Novel alcohol/aldehyde dehydrogenases

Abstract

The present invention is directed to a recombinant enzymes having alcohol and aldehyde dehydrogenase activity which comprises one or more recombinant polypeptides selected from the group consisting of polypeptides which are identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and chimeric recombinant polypeptides that are a chimeric combination of at least two of the following amino acid sequences identified by SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 and functional derivatives of the polypeptides identified above which contain addition, insertion, deletion and/or substitution of one or more amino acid residues, wherein said enzymatic polypeptides have said alcohol and aldehyde dehydrogenase activity. DNA molecules encoding the recombinant polypeptides, vectors comprising such DNA molecules, host cells transformed by such vectors and processes for the production of such recombinant enzymes are provided. Furthermore, the recombinant enzymes having alcohol and aldehyde dehydrogenase activity are used for obtaining aldehydes, ketones or carboxylic acids, and specifically, 2-keto-L-gulonic acid an intermediate for the production of L-ascorbic acid (vitamin C).

1 . An enzyme comprising a recombinant polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and amino acid sequences with at least 80% identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 said recombinant polypeptide having alcohol and aldehyde dehydrogenase activity.  
     
     
         2 . An enzyme of  claim 1 , wherein the recombinant polypeptide is a chimeric polypeptide including a combination of at least two amino acid sequences each of said sequences being selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and amino acid sequences with at least 80% identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, said recombinant polypeptide having alcohol and aldehyde dehydrogenase activity.  
     
     
         3 . An enzyme of  claim 1 , wherein the enzyme includes at least two recombinant polypeptides in the form of a homodimer or a heterodimer.  
     
     
         4 - 8 . (canceled)  
     
     
         9 . An enzyme produced by vector selected from the group consisting of pSSA102R, pSSA′101R, pSSA″102, pSSB103R, pSSAP-B, pSSA/B101R, pSSA/B102R, pSSA/B103R, pSSB/A101R, pSSB/A102R, pSSB/A103R, pSSsA2, pSSsA21, PSSsA22 and PSSsB.  
     
     
         10 - 19 . (canceled)  
     
     
         20 . A process for producing an aldehyde product from a substrate which comprises incubating a reaction mixture containing an enzyme of  claim 1  and said substrate wherein said substrate is selected from the group consisting of n-propanol, isopropanol, D-sorbitol and D-mannitol, and recovering the aldehyde product.  
     
     
         21 . A process for producing a ketone product from a substrate which comprises incubating a reaction mixture containing an enzyme of  claim 1  and said substrate wherein said substrate is selected from the group consisting of n-propanol, isopropanol, D-sorbitol and D-mannitol, and recovering the ketone product.  
     
     
         22 . A process for producing a carboxylic acid product from a substrate which comprises incubating a reaction mixture containing an enzyme of  claim 1  and said substrate wherein said substrate is selected from the group consisting of L-sorbose, D-glucose, D-fructose and L-sorbosone, and recovering the carboxylic acid product.  
     
     
         23 - 24 . (canceled).  
     
     
         25 . A process for producing 2-keto-L-gulonic acid which comprises: 
 (a) incubating a reaction mixture containing a substrate selected from the group consisting of D-sorbitol and L-sorbose, and a recombinant enzyme including a recombinant polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and amino acid sequences with at least 80% identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, said recombinant polypeptide having alcohol and aldehyde dehydrogenase activity, and    (b) converting the substrate to 2-keto-L-gulonic acid.    
     
     
         26 - 27 . (canceled).  
     
     
         28 . A process for the production of L-ascorbic acid from 2-keto-L-gulonic acid comprising obtaining 2-keto-L-gulonic acid by a process of  claim 25  and transforming the 2-keto-L-gulonic acid into L-ascorbic acid.  
     
     
         29 . An enzyme according to  claim 1  wherein the amino acid sequence is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.  
     
     
         30 . An enzyme encoded by a recombinant expression vector comprising a DNA sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and DNA sequences which encode a polypeptide with at least 80% identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, wherein the DNA sequence is functionally linked to one or more genetic control sequences and is capable of expression of an enzyme including at least one recombinant polypeptide having alcohol and aldehyde dehydrogenase activity.  
     
     
         31 . An enzyme encoded by a recombinant expression vector comprising a DNA sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and DNA sequences which encode a polypeptide with at least 80% identity to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, and having alcohol and aldehyde dehydrogenase activity.