43 results about "Double mutant" patented technology

The invention discloses a TAPC esterase mutant with improved thermal stability as well as a construction method and application of the TAPC esterase mutant. The TAPC esterase mutant is obtained by mutating on an amino acid sequence as shown in SEQ ID NO.2, and a mutation site is selected from one or a combination of more than one of S275Y, S326F, S326M, G266M, R321G, H385L, S84E and H297L. The TAPC esterase mutant provided by the invention comprises eight single-site mutants and two double mutants, and compared with wild type TAPC esterase, the mutant has a longer half-life period at 49 DEG C; the double mutant has a better effect, and the half-life period of the double mutant is about 13 times of that of wild TAPC esterase. The TAPC esterase mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when an ester-containing compound is catalytically hydrolyzed at a higher temperature, and has higher industrial application potential.

The application discloses a 17beta-hydroxysteroid dehydrogenase mutant, an engineering bacterium and application, and uses the 17beta-hydroxysteroid dehydrogenase mutant to biotransform methyl diketone as a substrate to prepare nandrolone. The 17beta-hydroxysteroid dehydrogenase is 17beta-hydroxysteroid dehydrogenase (17beta-HSDcl) from a fungus Cochliobolus lunatus, and the mutant is a single mutant in which histidine His at the 164th position is mutated into valine Val, a single mutant in which threonine Thr at the 151st position is mutated into alanine Ala, or a double mutant in which the 164th position and the 151st position are jointly mutated. The application uses pET28a as an expression plasmid, realizes heterologous expression of the 17beta-HSDcl mutant in E.coli BL21 (DE3), and through 24h whole cell transformation of the engineering bacterium, the nandrolone yield of the single mutant H164V, the single mutant T151A and the double mutant H164V / T151A is 1.27 times, 1.30 times and 1.34 times that of before mutation, respectively. The application provides a basis for industrialized production of nandrolone by microorganisms.

The application discloses application of a PRR5 gene in regulating early flowering of soybean under high temperature, and belongs to the field of genetic engineering. The application is directed to a soybean PRR5 gene, and a double mutant prr5a prr5b in which PRR5a shown in a nucleotide sequence as shown in SEQ ID NO. 1 and PRR5b shown in a nucleotide sequence as shown in SEQ ID NO. 2 are simultaneously knocked out is constructed by using CRISPR technology. Experimental results show that the double mutant has a flowering time under high temperature (30 DEG C) consistent with a flowering time under normal temperature (25 DEG C), and the response mechanism of the soybean in which the PRR5 gene is knocked out is relieved from early flowering under high temperature. The application provides a new idea, technical means and biological material for changing early flowering of the soybean under high temperature and enhancing production management of the soybean under high temperature. The application has a wide application prospect in research and high-yield breeding of the soybean in response to high temperature stress, and is helpful to promoting development of a soybean breeding industry.

The application belongs to the technical field of biology, and specifically provides a reverse transcriptase UCRT vZ mutant with improved thermal stability, the reverse transcriptase UCRT vZ mutant includes four single-point mutants, six double mutants and four triple mutants, compared with a wild-type reverse transcriptase UCRT vZ, the half-life of the mutants is longer at 65 DEG C, the effect of the double mutants is better, and the half-life is about 4 times that of the wild-type reverse transcriptase UCRT vZ. The application further provides a genetically engineered bacterium containing the mutant. The reverse transcriptase UCRT vZ mutant obtained by the construction method has better thermal stability, and when used for RNA reverse transcription into cDNA at a higher temperature, the reverse transcriptase UCRT vZ mutant has higher thermal stability and great application potential.

The invention relates to a method for the preparation of industrially useful strains of yeast Saccharomyces cerevisiae and the strain prepared by the method. In particular the invention relates to the creation of double mutant strains of the yeast that shows high ethanol conversion efficiency and substantially reduced acetic acid and glycerol production during fermentative production of ethanol from a sugar-based feedstock.

The invention belongs to the field of biotechnology and plant breeding, and discloses a method for creating haploids by using rice endogenous genes. The invention relates to an application of an OsKPL1 gene or an OsKPL1 gene and an OsKPL2 gene in creating a rice haploid induction system or creating a rice haploid, and the gene number of the OsKPL1 gene is LOCOs04g51260, and the gene number of the OsKPL2 gene is LOCOs04g51290. Researches show that a single mutant of the oskpl1 has haploid inducibility of about 0.5%, double mutants of the oskpl1 and the oskpl2 have haploid inducibility of about 1.6%, and additional gene resources are provided for haploid breeding in rice.

The invention discloses application of double-mutant zebrafish in preparation of a mast cell function defect animal model. The double-mutant zebra fish disclosed by the invention is cpa1- / -cpa5- / -double-mutant zebra fish, and is obtained by further knocking out a cpa1 gene on the basis of a cpa5- / -zebra fish mutant. According to the method, potential compensation effects of two key carboxypeptidases are eliminated at the same time, and the storage and release capacity of mast cell protease is remarkably weakened from the genetics level, so that the zebrafish animal model with the mast cell function defect, which is clear in phenotype and stable in function, is obtained. The animal model can be used for mast cell function mechanism research, related disease mechanism exploration and drug screening and evaluation, and has a good application prospect.

This invention discloses the application of reducing the expression of negatively regulated genes in improving resistance to Phytophthora sojae, belonging to the field of plant genetic engineering. Research has found that the soybean genes GmTIFY5a or GmTIFY5b have a negative regulatory function on resistance to Phytophthora sojae. Knocking out GmTIFY5a or GmTIFY5b, or a combination of GmTIFY5a and GmTIFY5b, can enhance soybean resistance to Phytophthora sojae. This can be applied to improving soybean resistance to diseases caused by Phytophthora sojae or to breeding soybean varieties resistant to these diseases. In the soybean cultivar "Williams 82," a double mutant lacking both GmTIFY5a and GmTIFY5b, the accumulation of Phytophthora sojae was reduced by 50-60% compared to the wild type, the lesion infection area was reduced by 80-90%, and the survival rate was increased by 20-30%. Knocking out GmTIFY5a and GmTIFY5b significantly improved plant resistance to Phytophthora sojae. By genetically engineering the above genes to combat plant root rot caused by septicemia, we can not only provide important genetic resources for plant molecular breeding, but also provide an effective means to achieve high plant yields.

The invention discloses a sucrose isomerase mutant with improved thermal stability and a construction method thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the sucrose isomerase mutant, the thermal stability and the expression quantity are simultaneously improved, the mutant is used for preparing isomaltulose, the half-life period of the single mutant V447E at the temperature of 40 DEG C is prolonged by 1.38 times, the optimum temperature is increased to 35 DEG C from 30 DEG C, and the expression quantity is increased to 33.06 U * mL <-1 > from 18.89 U * mL <-1 >; other mutations are superposed on the basis of the mutant V447E, and the thermal stability of the constructed double mutant V447E / D232P is further improved. Compared with a wild type, the V447E / D232P has the advantages that the optimal temperature is increased by 5 DEG C, the half-life period at 40 DEG C is prolonged by 1.52 times, and the expression quantity is increased from 18.89 U.mL <-1 > to 30.73 U.mL <-1 >.

The invention relates to the technical field of biology, in particular to a Zymomonas mobilis transcriptional activation system and application thereof.The transcriptional activation system comprises crRNA of a target gene promoter regulatory region and a sequence of a coding effect protein complex, dCas12a losing nuclease activity and a transcriptional activation factor SoxS double mutant (R93A / S101A) are subjected to fusion expression, and the Zymomonas mobilis transcriptional activation system is obtained. Or through mediation of SpyTag / SpyCatcher, the two are coupled, so that an effect protein complex is constructed, and effective transcriptional activation can be carried out on different exogenous promoters and zymomonas mobilis endogenous promoters.

The application belongs to the technical field of biology, and specifically provides a reverse transcriptase UCRT vL mutant with improved thermal stability, which includes four single-point mutants, six double mutants and four triple mutants, and the half-life of the mutants is longer than that of the wild-type reverse transcriptase UCRT vL at 65 DEG C; the triple mutants have better effects, and the half-life is about 3 times that of the wild-type reverse transcriptase UCRT. The application also provides a genetically engineered bacterium containing the mutants. The reverse transcriptase UCRT vL mutant obtained by the construction method has better thermal stability, and when used for RNA reverse transcription into cDNA at a higher temperature, the reverse transcriptase UCRT vL mutant has higher thermal stability and great application potential.

The invention discloses an application of FLOE2 protein and FLOE3 protein in regulation and control of plant disease resistance. A floe2 single mutant and a floe3 single mutant are hybridized to obtain a floe23 double-mutant material in which both an FLOE2 gene and an FLOE3 gene are mutated, and the analysis on the disease resistance of the floe23 double-mutant material shows that compared with wild arabidopsis thaliana, when the floe23 double-mutant material is infected by pseudomonas syringae carrying Hopz1 effect protein, the disease resistance of the floe23 double-mutant material is remarkably improved, and the disease resistance of the floe23 double-mutant material is remarkably improved. The resistance to pseudomonas syringae is obviously reduced. It is shown that the FLOE2 protein and the FLOE3 protein or biological materials related to the FLOE2 protein and the FLOE3 protein can regulate and control the resistance of plants to the pseudomonas syringae induced by the Hopz1 effect protein and can be used for cultivating disease-resistant plant varieties in agricultural production. The invention is of great significance to the cultivation of disease-resistant plant varieties.

The invention discloses a sorbose dehydrogenase mutant capable of efficiently degrading vomitoxin, and belongs to the technical field of agricultural biology. The mutant is based on wild type SDH, through rational design and a directed evolution strategy, the mutant comprises three mutation sites F103A, T492E and N427A, the amino acid sequence and the nucleotide sequence of the mutant are SEQ ID NO.1 and SEQ ID NO.2 respectively, the mutant SDHF103A / T492E / N427A can efficiently degrade DON without depending on phenazine methyl sulfate (PMS), and the degradation activity of the mutant SDHF103A / T492E is improved by about two times compared with that of a double mutant SDHF103A / T492E under the condition that PMS does not exist. The method can be applied to preparation of the vomitoxin detoxification enzyme, and has a good application prospect in the aspect of biological detoxification of DON in feeds, grains and food.

The present invention discloses a TAPC esterase mutant with improved thermal stability, a construction method, and applications thereof. The TAPC esterase mutant is mutated in the amino acid sequence shown in SEQ ID NO. 2, and the mutation site is selected from one or a combination of more than one of S275Y, S326F, S326M, G266M, R321G, H385L, S84E, and H297L. The TAPC esterase mutants provided by the present invention include 8 single-point mutants and 2 double mutants. Compared with the wild-type TAPC esterase, the mutants have a longer half-life at 49°C; the double mutant is even more effective, with a half-life approximately 13 times that of the wild-type TAPC esterase. The TAPC esterase mutant obtained by the construction method provided by the present invention has improved thermal stability and exhibits high thermal stability when catalyzing the hydrolysis of ester-containing compounds at higher temperatures, thus having great potential for industrial application.

The present invention provides a nitrilase mutant and application thereof in the synthesis of 1-cyanocyclohexyl acetic acid, the nitrilase mutant is obtained by mutating one or two of the amino acids at position 180 and 205 of the amino acid sequence shown in SEQ ID No. 2. In the present invention, by semi-rational design and protein molecular modification, the specific enzyme activity of the nitrilase double mutant AcN-G180D / A205C was increased by up to 1.6 folds, and the conversion rate>99%. And the reaction time was shortened to a quarter of the original using the recombinant Escherichia coli containing the nitrilase mutant to hydrolyze 1-cyanocyclohexylacetonitrile at high temperature (50° C.). Therefore, the mutants obtained by the present invention have a good application prospect in efficiently catalyzing 1-cyanocyclohexylacetonitrile to synthesize gabapentin intermediate, 1-cyanocyclohexyl acetic acid.

The invention discloses a leucine transporter mutant capable of reducing methionine transport activity and application of the leucine transporter mutant, and belongs to the technical field of bioengineering. Compared with a wild leucine transporter with an amino acid sequence as shown in SEQ ID NO.1, the mutant provided by the invention contains amino acid mutation at the 30th site, the 108th site or the 123th site, and the amino acid mutation is F30K, F30P, S108F, S108Y or L123R respectively. In order to improve the ACC transfer efficiency, the invention also provides double mutations of S108F / L160Y. Experiments show that compared with a wild type, the Escherichia coli strain of the leucine transporter mutant has the advantages that the sensitivity to methionine analogues is obviously improved, and the concentration of methionine in the strain can be obviously improved. Meanwhile, the double mutants can effectively improve the ACC tolerance of escherichia coli. Therefore, the method has a good application prospect in the field of ACC fermentation preparation.

The present invention relates to a double mutant heat labile enterotoxin gene comprising a spacer and, more specifically, to a use of a heat labile enterotoxin protein, encoded by the gene, for enhancing immunity. It was found that when the double mutant heat labile enterotoxin gene comprising a spacer according to the present invention is introduced to host cells, the translations of LTA and LTB constituting the labile enterotoxin gene do not affect each other, and thus LTB expression increases more than fivefold. In addition, it was experimentally confirmed that the expressed double mutant heat labile enterotoxin protein exhibits weak enterotoxic activity and simultaneously activates humoral and cellular immunity. Thus, the double mutant heat labile enterotoxin gene according to the present invention can be variously utilized in the fields of vaccines and vaccine adjuvants.

The invention belongs to the technical field of plant biotechnology and crop molecular breeding, and particularly relates to an application and a method of a replication gene pair for inducing development of maize spontaneous endosperm and embryos, and the application of the replication gene pair for inducing development of maize spontaneous endosperm and embryos to ZmFIE develops a new approach for apomixis of maize. It is proved that ZmFIE2 (and the synergistic effect of ZmFIE2 and ZmFIE1) is a key factor for maintaining fertilization dependence of corn seed development, corn can be induced to generate autonomously developed endosperm and embryoid structures under the condition that exogenous genes are not introduced through mutation of the gene, and a core element is provided for fixing heterosis. An efficient germplasm innovation material is provided, the zmfie1 and zmfie2 double mutant shows more severe phenotype than a single mutant, endosperm cell differentiation in mutant grains is blocked, intracellular replication is remarkably reduced, mitotic activity is enhanced, and the strong cell cycle change is a characteristic necessary for constructing an efficient haploid induction line.

This invention discloses a strain for the efficient synthesis of D-tagatose, its preparation method, and its applications. Through strategies such as the discovery of novel galactitol dehydrogenase and promoter engineering, this invention initially increased tagatose yield by 28.48%. Simultaneously, a highly efficient and sensitive tagatose biosensor was developed, which was used as a screening tool for xylose reductase. Ss XR molecularly modified the strain to obtain the F128M-Q219K double mutant, which achieved a tagatose yield of 5.39 g / L in the producing strain. Based on this, amino acid complementation optimization was performed on the high-yielding strain, and the final engineered strain achieved a tagatose yield of 44.61 g / L in a 5 L bioreactor. This study provides a new strategy and core technology support for the efficient biosynthesis of tagatose.

The invention belongs to the technical field of soybean genetic breeding, and particularly relates to application of a soybean BRC1 gene and an editing site thereof to improvement of soybean yield traits and / or plant type traits. The invention provides an application of a BRC1 gene in improvement of soybean yield traits. The BRC1 gene is a BRC1c gene and / or a BRC1d gene. The sequence of the BRC1c gene is as shown in SEQ ID NO. 1; and the sequence of the BRC1d gene is as shown in SEQ ID NO. 3. The soybean brc1c brc1d double mutant is obtained through a CRISPR / Cas9 technology, and research results show that the plant height of the soybean brc1c brc1d double mutant is reduced, the internode spacing is shortened, and the number of branches, the number of pods of a single plant and the grain weight of the single plant are increased. The BRC1c and BRC1d genes are utilized to improve soybeans, internode short density-tolerant soybean germplasm resources can be created, high-yield soybean varieties can be cultivated, and the BRC1c and BRC1d genes have good application prospects and value.

The application belongs to the technical field of biology, and specifically provides a UCRT heat-resistant reverse transcriptase mutant with improved thermal stability, the mutant including four single-point mutants, six double mutants and four triple mutants, compared with the wild-type UCRT III heat-resistant reverse transcriptase, the mutant has a longer half-life at 65 DEG C; the double mutant has a better effect, and the half-life thereof is about 4 times that of the wild-type DNA polymerase. The application further provides a genetically engineered bacterium containing the mutant. The UCRT heat-resistant reverse transcriptase mutant obtained through the construction method has better thermal stability, and when reverse transcription and DNA synthesis are performed at a higher temperature, the mutant has higher thermal stability, and has great application potential.

The invention discloses a glucosyltransferase mutant and application thereof, and the glucosyltransferase mutant comprises six single mutants and six double mutants; the six single mutants are A146S, T286R, T286W, L344V, F378G and F378S, the six double mutants are A146S / T286R, A146S / L344V, A146S / F378G, T286R / L344V, T286R / F378G and L344V / F378G, and the gene sequences of the double mutants are respectively as shown in SEQ ID NO. 1 to SEQ ID NO. 12. According to the method, mutation sites are predicted through computer-aided semi-rational design; the predicted mutant is expressed in an escherichia coli chassis and used for preparing the anthocyanin, the glycosylation efficiency is remarkably improved, efficient production of the anthocyanin is achieved, a new technical route is provided for industrial production of the anthocyanin, and the method is safe and environmentally friendly.

The application discloses a leucine transporter mutant with reduced methionine transport activity and application thereof, and belongs to the technical field of bioengineering. The mutant provided by the application contains amino acid mutations at positions 30, 108 or 123, i.e. F30K, F30P, S108F, S108Y or L123R, relative to a wild-type leucine transporter with an amino acid sequence as shown in SEQ ID NO. 1. In order to improve the ACC transport efficiency, the application simultaneously provides a double mutation of S108F / L160Y. Experiments show that, compared with the wild type, the E. coli strain of the leucine transporter mutant has significantly improved sensitivity to methionine analogs, and can significantly improve the methionine concentration in the strain. Meanwhile, the double mutant can effectively improve the tolerance of E. coli to ACC. Therefore, the application has a good application prospect in the field of ACC fermentation preparation.

The application discloses a kind of xylanase activity enhanced mutant optimized by gating adjacent residues and application thereof.The application takes GH11 family xylanase XynLC9 derived from Bacillus subtilis as research object, carries out modeling to its structure using AlphaFold 3, carries out site-directed saturation mutation and combination mutation to candidate site by combining gating region adjacent residue analysis, and obtains xylanase mutant with enhanced activity.The mutant is mutated at the 123th and / or 124th position of the amino acid sequence shown in SEQ ID NO:1, preferably D123G and / or R124E, more preferably double mutant D123G / R124E.Compared with wild type, the relative enzyme activity of double mutant D123G / R124E is increased by 4.0 times, and it can be used for xylan degradation and xylo-oligosaccharide preparation.

The invention concerns a variant (double mutant form) of the survivin polypeptide; nucleic acid molecules encoding the survivin variant; antigen presenting cells (APCs) such as dendritic cells, or APC precursors, comprising the variant survivin polypeptide or encoding nucleic acid sequence; and methods for treating a malignancy, such as myeloma, or for inducing an immune response, utilizing a variant survivin polypeptide, nucleic acid molecule, or APC.

The application discloses application of an OsCATA gene in controlling rice heading stage and belongs to the technical field of genetic engineering. The application finds for the first time that the OsCATA gene (cDNA sequence as shown in SEQ ID NO:1) derived from rice Nipponbare is involved in the regulation of the rice heading stage, and knocking out the gene can significantly delay the heading of rice under short-day and long-day conditions; under long-day, the OsCATA gene regulates the heading stage in a manner dependent on OsELF3-1, down-regulates Ghd7 to inhibit the expression of Ehd1, Hd3a and RFT1, and finally delays the heading. The application also provides a knocking-out vector and method of the OsCATA gene and obtained mutant plants, the oscata single mutant delays the heading by 12.4 days under the long-day in Hangzhou and by 7.2 days under the short-day in Hainan, and the oself3-1 oscata double mutant has a more significant delay effect. The application provides a new gene target for the regulation of the rice heading stage, perfects the photoperiodic flowering regulation network of rice, and the provided method and mutant can be used for the directional breeding of new rice varieties suitable for different photoperiod regions, and has important breeding application value.

A mutant Taq DNA polymerase and its application belong to the field of biotechnology. On the one hand, the present invention provides a mutant Taq DNA polymerase. Compared with the amino acid sequence of the wild-type Taq DNA polymerase, the mutant Taq DNA polymerase includes the mutation site D785 and / or the mutation site R573. On the other hand, the present invention provides the application of the mutant Taq DNA polymerase in the preparation of PCR reaction reagents. The Taq DNA polymerase mutant described in the present invention is a mutant obtained by predicting the activity of key mutation sites by AlphaFold3 and then verifying the expression. The double mutant strain obtained in the present invention provides a better Taq enzyme selection for genomic amplification.

The application discloses a sucrose isomerase mutant with improved thermal stability and a construction method thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The application constructs a sucrose isomerase mutant with improved thermal stability and expression amount, and uses the mutant for preparation of isomaltulose, wherein a single mutant V447E has a half-life at 40 DEG C prolonged by 1.38 times, an optimum temperature increased from 30 DEG C to 35 DEG C, and an expression amount increased from 18.89 U.mL ‑1 -1 to 33.06 U.mL ‑1 -1; a double mutant V447E / D232P with other mutations superimposed on the basis of the mutant V447E has further improved thermal stability; compared with a wild type, the double mutant V447E / D232P has an optimum temperature increased by 5 DEG C, a half-life at 40 DEG C prolonged by 1.52 times, and an expression amount increased from 18.89 U.mL ‑1 -1 to 30.73 U.mL ‑1 -1.

The application discloses an L-homoserine phosphotransferase mutant, a coding gene, a plasmid, a genetically engineered bacterium and application, and belongs to the technical field of bioengineering, wherein a cysteine at the 223th position of L-homoserine phosphotransferase is mutated into a glycine single mutation or a double mutation of mutating isoleucine at the 180th position into serine on the basis of the single mutation, namely the L-homoserine phosphotransferase mutant, a corresponding coding gene is obtained, and then a corresponding plasmid and genetically engineered bacterium are obtained; the enzyme mutant not only has high enzyme activity, but also is resistant to feedback inhibition of alpha-ketoglutarate; compared with simply improving enzyme activity, the application can further promote the synthesis of L-homoserine by affecting the reversible reaction direction. The application is suitable for applying the enzyme mutant to industrial fermentation production of L-homoserine, can significantly improve production efficiency, and can improve the yield of L-homoserine by about 60% by adding L-glutamine.

The invention concerns a variant (double mutant form) of the survivin polypeptide; nucleic acid molecules encoding the survivin variant; antigen presenting cells (APCs) such as dendritic cells, or APC precursors, comprising the variant survivin polypeptide or encoding nucleic acid sequence; and methods for treating a malignancy, such as myeloma, or for inducing an immune response, utilizing a variant survivin polypeptide, nucleic acid molecule, or APC.