The present invention generally relates to processes for the enzymatic production of a phosphinothricin product or precursor thereof from a nitrile-containing substrate.

The present invention provides a nitrilase mutant protein with increased thermal stability and its application in the synthesis of an anti-epileptic drug intermediate, wherein the mutant is obtained by mutating one or two of the amino acids at position 151, 223 and 250 of the amino acid sequence shown in SEQ ID No. 2. the thermal stability of the nitrilase mutant AcN-T151V/C223A/C250G was increased by up to 1.73 folds. The yield of the final product was up to 95% using the recombinant Escherichia coli containing the nitrilase mutant to hydrolyze 1M 1-cyanocyclohexylacetonitrile to produce 1-cyanocyclohexyl acetic acid at 35? C. And the yield of the final product was up to 97% when hydrolyzing 1.2M 1-cyanocyclohexylacetonitrile at 35? C. The final yield was up to 80% when using the nitrilase mutants obtained by the present invention to synthesize gabapentin.

Combined therapy of cancer using an immune check point modulators (e.g., an immune checkpoint inhibitor) and a fermented product, which may be prepared using symbiotic microbiota.

A method for converting vanillin to bis(cyanate) ester monomers, comprising treating vanillin with a reductive coupling agent to form at least one olefin. The olefin or olefins are treated with hydrogen and a metal catalyst to hydrogenate said olefin. The hydrogenated olefin or olefins are treated with at least one cyanogen halide and a base in an organic solvent to afford at least one olefin monomer. The olefin monomer or monomers are purified by recrystallization or precipitation from an organic solvent.

The disclosure provides a nitrilase from Arabis alpina, which belongs to genus Arabis, family brassicaceae. The disclosure further provides the encoding gene, vector, recombinant bacterial strain, and the application in the manufacturing of (S)-3-cyano-5-methylhexanoic acid. The wet resting cells containing nitrilase Aa-Nit can kinetically resolve racemic IBSN at 1.2 M with a 42% conversion rate in 15 hr and >99% ee value. The disclosure provides a regio- and stereoselective method for the preparation of (S)-3-cyano-5-methylhexanoic acid. This method provides an atom economical, mild, environmental friendly industrial method to manufacture (S)-3-cyano-5-methylhexanoic acid.

A process is provided for preparing a compound having the formula (I):

##STR00001##

said process being conducted by:

a) the addition of an oxygen source into a solution of a compound of formula (II)

##STR00002##

in a water-miscible solvent,

b) the addition of a laccase in the solution obtained in a); and

c) the possible recovering of the compound of formula (I) thus obtained.

The disclosure relates to engineered enone reductase polypeptides having improved properties, polynucleotides encoding the engineered polypeptides, related vectors, host cells, and methods for making the engineered enone reductase polypeptides. The disclosure also provides methods of using the engineered enone reductase polypeptides for chemical transformations.

The disclosure provides a nitrilase from Arabis alpina, which belongs to genus Arabis, family brassicaceae. The disclosure further provides the encoding gene, vector, recombinant bacterial strain, and the application in the manufacturing of (S)-3-cyano-5-methylhexanoic acid. The wet resting cells containing nitrilase Aa-Nit can kinetically resolve racemic IBSN at 1.2 M with a 42% conversion rate in 15 hr and >99% ee value. The disclosure provides a regio- and stereoselective method for the preparation of (S)-3-cyano-5-methylhexanoic acid. This method provides an atom economical, mild, environmental friendly industrial method to manufacture (S)-3-cyano-5-methylhexanoic acid.

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 (V) to racemic 3-cyano-5-methyl-hexanoic acid or salt thereof of formula (VI) with a genetically modified nitrilase enzyme (Nit 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. ##STR00001##

This document describes biochemical pathways for producing one or more of pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine and 1,7-heptanediol by forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl groups, in a C7 aliphatic backbone substrate produced from succinate semialdehyde or pyruvate. These pathways, metabolic engineering and cultivation strategies described herein rely on the aldol condensation of succinate semialdehyde and pyruvate.