The present invention provides an engineered nitrilase polypeptide capable of converting (1-cyanomethyl) cyclohexane-1-carbonitrile into (1-cyanocyclohexyl)-acetic acid. Utilizing advanced enzyme engineering techniques, the nitrilase exhibits enhanced stability and activity over natural variants. These engineered enzymes can hydrolyze a wide range of nitrile-containing compounds, including cyclic and aliphatic substrates. They handle higher substrate concentrations (200 g/L to 300 g/L), are thermostable above 50 C., and remain stable within a pH range of 5.5 to 8.0, making them suitable for various industrial applications. Their ability to convert substrates such as mandelonitrile, 2-(2-chlorophenyl)-2-hydroxyacetonitrile, 2-(6-methoxynaphthalen-2-yl)propanenitrile, and 2-[1-(aminomethyl)cyclohexyl]acetonitrile into corresponding carboxylic acids enables efficient and cost-effective production from diverse starting materials. This invention offers an engineered nitrilase enzyme as an alternative to alkaline or acid hydrolysis for converting nitrile substrates into carboxylic acids. It has applications in pharmaceuticals, agrochemicals, fine chemicals, waste treatment, and bioremediation, making these engineered polypeptides valuable for chemical production.

A method for synthesizing a brivaracetam chiral intermediate (R)-3-cyanohexanoic acid by catalyzing the hydrolysis of 3-cyanohexanitile using an enzyme with nitile hydrolysis activity, and the enzyme with nitrile hydrolysis activity is obtained by carrying out a single mutation or a double mutation on an amino acid at position 140 or an amino acid at position 175 in an amino acid sequence as set forth in SEQ ID NO.2. Compared with a wild type, the nitrilase mutant has the activity increased by 10 times, an ee value increased to 300 or more from 39, a substrate conversion rate of 45%, and a product ee which can reach 98.5%, and the yield of (R)-3-aminomethyl-hexanoic acid by catalytic hydrogenation synthesis using (R)-3-cyanohexanoic acid reaches 85% or more. This features a short synthesis route, mild reaction conditions, and high atom economy, and can be applied to the industrial synthesis of the brivaracetam intermediate.

The present invention provides a nitrilase mutant and application thereof in the synthesis of 1-cyanocyclohexyl acetic acid, the nitrilase mutant is obtained by mutating one or two of the amino acids at position 180 and 205 of the amino acid sequence shown in SEQ ID No. 2. In the present invention, by semi-rational design and protein molecular modification, the specific enzyme activity of the nitrilase double mutant AcN-G180D/A205C was increased by up to 1.6 folds, and the conversion rate>99%. And the reaction time was shortened to a quarter of the original using the recombinant Escherichia coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at high temperature (50 C.). Therefore, the mutants obtained by the present invention have a good application prospect in efficiently catalyzing 1-cyanocyclohexylacetonitrile to synthesize gabapentin intermediate, 1-cyanocyclohexyl acetic acid.

The present invention discloses a nitrilase mutant and application thereof in catalytic synthesis of 2-chloronicotinic acid, and belongs to the technical field of enzyme engineering. The nitrilase mutant has an amino acid sequence shown in SEQ ID NO. 4, that is, 167th tryptophan W of a parent nitrilase is mutated into glycine G. According to the nitrilase mutant provided by the present invention, the hydration activity of the parent nitrilase to 2-chloronicotinonitrile is eliminated, a byproduct of 2-chloronicotinamide is not generated in the catalytic process, the nitrile hydrolysis activity is greatly improved, and 2-chloronicotinonitrile can be specifically subjected to catalytic hydrolysis to synthesize 2-chloronicotinic acid. Therefore, the nitrilase mutant has an important potential in enzymatic industrial synthesis of 2-chloronicotinic acid.