Gluconobacter oxydans 2-ketoreductase enzyme and applications thereof

Abstract

This invention relates to an novel Gluconobacter oxydans 2-ketoreductase useful for the synthesis of chiral alcohols. Also related are isolated nucleic acids encoding G. oxydans 2-ketoreductase enzymes, and enzyme fragments and variants thereof, as well as vectors and host cells comprising these nucleic acids. Further related are isolated G. oxydans 2-ketoreductase polypeptides, and fragments and variants thereof, and antibodies that specifically bind to G. oxydans 2-ketoreductase polypeptides, fragments, or variants. The invention also relates to methods of obtaining isolated G. oxydans 2-ketoreductase nucleic acids, polypeptides, and antibodies, and methods of using G. oxydans 2-ketoreductase in various reactions for industrial or pharmaceutical applications.

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         13 . An isolated polypeptide comprising amino acid sequence SEQ ID NO:2.  
     
     
         14 . An isolated polypeptide comprising the short chain dehydrogenase domain of amino acid sequence SEQ ID NO:2, wherein said isolated Polypeptide has 2-ketoreductase activity, and wherein the short chain dehydrogenase domain is amino acids 4-255 of SEQ ID NO:2.  
     
     
         15 . An isolated polypeptide comprising an amino acid sequence that shares at least 95% sequence identity with the isolated polypeptide of  claim 13 , wherein said isolated polypeptide has 2-ketoreductase activity.  
     
     
         16 . An isolated polypeptide that is a 2-ketoreductase encoded by the nucleotide sequence contained in the plasmid in the ATCC deposit designated PTA-3864.  
     
     
         17 . An isolated polypeptide of  claim 13 , wherein said isolated polypeptide is recombinant.  
     
     
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         20 . A method of producing a chiral alcohol comprising: incubating the isolated polypeptide of  claim 13  with a ketone substrate under conditions to allow reduction of the ketone substrate, thereby producing a chiral alcohol.  
     
     
         21 . The isolated polypeptide of  claim 14 , wherein said isolated polypeptide shares at least 95% sequence identity with the amino acid sequence SEQ ID NO:2.  
     
     
         22 . A method of producing a chiral alcohol comprising: incubating the isolated polypeptide of  claim 14  with a ketone substrate under conditions to allow reduction of the ketone substrate, thereby producing a chiral alcohol.  
     
     
         23 . A method of producing a chiral alcohol comprising: incubating the isolated polypeptide of  claim 15  with a ketone substrate under conditions to allow reduction of the ketone substrate, thereby producing a chiral alcohol.  
     
     
         24 . A method of producing a chiral alcohol comprising: incubating the isolated polypeptide of  claim 16  with a ketone substrate under conditions to allow reduction of the ketone substrate, thereby producing a chiral alcohol.  
     
     
         25 . A method of producing a chiral alcohol comprising: incubating the isolated polypeptide of  claim 21  with a ketone substrate under conditions to allow reduction of the ketone substrate, thereby producing a chiral alcohol.  
     
     
         26 . The method of  claim 22 , wherein said ketone is selected from the group consisting of 2-pentanone, 2-heptanone, 2-octanone, 2-decanone, and 2-hexanone.  
     
     
         27 . The method of  claim 23 , wherein said ketone is selected from the group consisting of 2-pentanone, 2-heptanone, 2-octanone, 2-decanone, and 2-hexanone.  
     
     
         28 . The method of  claim 26 , wherein said ketone is 2-pentanone.  
     
     
         29 . The method of  claim 27 , wherein said ketone is 2-pentanone.  
     
     
         30 . An isolated polypeptide of  claim 14 , wherein said isolated polypeptide is recombinant.  
     
     
         31 . An isolated polypeptide of  claim 15 , wherein said isolated polypeptide is recombinant.  
     
     
         32 . An isolated polypeptide of  claim 16 , wherein said isolated polypeptide is recombinant.  
     
     
         33 . An isolated polypeptide of  claim 13 , wherein said isolated polypeptide consists of the amino acid sequence of SEQ ID NO:2.