The present invention discloses a recombinant vector constructed from an encoding gene of a nitrilase mutant, a recombinant genetic engineered strain and application thereof the nucleotide sequence of the gene is shown in SEQ ID No. 5, and the amino acid sequence of the mutant is shown in SEQ ID No. 6. In the present invention, by the protein molecular modification, thermostability of the purified nitrilase LNIT5 is increased by up to 4.5 folds; and by utilizing recombinant E. coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at a high temperature (45° C.), product tolerance is increased, activity of NITS-L201F is increased by 20%, and the mutant NITLNIT5-AcN can completely hydrolyze 750 mM 1-cyanocyclohexylacetonitrile within 8 hours and achieve an doubled conversion rate. 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 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 its construction method and its application in the synthesis of chiral intermediate of pregabalin in the technical field of bioengineering. The present invention, respectively, takes turnip nitrilase BrNIT and arabidopsis nitrilase AtNIT as parent, using peptide fragment displacement method, displaces the sites 226-286 of BrNIT amino acid sequence and sites 225-285 of AtNIT amino acid sequence with sites 225-285 of Arabis alpina L. nitrilase AaNIT, obtain nitrilase mutants BrNIT.sub.225-285 and AtNIT.sub.225-285 of which the amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.3. Compared with wild type nitrilase, the activity of the nitrilase mutant provided by the present invention in catalyzing and hydrolyzing racemic IBSN and the stereoselectivity of the product show substantial improvement, it can satisfy the requirements of industrial application, and has good application prospect in efficient catalysis of racemic IBSN to synthesize 3-cyano-5-methylhexanoic Acid.

A strain of Rhodococcus rhodochrous in which a gene coding at least part of a nitrile hydratase enzyme or any gene coding a protein involved in the transcription, translation or formation of at least part of the nitrile hydratase enzyme has been deactivated or rendered ineffective or a strain of Rhodococcus rhodochrous cultured under condition wherein the nitrile hydratase enzyme is been inhibited.

Described herein are an immobilized enzyme, and a preparation method therefor and a use thereof. The immobilized enzyme includes activated PEI and an enzyme covalently bonded to the activated PEI, where the enzyme is selected from any one of a transaminase, a ketoreductase, a monooxygenase, an ammonia lyase, an ene-reductase, an imine reductase, an amino acid dehydrogenase and a nitrilase.

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 present invention relates to means and methods for producing an amide compound from a nitrile compound with less acrylic acid as by-product using a Nitrile hydratase (NHase) and Amidase producing microorganism as biocatalyst. Also provided is an aqueous amide compound obtained by the methods of the invention as well as a composition comprising acrylamide or polyacrylamide as well as a dried microorganism exhibiting a NHase/Amidase activity ratio of at least 400 when being brought into contact with a nitrile compound to convert said nitrile compound into an amide compound.

The present disclosure relates to processes for preparing (cyclopentyl[d]pyrimidin-4-yl)piperazine compounds, and more particularly relates to processes for preparing (R)-4-(5-methyl-7-oxo-6,7-dihydro-5H-cyclopenta[d] pyrimidin-4-yl)piperazine and N-protected derivatives thereof, which may be used as an intermediate in the synthesis of Ipatasertib (i.e., (S)-2-(4-chlorophenyl)-1-(4-((5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)piperazin-1-yl)-3-(isopropylamino)-propan-1-one). The present disclosure additionally relates to various compounds that are intermediates employed in these processes.

The present invention pertains to a process for preparing 3-hydroxy-3-methylbutyrate (HMB) or a salt thereof, the method comprising (a) reacting isobutylene oxide with cyanide in order to obtain 3-hydroxy-3-methylbutyronitrile, and (b) hydrolyzing the 3-hydroxy-3-methylbutyronitrile obtained in step (a) in order to obtain HMB, wherein hydrolysis step (b) is performed using either at least one nitrilase enzyme or, alternatively, using a combination of enzymes, said combination comprising at least one nitrile hydratase and at least one amidase.

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(ll) 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.