81 results about "Nitrilase" patented technology

The invention provides a nitrilase mutant and application of the nitrilase mutant in synthesis of p-cyanobenzoic acid. The invention provides a nitrilase mutant capable of improving the activity of catalytic synthesis of p-cyanobenzoic acid, the mutant protein is a non-natural protein, and the mutant protein can significantly improve the activity of catalyzing p-phthalonitrile to generate p-cyanobenzoic acid. And the mutant protein mutates in two or more core amino acids related to enzyme catalytic activity of the wild nitrilase. The nitrilase mutant provided by the invention can significantly improve the yield of a nitrilase catalytic synthesis product.

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).

The invention discloses a nitrilase mutant and application of the nitrilase mutant in synthesis of a brivaracetam chiral intermediate, and provides an efficient (R)-3-cyanohexanoic acid nitrilase synthesis process by constructing the nitrilase mutant with improved catalytic activity and stereoselectivity. The requirement of industrially synthesizing the brivaracetam key chiral intermediate is met, and a foundation is laid for industrial production of brivaracetam synthesized by a chemical-enzyme method.

The invention discloses construction and application of a novel nitrilase with improved amide generation capability and a mutant thereof. The amino acid sequence of the nitrilase disclosed by the invention is as shown in SEQ ID NO: 1, and the mutant of the nitrilase is obtained by performing single mutation on the 51st site, the 162nd site and the 185th site of the sequence as shown in SEQ ID NO: 1. The nitrilase provided by the invention has excellent amide generation capability, and has important industrial application potential for green and efficient preparation of amide compounds.

The invention relates to the technical field of enzyme catalysis, relates to the technical field of bioengineering application, and in particular relates to a magnetic nitrilase catalyst for green cyclic synthesis of organic acid as well as a preparation method and application of the magnetic nitrilase catalyst. According to the method, nitrilase escherichia coli is fixed on a magnetic composite carrier in a covalent binding and adsorption-crosslinking manner to form a magnetic nitrilase catalyst, and the catalyst is used for catalytic synthesis of organic acid; after the reaction is finished, the magnetic nitrilase catalyst is recovered by magnetic attraction, and the reaction waste liquid is acidized to realize the purpose of green cyclic synthesis of the organic acid. The prepared magnetic nitrilase catalyst can stably exist in nitrile substances and has the advantages of being high in mechanical strength, low in enzyme activity loss rate and easy to store, the magnetic nitrilase catalyst and reaction liquid can be recycled, beneficial reference is provided for promoting clean, environment-friendly and efficient enzyme method production of organic acid, and the application prospect is wide. The method has huge application potential in continuous separation and industrial application.

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 nitrilase mutant with improved activity and thermal stability, and belongs to the field of enzyme engineering. The nitrilase mutant capable of improving the catalytic activity is obtained through a PROSS design tool, and the enzyme activity respectively reaches 300%, 230%, 115% and 165% of that of wild type Nit6803. Besides, after the nitrilase mutant capable of improving the thermal stability is incubated for 8 hours at the temperature of 50 DEG C, compared with 40% of relative activity of wild type Nit6803, the relative enzyme activity of the mutant is 76%, and the relative enzyme activity of the mutant is 61%. Therefore, the invention uses the enzyme to catalyze nitrile substances to produce carboxylic acid substances in industry.

The invention discloses an ancestor nitrilase design based on multi-sequence alignment and machine learning and application of a mutant, and belongs to the technical field of enzyme engineering. The invention designs an ancestor enzyme sequence-structure-molecular dynamics strategy so as to obtain information for eradicating ancestor enzyme in an evolutionary landscape, specifically, a nitrilase sequence is collected for evolutionary tree analysis to obtain the evolutionary landscape, an ancestor enzyme reconstruction algorithm is combined to obtain an amino acid sequence of the ancestor enzyme, and an ancestor enzyme primary structure sequence library is constructed; predicting the tertiary structures of all ancestor enzymes to obtain a structural library; and finally, carrying out molecular dynamics simulation on all ancestor enzyme tertiary structures to obtain a kinetic parameter library, and screening through specific kinetic parameters. Based on the strategy, ancestor nitrilase capable of tolerating 90 DEG C is obtained, the thermal stability of the ancestor nitrilase is evolved, and a series of dominant mutants are obtained.

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.

The invention discloses a nitrilase mutant capable of reducing the selectivity of 5-chloronicotinonitrile and improving the selectivity of 6-chloronicotinonitrile and application of the nitrilase mutant. The nitrilase mutant is obtained by performing single mutation or multiple mutation on the 128 site, the 144 site and the 146 site of an amino acid sequence shown in SEQ ID No.2. The nitrilase mutant with obviously improved selectivity on 6-chloronicotinonitrile is obtained by screening, the 6-chloronicotinonitrile can be selectively catalyzed by taking recombinant cells containing nitrilase mutant genes or broken crude enzyme liquid of the recombinant cells as a catalyst and 6-chloronicotinonitrile and 5-chloronicotinonitrile as a common substrate, the high-purity 6-chloronicotinic acid can be synthesized in one step, the process is simple, the cost is low, and the method is suitable for industrial production. And the industrial cost is low. Compared with a wild type, the mutant has the advantages that the catalytic activity on 5-chloronicotinonitrile is reduced, and the selectivity on 6-chloronicotinonitrile is improved.

The invention discloses a nitrilase mutant and an application of the nitrilase mutant in synthesis of chiral cyanamide. Compared with an amino acid sequence of wild nitrilase amine, the nitrilase mutant protein has the advantages that the nitrilase mutant protein has a higher amino acid sequence; mutation exists at any one or more of the following amino acid sites corresponding to the amino acid sequence as shown in SEQ ID NO.1: the 54th site, the 118th site, the 120th site, the 139th site, the 148th site, the 168th site, the 170th site, the 194th site, the 197th site, the 198th site, the 202 site and / or the 225th site. The mutant constructed by the method disclosed by the invention can be used for efficiently catalyzing a 3-substituted-glutaronitrile compound to generate an (S)-3-substituted-4-cyanobutyramide compound. According to the invention, the problem of insufficient catalytic activity of the existing enzyme is solved, and an efficient scheme is provided for green synthesis of chiral cyanamide.

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 relates to the technical field of bioengineering, in particular to a nitrilase mutant containing non-natural amino acid, and is characterized in that the nitrilase mutant comprises one of the following mutation modes: (1) proline at the 340th site of an amino acid sequence as shown in SEQ ID NO.1 is mutated into L-(7-hydroxycoumarin-4-yl) ethylglycine; (2) proline at the 367 site of the amino acid sequence as shown in SEQ ID NO.6 is mutated into L-(7-hydroxycoumarin-4-yl) ethyl glycine; and (3) the proline at the 314th site of the amino acid sequence as shown in SEQ ID NO.8 is mutated into L-(7-hydroxycoumarin-4-yl) ethyl glycine. The nitrilase mutant NIT-P340G-Hco with high activity and stability is successfully screened out by establishing a high-throughput screening model, constructing a large-capacity mutant library and introducing non-natural amino acid on the basis of initial enzyme for enzyme modification, the whole-cell enzyme activity of the nitrilase mutant NIT-P340G-Hco is 15.84 times that of original nitrilase, and the nitrilase mutant NIT-P340G-Hco has high activity and stability. Therefore, the enzyme activity is enhanced while the enzyme stability is improved.

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 directed evolution method for improving thermal stability of nitrilase, and belongs to the technical field of enzyme engineering. The ancestor enzyme with improved thermal stability is obtained by the following method: collecting a sequence of nitrilase, carrying out evolutionary tree analysis to obtain an evolutionary landscape, obtaining an amino acid sequence of the ancestor enzyme in combination with an ancestor enzyme reconstruction algorithm, and constructing an ancestor enzyme primary structure sequence library; predicting the tertiary structures of all ancestor enzymes to obtain a structural library; and finally, carrying out molecular dynamics simulation on all the ancestor enzyme tertiary structures to obtain a kinetic parameter library, and screening through specific kinetic parameters to obtain the novel heat-resistant ancestor nitrilase. And then predicting and verifying mutation sites, and screening out a forward mutant with improved thermal stability and enzyme activity.

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 and application thereof in synthesis of a gamma-aminobutyric acid derivative, the nitrilase mutant with improved catalytic activity and stereoselectivity is constructed to efficiently catalyze hydrolysis of 2-substituted succinonitrile to synthesize 3-substituted-3-cyanopropionic acid, and the gamma-aminobutyric acid derivative is further synthesized through hydrogenation. And a foundation is laid for industrial production of gamma-aminobutyric acid drugs synthesized by a nitrilase method.

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 provides a nitrilase mutant and application thereof in feed. Castor cake meal is residues obtained after castor seeds are subjected to oil extraction, contains rich protein and cellulose, and can cause huge harm to livestock due to the fact that the castor cake meal contains highly toxic substances such as ricinine, and application of the castor cake meal in the fields of feed and fertilizer is limited. Therefore, the nitrilase is applied to degradation of ricinine in castor cakes, single-point mutation or combined mutation is carried out on the 165th site, the 190th site and the 221 site of the nitrilase from Streptomyces lunalactis, the specific enzyme activity of the obtained optimal nitrilase mutant is 840 times that of wild type nitrilase, most of ricinine can be hydrolyzed within 24 hours, and the optimal nitrilase mutant can be used for degrading ricinine in castor cakes. The challenge confronted by using the castor cake meal as a feed additive is solved, and the method has a relatively high industrial application value.

The invention discloses construction and application of a novel nitrilase with excellent amide generation capability and a mutant thereof. The amino acid sequence of the nitrilase disclosed by the invention is as shown in SEQ ID NO: 1, and a mutant of the nitrilase is obtained by performing single mutation on the 166th site, the 168th site and the 191th site of the sequence as shown in SEQ ID NO: 1. The nitrilase provided by the invention has excellent amide generation capability, and has important industrial application potential for green and efficient preparation of amide compounds.

The present invention relates to a method for preparing substituted 4-(N'-hydroxycarbamimidoyl)benzoic acid, which can be obtained by hydration of substituted terephthalonitrile of formula II in an aqueous medium catalyzed by nitrilase to provide 4-cyanobenzoic acid (ammonium) IIa. After hydration, the aqueous reaction medium is treated with hydroxylamine or a salt thereof to provide amide oxime I.

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 present invention discloses a nitrilase immobilization technology production equipment for nicotinic acid production, comprising an immobilization box, an immobilization mechanism arranged on the inner wall of the immobilization box, a liquid inlet pipe, the liquid inlet pipe is used to input nitrilase liquid into the immobilization box, a crosslinking agent input pipe, the crosslinking agent input pipe is used to input the crosslinking agent into the immobilization box, a processing mechanism, the processing mechanism is used to purify and discharge the residual liquid, and a storage mechanism. The present invention relates to the technical field of nitrilase immobilization equipment; after the nitrilase enters the immobilization box, the nitrilase can be immobilized and cleaned through the cooperation of a first driving motor, a transmission shaft, a stirring blade, a friction disk, a friction ring plate, a through hole, a heat-insulating ring plate, an electric push rod and a sliding ring, so as to avoid impurities adhering to the solid surface of the nitrilase to affect its storage, and at the same time, the soft pad is arranged to avoid damage to the nitrilase solid when the stirring blade rotates at a high speed.

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 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.

Disclosed is a method for enzymatic synthesis of a brivaracetam chiral intermediate, i.e., 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 of the present invention 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. The present invention features a short synthesis route, mild reaction conditions, and high atom economy, and can be applied to the industrial synthesis of the brivaracetam intermediate.