Cloning, Expression, Purification And Characterization Of Two NADPH-dependent Oxidoreductases From Gluconobacter Oxydans

Two genes from Gluconobacter oxydans 621H, gox0644 and gox1615, which show 60-70% similarities to members of aldo-keto reductase (AKR) superfamily, were cloned and expressed in Escherichia coli BL21(DE3). The recombinant proteins with apparent molecular mass of 32 kDa and 50 kDa in the soluble fractions were purified by one step affinity chromatography with a Ni-NTA agarose column.The substrate specificities of the recombinant enzymes were determined with the purified proteins. Using NADPH as a cofactor, GOX0644 exhibited better activity to aromatic aldehydes over aliphatic aldehydes. It showed no activity towards D-xylose, D-glucose, etc. The enzyme also did not show any activities whenβ-keto esters and a-keto carboxylic acids were used as substrates. Based on the catalytic capabilities, GOX0644 was classified as a new member of the aldehyde reductase family. GOX1615 showed the highest activity towards DL-glyceraldehyde, utilizing NADPH as a cofactor. As a biochemical comparison between NADP+glycerol dehydrogenases (GlyDHs; EC1.1.1.72) from Hypocrea Jecorina and GOX1615 revealed some similarities with respect to substrate specificity, GOX1615 was defined as a NADP+ glycerol dehydrogenase. Kinetic parameters of the proteins and the dependence of their activity on temperature and pH were also determined.