Purification, characterization, and gene cloning of glycerol dehydrogenase from Hansenula ofunaensis, and its expression for production of optically active diol

• Published in: Journal of bioscience and bioengineering 2006-12, Vol.102 (6), p.545-551
• Main Authors: Yamada-Onodera, Keiko, Nakajima, Akira, Tani, Yoshiki
• Format: Article
• Language: English
• Subjects: ALCOHOL DEHYDROGENASE; ALCOHOL DESHIDROGENASA; ALCOOL DESHYDROGENASE; Amino Acid Sequence; Ascomycota - enzymology; Ascomycota - genetics; Biological and medical sciences; Biotechnology; chiral alkanediol; chiral diol production; CLONACION MOLECULAR; CLONAGE MOLECULAIRE; Cloning, Molecular - methods; Enzyme Activation; Enzyme Stability; ESCHERICHIA COLI; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; GLICEROL; GLYCEROL; glycerol dehydrogenase; Glycols - chemical synthesis; HANSENULA; Hansenula ofunaensis; Hansenula polymorpha; Isomerism; long-chain secondary alcohol dehydrogenase; MOLECULAR CLONING; Molecular Sequence Data; optically active 1,2-octanediol production; ORGANISME TRANSGENIQUE; Oxidation-Reduction; Pichia angusta; Protein Engineering - methods; PURIFICACION; PURIFICATION; Sugar Alcohol Dehydrogenases - chemistry; Sugar Alcohol Dehydrogenases - isolation & purification; Sugar Alcohol Dehydrogenases - metabolism; TRANSGENICOS; TRANSGENICS
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1263/jbb.102.545

Optically active alcohol is an important building block as a versatile chiral synthon for the asymmetric synthesis of pharmaceuticals and agrochemicals. We purified and characterized glycerol dehydrogenase from Hansenula ofunaensis and prepared optically active 1,2-octanediol using a recombinant Escherichia coli harboring the glycerol dehydrogenase gene. The deduced amino acid sequence was investigated for identities with those of other alcohol dehydrogenases in the NCBI databank. The identification of the unknown product of a resting-cell reaction was performed by GC-MS. In the deduced amino acid sequence composed of 376 residues, the NAD(H) binding pattern and cysteine residues that correspond to the cysteine ligands at the zinc atom were conserved as they are in alcohol dehydrogenases from other origins. Glycerol dehydrogenase from Hansenula polymorpha DL-1 ( Pichia angusta, DDBJ/EMBL/GenBank accession no. BAD32688) had the highest identity to our enzyme, showing 73% identity. Our glycerol dehydrogenase catalyzed the NAD +-dependent oxidation of long-chain secondary alcohols such as 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, and 1,2-octanediol. Activities toward 2,4-pentanediol and 2,5-hexanediol were hardly detected. From these results, it was confirmed that our enzyme requires two hydroxyl groups on adjacent carbon atoms for oxidation. 2,3-Pentanedione, 2,3-hexanedione, and 3,4-hexanedione were significantly reduced. The transformants oxidized only ( R)-1,2-octanediol in 50 mM racemate ( R: S=52:48), and produced ( S)-1,2-octanediol (24 mM,