379 results about "Directed mutagenesis" patented technology

The present invention relates to a fusion protein that produces point mutations in cells, its preparation and use. Specifically, the fusion protein provided by the present invention contains a Cas enzyme that lacks cytosine deaminase and nuclease activity and retains helicase activity, or is formed by a Cas enzyme that lacks cytosine deaminase and nuclease activity and retains helicase activity. The present invention also relates to the coding sequence of the fusion protein, a polynucleotide sequence containing the coding sequence, a nucleic acid construct containing the polynucleotide sequence, a corresponding host cell, a method for producing point mutations in a cell, and a kit, etc. By using the present invention, it is possible to achieve site-directed mutagenesis while obtaining high mutation efficiency and a variety of mutation combinations in a specific gene region.

The invention discloses application of an upstream non-coding region uORF of an ABA2 gene in regulation and control of crop agronomic traits, in particular to application in improvement of pre-harvest sprouting resistance of rice and cultivation of pre-harvest sprouting resistant rice strains, and belongs to the technical field of gene engineering. The invention finds that uORF in the upstream non-coding region of the crop ABA2 gene can inhibit the translation level of downstream ABA2 protein on the basis of not influencing the transcriptional level, so that the uORF can participate in regulation and control of crop agronomic traits in which ABA participates in regulation and control, such as improvement of pre-harvest sprouting resistance of rice. Therefore, the upstream non-coding region uORF of the crop ABA2 gene can be modified through a hybridization or gene editing technology, so that the purpose of regulating and controlling crop agronomic traits in which ABA participates in regulation and control is achieved, for example, site-directed mutagenesis is performed on an initiation codon of the upstream non-coding region uORF of the rice ABA2 gene, and the pre-harvest sprouting resistance of rice is improved.

According to the invention, a glycosyl transferase mutant derived from Bacillus licheniformis ZSP01 is used as a research object, AlphaFold 2 is used for constructing a three-dimensional structure of a protein, then technologies such as molecular docking and molecular dynamics simulation are used for analyzing amino acids far away from a catalytic center in glycosyl transferase, site-directed mutagenesis is carried out on the amino acids, and the glycosyl transferase is obtained. The activity of the mutant for catalytically synthesizing the icariside D2 glycosyltransferase is improved.

The invention belongs to the field of gene engineering, and particularly relates to a PET hydrolase mutant with high catalytic activity and application thereof. According to the invention, amino acids at the 62nd site, the 65th site, the 66th site, the 71th site, the 154th site, the 156th site, the 177th site, the 183th site, the 184th site, the 209th site, the 213th site, the 214th site and the 215th site in the PES-H1L92F / Q94Y amino acid sequence of the PET hydrolase shown as SEQ ID NO.2 are mutated by utilizing a structural analysis and site-specific mutagenesis technology to obtain various mutants, and the mutants with improved catalytic activity are obtained. The PET hydrolase mutants promote the hydrolysis efficiency of ester bonds in PET so as to improve the degradation capacity of PET plastics, and can be applied to enzymatic depolymerization of PET plastics in life in the future.

According to the invention, glycosyl transferase UGTBL1 derived from Bacillus licheniformis ZSP01 is taken as a research object, AlphaFold 2 is used for modeling, molecular docking, molecular dynamics simulation and other technologies, amino acid in an active pocket region of glycosyl transferase is analyzed, amino acid in a substrate channel is analyzed by software CAVER3.0, site-directed mutagenesis is carried out, and the glycosyl transferase UGTBL1 is obtained. The regioselectivity and the activity of the mutant for catalytically synthesizing the icariside D2 glycosyltransferase are improved.

The invention belongs to the technical field of biosynthesis, and particularly relates to a strain for producing sclareol as well as a construction method and application of the strain. In order to solve the problem of microbial synthesis of sclareol, phosphoketolase in an NOG pathway is mutated and optimized, phosphoketolase NaXpk from nanoarchaea archaeon is subjected to site-directed mutagenesis, a sclareol synthesis pathway is constructed in an escherichia coli host, the influence of NaXpk before and after mutation on the yield of sclareol is compared, and the yield of sclareol is improved. The S472F mutant and the S472Y mutant are determined, the S472F mutant and the S472Y mutant are applied to a sclareol production path, the sclareol yield of the finally obtained production strains can reach 623.3 mg / L and 655.2 mg / L, and the effect is remarkable.

According to the present invention, glycosyl transferase UGTBL1 derived from Bacillus licheniformis ZSP01 is adopted as a research object, AlphaFold 2 is adopted to carry out modeling, molecular docking, molecular dynamics simulation and other technologies, amino acids in an active pocket region of glycosyl transferase are analyzed, site-directed mutagenesis is performed on the amino acids to obtain a series of glycosyl transferase mutants, and salidroside can be highly selectively catalyzed and synthesized.

The application discloses a kind of fungal laccase mutant Lcc5-G and its expression strain and application.The application is based on the fungal laccase from Coprinopsis cinerea heterologous expression since, by site-directed mutagenesis, obtain mutant gene.After fungal induction expression containing mutant plasmid, obtain laccase mutant enzyme Lcc5-G with improved specific activity, stability and AFB1 removal efficiency.When ABTS is used as substrate, the specific activity of the mutant is improved by 3 times, and the stability of the mutant enzyme is improved to 5 times of the original enzyme at 45 DEG C and pH 7.The mutant has potential application value in oxidizing aflatoxin.

The present invention belongs to the field of enzyme engineering and food engineering technology, and specifically relates to the directed evolution and site-directed mutagenesis of a lactase enzyme molecule and its application in fresh milk. The amino acid sequence of the lactase mutant obtained by the present invention is shown in SEQ ID NO.4. Through the directed artificial evolution of the lactase molecule, a new lactase molecule is obtained that can efficiently convert lactose in fresh milk and synthesize oligosaccharides at a relatively low temperature, and is rapidly inactivated at a subsequent elevated temperature (such as pasteurization). This facilitates the highly nutritious in situ bioconversion of lactose in fresh milk into prebiotic oligosaccharides, further improving the nutritional value of fresh milk and expanding the fresh milk consumer group.

According to the scheme, the invention provides the recombinant macrolide enzyme as well as the preparation method and the application thereof, the amino acid sequence of the recombinant macrolide enzyme is as shown in SEQ ID NO.1, and the recombinant macrolide enzyme is derived from an EreC esterase family of enterobacter hormaechei and is obtained through site-specific mutagenesis; the mutation sites are as follows: glutamic acid at the 44th site is mutated into asparagine, tyrosine at the 55th site is mutated into proline, proline at the 76th site is mutated into arginine, phenylalanine at the 153rd site is mutated into alanine, and serine at the 219th site is mutated into glutamic acid. The method is expected to remove macrolide antibiotic pollution, and has good economic benefits and practical values.

Disclosed in the present invention are a sucrose synthase mutant and a method for preparing RD using same. The sucrose synthase mutant is based on the amino acid sequence shown in SEQ ID NO: 2 and contains a mutation in at least one of the following sites: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R. By means of site-directed mutagenesis of sucrose synthase, the enzyme activity of the sucrose synthase mutant is improved compared with that of the wild type. Also, some mutants can still maintain a high conversion rate in a wide pH range.

The application provides a preparation method of a functionalized phage probe and application of the functionalized phage probe in detection of escherichia coli. The functionalized phage is a recombinant phage (M13KO7@SaBP) in which streptavidin binding peptide (SaBP) is fused and expressed at the N terminal of the main capsid protein P8 protein of M13K07 phage. In the application, the gene encoding SaBP (MDVEAWLGAR) is inserted into the N terminal of the P8 protein gene of M13K07 phage by site-directed mutagenesis to prepare M13K07@SaBP. The stable signal amplification is realized by using the abundant P8 protein (about 2700) of M13 phage and the super strong binding force of biotin-streptavidin, the interference of food sample matrix is reduced by combining the magnetic separation technology, and a detection method of escherichia coli with high sensitivity and small sample matrix interference is constructed.

The invention discloses an incision beta-1, 3-glucanase mutant with improved enzyme activity and thermal stability, and belongs to the technical field of gene engineering and enzyme engineering. According to the invention, a site-directed mutagenesis technology is utilized to carry out molecular modification on wild type beta-1, 3-glucanase, and a series of mutants S22F, N226D and N187S are obtained. Compared with a wild enzyme, the beta-1, 3-glucanase mutant constructed by the invention has the advantages that the enzyme activity is improved by 1.04-1.81 times, the Tm value is improved by 0.97-1.19 DEG C, and the half-life period at 70 DEG C is prolonged to 1.08-1.28 times. According to the invention, the enzyme activity and the thermal stability of the wild beta-1, 3-glucanase are improved at the same time, and better use conditions are created for the enzyme in practical application.

The invention relates to the technical field of biology, in particular to a mutant of difunctional glutathione synthetase and application of the mutant. The amino acid sequence of the mutant of the difunctional glutathione synthetase provided by the invention is obtained by performing site-directed mutagenesis on at least one of the 597th site, the 607th site, the 609th site, the 610th site, the 728th site and the 733th site of the amino acid sequence of the wild type difunctional glutathione synthetase, and the amino acid sequence of the wild type difunctional glutathione synthetase is shown as SEQ ID NO: 1. The mutant of the bifunctional glutathione synthetase provided by the invention has relatively high enzyme activity and enzyme stability, is more suitable for industrial production and is suitable for preparing glutathione, and the efficiency of preparing glutathione is improved.

The invention relates to a beta-galactosidase mutant with improved thermal stability and pH stability, and belongs to the technical field of enzyme engineering. The beta-galactosidase derived from Kluyveromyces lactis is subjected to molecular modification, a potential mutation site is determined by using an alanine scanning strategy, beta-galactosidase mutants Pro433Ala and Asp441Ala of which the thermal stability and the pH stability are simultaneously improved are obtained through site-directed mutagenesis screening, the catalytic efficiency of the mutants is improved to 1.03-1.18 times compared with that of the mutants before optimization, and the beta-galactosidase mutant can be used for preparing beta-galactosidase. Wherein the maximum half-life period of the mutant Asp441Ala under the condition of 50 DEG C reaches 91.5 minutes and is increased by 213.4% compared with that of a wild type, the problems of poor catalytic thermal stability and poor pH stability of beta-galactosidase in the prior art are solved, and a brand-new tool is provided for obtaining galactooligosaccharide by using beta-galactosidase to hydrolyze lactose.

The invention provides a tyrosine ammonia lyase mutant and application thereof in production of p-coumaric acid. The tyrosine ammonia lyase mutant is obtained by mutating wild type tyrosine ammonia lyase with an amino acid sequence as shown in SEQ ID No.1, and a mutation site is selected from at least one of Q108S, Y340Q and P375H. The catalytic activity center of wild type tyrosine ammonia lyase is analyzed, and site-directed mutagenesis is carried out on five catalytic activity center sites, so that the mutant shows different enzyme catalytic activity when the mutation sites are different, the mutation sites are the same but the mutation targets are different; q108S, Y340Q and P375H single mutations can significantly improve the enzyme catalytic activity of the tyrosine ammonia lyase mutant, and the tyrosine ammonia lyase mutant having any two mutation sites, especially having three mutation sites, also has better enzyme catalytic activity than the wild type, and has great application potential.

The invention relates to the technical field of bioengineering, in particular to a penicillin G acylase mutant, polynucleotide, an expression vector and application. The penicillin G acylase mutant is obtained by carrying out site-directed mutagenesis on a wild type penicillin G acylase gene of parent Escherichia coli, and carrying out site-directed mutagenesis on the wild type penicillin G acylase gene to obtain the penicillin G acylase mutant. Phenylalanine (F) at the 24th site of an alpha chain, proline (P) at the 383rd site of a beta chain, threonine (T) at the 384th site of the beta chain, glutamic acid (G) at the 385th site of the beta chain and serine (S) at the 386th site of the beta chain of the penicillin G acylase are introduced and mutated by a whole plasmid PCR (Polymerase Chain Reaction) technology to obtain mutants. When the penicillin G acylase mutant obtained by the invention is used for catalyzing methyl mandelate and 3-(1-methyl-1H-tetrazole-5-yl) thiomethyl-7-aminocephalosporanic acid to synthesize cefamandole, the synthesis activity is improved, and meanwhile, the side reaction rate is greatly reduced.

The invention discloses a bergamot alkene synthase mutant and application thereof, and belongs to the technical field of enzyme engineering and bioengineering. According to the method, bergamotene synthase SanSyn which is obtained from plant clausena lansium and can catalyze FPP to generate alpha-trans-bergamotene is taken as an object, semi-rational design and site-specific mutagenesis are carried out on the bergamotene synthase SanSyn, a series of obtained SanSyn mutants are remarkably improved in the aspect of FPP substrate conversion efficiency, and the optimal bergamotene synthase mutant SanSynI273C-L514I has the advantages that the optimal bergamotene synthase mutant SanSynI273C-L514I can be used for catalyzing the FPP to generate alpha-trans-bergamotene; the unit cell productivity of the alpha-trans-bergamotene generated by catalyzing FPP is 6.9 times of that of a wild type, and the yield is 6.8 times of that of the wild type and reaches 43.58 mg / L. According to the bergamot alkene synthase mutant, the performance of alpha-trans-bergamot alkene synthesized by microorganisms is enhanced, and an effective technical route is provided for green production of industrial high-purity bergamot alkene.

A method for treating tumors using a combination of an oncolytic virus vaccine and immune cells. The method specifically includes the following step: treating the tumors by using a combination of the immune cells and the oncolytic virus vaccine; the oncolytic virus vaccine includes a recombinant oncolytic virus expressing a tumor antigen and is used for targeting the tumor cells; the immune cells express a chimeric antigen receptor paired with the tumor antigen, and are used for killing or destroying the tumor cells targeted; the recombinant oncolytic virus includes an M protein, G protein, N protein, P protein and L protein subjected to site-directed mutagenesis. The tumor antigen expressed by the oncolytic virus vaccine can guide the immune cells to reach the target tumor tissue center, achieving a curative effect where 1+1 is greater than 2, with a tumor cell killing rate being up to 100% at most.

The invention discloses a polypeptide (NP1706, SEQ ID NO: 6) derived from the skin of nanorana parkeri, a polypeptide derivative as well as a composition and application of the polypeptide derivative. A natural parent peptide (NP1711, SEQ ID NO: 11) is optimized by combining a polypeptide activity prediction system with a molecular design technology, systematic mutation screening finds that the 5th site, the 9th site and the 14th site are active non-essential residues, and variant derivatives obtained by performing site-directed mutagenesis on the 5th site, the 9th site and the 14th site do not significantly change the original biological activity. The pharmacological activity and safety parameters of the NP1706 show optimal balance, so that the NP1706 is determined as the optimal candidate polypeptide. Based on the polypeptide and the polypeptide derivative, the activities of promoting cell proliferation and migration, resisting oxidation, resisting inflammation and the like are provided, and the application of the polypeptide and the polypeptide derivative in cosmetics and medicines is provided. The invention has the advantages of simple synthesis, good safety and excellent biological activity, and can be widely applied to the fields of skin repair, aging resistance, wound healing, inflammation resistance and the like.

The invention belongs to the field of plant biotechnology breeding, and relates to an application of a ZmGalAT1 gene and an encoding protein thereof in corn fertility control. According to the invention, a corn male sterility mutant galat1 is obtained by EMS mutagenesis, and a gene ZmGalAT1 for controlling corn male flower development is identified through map-based cloning. The gene is subjected to site-directed mutagenesis in wild type corn through a CRISPR / Cas9 gene editing technology, three galat1 allelic corn male sterility mutants are created, corn male flowers of the mutants are completely aborted, sterile mutants without transgenic ingredients can be obtained through offspring screening, and the mutants are further applied to corn sterile breeding and seed production. The invention also designs functional molecular markers for three allelic male sterile mutants of GalAT1, and has important application value in maize male sterile line cultivation, sterile hybrid seed production and molecular marker-assisted selection.

The invention belongs to the technical field of enzyme gene engineering, and particularly relates to a methionine gamma-lyase mutant with improved enzyme activity and application thereof. The methionine gamma-lyase mutant CqMGLV160I / V265I with high activity is obtained by taking methionine gamma-lyase (CqMGL) which is derived from Calorama quimayensis and of which cysteine residues at 116 sites are replaced by histidine residues as a parent and carrying out site-specific mutagenesis on amino acid residues at key sites through transformation. The methionine gamma-lyase mutant CqMGLV160I / V265I with high activity can be obtained by using the methionine gamma-lyase mutant CqMGLV160I / V265I as the parent. By adopting the CqMGLV160I / V265I mutant disclosed by the invention, the conversion rate of enzyme catalysis can be improved, allicin is efficiently synthesized, and the mutant is successfully applied to the aspects of food, feed, chemical industry, medicine preparation and the like.

The invention belongs to the technical field of gene engineering, and discloses a promoter and application thereof in enhancing target gene expression and promoting mannitol metabolism to synthesize bacterial cellulose. The nucleotide sequence of the promoter P00300 is as shown in SEQ ID NO: 2. The promoter is obtained by site-directed mutagenesis of a wild type promoter P06915 of gluconacetobacter xylinum and screening. The promoter provided by the invention can be used for enhancing the expression of a target gene and producing a target compound. For example, the expression level of fk, mdh1 and mdh2 three-gene combination is remarkably improved, the recombinant strain can efficiently synthesize BC by using mannitol, and the yield reaches 6.1 g / L, is about 2.87 times of that of a wild strain, and is 1.27 times of that of a bla promoter in pBla. The result lays a foundation for further understanding mannitol metabolism of gluconacetobacter xylinum and for high-value conversion and utilization of marine carbon resource mannitol and BC synthesis.

The invention relates to a phospholipase D mutant Lip-Gri / NQ, a plasmid, a recombinant bacterium and application of the phospholipase D mutant Lip-Gri / NQ and belongs to the field of enzyme engineering, the amino acid sequence of the phospholipase D mutant is shown as SEQ ID NO: 1, and the nucleotide sequence of a gene for coding the phospholipase D mutant Lip-Gri / NQ is shown as SEQ ID NO: 2. The invention also provides a recombinant plasmid and an engineering bacterium containing the gene of the recombinant plasmid phospholipase D mutant Lip-Gri / NQ, and application of the phospholipase D mutant Lip-Gri / NQ in catalyzing phospholipids to generate specific-configuration functional lipids including phosphatidylserine. According to the phospholipase D Lip-Gri disclosed by the invention, wild phospholipase D Lip-Gri from Streptomyces griseofuscus is transformed through site-directed mutagenesis, and the enzyme activity of the obtained mutant is remarkably improved and is 11 times that of the wild phospholipase D Lip-Gri.

The application provides an arginine mutant and application thereof in 1,4-butanediamine biosynthesis, and belongs to the field of bioengineering. Escherichia coli The mutant is obtained by site-directed mutagenesis of key site amino acid residues based on wild-type EcAdiA from strain The application improves the enzyme activity stability of arginine decarboxylase in neutral environment, improves the conversion rate and conversion speed of 1,4-butanediamine in neutral environment, and lays a foundation for high-efficiency biosynthesis of 1,4-butanediamine.

The invention discloses a sucrose synthase mutant and a method for preparing RD (Reduced Disorder) by using the sucrose synthase mutant. The sucrose synthase mutant is obtained by mutation of at least one of the following sites on the basis of an amino acid sequence as shown in SEQ ID NO: 2: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R; site-directed mutagenesis is carried out on sucrose synthetase, so that the enzyme activity of the sucrose synthetase mutant is improved compared with that of a wild type, and meanwhile, a part of the mutant still maintains a relatively high conversion rate in a relatively wide pH range.

The application discloses a thermophilic bacteria-derived esterase mutant and application thereof. The application provides a protein, wherein the amino acid residue at the 211th position in the amino acid sequence shown in SEQ ID NO: 4 is mutated, and the amino acid residues at other positions are not changed, so that a protein with PBAT hydrolytic enzyme activity is obtained; or the amino acid residues at the 211th and 256th positions in the amino acid sequence shown in SEQ ID NO: 4 are mutated, and the amino acid residues at other positions are not changed, so that a protein with PBAT hydrolytic enzyme activity is obtained; the wild type Tcur-lipase is mutated by using structure analysis and site-directed mutagenesis technology, and the Tcur-lipase mutants N211A and N211A / D256A are obtained; the Tcur-lipase mutant improves the degradation efficiency of PBAT, and has a good industrial application prospect.

The present invention discloses the use of an oxidoreductase and its mutants in the biosynthesis of naringol, belonging to the field of bioengineering technology. The present invention, for the first time, obtains an oxidoreductase with high conversion capacity for naringol from the marine killer yeast (Wickerhamomyces anomalus M15), and obtains an oxidoreductase mutant with even higher capacity through site-directed mutagenesis. Compared with existing oxidoreductases capable of catalyzing the conversion of naringol, the oxidoreductase and its mutants have good substrate tolerance, high conversion rate, and high salt tolerance. The oxidoreductase and its mutants provided by the present invention provide conditions for the in vitro enzymatic synthesis of naringol, achieving an environmentally friendly and efficient catalytic conversion of naringol to the naringol product. The present invention will provide an important tool enzyme for the synthesis of naringol and bring significant economic benefits to the naringol synthesis industry.

The application discloses a glycerol dehydrogenase mutant and application thereof, and the glycerol dehydrogenase is derived from Klebsiella pneumoniae and is encoded by gene dhaD. The application improves the molecular structure of the glycerol dehydrogenase by rational design and a site-directed mutagenesis technology to obtain a mutant enzyme F245Q / GDH, and the mutant enzyme is back-supplemented into Klebsiella pneumoniae which is deficient in glycerol dehydrogenase. Compared with a wild-type strain, the yield of a main product 1,3-propanediol of the back-supplemented mutant enzyme F245Q / GDH strain is improved, and the yield of a by-product 2,3-butanediol is significantly reduced.

The invention discloses a plurality of mutants of esterase E7, comprising: esterase E7-F145L, the amino acid sequence of which is as shown in SEQ ID NO.5; the amino acid sequence of the esterase E7-F145A is as shown in SEQ ID NO. 4; the amino acid sequence of the esterase E7-Q199F is as shown in SEQ ID NO. 8. The mutant of the esterase E7 is applied to preparation of glyceride, preferably EPAMG. Through semi-rational design, site-directed mutagenesis is carried out on a bridge-shaped structure of esterase E7 from stenotrophomonas maltophilia, a plurality of mutants with remarkably improved catalytic activity are obtained, the effect of the mutant E7-F145L is optimal, the reaction efficiency is greatly improved, the high specificity of a catalytic product is reserved, and the application potential is huge.