| 摘要 |
The alcohol dehydrogenase has an amino acid sequence of a hydroxybutyrate dehydrogenase, and a sequence motif, where the hydroxybutyrate dehydrogenase is used for producing a new enzyme. The alcohol dehydrogenase has a sequence motif, which is (Gln or Mut)-X 1 6 - 2 2-N-X 2 4 - 3 0-S-X-(His or Mut)-X 6 - 8-(Lys or Mut)-X 2-Y-X 3-Lys-X 3 3 - 3 9-(Gln or Mut), where Mut is methionine or a nonpolar amino acid selected from isoleucine, leucine, alanine or valine and X is a mixture of arbitrary amino acid. The naturally occurring amino acids Gln, His or Lys are replaced by Mut in the three, preferably all four positions of the amino acids. Independent claims are included for: (1) a gene sequence coding for an alcohol dehydrogenase and an expression vector, which is a nucleic acid sequence of SEQ ID NO: 10-12 for the alcohol dehydrogenase and of SEQ ID NO: 13-15 for the expression vector, where the nucleic acid sequence is present in prokaryotic or eukaryotic unicellular host organism, which is used for producing R-enantiomeric secondary alcohol and separating the secondary alcohol from racemic mixture; (2) a method for producing new enzymes, which involves cloning a gene sequence, mutating three of the naturally occurring amino acids, glutamine, histidine and a lysine in the sequence motif and inserting the mutated gene sequence in the expression vector, where the cloned gene sequence codes for hydroxybutyrate dehydrogenase, and the sequence motif is Gln-X 1 6 - 2 2-N-X 2 4 - 3 0-S-X-His-X 6 - 8-Lys-X 2-Y-X 3-Lys-X 3 3 - 3 9-Gln, that codes nucleotide triplet by site-directed mutagenes and the triplets that code for asparagine, serine, tyrosine and lysine remain unchanged, and transfection and expression of the enzyme takes place in the prokaryotic or eukaryotic unicellular host organism; (3) a method for producing R-enantiomeric secondary alcohols of formula (I), which involves introducing corresponding prochiral ketone of formula (II) with the alcohol dehydrogenase or the host organism; and (4) a method for separating the S-enantiomer of formula (III) from the racemic mixture of chiral secondary alcohol of formula (IV), which involves introducing the racemic mixture with the alcohol dehydrogenase, oxidizing the R-enantiomer of the formula (I) of the mixture to ketone of the formula (II), and separating the unreacted enantiomer from the ketone. R 1,R 2saturated, unsaturated, straight chain or branched 1-10C hydrocarbon residue, where R 1and R 2are not equal. [Image] [Image] [Image] [Image]. |
