Codon-optimized NADH oxidase gene expression and gene fusion with glycerol dehydrogenase for bienzyme system with cofactor regeneration

Abstract

NADH oxidases (NOXs) play an important role in maintaining balance of NAD NAD + /NADH by catalyzing cofactors regeneration. The expression of nox gene from Lactobacillus brevis in Escherichia coliBL21 (BL21 (DE3)) was studied. Two strategies, the high AT-content in the region adjacent to the initiation codon and codon usage of the whole gene sequence consistent with the host, obtained the NOX activity of 59.9 U/mg and 73.3 U/mg (crude enzyme), with enhanced expression level of 2.0 and 2.5-folds, respectively. Purified NOX activity was 213.8 U/mg. Gene fusion of glycerol dehydrogenase (GDH) and NOX formed bifuctional multi-enzymes for bioconversion of glycerol coupled with coenzyme regeneration. Kinetic parameters of the GDH-NOX for each substrate, glycerol and NADH, were calculated as V max(Glycerol) 20 μM/min, K m(Glycerol) 19.4 mM, V max (NADH) 12.5 μM/min and K m(NADH) 51.3 μM, respectively, which indicated the potential application of GDH-NOX for quick glycerol analysis and dioxyacetone biosynthesis.