12 results about "Nitrilase" patented technology

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 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 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 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 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.

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.

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.

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.

The invention discloses a nitrilase mutant and application thereof in synthesis of nicotinic acid, and belongs to the technical field of bioengineering. Comprising a nitrilase mutant of a new amino acid sequence formed by replacing one or two amino acid residues in 95 alanine and 124 asparagine of an original nitrilase BbNit amino acid sequence with other amino acid residues, a coding gene of the nitrilase mutant, and a recombinant expression vector and a recombinant expression transformant containing the coding gene sequence, the invention relates to a catalyst containing a recombinant nitrilase mutant and application of the catalyst to catalytic hydrolysis of 3-cyanopyridine to synthesize nicotinic acid. Compared with the prior art, the nitrilase mutant prepared by the invention has the advantages that the enzyme catalytic activity is improved, the tolerance to a substrate 3-cyanopyridine is also improved, and the problems of long reaction time, low space time yield and the like in synthesis of nicotinic acid by biologically catalyzing hydrolysis of 3-cyanopyridine are solved, so that the nitrilase mutant is beneficial to practical application in industrial preparation of nicotinic acid.