Thermostable NADP(+)-dependent medium-chain alcohol dehydrogenase from Acinetobacter sp. strain M-1: purification and characterization and gene expression in Escherichia coli.
NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C(2) to C(14) and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes.
Tani
A
A
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.
Sakai
Y
Y
Ishige
T
T
Kato
N
N
D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics*; D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000447:Aldehydes; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D013696:Temperature
D000447:Aldehydes; D017931:DNA Primers; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+)