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 concerns new strains of cyanogenic bacteria, and more particularly alkali-tolerant Pseudomonas bacteria, that are useful to gold mining activities. These bacteria are useful to leach gold from any source including ores, concentrates or waste material. They have the ability to effectively produce cyanide at alkaline pH.

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.

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 relates to a process for the preparation of substituted 4-(N-hydroxy-carbamimidoyl)benzoic acids, which can be obtained by nitrilase catalyzed hydration of substituted terephthalonitriles of formula (II) in an aqueous medium to afford (ammonium) 4-cyanobenzoic acids (IIa). The hydration is followed by treatment of the aqueous reaction medium with hydroxylamine or a salt thereof to afford amidoximes (I). ##STR00001##

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 present invention generally relates to methods of biological reduction of metal-cyanide complexes after metal-cyanidation and methods of biologically hydrolysing cyanide. More particularly, the present invention allows the engineering of an integrated synthetic lixiviant biological system to be housed within a synthetic host (such as the cyanogenic Chromobacterium violaceum) for efficient precious metal recovery and toxic metal remediation of electronic waste; with up to four main components/modules in the design and engineering of the synthetic host: 1) synthetic cyanogenesis; 2) synthetic metal recovery; 3) synthetic cyanolysis; and 4) synthetic circuits for lixiviant biology. Bacteria capable of reducing ionic metal to ionic metal (such as gold or silver) as nanoparticles, comprising mercury(11) reductase (MerA) comprising a substitution mutation at position V317, Y441, C464, A323D, A414E, G415I, E416C, L417I, I418D, or A422N, are also disclosed. Processes of synthetic cyanide lixiviant production using genetically engineered bacterium transformed with a heterologous hydrogen cyanide synthase gene and a heterologous 3-phosphoglycerate dehydrogenase mutant gene are also disclosed. Processes of synthetic cyanolysis using a genetically engineered bacterium transformed with a heterologous nitrilase gene are also disclosed.