256 results about "Directed mutagenesis" patented technology

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.

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.

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 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.

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 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 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.

The application discloses a multi-enzyme immobilized enzyme for glucose-6-phosphate synthesis, which is obtained by immobilizing three enzymes, starch debranching enzyme mutant AcDBE_E131R, maltodextrin phosphorylase mutant AtMalP_N188D and glucose phosphomutase mutant TkPGM_F360P, on a resin. The application also discloses a preparation method and application of the multi-enzyme immobilized enzyme. The application screens enzyme mutants with good thermal stability and high catalytic activity by site-directed mutagenesis of starch debranching enzymes, maltodextrin phosphorylases and glucose phosphomutases from different sources, and prepares the multi-enzyme immobilized enzyme AcDBE_E131R+AtMalP_N188D+TkPGM_F360P. The multi-enzyme immobilized enzyme is used as a catalyst, maltodextrin is used as a substrate, and glucose-6-phosphate is efficiently synthesized by one-pot method, and the conversion rate reaches 71.2%. After 50 times of reuse, the relative enzyme activity of the multi-enzyme immobilized enzyme still remains 85.1%.

The invention relates to the field of hemostatic dressings, in particular to a preparation method of a sandwich type antibacterial hemostatic dressing. Collagen mutants are obtained through site-specific mutagenesis, the mutants with the good effect are screened to prepare the sandwich type antibacterial hemostatic dressing, the hemostatic capacity of the sandwich type antibacterial hemostatic dressing can be remarkably improved, the wound adhesion degree can be reduced, meanwhile, the sandwich type antibacterial hemostatic dressing has the good antibacterial effect, and the sandwich type antibacterial hemostatic dressing has the good application prospect in biological hemostatic materials.

The application provides a kind of aromatic isopentenyl transferase mutant, which is based on the aromatic isopentenyl transferase gene MenA derived from Bacillus subtilis ( Bacillus subtilis ) and six aromatic isopentenyl transferase mutants MenA-1 to MenA-6 are obtained by site-directed mutagenesis PCR technology, and the recombinant bacteria Bacillus subtilis expressing the above mutants are constructed, respectively. The yield of heptamethylnaphthoquinone fermented by the recombinant bacteria is 64.49 mg / L, 69.30 mg / L, 83.86 mg / L, 105.84 mg / L, 119.23 mg / L and 125.45 mg / L, respectively, which is 133%, 143%, 173%, 219%, 246% and 259% of the yield of heptamethylnaphthoquinone fermented by the Bacillus subtilis expressing the wild-type aromatic isopentenyl transferase MenA.

The invention provides an in-vitro transcription method based on RNA polymerase and a kit, which are suitable for the fields of mRNA synthesis, gene research and biological pharmacy. According to the method, the problems of low efficiency, poor stability and the like of traditional in-vitro transcription are solved through an optimization technology. The method comprises the following specific steps: providing a linear DNA template containing a T7 promoter; according to the method, site-directed mutagenesis or fusion protein modified high-activity T7RNA polymerase is combined with chemically modified NTPs and a spermine-BSA compound stabilizer to react under the conditions that the temperature is 37-42 DEG C and the pH is 7.5-8.5, and formation of double-stranded RNA is promoted through dynamic temperature control. A reaction system contains Mg < 2 + > / Mn < 2 + > and DTT, and enzyme activity and product integrity are optimized. The kit comprises an improved reagent and a standardized process, the transcription efficiency is improved by 2-5 times, the product yield is greater than or equal to 80%, and the activity is not lost after the kit is stored at 4 DEG C for 1 month. The method has the technical advantages that NTPs degradation resistance is achieved, template preparation is simplified, universality is high, the method is suitable for large-scale production of mRNA vaccines, gene therapy and the like, the production cost is remarkably reduced, and wide application of the in-vitro transcription technology is promoted.

This invention discloses the application of arginine-depleted engineered bacteria for the treatment of soft tissue sarcomas, including preliminary validation, obtaining qualified engineered bacteria SGR1, obtaining drug compositions and regimens, experimental conclusions, and clinical application promotion. During use, clinical soft tissue sarcoma samples are collected, including undifferentiated pleomorphic sarcoma, synovial sarcoma, and liposarcoma. Immunohistochemistry (IHC) is used to detect ASS1 protein expression, qPCR is used to detect ASS1 gene transcription levels, and the proportion of ASS1 deletion / low expression is statistically analyzed. Arginine auxotrophic sarcoma subpopulations are confirmed based on literature. Cells are cultured in arginine-containing or arginine-free media, and cell viability, colony formation, cell cycle, and apoptosis are detected. The sensitivity of ASS1-deficient cells to arginine deprivation is verified, and therapeutic targets are identified. The enzyme activity, substrate specificity, and immunogenicity of human ARG I / II, mycoplasma ADI, and Pseudomonas ADI are compared. High-activity, low-immunogenic enzymes or enzyme combinations (such as human ARG I + microbial ADI) are selected, and site-directed mutagenesis or codon optimization is performed on microbial ADI to increase expression levels.

This invention discloses a nicotinamide phosphoribosyltransferase mutant, a recombinant strain, and a method for constructing the same. The mutants are I457V, G344A, Q378A, T283G, or F182A, obtained by single-point mutation of the wild-type nicotinamide phosphoribosyltransferase shown in SEQ ID NO:1. This invention utilizes a method derived from... Schizotequatrovirus By rationally designing and site-directed mutagenesis of KVP40 nicotinamide phosphoribosyltransferase, combined with metabolic engineering to block the NMN degradation pathway and enhance substrate synthesis and product transport, the yield of β-NMN by the recombinant strain during shake-flask fermentation reached 4.82 g / L. Through optimization of fed-batch fermentation conditions, the highest yield of β-NMN during fed-batch fermentation in shake flasks reached 15.60 g / L.

This invention provides the ketone reductase RasADH-E189D and its applications. The RasADH-E189D enzyme was obtained through site-directed mutagenesis, and its amino acid sequence is shown in SEQ ID NO.4. RasADH-E189D can efficiently and stereoselectively catalyze the asymmetric reduction reaction of aryl-substituted alkenyl 3,5-dicarbonyl esters, thereby preparing high-purity chiral aryl-substituted alkenyl β-hydroxy esters, which has important application value in the field of chiral drug synthesis.

This application relates to the biomedical technology field, and more specifically to a tumor therapy method combining recombinant oncolytic viruses and small molecule anticancer drugs. Specifically, it includes the step of treating a tumor using recombinant oncolytic viruses and small molecule anticancer drugs in combination, wherein the small molecule anticancer drugs include ALK-targeting small molecule anticancer drugs, BTK-targeting small molecule anticancer drugs, EGFR-targeting small molecule anticancer drugs, FGFR-targeting small molecule anticancer drugs, HER2-targeting small molecule anticancer drugs, Parp-targeting small molecule anticancer drugs, PI3K-targeting small molecule anticancer drugs, VEGFR-targeting small molecule anticancer drugs, CDK4 / 6-targeting small molecule anticancer drugs, and KRAS-targeting small molecule anticancer drugs, and the recombinant oncolytic viruses include site-directed mutagenesis of M protein, G protein, N protein, P protein, and L protein. In this application, it is possible to achieve an effect greater than 1+1 by using recombinant oncolytic viruses and low-molecular-weight anticancer drugs in combination to attack and kill tumor cells.

The invention relates to the technical field of enzyme engineering, and particularly discloses a phytase mutant and application thereof. The mutant T273K with improved heat resistance is obtained through rational design and site-directed mutagenesis screening on the basis of aspergillus tubingensis phytase. The thermal stability and catalytic ability of the mutant are obviously improved compared with those before mutation; after heat preservation is carried out for 90 min at the temperature of 60 DEG C, the relative enzyme activity is 69.4%, and compared with WT, the relative enzyme activity is improved by 20.4%; the specific activity is improved by about 77.78% compared with that of WT, and the catalytic efficiency kcat / Km is improved by 50.56% compared with that of WT; and the enzyme activity retention rate reaches 33.8% under the condition of 10% salinity. The phytase mutant T273K has the comprehensive characteristics of salt resistance, heat resistance and the like, the phytase mutant T273K can better exert the phytic acid enzymolysis effect in the food processing process, turbidity generated by phytic acid in liquid food such as soy sauce, vinegar, cooking wine and the like can be remarkably reduced, so that the product quality is improved, and the phytase mutant T273K has a wide application prospect.