22 results about "Mutation screening" patented technology

This invention provides a 7β-HSDH enzyme mutant, its encoding gene, and its applications. Through artificial mutation screening, 7α-HSDH and 7β-HSDH enzyme mutants with specific amino acid sequences were identified, resulting in improved enzyme activity and catalytic efficiency compared to wild-type 7α-HSDH and wild-type 7β-HSDH. This invention also provides a method for preparing ursodeoxycholic acid using the aforementioned 7α-HSDH and 7β-HSDH enzyme mutants, specifically using NADP... + A one-pot catalytic method for the conversion of chenodeoxycholic acid to ursodeoxycholic acid using NADPH-dependent 7α-HSDH enzyme mutants and NADPH-dependent 7β-HSDH enzyme mutants is employed. This method is simple, yields high product conversion, and can also achieve the conversion of the coenzyme NADP. + / NADPH's self-circulating regeneration eliminates the need for auxiliary enzymes and raw materials used in coenzyme regeneration, greatly reducing production costs and environmental pressure, and possessing enormous potential for industrial applications.

The invention discloses a device and method for screening key mutations affecting virus host adaptability based on a deep learning model, and the method comprises the following steps: 1) based on a specified pathogen type, collecting gene sequence data of a pathogen and adopted host information, and preprocessing the collected data, constructing a model training data set and a test data set; 2) performing feature extraction on the collected sequence data to obtain a feature matrix of each sequence; 3) constructing a deep learning convolutional neural network model to perform data training and feature learning on the training data set; according to the method, the problem that measurement is inaccurate due to the fact that homology between virus hosts is high is solved, and the method has more accurate host adaptability prediction capacity and key mutation screening capacity.

The invention relates to the technical field of enzyme engineering, in particular to a malic enzyme mutant and application thereof. Specifically, the malic enzyme derived from Escherichia coli is subjected to site mutation screening, and results show that after methionine at the 144th site is mutated into isoleucine (M144I), glutamine at the 253rd site is mutated into isoleucine (Q253I), and threonine at the 327th site is mutated into phenylalanine (T327F), the catalytic efficiency and the catalytic activity are obviously improved, which is of great significance to industrial application.

The application discloses a base editor ACGBEmax realizing simultaneous replacement of purine and pyrimidine, relates to the technical field of gene editing, and comprises, from N end to C end, an HMCES protein, a bifunctional deaminase TadDual, an nCas9(D10A) protein and an engineered N-methyl purine DNA glycosylase eMPG. The base editor can realize simultaneous replacement of purine and pyrimidine, significantly increases the diversity of mutants after targeted editing, has the advantages of high editing efficiency and low Indels rate, and has great application value in aspects of in vitro and in vivo protein mutation screening and identification of oncogenic amino acid mutations.

This invention discloses a STAT3-mutated cell product and its uses, belonging to the interdisciplinary field of genetic engineering and tumor immunotherapy. Through saturation mutation screening, this invention is the first to discover that STAT3 gain-of-function mutations promote CAR-T anti-tumor responses. Furthermore, through cell and animal experiments, it is demonstrated that activating STAT3 can alleviate CAR-T cell immune exhaustion and significantly enhance the in vivo and in vitro killing ability of CAR-T cells against tumor cells, thereby improving the efficacy of anti-tumor therapy. This invention provides a new therapeutic target and strategy for the field of tumor immunotherapy, possessing significant scientific and clinical application value.

The invention belongs to the technical field of agricultural biology, and particularly discloses a grape germplasm innovation method based on X-ray gradient mutagenesis and an LD50 precise prediction model, and the method comprises the following steps: selecting an annual grape branch for vernalization treatment, and trimming the annual grape branch into a single-bud stem segment; setting an irradiation time gradient, accurately calculating an LD50 value through a cubic spline interpolation model, and carrying out X-ray irradiation on the single-bud stem segment according to the LD50 value; after irradiation, performing cutting and liquid paraffin sealing, and then performing cuttage, potting and field planting treatment; performing secondary vernalization on mutagenesis plant branches, trimming the mutagenesis plant branches into single-bud stem segments, collecting tender leaves after cutting survival, extracting genome DNA, performing whole genome sequencing, and accurately distinguishing mutagenesis specific variation and natural variation. By establishing a complete technical system of standardized vernalization, gradient irradiation, LD50 accurate calculation and whole genome verification, the problems of inaccurate irradiation parameters, nonstandard material treatment, low mutation screening accuracy and the like in the prior art are solved.

The invention relates to the technical field of gene engineering and protein modification, in particular to a lipase mutant and application thereof in synthesis of a pregabalin key chiral intermediate. According to the invention, on the basis of thermomyces lanuginosus lipase TL, a large amount of mutation screening is carried out, so that the lipase mutant of which the catalytic efficiency of hydrolysis of 2-carboxyethyl-3-cyano-5-methyl ethyl hexanoate (CNDE) is remarkably improved is finally obtained. Wherein the catalytic constant Kcat of the single-point mutant containing S254A reaches 100.2 and is 45 times of that of a wild type, the enantiomer selectivity ee value of the single-point mutant containing S254A is also higher than that of the wild type, and an unexpected technical effect is achieved. The lipase mutant can be applied to efficient synthesis of a pregabalin key chiral intermediate, and has a wide prospect.

This invention relates to the fields of genetic engineering and protein modification technology, specifically to a lipase mutant and its application in the synthesis of a key chiral intermediate of pregabalin. This invention relates to *Thermophilus spp.* (…). Thermomyces lanuginosus Based on lipase TL, through extensive mutation screening, a lipase mutant with significantly improved catalytic efficiency for the hydrolysis of ethyl 2-carboxyethyl-3-cyano-5-methylhexanoate (CNDE) was finally obtained. Among them, the single-point mutant containing S254A has a catalytic constant... K The cat value reached 100.2, which is 45 times that of the wild type, and the enantioselectivity ee value was also higher than that of the wild type, achieving unexpected technical results. This lipase mutant has broad prospects for the efficient synthesis of key chiral intermediates of pregabalin.

The invention relates to the technical field of enzyme engineering, in particular to a malic enzyme mutant and application thereof. Specifically, the malic enzyme derived from Escherichia coli is subjected to site mutation screening, and the result shows that after valine at the 7th site is mutated into lysine (V7K), the catalytic efficiency and the catalytic activity are obviously improved, which is of great significance to industrial application.

This invention provides a 7α-HSDH enzyme mutant and a 7β-HSDH enzyme mutant, their encoding genes, and their applications. Through artificial mutation screening, 7α-HSDH and 7β-HSDH enzyme mutants with specific amino acid sequences were identified, resulting in improved enzyme activity and catalytic efficiency compared to wild-type 7α-HSDH and wild-type 7β-HSDH. This invention also provides a method for preparing ursodeoxycholic acid using the aforementioned 7α-HSDH and 7β-HSDH enzyme mutants, specifically using NADP... + A one-pot catalytic method for the conversion of chenodeoxycholic acid to ursodeoxycholic acid using NADPH-dependent 7α-HSDH enzyme mutants and NADPH-dependent 7β-HSDH enzyme mutants is employed. This method is simple, yields high product conversion, and can also achieve the conversion of the coenzyme NADP. + / NADPH's self-circulating regeneration eliminates the need for auxiliary enzymes and raw materials used in coenzyme regeneration, greatly reducing production costs and environmental pressure, and possessing enormous potential for industrial applications.

The application belongs to the technical field of molecular markers, and provides a Hu sheep 5K low-density SNP chip and application thereof.The chip comprises 5000 SNP molecular markers, and the SNP molecular markers are shown in Table 1.Based on resequencing data of Hu sheep, the application screens SNP markers with rich polymorphic information content of Hu sheep breeds, integrates functional variation sites such as nonsense mutations and splicing mutations, and customizes the 5K low-density chip of Hu sheep, which can be used for Hu sheep kinship identification and functional harmful mutation screening.

The invention discloses a construction method and application of an adeno-associated virus vector of a targeted vascular endothelial cell, and belongs to the technical field of genetic engineering. The construction method and the application comprise the following steps: S1, vector construction: cloning a Tie2-eNOS promoter to a pAAV-MCS plasmid, so as to obtain pAAV-Tie2-MCS; the preparation method comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification on a VEGF (Vascular Endothelial Growth Factor) gene, and inserting the VEGF gene into a pAAV-MCS plasmid to obtain an expression plasmid pAAV-Tie2- The method comprises the following steps: carrying out combined mutation on a VP1 gene of a pAAV-RC6 plasmid to obtain a mutant plasmid pAAV-RC6; s2, virus packaging and purification: culturing an HEK293 cell, and transfecting a pAAV-Tie2-VEGF (Vascular Endothelial Growth Factor), a mutant plasmid pAAV-RC6 and a pHelper plasmid; performing chromatographic purification by combining iodixanol gradient centrifugation with Heparin, so as to obtain rAAV; and S3, carrying out titer determination, SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) and in vitro and in vivo experiment detection on the rAAV. The preparation process is simple, the titer greater than or equal to 1 * 10 < 13 > vg / mL can be obtained through single transfection, and the production cost is reduced; and mutation screening is realized through a directed evolution technology, so that the experimental period is shortened. And the vector provided by the invention has relatively high specificity on vascular endothelial cells.

The application discloses a cembranoid alcohol synthase mutant and application thereof in biosynthesis of cembranoid alcohol, and belongs to the field of enzyme engineering and synthetic biology. The mutant with significantly improved catalytic performance is obtained by rational design and mutation screening of cembranoid alcohol synthase (NtCBTS) from tobacco. The cembranoid alcohol yield of the strain containing the mutant NtCBTS_K208R-A268C-K353R is increased by 231.18% compared with the strain containing NtCBTS. The mutant is applied to a Saccharomyces cerevisiae expression system, and efficient biosynthesis of cembranoid alcohol is realized, and the generation level of the target product is significantly improved. The high-activity enzyme mutant and the application strategy provided by the application effectively improve the biosynthesis efficiency of cembranoid alcohol, and provide important technical support for constructing a high-yield yeast cell factory and promoting industrialized production.

The application discloses a glycosyltransferase UGT91C1 mutant and a method for catalyzing synthesis of rebaudioside D, and belongs to biological catalytic synthesis. The glycosyltransferase UGT91C1 mutant is any one of the following (A)-(C): A) a protein obtained by making any one or more mutations in the amino acid sequence shown in SEQ ID NO. 1: the 89th amino acid is mutated from N to Y; the 155th amino acid is mutated from M to L; the 274th amino acid is mutated from S to T; and the 361st amino acid is mutated from N to S; (B) a protein having 95% or 98% or above identity with the amino acid sequence defined in (A) and having the same function; and (C) a fusion protein obtained by connecting a tag to the end of the protein defined in (A) or (B). The glycosyltransferase UGT91C1 mutant is obtained by mutation screening from a wild-type glycosyltransferase UGT91C1, has higher enzyme activity and catalytic rate, and can efficiently synthesize rebaudioside D by taking rebaudioside A as a substrate.

The invention relates to the technical field of bioinformatics, in particular to a mutation screening method of cellobiose epimerase and related equipment. According to the method provided by the embodiment of the invention, a protein diffusion model is adopted to reconstruct the key catalytic region to obtain a regenerated mutant protein skeleton; performing side chain optimization on the mutant protein skeleton by using a deep learning neural network model to obtain a predicted amino acid sequence of the mutant protein skeleton; obtaining a three-dimensional structure of a cellobiose epimerase mutant-substrate compound according to the predicted amino acid sequence and a molecular structure of a substrate, and screening the three-dimensional structure of the cellobiose epimerase mutant-substrate compound; through the mode, the mutation screening efficiency of the key catalytic region can be improved, the mutant capable of improving the conversion efficiency of lactulose can be rapidly screened, and the screening efficiency of the cellobiose epimerase mutant with the improved conversion efficiency of lactulose is improved.

The invention belongs to the technical field of bioengineering, and particularly relates to cellulase for degrading waste plant fibers and a mutant thereof. The amino acid sequence of the cellulase mutant is as shown in SEQ ID NO.3, and the nucleotide sequence of the cellulase mutant is as shown in SEQ ID NO.4. The cellulase mutant is obtained through mutation screening, compared with wild type cellulase, the enzyme activity of the cellulase mutant is obviously improved, and the cellulase mutant has more excellent pH stability and heat stability and larger industrial application potential. In addition, the high-performance super absorbent resin is prepared by grafting acrylic acid after the waste plant fibers are pretreated by the cellulase mutant, and an innovative solution is provided for realizing resource utilization of agricultural wastes.

The application provides a lytic polysaccharide monooxygenase mutant, a preparation method and application thereof, relates to the technical field of enzyme engineering, and is based on wild-type Myceliophthora thermophila lytic polysaccharide monooxygenase, and a V143I mutation is generated to obtain the lytic polysaccharide monooxygenase, wherein the amino acid sequence of the wild-type Myceliophthora thermophila lytic polysaccharide monooxygenase is shown in SEQ ID NO. 1. In the application, the lytic polysaccharide monooxygenase gene derived from Myceliophthora thermophila C1 is rationally designed, and a lytic polysaccharide monooxygenase mutant gene with improved thermal stability is obtained through mutation screening. Subsequently, the Pichia pastoris expression system is used to express and prepare the lytic polysaccharide monooxygenase mutant with improved heat resistance, the lytic enzyme is modified through engineering, the enzymatic properties such as activity and stability of the lytic enzyme are effectively improved, and the lytic enzyme can be successfully applied in the degradation of pulp cellulose.

The invention discloses a computer-aided enzyme design method for improving enzyme stereoselectivity, and relates to the field of computer science, bioinformatics and biosynthesis, and the computer-aided enzyme design method comprises the following steps: screening and determining different selective binding conformation distributions of an enzyme and a substrate; calculating combination conformations of enzymes with different selectivity and substrates to determine residue sites; determining parameters such as the optimal distance between the substrate in the target conformation and the amino acid residue of the enzyme; performing mutation prediction by using Rosetta software according to a target conformation constraint condition to generate an enzyme mutant library; and performing mutation screening on the enzyme mutant library, calculating the near-attack conformation percentage of the mutant and the wild type conformation, comparing the difference, and evaluating the stereoselectivity improvement proportion of the enzyme mutant. According to the method disclosed by the invention, the amino acid residue site with the optimal transformation potential is accurately positioned, the screening range of mutants is further narrowed, and the accuracy and speed of high-throughput screening of enzymes are remarkably improved.

The invention relates to the technical field of gene engineering and protein modification, in particular to a lipase mutant and application thereof in synthesis of a pregabalin key chiral intermediate. According to the invention, on the basis of thermomyces lanuginosus lipase TL, a large amount of mutation screening is carried out, so that the lipase mutant of which the catalytic efficiency of hydrolysis of 2-carboxyethyl-3-cyano-5-methyl ethyl hexanoate (CNDE) is remarkably improved is finally obtained. Wherein the catalytic constant Kcat of the single-point mutant containing S254A reaches 100.2 and is 45 times of that of a wild type, the enantiomer selectivity ee value of the single-point mutant containing S254A is also higher than that of the wild type, and an unexpected technical effect is achieved. The lipase mutant can be applied to efficient synthesis of a pregabalin key chiral intermediate, and has a wide prospect.

This invention discloses a cephalothinyl synthase mutant and its application in the biosynthesis of cephalothinyl synthase, belonging to the fields of enzyme engineering and synthetic biology. Through rational design and mutation screening of tobacco-derived cephalothinyl synthase (NtCBTS), this invention obtained a mutant with significantly improved catalytic performance. Specifically, the cephalothinyl synthase yield of strain containing the mutant NtCBTS_K208R-A268C-K353R was increased by 231.18% compared to strains containing NtCBTS. Applying this mutant to a *Saccharomyces cerevisiae* expression system achieved highly efficient biosynthesis of cephalothinyl synthase, significantly improving the production level of the target product. The highly active enzyme mutant and its application strategy provided by this invention effectively improve the biosynthetic efficiency of cephalothinyl synthase, providing important technical support for constructing high-yield yeast cell factories and promoting its industrial production.

The P450 monooxygenase derived from Spongiibacter sp IMCC 21906 is subjected to molecular modification by utilizing a protein engineering rational design method, a mutant of which the enzyme activity and the thermal stability are simultaneously improved is obtained through consensus sequence analysis and FoldX virtual saturation mutation screening, the enzyme activity is improved by 14.25 times compared with that of an original enzyme, and the half-life period of the P450 monooxygenase at 45 DEG C is prolonged by 4.6 times. A reasonable strategy is provided for improving the activity and the thermal stability of the P450 monooxygenase by utilizing a directed evolution technology, and a basis is provided for industrial application of the P450 monooxygenase.

The invention discloses a method for batch detection of gene editing events and a related sequencing library construction method thereof. According to the method, different sample PCR reaction systems are packaged into microdroplets and mixed in advance, and then amplification of a target detection sequence and embedding of a sample specific bar code are completed in one reaction tube in one step by utilizing the binding characteristics of an LNA modified primer at different temperatures; and finally, performing high-throughput sequencing on a sequencing library obtained by mixing the PCR products of all the samples to obtain mixed sequencing data, and performing efficient sample splitting and analysis on the mixed sequencing data based on bar codes to realize low-cost and high-throughput detection of gene editing events and gene editing efficiency. Meanwhile, the experiment period is remarkably shortened while the detection specificity and accuracy are improved, the risk of manual operation errors and cross contamination between samples is reduced, and the method can be widely applied to mutation screening in multiple fields of animal and plant breeding, gene function research, clinical diagnosis and the like.