BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, v.19, no.4, pp.613 - 620
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 beta-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 alpha-subunit. The beta-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 mu M 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 mu M, 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 mu M 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)