2471 results about "Mutant" patented technology

The invention discloses a variation test method and device based on an intelligent automation script. The method comprises the following steps: extracting a statement execution sequence and a branch dependency relationship in an original source code by using a context-aware syntax tree, and generating a control flow diagram and a data flow diagram; performing code semantic analysis on the multi-dimensional code features by using a large language model, identifying potential defect types and variation rules, predicting the test efficiency of variants in combination with a deep Q learning model, and generating to-be-processed variants; performing grammar check and equivalence analysis on the to-be-processed variants to obtain effective variants, and testing the effective variants; variation test indexes are calculated, code weak points are positioned, and test blind areas are identified according to survival variants, so that a visual analysis report is generated; and iteratively generating a new test case according to the current survival variant and the visual analysis report, and testing the current survival variant by using the new test case. According to the invention, the variation test efficiency can be improved.

The present invention relates to the fields of genetic engineering and plant genetic breeding, and specifically to the application of wheat TaPND gene. In the present invention, by taking gene TaPND as a target, designing a sgRNA sequence based on CRISPR / Cas9, connecting a DNA fragment containing the encoding sgRNA sequence to a vector carrying CRISPR / Cas, a plant binary expression vector capable of knocking out the TaPND gene in wheat is constructed; by genetically transforming wheat, offspring with TaPND gene knockout are obtained, and wheat mutants with early flowering time are obtained. Experiments have confirmed that transgenic wheat with TaPND gene knockout achieves the advancement of wheat flowering time. The technical scheme of the present invention provides a practical method for realizing rapid wheat breeding using genetic engineering technology, which has important breeding application value and broad market application prospects.

The invention discloses a glycosyl transferase UGT76G1 mutant and a method for catalytically synthesizing rebaudioside M by using the glycosyl transferase UGT76G1 mutant, and relates to the technical field of biological catalytic synthesis. The glycosyl transferase UGT76G1 mutant is any one of the following components (A)-(C): (A) a protein obtained by performing any one or more of the following mutations on the basis of an amino acid sequence as shown in SEQ ID NO.1: the 89th amino acid is mutated into H from M; the 380th amino acid is mutated from L to M; the 411th amino acid is mutated from A to Y; (B) a protein which has 95% or 98% or more of identity with the amino acid sequence defined by (A) and has the same function as the amino acid sequence defined by (A); and (C) a fusion protein obtained by connecting a tag to the terminal of the protein defined in (A) or (B). The glycosyl transferase UGT76G1 mutant disclosed by the invention is obtained by mutating and screening glycosyl transferase UGT76G1, has higher catalytic activity, and can be used for efficiently synthesizing rebaudioside M by taking rebaudioside D and UDPG as substrates.

The invention relates to the field of gene engineering, and discloses a D-3-phosphoglycerate dehydrogenase mutant, a coding gene thereof and application of the D-3-phosphoglycerate dehydrogenase mutant and the coding gene. Compared with a protein with an amino acid sequence as shown in SEQ ID NO: 1, the mutant comprises mutation at the 131 site and / or the 285 site. The mutant can specifically catalyze a substrate 3-phosphoglycerate to synthesize 3-phosphate-hydroxypyruvic acid, can effectively promote synthesis of L-serine in a metabolic pathway of microorganisms, improves the yield and conversion rate of the L-serine, and reduces the yield of byproducts at the same time.

The present invention discloses a Gs12-7 endonuclease variant and a gene editing system mediated thereby. Specifically, by means of a rational mutation strategy, two Gs12-7 mutants were constructed and compared, and it was found that after the 157th amino acid of the Gs12-7 endonuclease was mutated from Glu to Arg, the activity of this gene editing enzyme could be significantly improved, and this variant was designated as Gs12-7MAX. The present invention provides an efficient gene editing technology mediated by the CRISPR-Gs12-7MAX system, which has broad application prospects in the field of genome site-directed modification.

The invention provides an UTR (Untranslated Region) element H2202 P1-G as well as a construction method and application thereof, and relates to the technical field of mRNA (messenger ribonucleic acid). According to the present invention, the ribosome load prediction and the secondary structure optimization are performed on the natural 5 'UTR of the HIV TAT 202 gene through the BaidleHelix platform, and the obtained HTAT 202 P1 sequence avoids the inhibitory hairpin structure so as to significantly improve the luciferase expression quantity compared to the natural UTR; an ncRNA sequence without a secondary structure is introduced on the basis of the HTAT 202 P1, translation inhibition of a 5 'cap region is further relieved, and the protein expression quantity of the constructed H2202 P1-G mutant (the DNA sequence of the H2202 P1-G is as shown in SEQ NO 1, and the RNA sequence is as shown in SEQ NO 2) is further improved.

The invention discloses a bovine I-type alpha interferon-ferritin fusion protein, a mutant thereof, a preparation method and an application of the bovine I-type alpha interferon-ferritin fusion protein. The bovine I-type alpha interferon is fused with a ferritin subunit, and interferon molecules are highly repeatedly and orderly displayed on the surface of a ferritin nanocage by utilizing the self-assembly characteristic of ferritin, so that the expression level, the structural stability and the antiviral activity of the interferon are remarkably improved. The fusion protein is further subjected to single-site or multi-site rational design mutation, and a mutant with significantly improved antiviral activity and stability is obtained. According to the invention, a silkworm or insect cell eukaryotic expression system is adopted to express the fusion protein or the mutant thereof, and the expression system is safe to operate, simple and convenient in procedure, low in cost and extremely beneficial to large-scale industrial production; the prepared fusion protein or mutant nanoparticles have application prospects in preparation of drugs or reagents for preventing or treating bovine viral diseases.

The embodiment of the invention provides a tyrosine ammonia lyase mutant and application thereof in synthesis of p-coumaric acid. The mutant is subjected to any one of the following amino acid mutations on an amino acid sequence as shown in SEQ ID NO. 2: F497I, F497I + L248M, F497I + L248M + M4T, and L248M + F497I + M4T + V482L. According to the embodiment of the invention, an amino acid sequence as shown in SEQ ID NO. 2 is taken as a template, a key active site of the template is subjected to mutation transformation, a mutant library is constructed, and a series of mutants with remarkably improved catalytic activity and / or conversion rate are screened; the tyrosine ammonia lyase mutant provided by the embodiment of the invention can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield.

The invention provides a keto-alcohol acid reductoisomerase mutant and application of the keto-alcohol acid reductoisomerase mutant in construction of genetically engineered bacteria for high yield of D-pantothenic acid. In order to solve the technical problem that in the prior art, the yield of D-pantothenic acid synthesized through microbial fermentation is not high, semi-rational design of enzyme is adopted, and a keto-alcohol acid reductoisomerase mutant with substrate specificity is obtained and applied to construction of genetically engineered bacteria for high yield of D-pantothenic acid. According to the invention, a genetically engineered bacterium with high yield of D-pantothenic acid is constructed by utilizing the keto-alcohol acid reductoisomerase mutant through a CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04%, and compared with an original strain, the valine accumulation amount in obtained fermentation liquor is almost unchanged compared with that of a control, and the yield of D-pantothenic acid is greatly improved. However, the accumulation of branched chain amino acid isoleucine is less, so that the modification of the substrate specificity of the keto-alcohol acid reductoisomerase is achieved.

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 belongs to the field of bioengineering, and particularly discloses a tryptophan synthetase mutant with improved enzymatic activity and application of the tryptophan synthetase mutant in production of L-cysteine. Compared with wild tryptophan synthetase from escherichia coli, the amino acid sequence of the tryptophan synthetase mutant has the advantages that a beta catalytic subunit has T69A, S143A, R219E and F280H site combined mutation. Researches show that the enzyme activity of the mutant is increased by 2.4 times compared with that of a wild type through enzyme activity determination and analysis of inducible expression and purification. The beneficial tryptophan synthetase mutant provided by the invention can efficiently biocatalyze L-serine and sodium hydrosulfide to synthesize L-cysteine, and has a better industrial application prospect.

The invention belongs to the technical field of enzyme engineering, and discloses an MHETase-EstD9 mutant and an application of the MHETase-EstD9 mutant. The invention provides an MHETase-EstD9 mutant, the amino acid sequence of the MHETase-EstD9 mutant is shown as SEQ ID No.2, and on the basis of the mutant, the amino acid of the MHETase-EstD9 mutant is further mutated to design five MHETase-EstD9 mutants. At 70 DEG C, compared with wild type MHETase-EstD9, the BHET degradation activity of the MHETase-EstD9 mutant provided by the invention is improved by 15% or more, and the highest BHET degradation activity can be improved by 24 times; meanwhile, the thermal stability of the MHETase-EstD9 mutant is also improved to a certain extent, and the Tm value is maximally improved by 18.0 DEG C. The method can be applied to the fields of degradation of PET, MHET or BHET, preparation of degradation agents of PET and degradation products thereof, TPA recovery and the like, and has important environmental benefits, economic benefits and social benefits.

The invention discloses a tryptophan synthase mutant as well as a coding gene, a vector, recombinant bacteria and application thereof. Comprising the following steps: carrying out error-prone PCR (Polymerase Chain Reaction) on a coding gene trpBA of TrpS from EscherichiaoliK12MG1655, screening mutants by combining high-throughput screening, and finding that the affinity of the TrpS to a substrate indole can be effectively improved by mutating 114-site Gln of a beta subunit of the TrpS into Met and mutating 84-site Gly into Ser, so that the synthesis of L-tryptophan is promoted. The efficiency of the TrpS for converting indole and L-serine into L-tryptophan is higher due to the improvement of the affinity of indole. A new idea is provided for efficient production of L-tryptophan in the fermentation industry.

The embodiment of the invention provides a method for obtaining a tyrosine ammonialyase mutant based on high-throughput screening, the tyrosine ammonialyase mutant and application of the tyrosine ammonialyase mutant. The mutant is subjected to any one of the following amino acid mutations on an amino acid sequence as shown in SEQ ID NO.2: A245G + A247T, and D192V + A245G + A247T. According to the embodiment of the invention, an amino acid sequence as shown in SEQ ID NO.2 is taken as a template (basis), a key active site of the template is subjected to mutation transformation, a mutant library is constructed, and a series of mutants with remarkably improved catalytic activity and / or conversion rate are screened; the tyrosine ammonia lyase mutant provided by the embodiment of the invention can selectively catalyze L-tyrosine to generate p-coumaric acid, and has excellent yield and / or higher yield, which indicates that the mutant has more significant application prospects in catalysis of L-tyrosine to generate p-coumaric acid.

The invention discloses a haloalkane dehalogenase mutant and an application thereof in degradation of 1, 2, 3-tribromopropane, haloalkane dehalogenase DhaA31 coded by dhaA gene of a Rhodococcus rhodococcus NCIMB 13064 strain is modified to obtain the haloalkane dehalogenase mutant F168W / I246C, the haloalkane dehalogenase mutant has high degradation efficiency on haloalkane pollutant 1, 2, 3-tribromopropane, and the haloalkane dehalogenase mutant has high degradation efficiency on haloalkane pollutant 1, 2, 3-tribromopropane under the condition of water phase normal temperature. The degradation rate of the alkyl halide dehalogenase mutant to 10 mM of 1, 2, 3-tribromopropane within 1 h reaches 100%, and the degradation rate of the alkyl halide dehalogenase mutant to 10 mM of 1, 2, 3-tribromopropane reaches 100%. The haloalkane dehalogenase mutant has high stereoselectivity on (R)-2, 3-dibromo-1-propanol, and the e.r. Value of the haloalkane dehalogenase mutant reaches 6: 94. The haloalkane dehalogenase mutant provided by the invention has high catalytic efficiency and high stereoselectivity, is beneficial to recognition and further degradation of downstream enzymes, and has a good application prospect in bioremediation of emerging haloalkane pollutants.

The invention discloses a porcine epidemic diarrhea virus antigen fusion protein, a coding gene and a porcine epidemic diarrhea vaccine prepared from the porcine epidemic diarrhea virus antigen fusion protein. The method comprises the following steps: screening to obtain a homosensory sequence of the spike protein of the porcine epidemic diarrhea virus, and fusing the homosensory sequence with a monomeric porcine ferritin heavy chain subunit to obtain an antigen fusion protein; in order to improve the expression quantity or the titer of the antigen fusion protein, the obtained antigen fusion protein is subjected to sequence optimization and then subjected to single-site or multi-site mutation, and the expression quantity and the titer of the obtained mutant are remarkably improved. According to the invention, a recombinant protein is expressed by using a silkworm or AcMNPV-insect cell and other eukaryotic expression systems to obtain a self-assembled ferritin nanoparticle antigen for displaying an S protein antigen protein on the surface of a cage structure of porcine ferritin, and a vaccine prepared from the prepared nanoparticle antigen can induce a widely neutralized anti-porcine epidemic diarrhea virus antibody; the porcine epidemic diarrhea virus vaccine has the potential of becoming a universal porcine epidemic diarrhea virus vaccine with cross immune efficacy.

The invention belongs to the field of gene engineering, and relates to a rice BRW1 gene and application thereof. The cDNA (complementary deoxyribonucleic acid) sequence of the rice BRW1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the RNA binding protein coded by the rice BRW1 gene is shown as SEQ ID NO.2. The invention discloses the rice RNA binding protein gene BRW1 and the protein coded by the rice RNA binding protein gene BRW1. The rice BRW1 gene controls the grain weight of grains, the thousand grain weight of a knockout mutant of the gene is remarkably increased, and meanwhile the grain length and the grain width are increased. By mutating the BRW1 gene in the invention, the grain weight of rice grains can be increased, which indicates that the genetic improvement of the grain weight and grain shape of rice is facilitated by using the gene.

The invention belongs to the technical field of gene engineering, and particularly relates to a mutant of polymyxin efflux transporter and application of the mutant. The mutant is a PmxD transporter mutant, the amino acid sequence of the PmxD transporter mutant is shown as SEQ ID NO: 1, and compared with wild type PmxD, the polymyxin transport capacity of the PmxD transporter mutant (T38W) is improved by 450.46%; and the total discharge amount of polymyxin is increased by 85.72%. Meanwhile, the mutant can significantly improve the growth ability of the strain on a plate containing 250 [mu] g / mL of polymyxin B, namely significantly improve the autoresistance of paenibacillus polymyxa to polymyxin.

The invention discloses an inositol dehydrogenase mutant and a preparation method of D-chiral inositol, and belongs to the technical field of genetic engineering. The inositol dehydrogenase mutant is obtained by mutating an amino acid sequence as shown in SEQ ID NO.2 through the following mutations: the 200th amino acid is mutated into C from V, the 234th amino acid is mutated into Q from V, and the 256th amino acid is mutated into E from R. According to the invention, the amino acid sequence of wild type inositol dehydrogenase is mutated to obtain the inositol dehydrogenase mutant which is a high-activity mutant capable of catalyzing conversion of myo-inositol into 2-keto-myo-inositol; therefore, when the inositol dehydrogenase mutant and keto isomerase act together to prepare D-chiral inositol by taking myo-inositol as a substrate, the yield of the D-chiral inositol is effectively improved.

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.

Glycosyltransferase UGT76G1 and UGT91C1 mutants and a method for catalytic synthesis of rebaudioside A, D, and M by using same, pertaining to the technical field of biocatalytic synthesis. The glycosyltransferase UGT76G1 mutant can effectively synthesize rebaudioside A using stevioside STV as a substrate. The glycosyltransferase UGT91C1 mutant is obtained by mutation screening of wild-type glycosyltransferase UGT91C1, has higher enzyme activity and catalytic rate, and can effectively synthesize rebaudioside D using rebaudioside A as a substrate. The glycosyltransferase UGT76G1 mutant can also be obtained by mutation and subsequent screening of glycosyltransferase UGT76G1, has higher catalytic activity, and can effectively synthesize rebaudioside M using rebaudioside D and UDPG as substrates.

The invention provides a BaP4H mutant and application thereof, and belongs to the technical field of enzyme engineering, the BaP4H mutant is mutated on an amino acid sequence as shown in SEQ ID NO.1; the invention further provides a recombinant expression vector for expressing the BaP4H mutant, a recombinant cell, a preparation method and application. According to the BaP4H mutant provided by the invention, the recombinant collagen proline is efficiently hydroxylated, and the stability of the recombinant collagen is enhanced.

The invention discloses a group of amide hydrolase mutants capable of efficiently degrading ochratoxin A as well as a coding gene, a recombinant vector and application of the amide hydrolase mutants, and belongs to the technical field of enzyme engineering. The invention also provides application of the mutant of the group of recombinant amide hydrolase in detoxification of ochratoxin A in beer or corn flour polluted by ochratoxin A. The activity of the mutant on ochratoxin A is obviously improved compared with the activity of the existing recombinant amide hydrolase ADH3; beer or corn flour containing ochratoxin A is treated for 10-15 minutes, the degradation rate of ochratoxin A can reach 100%, and the degradation efficiency is high in the field.

The invention belongs to the technical field of gene engineering, and particularly relates to an RNA polymerase beta subunit mutant and application thereof. The RpoB mutant is obtained through a gene editing technology, after the 560th amino acid of the RpoB is changed into leucine from proline, the number of living cells during strain fermentation is greatly increased, the tolerance of the RpoB mutant in the later environment of a fermentation tank is improved, the RpoB mutant is applied to escherichia coli for producing human milk oligosaccharide, and finally the yield of the human milk oligosaccharide is greatly increased.

Provided is a glycosyltransferase UGTSL2 mutant, which is produced through any one or more selected from the group consisting of mutations based on an amino acid sequence shown in SEQ ID NO: 1. Also provided is a glycosyltransferase mutant, which is the following A1) or A2): A1) a protein produced through an amino acid residue substitution in an amino acid sequence shown in SEQ ID NO: 66, where the protein has an identity of 90% or more with and the same function as the amino acid sequence shown in SEQ ID NO: 66; and A2) a fusion protein produced by linking a tag to an N-terminus and / or a C-terminus of the protein in the A1).

Described herein are programmable DNA proteolytic target chimera complexes that can be used both for the direct treatment of cancer by inhibiting biochemical pathways that are overexpressed in cancer cells, and for the indirect treatment of cancer by recruiting the E3 ligase complex to engage with a protein of interest or a mutant thereof and initiating proteolysis. Also described herein are methods of using the complexes in the treatment of cancer, as well as compositions comprising the complexes.

The invention provides formaldehyde lyase as well as a mutant and application thereof. The amino acid sequence of the formaldehyde lyase mutant is shown as SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and contains at least one of the following sites: S26, W86, N87 or Y87, L109 or M109, L110, H281, Q282 or E282 and A460, and the amino acid sequence is subjected to amino acid mutation and has a formaldehyde lyase activity function. The amino acid sequences of the formaldehyde lyase and the mutants of the formaldehyde lyase have at least 99% of sequence identity with SEQ ID NO.1-7. The formaldehyde lyase and the mutant thereof can catalyze formaldehyde to synthesize 1, 3-dihydroxyacetone, and a new way for converting formaldehyde into 1, 3-dihydroxyacetone is provided.

The present invention discloses a humanized human-porcine chimeric uricase chimeric mutant. The amino acid sequence of the mutant is shown in SEQ ID NO. 1. The mutant has excellent uric acid level reduction and high homology with human uricase, providing a new option for drug treatment of hyperuricemia.

The present invention provides a yeast recombinant type XVII humanized collagen and its preparation method, in particular, a recombinant humanized collagen expressing the 15th helical region of type XVII collagen, a recombinant engineering bacterium and its preparation method, belonging to the technical fields of genetic engineering and synthetic biology. The present invention provides the full-length sequence of the 15th helical region of type XVII collagen, and mutants are obtained by mutating amino acid residues based on this sequence; for the first time, the present invention expresses the full-length sequence of the 15th helical region of type XVII collagen in Pichia pastoris, establishes a complete set of fermentation processes and purification processes, improves the protein expression level and reduces degradation, and through experiments, it is verified that the obtained protein has biological activities such as cell adhesion activity and cell migration promotion activity that reach or exceed those of natural human collagen, can achieve the purpose of real product application, and can be widely applied in the fields of medicine, medical devices, biological materials, tissue engineering, cosmetics, etc.

The invention provides a keto-alcohol acid reductoisomerase mutant and application of the keto-alcohol acid reductoisomerase mutant in construction of genetically engineered bacteria for high yield of D-pantothenic acid. In order to solve the technical problem that in the prior art, the yield of D-pantothenic acid synthesized through microbial fermentation is not high, semi-rational design of enzyme is adopted, and a keto-alcohol acid reductoisomerase mutant with substrate specificity is obtained and applied to construction of genetically engineered bacteria for high yield of D-pantothenic acid. According to the invention, a genetically engineered bacterium with high yield of D-pantothenic acid is constructed by utilizing the keto-alcohol acid reductoisomerase mutant through a CRISPR-Cas9 gene editing technology, so that the yield of D-pantothenic acid is increased by about 12.04%, and compared with an original strain, the valine accumulation amount in obtained fermentation liquor is almost unchanged compared with that of a control, and the yield of D-pantothenic acid is greatly improved. However, the accumulation of branched chain amino acid isoleucine is less, so that the modification of the substrate specificity of the keto-alcohol acid reductoisomerase is achieved.