190 results about "Nitrilase" patented technology

The invention provides a nitrilase gene coding nitrilase, a recombinant vector containing the gene, a recombinant gene engineering bacteria obtained by converting the recombinant vector and application thereof in preparing recombinant nitrilase. The nitrilase gene can be connected with an expression vector for construction to obtain endoenzyme expression recombinant plasmid containing the gene or secretion expression recombinant plasmid, and then the endoenzyme expression recombinant plasmid containing the gene or the secretion expression recombinant plasmid is respectively and correspondingly converted to a colibacillus bacterial strain to obtain recombinant colibacillus; the recombinant colibacillus contains recombinant nitrilase and can recombine colibacillus into an enzyme resource for biological catalysis and conversion. The recombinant nitrilase serves as the enzyme for conversion, and racemisation mandelonitrile, acrylonitrile, iminodiacetonitrile or 2,2-dimethylcyclopropane carbonitrile and the like serve as a substrate for converting to react and prepare corresponding R-mandelic acid, crylic acid, iminodiacetic acid or chiral 2,2-dimethylcyclopropane formic acid and the like.

The present invention provides an improved process for the preparation of a compound of formula (I), which comprises the steps of: formula (I), (a) reacting isovaleraldehyde of formula (II) and alkyl cyanoacetate of formula (III) optionally in presence of salts of weak acid and weak base or weak base in a suitable solvent to get 2-cyano-5-methyl-hex-2-enoic acid alkyl ester of formula (IV); (b) reacting 2-cyano-5-methyl-hex-2-enoic acid alkyl ester of formula (IV) with a suitable cyanide source in water or in an organic solvent or mixture thereof to get 2-isobutylsuccinonitrile of formula (V); (c) obtaining optionally 2-isobutylsuccinonitrile of formula (V) by reacting isovaleraldehyde of formula (II) and alkyl cyanoacetate of formula (III) in presence of suitable cyanide source in water or in an organic solvent or mixture thereof in single step; (d) converting 2-isobutylsuccinonitrile of formula pa (V) to racemic 3-cyano-5-methyl-hexanoic acid or salt thereof of formula (VI) with a genetically modified nitrilase enzyme (Nit pt 9N_56_2) in water or optionally with an organic co-solvent at appropriate pH and temperature; (e) converting racemic 3-cyano-5-methyl-hexanoic acid or salt thereof of formula (VI) to racemic alkyl 3-cyano-5-methyl-hexanoate of formula (VII) by treatment with alcohol (R3OH) and acidic catalyst or alkyl halide (R3X) in presence of a base in a suitable solvent or a mixture of solvents thereof; (f) obtaining (S)-alkyl 3-cyano-5-methyl-hexanoate of formula (VIII) and (R)-3-cyano-5-methyl-hexanoic acid or salt thereof of formula (X) by enzymatic enantioselective hydrolysis in water or organic solvent or a mixture thereof from racemic alkyl 3-cyano-5-methyl-hexanoate of formula (VII); (g) obtaining optionally the compound of formula (VII) by racemizing unwanted (R)-3-cyano-5-methyl-hexanoic acid or salt thereof of formula (X) or substantially enriched (R)-3-cyano-5-methyl-hexanoic acid salt thereof of formula (X) in presence of a base in organic solvent or a mixture thereof; (h) converting (S)-alkyl 3-cyano-5-methyl-hexanoate of formula (VIII) to pregabalin of formula (I) by hydrolyzing ester group with suitable alkali or alkaline earth metal base followed by hydrogenation optionally in one pot in a solvent selected from water or other organic solvents or a mixture thereof in presence of a suitable hydrogenation catalyst.

The invention discloses a nitrilase N1 derived from pantoea sp.AS-PWVM4 and a gene thereof, a nitrilase N2 derived from arabidopsis thaliana and a gene thereof, a nitrilase N3 derived from acidovoraxfacilis 72W and a gene thereof, a nitrilase N4 derived from leptolyngbya sp. and a gene thereof, a nitrilase N5 derived from brassica oleracea var.oleracea and a gene thereof and a nitrilase N6 derived from camelina sativa and a gene thereof, and a method for preparing paracyanobenzoic acid as p-aminomethylbenzoic acid intermediate by using the nitrilase as a biological catalyst; resting cells ofthe corresponding nitrilases can be used for catalyzing 100g / L of substrate; the conversion rate is greater than 99%; the method has the obvious characteristics of mild reaction conditions, no pollution and simple process route, and has broad industrial application prospects.

The invention relates to nitrilases and to nucleic acids encoding the nitrilases. In addition methods of designing new nitrilases and method of use thereof are also provided. The nitrilases have increased activity and stability at increased pH and temperature.

The invention provides an immobilized enzyme as well as a preparation method and an application thereof. The immobilized enzyme comprises an enzyme and an amino resin carrier for immobilizing the enzyme, the enzyme is selected from any one of transaminase, ketoreductase, monooxygenase, ammonia lyase, alkene reductase, imine reductase, amino acid dehydrogenase and nitrilase, the amino resin carrieris the cross-linking agent modified amino resin carrier, and a cross-linking agent is a cross-linking agent treated by a high polymer. The amino resin carrier is modified by adopting the cross-linking agent treated by the high polymer, so that enzymes immobilized on the amino resin carrier can form netted cross-linking, the immobilization effect of the enzymes is more stable, and the cyclic utilization efficiency of the enzymes is improved.

The invention discloses a nitrilase mutant, gene, carrier, engineering bacteria and application in preparation of R-mandelic acid. The nitrilase mutant is obtained by carrying out site-specific mutagenesis on a 190th serine of an amino acid sequence shown in SEQ ID NO.2. The nitrilase mutant (with the amino acid sequence shown in SEQ ID NO.12) disclosed by the invention has the advantages that specific enzyme activity is improved by 3.0 times and enantiomer selectivity is increased to more than 99% from 94% compared with parent nitrilase (with the amino acid sequence shown in SEQ ID NO.2). Results show that the nitrilase mutant (with the amino acid sequence shown in SEQ ID NO.12) can completely hydrolyze 100mM mandelonitrile within 30min with a small amount of wet thalli (10g / L), and enantiomer selectivity is more than 99%, so that the nitrilase mutant disclosed by the invention can be applied to industrialized production of a medical intermediate R-mandelic acid.