016-1542-8

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