Purification and characterization of NAD-dependent n-butanol dehydrogenase from solvent-tolerant n-butanol-degrading Enterobacter sp. VKGH12.
The solvent-tolerant bacterium Enterobacter sp. VKGH12 is capable of utilizing n-butanol and contains an NAD+-dependent n-butanol dehydrogenase (BDH). The BDH from n-butanol-grown Enterobacter sp. was purified from a cell-free extract (soluble fraction) to near homogeneity using a 3-step procedure. The BDH was purified 15.37-fold with a recovery of only 10.51, and the molecular mass estimated to be 38 kDa. The apparent Michaelis-Menten constant (Km) for the BDH was found to be 4 mM with respect to n-butanol. The BDH also had a broad range of substrate specificity, including primary alcohols, secondary alcohols, and aromatic alcohols, and exhibited an optimal activity at pH 9.0 and 40oC. Among the metal ions studied, Mg2+ and Mn2+ had no effect, whereas Cu2+, Zn2+, and Fe2+ at 1 mM completely inhibited the BDH activity. The BDH activity was not inhibited by PMSF, suggesting that serine is not involved in the catalytic site. The known metal ion chelator EDTA had no effect on the BDH activity. Thus, in addition to its physiological significance, some features of the enzyme, such as its activity at an alkaline pH and broad range of substrate specificity, including primary and secondary alcohols, are attractive for application to the enzymatic conversion of alcohols.
Veeranagouda
Y
Y
Department of Biochemistry, Gulbarga University, Gulbarga 585 106, India. yvgoudar@naver.com
Benndorf
Dirk
D
Heipieper
Hermann J
HJ
Karegoudar
T B
TB
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000440:Butanols / Q000378:metabolism*; D004754:Enterobacter / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D009243:NAD / Q000378:metabolism*; D010449:Peptide Mapping; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D000440:Butanols; D009243:NAD; D000429:Alcohol Oxidoreductases; C060833:butanol dehydrogenase