644 results about "Site-directed mutagenesis" patented technology

The invention relates to an engineering bacterium of high-yield ornamental blue as well as a construction method and application of the engineering bacterium, and belongs to the technical field of natural dye synthesis by a biological method. The engineering bacterium capable of highly producing the ornamental blue, disclosed by the invention, expresses icd, bpsA, glnAY405F and gd hA; acnR, yggB, glsK, aceA and ldh are knocked out from the engineering bacteria. According to the application, yggB and aceA genes are knocked out, site-directed mutagenesis (Y405F) is carried out on a glnA gene, a glsK gene is replaced by a glnAY405F gene, an acnR gene is replaced by an icd gene, an ldh gene is replaced by a gdhA gene, and a bpsA gene is integrated, so that the formation of by-products such as lactate and succinic acid in a blue-viewing synthesis process can be blocked; the flow direction of a passage from citric acid to isocitric acid to alpha-ketoglutaric acid to glutamic acid to glutamine is improved, so that the concentration of glutamine in cells is improved, and the yield of the ornamental blue is improved.

The invention discloses a high temperature resistant xanthan gum lyase mutant and a preparation method thereof, wild type xanthan gum lyase comes from Paenibacillus anensis, site-directed mutagenesis is carried out on the wild type xanthan gum lyase to obtain the high temperature resistant xanthan gum lyase mutant, and the mutant comprises at least one of N66V, R292A and S676V. The enzymatic activity of the xanthan gum lyase mutant treated in a buffer solution with the pH value of 5.0 at the temperature of 65-85 DEG C for 2 h is remarkably improved compared with that of an original enzyme, and the enzymatic activity of N66V / R292A / S676V is improved by 3.8 times compared with that of the original enzyme at the temperature of 85 DEG C.

The invention belongs to the field of bioengineering, and particularly discloses a glutamate oxidase mutant with improved enzymatic activity. According to the invention, site-specific mutagenesis and saturation mutation are carried out on glutamate oxidase coding genes derived from streptomyces mobaraensis, wherein mutations are C536R, P532R, Q378D, V548E and G236D. After the mutant obtained by the invention is induced and purified, the specific enzyme activity of the optimal glutamate oxidase mutant is 1.69 U / mg and is improved by about 30% compared with that of wild bacteria, which shows that the 378-site amino acid residue has great influence on the catalytic action and stability of the enzyme. In addition, the invention also discloses a construction method of the glutamate oxidase mutant with improved enzymatic activity and application of the glutamate oxidase mutant in production of amino acid derivatives such as alpha-ketoglutaric acid. Therefore, the beneficial mutant provided by the invention can lay a good foundation for industrial fermentation production of alpha-ketoglutaric acid and other products.

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 tyrosine phenol lyase mutant and application of the tyrosine phenol lyase mutant in synthesis of levodobar. The tyrosine phenol lyase mutant is obtained by carrying out site-specific mutagenesis on the basis of wild type tyrosine phenol lyase. The mutant obtained through transformation can transform more substrates, the yield is also obviously improved, and the method is suitable for industrial production of levodopa.

The invention relates to the technical field of gene engineering and enzyme engineering, in particular to an esterase mutant for degrading ethyl carbamate and application of the esterase mutant, the esterase mutant has an amino acid sequence as shown in SEQ ID NO.1 which is subjected to site mutation, the site mutation is any one or more site combined mutation of V129S, V161L or I229M, and the amino acid sequence of the esterase mutant is shown in SEQ ID NO.1. The V129S site mutation is that valine at the 129 site is mutated into serine, the V161L site mutation is that valine at the 161 site is mutated into leucine, and the I229M site mutation is that isoleucine at the 229 site is mutated into methionine. Mutation is carried out through a site-directed mutagenesis method, so that the amino acid sequence of the esterase mutant V129S / I229M is changed, the autoplasmic structure and function of eggs are changed, the esterase mutant V129S / I229M with the ethyl carbamate hydrolysis rate increased to 6.55 times in an acid environment is finally obtained, and the esterase mutant V129S / I229M has high industrial application value.

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 relates to laccase as well as a mutant and application thereof, and belongs to the technical field of biology. The amino acid sequence of the laccase PnLac1 is as shown in SEQ ID NO. 2, and the nucleotide sequence of the laccase PnLac1 is as shown in SEQ ID NO. 1. The mutant is characterized in that aspartic acid at the 166th position of laccase PnLac1 is subjected to site-specific mutagenesis, histidine at the 348th position of laccase PnLac1 is subjected to site-specific mutagenesis, methionine at the 403th position of laccase PnLac1 is subjected to site-specific mutagenesis, or leucine at the 467th position of laccase PnLac1 is subjected to site-specific mutagenesis, amino acid sequences are shown as SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and SEQ ID NO.10, and nucleotide sequences are shown as SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9. The decoloring efficiency of the laccase PnLac1 on crystal violet reaches 5h, and the degradation rate reaches 75%. When the ABTS is used as a substrate, the enzyme activities of the laccase mutants H348F and L467M are respectively improved by 1.89 times and 1.65 times, and the ABTS has the potential of degrading stubborn substances; when guaiacol is used as a substrate, the enzyme activities of the laccase mutants D166A and M403F are respectively improved by 2.07 times and 3.72 times, and the guaiacol has a relatively high application prospect in the field of lignin.

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 belongs to the technical field of bioengineering, and particularly relates to an alkaline protease mutant which is obtained through molecular modification and has improved enzyme activity and thermal stability and application of the alkaline protease mutant. An ALK wild type is mutated by utilizing a site-directed mutagenesis technology to obtain a mutant T56C / A90C of which the enzyme activity is improved relative to that of the wild type at 40 DEG C, and the mutant T56C / A90C is subjected to heat preservation at 60 DEG C for 4 hours to determine the protease activity and residual enzyme activity and has certain activity in a high-temperature environment, so that the heat stability of alkaline protease is improved, and the alkaline protease can play a role in a relatively high alkaline environment; therefore, the method can be widely applied to a plurality of industries such as washing, leather, medicine, feed, silk, food and the like.

The invention discloses an L-aspartic acid transaminase mutant and application thereof. The L-aspartic acid transaminase mutant is obtained by performing single mutation on the 104th amino acid of the amino acid sequence of L-aspartic acid transaminase. The L-aspartic acid transaminase mutant is used for synthesizing an L-phenylglycine derivative. The L-aspartic acid transaminase mutant is from escherichia coli, homologous sequence analysis and molecular docking simulation are carried out, site-specific mutagenesis verification is combined, and single-point mutation is carried out on the 104 amino acid to obtain the T104S mutant. The L-aspartic acid transaminase mutant disclosed by the invention can be used for efficiently catalyzing to generate an L-phenylglycine derivative, and compared with a wild type, the specific activity of the T104S mutant to o-chlorophenoxyacetic acid is 3.2 times that of the wild type.

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 invention discloses a diene lactone hydrolase mutant and an application thereof. The protein provided by the invention is a diene lactone hydrolase mutant, the 184th amino acid residue on an amino acid sequence shown as SEQ ID NO: 2 is mutated, and the amino acid residues at other positions are not changed, so that the protein with PBAT hydrolase activity is obtained; the PmDLH mutant T184H is obtained by mutating wild type PmDLH by utilizing a structural analysis and site-directed mutagenesis technology, and compared with the wild type PmDLH, the PmDLH mutant T184H has the advantages that the problem of low enzyme activity of the wild type PmDLH is solved, the activity of the PmDLH in degrading polyester plastic PBAT is effectively improved, the degradation effect of the PmDLH is improved, and the industrial application prospect is good.

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.

The invention belongs to the technical field of gene engineering, and relates to high-temperature-resistant laccase, a gene and application of the high-temperature-resistant laccase in mycotoxin degradation. The amino acid sequence of the laccase TtLac-4 is as shown in SEQ ID NO: 1. On the basis of wild laccase, site-directed mutagenesis modification is carried out through artificial rational design, and an amino acid sequence of the modified high-temperature-resistant laccase and a nucleotide sequence of the artificially optimized high-temperature-resistant laccase gene are obtained. The high-temperature-resistant laccase obtained through gene coding of the high-temperature-resistant laccase has the remarkably-improved high-temperature-resistant characteristic, and the optimum temperature reaches 90 DEG C. The degradation rate of the laccase TtLac-4 obtained by the invention on mycotoxin aflatoxin B1 and zearalenone reaches 100%. The invention lays a foundation for a green degradation process and application of mycotoxin degradation.

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 technical field of bioengineering, and particularly relates to an activity-improved tyrosinase mutant obtained by site-directed mutagenesis through an overlapping PCR technology, and preparation and application thereof. The tyrosinase mutant is obtained by mutation containing F337I / F352I on the basis of wild type tyrosinase as shown in SEQ ID NO.1, the enzyme activity of the tyrosinase mutant is obviously improved compared with that of the wild type tyrosinase, the F337I / F352I mutant gene is used for constructing a recombinant vector, and the recombinant vector is successfully expressed in bacillus subtilis WB600, bacillus amyloliquefaciens CGMCC No.11218 and bacillus licheniformis 2709, so that the tyrosinase mutant is obtained. The recombinant strain with improved enzyme production activity is further optimized through a fermentation process to obtain the novel tyrosinase.

The invention provides a glucamylase mutant and application thereof, and belongs to the technical field of bioengineering. The method comprises the following steps: training an amino acid sequence and three-dimensional structure data of GLL by utilizing a machine learning algorithm, and selecting specific amino acid sites according to a prediction result of a machine learning model to carry out site-specific mutagenesis of six amino acids to obtain a glucamylase mutant; the amino acid sequence of the glucamylase mutant is as shown in SEQ ID NO: 5, and the nucleotide sequence for coding the glucamylase mutant is as shown in SEQ ID NO: 6. Compared with a wild type, the glucamylase mutant has higher hydrolytic activity and thermal stability, has important significance in improving the production efficiency and reducing the cost, and has good practicability.

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.

The invention relates to pectin lyase as well as a mutant, a recombinant expression vector and application thereof. The nucleotide sequence of the pectin lyase is as shown in SEQ ID NO.1, the full length of the gene is 1056 bp, 352 amino acids are coded in total, and the amino acid sequence is as shown in SEQ ID NO.2; the pectin lyase mutant is formed by site-directed mutagenesis on the basis of an amino acid sequence of pectin lyase, a mutated nucleotide sequence is as shown in SEQ ID NO.3, and the amino acid sequence is as shown in SEQ ID NO.4; compared with the existing pectin lyase, the pectin lyase and the mutant thereof provided by the invention have the characteristics of high activity and temperature stability, and can be widely applied to fruit juice extraction, fruit juice clarification and fruit and vegetable juice beverage processing.

The invention provides an L-tryptophan production strain as well as a construction method and application thereof. The strain lacks tnaA, serA, trpR and trpL genes; the transcription of aroC and tktA genes is enhanced by using a tac promoter, and the aroC and tktA genes are respectively integrated to tnaA and trpR sites; according to the present invention, the trpE gene in the genome is subjected to site-directed mutagenesis solution feedback, the tryptophan feedback inhibition is relieved, the constructed strain has advantages of good tryptophan synthesis ability, stable performance and high acid production efficiency, especially by carrying the medium copy plasmid pTtas, the finally obtained L-tryptophan production strain has the clear genetic background, the plasmid pTtas carried by the strain has the antibiotic-free screening tag, and the obtained L-tryptophan production strain has the broad application prospect. The strain is high in yield, free of antibiotic pressure screening, good in L-tryptophan production performance and high in sugar-acid conversion rate, and L-tryptophan can be stably and efficiently produced or accumulated through a microorganism direct fermentation method.

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 provides a site-directed mutated influenza virus gene non-coding region nucleic acid fragment and its application in improving the expression efficiency of mRNA vaccines. The provided site-directed mutated influenza virus gene non-coding region nucleic acid fragment has a sequence of SEQ ID NO: 3. The present invention also provides an mRNA fragment for translating and expressing in a host, which contains a coding frame for encoding a target protein, and the 5′UTR and / or 3′UTR of the mRNA fragment is the site-directed mutated influenza virus gene non-coding region nucleic acid fragment. In view of the problems that the existing mRNA vaccine has low translation efficiency, relatively insufficient activation of the immune system, and difficulty in producing the expected immune protection effect, the present invention provides a method for site-directed mutagenesis of a non-coding region derived from an influenza virus M gene fragment, which can be used as the 5′UTR and / or 3′UTR of the target expression sequence, and can improve the translation efficiency of the target mRNA.

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.