33 results about "Nitrilase" patented technology

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).

This invention provides a nitrile hydrolase mutant and its application in the synthesis of p-cyanobenzoic acid. The invention provides a nitrile hydrolase mutant that enhances the catalytic activity for the synthesis of p-cyanobenzoic acid. The mutant protein is a non-natural protein, and it exhibits significantly enhanced activity in catalyzing the conversion of terephthalonitrile to p-cyanobenzoic acid. Furthermore, the mutant protein contains mutations in two or more core amino acids related to the enzyme's catalytic activity in the wild-type nitrile hydrolase. The nitrile hydrolase mutant of this invention can significantly improve the yield of the synthesized product catalyzed by the nitrile hydrolase.

The invention discloses a nicotinic acid immobilized enzyme conversion method, which comprises the following steps: taking wet thalli containing nitrilase, preparing a flocculation clear liquid, passing the flocculation clear liquid through an ultrafiltration membrane, carrying out embedding immobilization to obtain embedded particles not greater than 2mM, and after the embedded particles are hardened, separating and purifying the obtained conversion liquid by adopting a membrane separation method to obtain the nicotinic acid immobilized enzyme. Sequentially carrying out microfiltration, activated carbon decoloration, ultrafiltration, nanofiltration decoloration, acidification crystallization and centrifugation to finally obtain a nicotinic acid finished product. The immobilized enzyme prepared by the method disclosed by the invention can stably exist in nitrile substances, can be repeatedly used for many times, and is high in stability and low in enzyme activity loss rate, and a separated and purified product is free of impurities and high in yield.

This invention belongs to the field of enzyme engineering technology, specifically relating to an enzyme engineering method and its application in biocatalysis. Based on the excellent performance optimization capabilities of the deep learning-guided protein sequence design model ProteinMPNN, this invention developed a computational design framework called DeepPCD. This framework integrates deep learning-guided sequence design, physical energy calculation, and molecular dynamics simulation, constructing a three-level intelligent screening process from "massive generation" to "high-precision prediction." Using this strategy, nitrile hydrolase (EC 3.5.5.1) was mutated, resulting in a nitrile hydrolase mutant with improved stability, specificity, and enzyme activity, capable of efficiently and specifically catalyzing the hydrolysis of nitrile compounds to carboxylic acids.

The invention discloses a nitrilase mutant and an application of the nitrilase mutant in preparation of (S)-3-methylheptanoic acid. The nitrilase mutant disclosed by the invention has high catalytic activity and stereoselectivity, and can overcome the defects of low substrate concentration, low stereoselectivity, high cost, environmental pollution and the like in the existing method, so that 3-methylheptonitrile can be quickly catalyzed to generate (S)-3-methylheptanoic acid under high substrate concentration. In addition, the method also has the effects of good enzyme catalysis effect, high product purity, high recovery rate, simple steps, greenness, environmental protection and the like.

The invention discloses a nitrilase mutant and an application technology thereof in synthesis of nicotinic acid, and belongs to the technical fields of gene engineering, enzyme engineering and biological catalysis. The nitrilase mutant disclosed by the invention is obtained by mutating nitrilase from a source of Pseudomonas putida CGMCC (China General Microbiological Culture Collection Center) 3830, and the heterologous expression of the nitrilase mutant in corynebacterium glutamicum is successfully realized. The optimal mutant can efficiently catalyze a substrate 3-cyanopyridine to synthesize 550.68 g / L of nicotinic acid, and the conversion rate is 91.37%.

The invention discloses a nitrilase mutant with high activity and stereoselectivity and application of the nitrilase mutant in synthesis of chiral carboxylic acid. The mutant is obtained by performing single mutation or multiple mutation on 79th, 166th, 167th and 246th amino acids of an amino acid sequence shown as SEQ ID NO.1. The invention further discloses a preparation method of the nitrilase mutant. According to the invention, the nitrilase OsNIT-mut is subjected to molecular modification through (semi) rational design, and a mutant with synergistically improved hydrolysis reaction specificity and stereoselectivity is obtained. The content of mandelic acid in a product of the mutant OsNIT-mut / V246N / T166G / R79Y / A167N for catalyzing mandelonitrile is 90.5%, the e.e. Value of R-mandelic acid is increased to 95.2%, and the relative hydrolytic activity is 399.1%. The nitrilase mutant can be applied to green industrial catalytic synthesis of chiral mandelic acid through regulation and control of hydrolysis reaction specificity and stereoselectivity, and has important significance.

The invention relates to the technical field of organic synthesis, in particular to a biological catalysis method for synthesizing 4-acetyl-2-methyl benzoic acid based on 4-acetyl-2-methyl cyanophenyl. Free or immobilized nitrilase is mostly adopted in a biological catalysis method, and the conversion rate of 4-acetyl-2-methylbenzonitrile is poor. In order to solve the technical problems, the invention provides the biological catalysis method for synthesizing the 4-acetyl-2-methyl benzoic acid based on the 4-acetyl-2-methyl cyanophenyl, and the method comprises the following steps: by taking the 4-acetyl-2-methyl cyanophenyl as a reaction substrate, resting cells of Rhodococcus sp. CCZU10-1 as a catalyst and a deep eutectic solvent (DES) as a cosolvent, synthesizing the 4-acetyl-2-methyl benzoic acid on the basis of the 4-acetyl-2-methyl cyanophenyl. A phosphoric acid buffer solution is used as a reaction solvent, 4-acetyl-2-methyl benzoic acid is obtained through catalytic stirring hydrolysis reaction, the reaction condition is mild, DES is green and low in toxicity and biodegradable, the substrate conversion rate is high (gt, 95%), the product selectivity is good, the process environment is friendly, and the green chemical principle is met.

The invention belongs to the field of enzyme engineering and agricultural waste conversion, and particularly relates to an acid-resistant and heat-resistant nitrilase mutant and application thereof in fermentation of rapeseed meal. Aiming at the conditions that the existing nitrilase is insufficient in acid resistance and heat resistance and relatively low in activity, the nitrilase RsNit anchored on the surface capsid protein cotG of bacillus subtilis DB403 is used as a starting target for modification; according to the nitrilase mutant disclosed by the invention, a catalytic hot spot existing in an active center is found by carrying out semi-rational design on amino acid at the active center, and combined mutation is carried out on the catalytic hot spot, so that it is found for the first time that RsNit has a catalytic hot spot on 4-pentenenitrile at the active center, and a nitrilase mutant of which the activity, the thermal stability and the acid tolerance are all remarkably improved is successfully screened out; the method is applied to fermentation of the rapeseed meal, so that full utilization of the rapeseed meal in livestock and poultry feed is promoted, and the method has a wide market application prospect.

The application discloses a nitrilase mutant and application thereof in synthesis of chiral cyanoamide, wherein the nitrilase mutant has mutations in any or any of the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID NO. 1: 54th, 118th, 120th, 139th, 148th, 168th, 170th, 194th, 197th, 198th, 202th and / or 225th, compared with the amino acid sequence of wild-type nitrilase amine. The mutant constructed by the application can efficiently catalyze 3-substituted-glutaronitrile compounds to generate (S)-3-substituted-4-cyanobutyramide compounds. The application solves the problem of insufficient enzyme catalytic activity in the prior art, and provides an efficient scheme for green synthesis of chiral cyanoamide.

The present application provides a nitrilase mutant and its application. Compared with a wild-type nitrilase with an amino acid sequence shown in SEQ ID NO: 2, the nitrilase mutant comprises one or more of the following amino acid mutation sites: S201F, G239M, K242T, V243Q and G246Q. The present application constructs a nitrilase mutant with high enzyme activity, high salt tolerance and high substrate tolerance by mutating and modifying a nitrilase derived from Alcaligenes.

The invention belongs to the technical field of enzyme engineering and biology, and discloses a nitrilase mutant as well as a coding gene, a recombinant vector, a recombinant gene engineering bacterium, an enzyme preparation and application thereof. The nitrilase mutant is obtained by carrying out single-point mutation or multi-point mutation on 8th, 17th, 35th or 150th amino acids of an amino acid sequence of the nitrilase as shown in SEQ ID NO.2. The invention further discloses a preparation method of the nitrilase mutant. Compared with wild type Gi-Nit, the nitrilase mutant disclosed by the invention has the advantages that the activity is improved by 32 times, the yield is improved by 15.8 times, and a foundation is laid for synthesizing 2-(4-nitrophenyl) butyric acid by an industrial enzyme method.

The application discloses application of a nitrilase in selective catalysis of 6-chloronicotinonitrile to synthesize 6-chloronicotinic acid and a mutant, wherein the nitrilase mutant is obtained by mutating alanine at the 55th position of the amino acid shown in SEQ ID NO. 2 into serine. In an optimal system, the hydrolysis activity of the nitrilase to 6-chloronicotinonitrile reaches 450 U / g (WCW), 300 g / L 6-chloronicotinonitrile can be catalyzed to synthesize 6-chloronicotinic acid, the yield reaches 92%, and the catalysis synthesizes 2-chloronicotinamide with a concentration of up to 4.8 g / L; when the nitrilase mutant is used to catalyze 6-chloronicotinonitrile, the activity reaches 580 U / g (WCW), 300 g / L 6-chloronicotinonitrile can be catalyzed to synthesize 6-chloronicotinic acid, the yield reaches 99%, and the hydration activity of 2-chloronicotinonitrile is eliminated, and 2-chloronicotinamide is not generated.

The invention relates to a novel process for generating N-acetylgalactosamine (GalNAc) clusters using a nitrilase and conjugating said GalNAc clusters to oligonucleotides, such as oligonucleotides for use in therapy. In particular, the nitrilase is used to catalyse the conversion of a trinitrile intermediate to a triacid intermediate.

The application discloses a nitrilase mutant PgNIT2-A190I, which is obtained by point mutation of alanine at the 190th position of nitrilase PgNIT2 of Paraburkholderia graminis C4D1M into isoleucine. Accordingly, the inventors also design a recombinant plasmid of a nitrilase mutant PgNIT2-A190I coding gene, and construct a corresponding recombinant genetically engineered bacterium. Researches show that, with 3-butenenitrile and 4-pentenenitrile as substrates, the enzyme activity of the nitrilase and the mutant PgNIT2-A190I of the application is 5.94 U / mg and 6.66 U / mg, 9.95 U / mg and 11.90 U / mg at pH 7.0 and 45 DEG C respectively, and the nitrilase mutant of the application is significantly improved. Under various substrate concentration conditions, the hydrolysis capacity of the mutant PgNIT2-A190I of the application to 3-butenenitrile and 4-pentenenitrile is also higher than that of the nitrilase PgNIT2. Moreover, the mutant xylanase has good pH and temperature tolerance, and has production application potential in efficient hydrolysis of rapeseed meal glucosinolate derived nitrile compounds.

The invention belongs to the technical field of synthetic biology and metabolic engineering, and particularly relates to a foreign protein expression system with low-degree interference on normal metabolism of a host as well as construction and application of the foreign protein expression system. The system is based on an orthogonal ribosome-mRNA pairing mechanism, an orthogonal SD sequence with complementarity lower than 20% with a host natural anti-SD sequence is obtained through design and screening, a candidate sequence with the host growth inhibition rate lower than 5% is screened from the orthogonal SD sequence, and the optimal orthogonal SD sequence and an orthogonal ASD sequence completely complementary with the optimal orthogonal SD sequence are determined. The system is suitable for high-efficiency expression of esterase, nitrilase and NADPH dependent oxidoreductase, and the enzyme activities of whole cells are respectively improved by 2.5 times, 3.4 times and 2.8 times, so that the system can effectively solve the problems of translation resource competition, high metabolic load, strong protein toxicity, inclusion body formation and the like in the foreign protein expression process; and a universal and stable technical platform is provided for efficient soluble expression of industrial enzymes.

The invention relates to a nitrilase combined mutant and application thereof in whole-cell biological catalysis, and belongs to the technical field of biological catalysis. Through semi-rational design, a double-combination mutant is constructed based on a loop engineering strategy, and the catalytic activity of nitrilase on methylaminoacetonitrile can be remarkably enhanced. The enzyme activity of the nitrilase combined mutant to methylaminoacetonitrile can reach 6.69 U.mL <-1 > and is increased by 8.5 times compared with that of an initial strain. Whole-cell catalysis conditions are further optimized, the nitrilase combined mutant has the highest activity under the conditions that the temperature is 30 DEG C and the pH is 7.2 through a PBS buffer solution, and 75 mM methylaminoacetonitrile can be efficiently converted at the cell concentration of 7.5 g.L <-1 >. The invention provides a novel enzyme tool and process support for efficient biological catalysis of methylaminoacetonitrile.

The application discloses a nitrilase mutant and application thereof in synthesis of gamma-aminobutyric acid derivatives, and relates to a nitrilase mutant with improved catalytic activity and stereoselectivity, which is used for efficiently catalyzing hydrolysis of 2-substituted succinonitrile to synthesize 3-substituted-3-cyanopropionic acid, and further synthesizing gamma-aminobutyric acid derivatives through hydrogenation, thereby laying a foundation for industrialized production of gamma-aminobutyric acid drugs synthesized by the nitrilase method.

The application provides a nitrilase mutant and application thereof in feed. Castor meal is the residue after castor seed oil is pressed, and contains rich protein and cellulose. Because the castor meal contains ricin and other toxic substances, the castor meal can cause great harm to poultry and livestock, and the application of the castor meal in the field of feed and fertilizer is limited. Therefore, the application of nitrilase in degrading ricin in castor meal is provided, and single-point mutation or combined mutation is carried out on the 165th, 190th and 221st sites of nitrilase from Streptomyces lunaelactis. The specific enzyme activity of the optimal nitrilase mutant is 840 times that of wild-type nitrilase, most of the ricin can be hydrolyzed in 24 hours, the challenge of adding castor meal as feed is solved, and the nitrilase mutant has great industrial application value.

The invention belongs to the technical field of enzyme engineering and biology, and discloses a nitrilase mutant as well as a coding gene, a recombinant vector, a recombinant gene engineering bacterium, an enzyme preparation and application thereof. The nitrilase mutant is obtained by performing single-point mutation or multi-point mutation on the 16th, 23rd or 47th amino acid of the amino acid sequence of the nitrilase as shown in SEQ ID NO.2. The invention further discloses a preparation method of the nitrilase mutant. The nitrilase mutant has the advantages that compared with AaNIT, the nitrilase mutant is greatly improved in activity and stereoselectivity, and when a crude extract or engineering bacterium whole cells of the nitrilase are used for catalysis, the reaction enzyme activity and stereoselectivity are still kept at a high level. Compared with a parent, the nitrilase mutant disclosed by the invention has the advantages that the activity is improved by 3.8 times, the stereoselectivity is improved by 10.3 times, and a foundation is laid for synthesizing (S)-2-(4-nitrophenyl) butyric acid by an industrial enzyme method.

The application discloses a nitrilase mutant with improved activity and thermal stability, and belongs to the field of enzyme engineering. The nitrilase mutant with improved catalytic activity is obtained through a PROSS design tool, and the enzyme activity reaches 300%, 230%, 115% and 165% of that of wild type Nit6803 respectively. In addition, the nitrilase mutant with improved thermal stability has a relative enzyme activity of 76% and 61% after incubation at 50 DEG C for 8 hours, compared with 40% of the relative activity of wild type Nit6803. Therefore, the application of the enzyme catalyzed nitrile substances to produce carboxylic acid substances in industry is utilized.

This invention belongs to the fields of enzyme engineering and biotechnology, and discloses a nitrile hydrolase mutant, its encoding gene, recombinant vector, recombinant genetically engineered bacteria, enzyme preparation, and applications. The nitrile hydrolase mutant is obtained by single-point or multi-point mutation of amino acids 8, 17, 35, or 150 of the nitrile hydrolase amino acid sequence shown in SEQ ID NO. 2. The nitrile hydrolase mutant of this invention exhibits 32-fold increased activity and 15.8-fold increased yield compared to the wild-type Gi-Nit, laying the foundation for the industrial enzymatic synthesis of 2-(4-nitrophenyl)butyric acid.

The application provides a nitrilase mutant and its application in synthesis of p-cyanobenzoic acid. The application provides a nitrilase mutant with improved activity in catalytic synthesis of p-cyanobenzoic acid, wherein the mutant protein is a non-natural protein, and one or more substitution mutations exist in the amino acid sequence shown in SEQ ID NO:1, V65, H137, M193, I197, Q201, H208 and R281 are catalysts, and the mutant protein has significantly improved activity in catalyzing p-xylyl cyanide to generate p-cyanobenzoic acid, and the mutant protein is mutated in two or more core amino acids of wild-type nitrilase which are related to enzyme catalytic activity. In a preferred example, the conversion rate is 99% at a p-xylyl cyanide concentration of 600 g / L.

The invention discloses a nitrilase mutant with high stereoselectivity and hydration activity and application of the nitrilase mutant. The mutant is obtained by performing single mutation or multiple mutation on amino acids at the 163rd, 166th and 201st sites of an amino acid sequence shown as SEQ ID NO.1. The invention further discloses a preparation method of the nitrilase mutant. According to the invention, molecular modification is carried out on reaction non-specific nitrilase OsNIT through (semi) rational design, so that a mutant with strict hydration activity and high stereoselectivity is obtained. When the mutant OsNIT-M0 / L163E / T166I / M201L is used for catalyzing mandelonitrile, the content of mandelamide is changed into 96.8%, the e.e. Value of R-mandelamide is increased to 92.7%, and the relative hydration activity is 163% of that before mutation. The reaction specificity and stereoselectivity of the nitrilase are regulated and controlled, so that the nitrilase can be applied to green industrial catalytic synthesis of amide and has important significance.

The application discloses a nitrilase mutant and application thereof in synthesis of a chiral intermediate of brivaracetam, and establishes a high-efficiency (R)-3-cyanohexanoic acid nitrilase synthesis process by constructing a nitrilase mutant with improved catalytic activity and stereoselectivity, so that the demand for industrial synthesis of a key chiral intermediate of brivaracetam is met, and a foundation is laid for industrial production of brivaracetam synthesized by a chemical-enzyme method.

The application discloses a nitrilase mutant and application thereof in hydrolysis of pyridine nitrile derivatives, wherein the nitrilase mutant is obtained by single mutation or multiple mutations of amino acids at positions 59, 134, 135 or 192 of the amino acid sequence shown in SEQ ID No. 2. The application is semi-rational design, and the protein is molecularly modified. The catalytic activity of the nitrilase four-mutant AcNR-W59A / T134G / H135L / S192G to 4-TCN is increased by about 142 times, and the catalytic activity to ricinine is increased by about 77 times, while the complete hydrolysis activity is maintained. The nitrilase mutant reported in the application has high catalytic activity to various pyridine nitrile substances, and has a wide and good application prospect in pesticide and feed production.

The application belongs to the technical field of detection and relates to a method for visual rapid detection of nitrilase. Functional monomers, cross-linking agents and initiators are mixed in a solvent to form a copolymer system, which is then ultrasonically treated to make the mixture uniform, so as to obtain a prepolymer solution, which is stored in cold storage for standby use. The functional monomers are double-bonded nitrile compounds. The prepolymer solution is dropped into the gap of a photonic crystal template and polymerized under a UV lamp. After polymerization is completed, the inverse opal nitrile-based polymer photonic crystal is obtained through etching, and then the inverse opal nitrile-based polymer photonic crystal is washed with deionized water so as to reach swelling equilibrium and be neutral. The inverse opal nitrile-based polymer photonic crystal is placed in an alkaline solution to obtain a mixed system. Nitrilase solutions with different concentrations are dropped into the mixed system. The nitrilase makes the nitrile groups on the inverse opal nitrile-based polymer photonic crystal catalytically hydrolyzed into carboxyl groups, so that the photonic crystal swells and the lattice constant changes, thereby realizing visual detection of the nitrilase.

This invention belongs to the fields of enzyme engineering and biotechnology, and discloses a nitrile hydrolase mutant, its encoding gene, recombinant vector, recombinant genetically engineered bacteria, enzyme preparation, and applications. The nitrile hydrolase mutant is obtained by single-point or multi-point mutation of amino acids 16, 23, or 47 in the amino acid sequence of the nitrile hydrolase shown in SEQ ID NO.2. The beneficial effects of this invention are mainly reflected in: the nitrile hydrolase mutant described in this invention is relatively... Aa The activity and stereoselectivity of NIT are significantly improved. When catalyzed using the crude extract of the nitrile hydrolase described in this invention or whole cells of engineered bacteria, the enzyme activity and stereoselectivity remain at a high level. The nitrile hydrolase mutant of this invention exhibits 3.8 times higher activity and 10.3 times higher stereoselectivity than the parent, facilitating industrial-scale enzymatic synthesis. S This laid the foundation for 2-(4-nitrophenyl)butyric acid.

The invention relates to a preparation method of an interleukin-23 receptor peptide inhibitor and a chiral intermediate thereof, in particular to a method for preparing an L-penicillamine derivative, and the L-penicillamine derivative can be used for preparing the interleukin-23 receptor peptide inhibitor. On one hand, the invention relates to a method for preparing a compound shown as a formula I. The method comprises the following steps: brominating isobutyraldehyde, substituting bromine atoms on the isobutyraldehyde with benzyl mercaptan and the like, then converting aldehyde groups into nitrile ammonia by using sodium cyanide, and finally converting nitrile groups into carboxylic acid by using nitrilase, so as to obtain the benzyl and the like substituted L-penicillamine derivative. The invention also relates to a penicillamine derivative shown as a formula I-Fmoc obtained by protecting free amino groups on the L-penicillamine derivative with Fmoc, and a polypeptide which is prepared from the penicillamine derivative shown as the formula I-Fmoc and has a general name of Icotrokinra. Some steps of the method have the characteristics of high purity and high yield.

This invention discloses an enzymatic method for preparing (R)-2-trifluoromethyl-2-hydroxypropionic acid. The method uses 3,3,3-trifluoro-2-hydroxy-2-methylpropionitrile as a raw material, which is hydrolyzed by a nitrile hydrolase to obtain (R)-2-trifluoromethyl-2-hydroxypropionic acid. The method disclosed in this invention has a short route, high efficiency, and simple operation, making it more suitable for industrial production.