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KMID : 1094720140190040613
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Biotechnology and Bioprocess Engineering
2014 Volume.19 No. 4 p.613 ~ p.620
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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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Choe Hyun-Jun
Lee Su-Mi
Hwang Hyo-Jin
Joo Jeong-Chan
Cho Dae-Haeng
Kim Yong-Hwan
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Abstract
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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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KEYWORD
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formate dehydrogenase, Methylobacterium extorquens AM1, NADH oxidase, iron-sulfur cluster, electron mediator
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