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Expression of the NAD-dependent FDH1 β-subunit from Methylobacterium extorquens AM1 in Escherichia coli and its characterization

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Abstract

The efficient regeneration of nicotinamide cofactors is an important process for industrial applications because of their high cost and stoichiometric requirements. In this study, the FDH1 β-subunit of NAD-dependent formate dehydrogenase from Methylobacterium extorquens AM1 was heterologously expressed in Escherichia coli. It showed water-forming NADH oxidase (NOX-2) activity in the absence of its α-subunit. The β-subunit oxidized NADH and generated NAD+. The enzyme showed a low NADH oxidation activity (0.28 U/mg enzyme). To accelerate electron transfer from the enzyme to oxygen, four electron mediators were tested; flavin mononucleotide, flavin adenine dinucleotide, benzyl viologen (BV), and methyl viologen. All tested electron mediators increased enzyme activity; addition of 250 μM BV resulted in the largest increase in enzyme activity (9.98 U/mg enzyme; a 35.6-fold increase compared with that in the absence of an electron mediator). Without the aid of an electron mediator, the enzyme had a substrate-binding affinity for NADH (K m) of 5.87 μM, a turnover rate (k cat) of 0.24/sec, and a catalytic efficiency (k cat/K m) of 41.31/mM/sec. The addition of 50 μM BV resulted in a 22.75-fold higher turnover rate (k cat, 5.46/sec) and a 2.64-fold higher catalytic efficiency (k cat/K m, 107.75/mM/sec).

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Authors and Affiliations

  1. Department of Chemical Engineering, Kwangwoon University, Seoul, 139-701, Korea

    Hyunjun Choe, Sumi Lee, Hyojin Hwang, Jeong Chan Joo, Dae Haeng Cho & Yong Hwan Kim

  2. Department of Chemical Engineering and Applied Chemistry, Toronto, ON, M5S 3E5, Canada

    Jeong Chan Joo

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  1. Hyunjun Choe
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Choe, H., Lee, S., Hwang, H. et al. Expression of the NAD-dependent FDH1 β-subunit from Methylobacterium extorquens AM1 in Escherichia coli and its characterization. Biotechnol Bioproc E 19, 613–620 (2014). https://doi.org/10.1007/s12257-014-0126-1

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