Overproduction and characterization of two distinct aldehyde-oxidizing enzymes from Gluconobacter oxydans 621H.
The Gluconobacter oxydans 621H genome contains two genes (gox1122 and gox0499) that encode putative cytosolic NAD(P)-dependent aldehyde dehydrogenases. Each gene was expressed in Escherichia coli, and the recombinant enzymes were purified and characterized. The native protein Gox1122 exhibited an apparent molecular mass of 50.1 kDa, and the subunit mass was 50.5 kDa, indicating a monomeric structure of the native enzyme. The preferred substrates were acetaldehyde and NADP. The enzyme also oxidized other short-chained aliphatic and aromatic aldehydes at lower rates. Recombinant protein Gox0499 was composed of a single subunit and had an apparent molecular mass of 49.5 kDa. The substrate spectrum of Gox0499 was broad with a preference for long-chained aliphatic and aromatic aldehydes. Highest activities were obtained using dodecanal and NAD as substrates. RT real-time PCR showed that genes gox0499 and gox1122 were expressed at an elevated level (about 3-fold) when the cells were exposed to ethanol and dodecanal in comparison to control cells.
Schweiger
Paul
P
Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA.
Volland
Sonja
S
Deppenmeier
Uwe
U
D000079:Acetaldehyde / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D009249:NADP; D000444:Aldehyde Dehydrogenase; D000079:Acetaldehyde