Gene cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols.
The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).
Inoue
Kousuke
K
Biotechnology Research Center, Toyama Prefectural University, Japan.
Makino
Yoshihide
Y
Dairi
Tohru
T
Itoh
Nobuya
N
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000438:Alcohols / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002474:Cell-Free System; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics*; D006859:Hydrogen / Q000737:chemistry* / Q000378:metabolism*; D008835:Micrococcaceae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism; D014170:Transformation, Genetic / Q000235:genetics
D000438:Alcohols; D011994:Recombinant Proteins; D006859:Hydrogen; D000426:Alcohol Dehydrogenase