The gene which encodes(R)-specific carbonyl reductase(rCR)from Candida parapsilosis CCTCC M203011 was cloned,sequenced and compared with genes from the GenBank.The results indicated that rCR gene was 1011 bp,encoding ...The gene which encodes(R)-specific carbonyl reductase(rCR)from Candida parapsilosis CCTCC M203011 was cloned,sequenced and compared with genes from the GenBank.The results indicated that rCR gene was 1011 bp,encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa,and its nucleotide sequence showed 99%similarity to those of other members of the alcohol dehydrogenase superfamily.The rCR gene could express in recombinant strain Escherichia coli JM109,and the expression plasmid could produce(R)-1-pheny-1,2-ethanediol(100%e.e.,80.14%yield)from b-hydroxyacetophenone without any additive to regenerate NAD+from NADH.展开更多
A novel NADPH-dependent carbonyl reductase was separated from Candida parapsilosis CCTCC 203011.The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),which was purified t...A novel NADPH-dependent carbonyl reductase was separated from Candida parapsilosis CCTCC 203011.The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),which was purified through ammonium sulfate,Diethylamino Ethanol(DEAE)sepharose Fast flow(FF),phenyl-sepharose FF and blue sepharose FF chromato graphy from cell-free extract.The molecular mass of the enzyme was about 30 kDa.The optimum pH and temperature for reduction were 4.5℃ and 35℃,respectively.The Cu2+had strong restrictive effect on enzyme activity.In addition,the carbonyl reductase was an enzyme with high substrate specificity and stereo-selectivity,and showed high asymmetric reduction activity towards a-hydroxyacetophenone and ethyl 4-chloro acetoacetate.For the asymmetric reduction of a-hydroxyacetophenone and ethyl 4-chloro acetoacetate,(S)-1-phenyl-1,2-ethanediol and(R)-ethyl 4-chloro-3-hydroxybutanoate were produced by the purified enzyme,with the 100% and 94.3%e.e.value,respec-tively.Therefore,the enzyme could be one of the effective biocatalysts for asymmetric synthesis of chiral alcohols.The amino acid sequences of one peptide from the purified enzyme were analyzed by LC-MASS-MASS,and the car-bonyl reductase showed some identity to the hypothetical protein CaO19.10414 reported.展开更多
基金This study was supported by the National Basic Research Program of China(No.2003CB716008)the Program for Changjiang Scholars and Innovative Research Team in University(PCSIRT,IRT0532).
文摘The gene which encodes(R)-specific carbonyl reductase(rCR)from Candida parapsilosis CCTCC M203011 was cloned,sequenced and compared with genes from the GenBank.The results indicated that rCR gene was 1011 bp,encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa,and its nucleotide sequence showed 99%similarity to those of other members of the alcohol dehydrogenase superfamily.The rCR gene could express in recombinant strain Escherichia coli JM109,and the expression plasmid could produce(R)-1-pheny-1,2-ethanediol(100%e.e.,80.14%yield)from b-hydroxyacetophenone without any additive to regenerate NAD+from NADH.
基金This work was supported by the National Natural Science Foundation of China(Grant No.20376031)the National Key Basic Research and Development Program of China(973 Program)(Grant No.2003CB716008)+1 种基金the Program for New Century Excellent Talents in University,Ministry of Education,China(Grant No.NCET-04-0498)the Program for Changjiang Scholars and Innovative Research Team in University(Grant No.PCSIRT,IRT0532).
文摘A novel NADPH-dependent carbonyl reductase was separated from Candida parapsilosis CCTCC 203011.The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),which was purified through ammonium sulfate,Diethylamino Ethanol(DEAE)sepharose Fast flow(FF),phenyl-sepharose FF and blue sepharose FF chromato graphy from cell-free extract.The molecular mass of the enzyme was about 30 kDa.The optimum pH and temperature for reduction were 4.5℃ and 35℃,respectively.The Cu2+had strong restrictive effect on enzyme activity.In addition,the carbonyl reductase was an enzyme with high substrate specificity and stereo-selectivity,and showed high asymmetric reduction activity towards a-hydroxyacetophenone and ethyl 4-chloro acetoacetate.For the asymmetric reduction of a-hydroxyacetophenone and ethyl 4-chloro acetoacetate,(S)-1-phenyl-1,2-ethanediol and(R)-ethyl 4-chloro-3-hydroxybutanoate were produced by the purified enzyme,with the 100% and 94.3%e.e.value,respec-tively.Therefore,the enzyme could be one of the effective biocatalysts for asymmetric synthesis of chiral alcohols.The amino acid sequences of one peptide from the purified enzyme were analyzed by LC-MASS-MASS,and the car-bonyl reductase showed some identity to the hypothetical protein CaO19.10414 reported.
