534 results about "Site-directed mutagenesis" patented technology

The invention discloses a hydroxysteroid dehydrogenase mutant with improved thermal stability, a screening method and application, key hotspot amino acid residues related to the thermal stability are rapidly identified based on computer-aided design technologies such as hydroxysteroid dehydrogenase structure prediction, PROSS and the like, so that a limited site-directed mutant library is constructed; and further screening to obtain a dominant mutant. The semi-inactivation temperature (T5105) of the obtained mutant is increased by 1.0-7.7 DEG C compared with that before mutation, wherein the semi-inactivation temperatures (T5105) of the mutants A126I and A132H are increased by 7.7 DEG C and 3.7 DEG C respectively. And the relative enzyme activity of the mutant reaches 99.86-136.73% of the relative enzyme activity before mutation. The invention provides a new method for rapidly improving the thermal stability of the enzyme, and lays a foundation for the industrial application of the hydroxysteroid dehydrogenase.

The invention discloses a formate dehydrogenase mutant and application of the formate dehydrogenase mutant in catalysis of carbon dioxide reduction, the 223 arginine (Arg) of an amino acid sequence of formate dehydrogenase PsFDH48 derived from Paracoccus sp. MKU1 is mutated into proline (Pro) or the 242 proline (Pro) is mutated into phenylalanine (Phe) through a site-specific mutagenesis technology, and the obtained mutants R223P and P242F have higher catalytic activity and can be applied to catalysis of carbon dioxide reduction. The catalyst can catalyze CO2 to generate formic acid more effectively in vitro, and has potential application value in the aspects of efficient activation of CO2 and further conversion of CO2 into other mono-carbon compounds through cascade reaction.

The invention discloses a high-stability hydroxysteroid dehydrogenase mutant, a co-immobilized enzyme construction method and application. According to the invention, a site-directed mutant library is constructed based on computer-aided design, and mutants with significantly improved thermal stability and greatly improved catalytic efficiency compared with wild type mutants are obtained through screening; meanwhile, a co-immobilized enzyme construction method based on electrostatic adsorption and chemical crosslinking is established, the hydroxysteroid dehydrogenase mutant and lactic dehydrogenase are co-immobilized, the co-immobilized enzyme can still keep high activity under high substrate concentration, and the conversion rate is still maintained to be 90% or above after 30 batches of co-immobilized enzyme are repeatedly used. And an important foundation is laid for industrial application of the hydroxysteroid dehydrogenase.

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to an alpha-2, 3-sialyltransferase mutant and application thereof. The a-2, 3-sialyltransferase mutants V175N, P198A, G173D, D174Q, F196K and A315H are obtained by performing site-specific mutagenesis on six amino acids in an a-2, 3-sialyltransferase amino acid sequence coded by a gene derived from Campylobacter jejuni (OH4384), the yield of 3 '-sialyllactose is remarkably increased by single-point mutagenesis of the V175N, the yield of 3'-sialyllactose is remarkably increased by single-point mutagenesis of the V175N, the yield of 3 '-sialyllactose is remarkably increased by single-point mutagenesis of the P198A, the G173D, the D174Q, the F196K and the A315H, the optimal recombinant microbial strain 3 '-SL-09 (V175N / P198A / D174Q) is obtained through combination of multiple mutation sites, the yield can reach 5.91 g / L, the bottleneck problem of low catalytic efficiency of sialyltransferase is effectively solved, and an enzyme element with excellent performance is provided for high-efficiency and low-cost biological manufacturing of 3'-SL.

The invention discloses a D-psicose 6-phosphate phosphatase mutant and application of the D-psicose 6-phosphate phosphatase mutant in preparation of psicose, and belongs to the technical field of enzyme engineering. According to the invention, site-directed mutagenesis is carried out on an amino acid sequence of D-psicose 6-phosphate phosphatase A6PP, and a combined mutant I21V / Y73F / R115M / T180V / L203I / Q193E is constructed, so that the substrate specificity, the thermal stability and the catalytic activity of the combined mutant are all remarkably improved. The invention further provides application of the combined mutant in preparation of psicose through multi-enzyme cascade catalysis, 150 g / L of maltodextrin can be converted into 97.7 g / L of D-psicose, the conversion rate reaches 65.1%, and the application prospect of the combined mutant in the field of food is widened.

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.

The application relates to an engineering bacterium for high yield of observation blue and a construction method and application thereof, and belongs to the technical field of biological synthesis of natural dyes. The engineering bacterium for high yield of observation blue expresses icd, bpsA, glnA Y405F and gdhA; the engineering bacterium knocks out acnR, yggB, glsK, aceA and ldh. By knocking out the yggB and aceA genes, performing site-directed mutation (Y405F) on the glnA gene, replacing the glsK gene with the glnA Y405F gene, replacing the acnR gene with the icd gene, replacing the ldh gene with the gdhA gene, and integrating the bpsA gene, the application can block the formation of by-products such as lactate and succinic acid in the observation blue synthesis process, improve the flow direction of the citric acid->isocitric acid->alpha-ketoglutaric acid->glutamic acid->glutamine path, and then improve the intracellular glutamine concentration, so that the yield of observation blue 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 discloses a male sterility gene ZmUCE4 and application thereof in creating a male sterility line of corn, and belongs to the field of plant biotechnology breeding. According to the invention, site-directed mutagenesis of the ZmUCE4 gene in wild type corn is carried out through a CRISPR / Cas9 gene editing technology, pollen development abnormality and abortion can be caused, complete male sterility is caused, and it is proved that the ZmUCE4 gene has a regulation and control function on corn male reproductive development. Through offspring screening, a sterile line without transgenic components can be obtained, a stable maize male sterile line is created, and the method has important significance on maize male fertility control and hybrid seed production. The invention also designs a functional molecular marker aiming at the obtained uce4 male sterility mutant gene, and has important application value in maize male sterility line cultivation, sterile hybrid seed production and molecular marker-assisted selection.

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 invention provides a 2-succinyl-5-enol acetonyl-6-hydroxy-3-cyclohexene-1-carboxylic acid (SEPHCHC) synthase mutant, which is characterized in that on the basis of a bacillus subtilis SEPHCHC synthase gene MenD, 12 SEPHCHC synthase mutants are obtained by virtue of a site-specific mutagenesis PCR (Polymerase Chain Reaction) technology, and the SEPHCHC synthase gene MenD is used as a base. Recombinant bacteria of bacillus subtilis or bacillus natto or bacillus licheniformis or bacillus amyloliquefaciens for expressing the mutant are respectively constructed, and the yield of heptaene menadione fermented by the recombinant bacteria is 74%-203% of the yield of heptaene menadione fermented by the recombinant bacteria of bacillus subtilis for expressing wild type SEPHCHC synthase MenD.

The application discloses a formic acid dehydrogenase mutant and application thereof in catalyzing carbon dioxide reduction. Paracoccus The formic acid dehydrogenase from sp.MKU1 is mutated by a site-directed mutation technology Ps The arginine (Arg) at the 223th position of the FDH48 amino acid sequence is mutated into proline (Pro) or the proline (Pro) at the 242th position is mutated into phenylalanine (Phe), and the obtained mutants R223P and P242F have higher catalytic activity, can more effectively catalyze CO2 to generate formic acid in vitro, and have potential application values in efficient activation of CO2 and further conversion into other one-carbon compounds through cascade reactions.

The invention discloses a chitosanase mutant which is derived from bacillus subtilis and has the capacity of hydrolyzing chitosan to produce chitosan disaccharide per unit and belongs to a GH46 family, and application of the chitosanase mutant in hydrolyzing chitosan to produce chitosan oligosaccharide. Compared with wild type chitosanase capable of producing chitodisaccharide and chitotriose, the chitosanase mutant has the advantage that the chitodisaccharide can be produced per unit when the chitodisaccharide is hydrolyzed by the chitosanase mutant. The invention provides eight kinds of chitosanase mutants, and the mutants are obtained by carrying out site-specific mutagenesis on 21st-site glycine on the basis of wild type chitosanase and respectively mutating the 21st-site glycine into lysine, arginine, leucine, histidine, methionine, tyrosine, proline and glutamine. The mutant can hydrolyze chitosan under mild conditions to produce chitosan oligosaccharide with single polymerization degree, and can greatly relieve the difficulty in separation and purification in chitosan oligosaccharide production.

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 invention discloses an antibacterial peptide IFD targeting gram-negative bacteria as well as a preparation method and application thereof. The amino acid sequence of the antibacterial peptide IFD is shown as SEQ NO.1, and a C terminal is amidated by adopting-NH2. The antibacterial peptide is obtained by modifying 119-132 fragments of human-derived MD-2 protein, and is prepared by site-specific mutagenesis, hydrophobic and hydrophilic sequence insertion optimization and solid-phase chemical synthesis. The antibacterial peptide has high antibacterial activity on Gram-negative bacteria (such as escherichia coli and salmonella typhimurium), the GM Gram-negative bacteria MIC reaches 3.63 mu M and is increased by 5.24 times compared with the original peptide, the targeting property is high, and the hemolysis rate 1t when the hemolysis activity is 128 mu M is low; 3%), the Gram-negative bacteria therapeutic index is as high as 76.1, and the Gram-negative bacteria can be used as a feed antibiotic substitute for preparing drugs for treating Gram-negative bacteria infectious diseases.

The invention discloses a penicillin G acylase mutant with high synthetic activity and low hydrolytic activity and application of the penicillin G acylase mutant, and belongs to the field of bioengineering. The penicillin G acylase gene derived from Escherichia coli is subjected to site-specific mutagenesis, so that the amino acid sequence coded by the penicillin G acylase gene mutates from phenylalanine F to alanine A at the 24th site of an alpha chain, the 146th site of the alpha chain mutates from phenylalanine F to valine V, and the 386th site of a beta chain mutates from serine S to alanine A to obtain the mutant. According to the penicillin G acylase mutant alphaF24A / betaS386A disclosed by the invention, compared with the conversion rate of cefamandole synthesized by catalyzing the reaction of methyl mandelate and 3-(1-methyl-1H-tetrazole-5-yl) thiomethyl-7-aminocephalosporanic acid (7-TMCA) within 4 hours before mutation, the conversion rate of cefamandole synthesized by catalyzing the reaction of methyl mandelate and 3-(1-methyl-1H-tetrazole-5-yl) thiomethyl-7-aminocephalosporanic acid (7-TMCA) is improved by 13.70%, and the yield of a side reaction product mandelic acid is reduced by 82%. The penicillin G acylase mutant disclosed by the invention can be used for catalyzing methyl mandelate and 7-TMCA to synthesize cefamandole while greatly reducing the occurrence of side reactions, and can be applied to preparation of cephalosporin antibiotics.

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 belongs to the technical field of microbial genetic engineering, and particularly relates to an alpha-2, 6-sialyltransferase mutant and application thereof. The a-2, 6-sialyltransferase mutants M133L, E140D, D228H, N289D and G322Q are obtained by carrying out site-specific mutagenesis on five amino acids in an a-2, 6-sialyltransferase amino acid sequence coded by a gene derived from Photobacterium sp.JT-ISH-224, and compared with wild type alpha-2, 6-sialyltransferase, the yield of the single-point mutant 6 '-SL of the E140D and the yield of the single-point mutant 6'-SL of the G322Q are increased by 87.80% and 78.01% respectively. The yield of the optimal recombinant microbial strain 6 '-SL-7 (E140D / G322Q) obtained by mutation site combination can reach 1.471 g / L, the bottleneck problem of low catalytic efficiency of sialyltransferase is effectively solved, and an enzyme element with excellent performance is provided for high-efficiency and low-cost biological manufacturing of 6'-SL.

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.

The invention relates to the field of biosynthesis of glycosaminoglycan compounds, and discloses a 2-O-sulfotransferase mutant and application of the 2-O-sulfotransferase mutant in heparin synthesis. On the basis of a chicken source 2-O-sulfotransferase protein sequence, site-directed mutagenesis design and modification are carried out, mutation sites comprise 6I, V60I, R120K, G118H, S157H, L227P, E270D, V258I and A275P, and the 2-O-sulfotransferase mutant is obtained. Compared with a wild type, the 2-O-sulfotransferase mutant provided by the invention has the advantages that the expression level, the thermal stability and the catalytic efficiency are obviously improved, and the 2-O-sulfotransferase mutant shows higher conversion rate and structural modification consistency in a reaction system taking N-sulfoheparin precursor polysaccharide as a substrate. The mutant has a wide application prospect in the structural precise control and industrial synthesis process of biological heparin.