Production Of 3-Hydroxycarboxylic Acid Using Nitrilase Mutants

Abstract

This invention relates to nitrilase mutants having improved nitrilase activity for converting 3-hydroxynitriles to 3-hydroxycarboxylic acids. More specifically, the Acidavorax facilis 72W (ATCC 55746) nitrilase gene was mutated using error-prone PCR and site-directed mutagenesis to create nitrilase enzymes having improved nitrilase activity for converting 3-hydroxynitriles (e.g., 3-hydroxybutyronitrile or 3-hydroxyvaleronitrile) to the corresponding 3-hydroxycarboxylic acids. A process using these improved mutants to produce the 3-hydroxycarboxylic acids is also provided.

1 . An isolated nucleic acid fragment encoding an enzymatically-active nitrilase polypeptide, the polypeptide having a polypeptide sequence selected from the group consisting of SEQ ID NO:6, 8, 12, 14, 16, and 18; and having nitrilase activity at least 1.8-fold higher relative to the activity of the  A. facilis  72W (ATCC 55746) nitrilase when converting 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid under identical reaction conditions. 
     
     
         2 . An isolated nucleic acid fragment, the nucleic acid fragment selected from the group consisting of SEQ ID NOs:5, 7, 11, 13, 15, and 17, wherein the isolated nucleic acid fragment encoding a polypeptide having an improvement in nitrilase activity at least 1.8-fold higher relative to the activity of  A. facilis  72W (ATCC 55746) nitrilase when converting 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid under identical aqueous reaction conditions. 
     
     
         3 . A polypeptide encoded by the nucleic acid fragment of  claim 1  or  claim 2 . 
     
     
         4 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 1  or  claim 2  operably linked to a suitable regulatory sequence. 
     
     
         5 . An expression cassette comprising the chimeric gene of  claim 4 . 
     
     
         6 . A transformed microorganism comprising the chimeric gene of  claim 4 . 
     
     
         7 . A transformed microorganism comprising the expression cassette of  claim 5 . 
     
     
         8 . The transformed microorganism of  claim 6  wherein the microorganism is selected from the group consisting of  Comamonas  sp.,  Corynebacterium  sp.,  Brevibacterium  sp.,  Rhodococcus  sp.,  Azotobacter  sp.,  Citrobacter  sp.,  Enterobacter  sp.,  Clostridium  sp.,  Klebsiella  sp.,  Salmonella  sp.,  Lactobacillus  sp.,  Aspergillus  sp.,  Saccharomyces  sp.,  Zygosaccharomyces  sp.,  Pichia  sp.,  Kluyveromyces  sp.,  Candida  sp.,  Hansenula  sp.,  Dunaliella  sp.,  Debaryomyces  sp.,  Mucor  sp.,  Torulopsis  sp.,  Methylobacteria  sp.,  Bacillus  sp.,  Escherichia  sp.,  Pseudomonas  sp.,  Rhizobium  sp., and  Streptomyces  sp. 
     
     
         9 . The transformed organism of  claim 8  wherein the transformed microorganism is an  Escherichia coli  strain selected from the group consisting of MG1655 (ATCC 47076), FM5 (ATCC 53911), W3110 (ATCC 27325), MC4100 (ATCC 35695), and W1485 (ATCC 12435). 
     
     
         10 . A process for hydrolyzing a 3-hydroxynitrile to a 3-hydroxycarboxylic acid comprising the steps of:
 (a) contacting a 3-hydroxynitrile in an aqueous reaction mixture with an improved nitrilase catalyst characterized by at least a 1.8-fold improvement in nitrilase activity relative to the activity of the  A. facilis  72W (ATCC 55746) nitrilase under identical reaction conditions, the improved nitrilase catalyst encoded by the isolated nucleic acid fragment of  claim 1  or  claim 2 ; and   (b) recovering the 3-hydroxycarboxylic acid produced in step (a).   
     
     
         11 . The process of  claim 10  wherein the nitrilase catalyst is characterized by at least a 5-fold increase in nitrilase activity relative to the activity of the  A. facilis  72W (ATCC 55746) nitrilase. 
     
     
         12 . A process for hydrolyzing 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid comprising the steps of:
 (a) contacting 3-hydroxyvaleronitrile in an aqueous reaction mixture with a nitrilase catalyst encoded by the isolated nucleic acid fragment of  claim 1  or  claim 2  characterized by at least a 1.8-fold improvement in nitrilase activity relative to the activity of the  A. facilis  72W (ATCC 55746) nitrilase under identical reaction conditions; and   (b) recovering the 3-hydroxyvaleric acid produced in step (a).   
     
     
         13 . A process for hydrolyzing 3-hydroxybutyronitrile to 3-hydroxybutyric acid comprising the steps of:
 (a) contacting 3-hydroxybutyronitrile in an aqueous reaction mixture with an enzymatically active nitrilase catalyst having the polypeptide sequence of SEQ ID NO:6 and characterized by at least a 1.9 fold improvement in nitrilase activity relative to the activity of the  A. facilis  72W (ATCC 55746) nitrilase under identical reaction conditions; and   (b) recovering the 3-hydroxybutyric acid produced in step (a).