Production Of 3-Hydroxycarboxylic Acid Using Nitrilase Mutants
AssigneeE. I. Du Pont de Nemours and Company
InventorsMark S. Payne, Robert DiCosimo, Daniel P. O'Keefe
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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.
Claims13
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.
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.
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).
Dependencies are inferred from the claim text.