76 results about "Site-directed mutagenesis" patented technology

The application provides an arginine mutant and application thereof in 1,4-butanediamine biosynthesis, and belongs to the field of bioengineering. Escherichia coli The mutant is obtained by site-directed mutagenesis of key site amino acid residues based on wild-type EcAdiA from strain The application improves the enzyme activity stability of arginine decarboxylase in neutral environment, improves the conversion rate and conversion speed of 1,4-butanediamine in neutral environment, and lays a foundation for high-efficiency biosynthesis of 1,4-butanediamine.

This invention relates to the fields of microbial genetic engineering and fermentation biomanufacturing technology, specifically disclosing a recombinant Corynebacterium glutamicum with high 5-aminolevulinic acid (ALA) production and its applications. Addressing the problems of existing strains such as crude enzyme activity regulation, inhibited cell growth, the need for external inhibitors, and low yield, this invention uses Corynebacterium glutamicum as a host and removes the gdhA gene without scarring to reduce precursor competitive metabolism. Site-directed mutations of the endogenous hemB gene (S288A, D128E, R231K) are performed to moderately reduce dehydratase activity. A high-expression plasmid pXMJ19-hemA containing the hemA gene from Rhodopsinus capsulatum is constructed and introduced into the modified strain to obtain a recombinant strain with optimized triple metabolism. This strain reduces product degradation without the need for external dehydratase inhibitors, does not affect cell growth, and achieves a yield up to 4.30 times higher than the control after 42 hours of fermentation, with ALA accumulation exceeding 8.9 g / L. The strain exhibits stable characteristics, the process is simple, and it has good industrial application value.

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

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

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

This invention discloses a pullulanase mutant with enhanced catalytic activity against highly branched dextrin substrates, its construction method, and its applications, belonging to the fields of enzyme engineering and biotechnology. This invention obtains two mutants, D139A and D139E, by site-directed mutagenesis of the pullulanase derived from Klebsiella pneumoniae at position 139 aspartic acid residue. Compared with the wild-type enzyme, both mutants maintain high hydrolytic activity against pullulan polysaccharides while significantly improving hydrolytic activity against dextrin substrates with high α-1,6 glycosidic bond content. Specifically, the hydrolytic activity against dextrin substrates with 6% and 10% α-1,6 bond content is increased by up to 242% and 110%, respectively. The pullulanase mutants obtained in this invention exhibit excellent catalytic performance in the enzymatic debranching reaction of highly branched dextrin substrates, showing promising application prospects in starch deep processing and related biomanufacturing fields.

The present application relates to the field of genetic engineering, in particular to L-tryptophan genetically engineered bacteria under the influence of regulatory factors and a construction method and application thereof, the Escherichia coli is reformed by using metabolic engineering means to improve the yield of L-tryptophan, mainly by inactivating the regulatory factor RpoS in the Escherichia coli, and simultaneously carrying out site-directed mutagenesis on FlhD and Crp, optimizing the metabolic flow of the Escherichia coli, and improving the activity of the regulatory factor capable of promoting L-tryptophan, further improving the ability of the Escherichia coli to produce L-tryptophan and the efficiency of the whole metabolic pathway, effectively improving the production of L-tryptophan, so that the yield of L-tryptophan is doubled compared with the original strain.

This invention relates to the field of biosynthesis technology, and particularly to engineered yeast strains and their application in the total fermentation production of steviol glycosides. This invention utilizes site-directed mutagenesis to mutate wild-type kaurenoic acid 13-hydroxylase, obtaining the G481L / Q159L mutant. This mutant is then applied to the production of steviol or steviol glycosides, constructing a steviol or steviol glycoside synthesis pathway in the *Saccharomyces cerevisiae* host to achieve efficient production of steviol or steviol glycosides. Simultaneously, by enhancing the level of diterpene precursor synthesis genes in chassis cells (MVA pathway, acetyl-CoA, GGPP, transport proteins, UDPG pathway), the yield of steviol glycosides is further increased, reducing costs for industrial production.

This invention discloses a high-activity, heat-resistant α-glucan phosphorylase mutant and its applications, belonging to the fields of genetic engineering and modern enzyme technology engineering. This invention utilizes site-directed mutagenesis technology to modify the wild-type... α‑ Dextran phosphorylase was modified to construct a series of mutants. Compared with the wild-type enzyme, the enzyme activity was increased by 1.68 to 2.49 times, and the mutants had a wider range of adaptability to temperature and pH. Among them, the mutant R282Y / H506A showed excellent thermostability. After incubation at 60℃ for 132 h, its enzyme activity was still 50.96%, and after incubation at 70℃ for 60 h, its enzyme activity was still 53.71%. The significantly improved thermostability creates better conditions for the use of this enzyme in practical applications.

The application discloses a sucrose synthetase mutant and a method for preparing RD by using the sucrose synthetase mutant, wherein the sucrose synthetase mutant contains at least one of the following mutations: D296Q, R636Q, G514L, H531L, R567T, D569Q, E610N, A642N, E663N, L667T and T742R on the basis of the amino acid sequence shown in SEQ ID NO:2; the sucrose synthetase mutant has higher enzyme activity than the wild type, and meanwhile, some mutants still maintain a high conversion rate in a wider pH range.

This invention discloses a sucrose isomerase site mutant, a genetically engineered bacterium, and a method for catalyzing the production of isomaltulose. This invention utilizes PROSS online analysis combined with sequence alignment analysis to screen key amino acid sites related to catalytic activity in sucrose isomerases. Through site-directed mutagenesis, the mutant WT-Gro7-Q474T was obtained, with a maximum specific enzyme activity of 683 U / mg. k cat (s ‑1 The concentration reached 718. By optimizing protein expression conditions, including temperature, IPTG concentration, bacterial concentration, and time, and combining this with the method of co-expression of molecular chaperone proteins, a highly efficient heterologous expression system for sucrose isomerase was established. This invention established an optimal isomaltulose-catalyzed reaction system: in a 2 L reactor, with 1 L of reaction solution and a substrate concentration of 800 g / L sucrose, after 6 hours of reaction, all sucrose was converted, yielding isomaltulose 792.50 g / L, with a conversion rate >99.5%.

This invention discloses a recombinant *E. coli* strain for producing phenyllactic acid, a lactate dehydrogenase mutant, a whole-cell catalytic method for preparing phenyllactic acid, and the application of the aforementioned recombinant *E. coli* strain or lactate dehydrogenase mutant in the catalytic preparation of phenylpyruvic acid from phenyllactic acid. This invention significantly improves catalytic efficiency by modifying the 52nd amino acid of lactate dehydrogenase through site-directed mutagenesis. After mutating glutamine (Q) to valine (V) at position 52 of the *Lactobacillus mucosa* lactate dehydrogenase, the catalytic efficiency is significantly improved even at low cell density (OD). 600 =15) and substrate 15 g / L conditions, almost complete conversion was achieved; and combined with high-density catalysis and surface display optimization, the yield of phenyl lactic acid was increased while the downstream separation and purification costs were significantly reduced, greatly enhancing the potential for industrial production.

This invention discloses a genetically engineered bacterium producing high levels of L-homoserine, its construction method, and its applications, belonging to the field of biotechnology. The genetically engineered bacterium uses *Escherichia coli* W3110 as the substrate cell. By knocking out the metA, lysA, thrBC, pflB, ldhA, poxB, adhE, iclR, sthA, tdcC, lacI, and ptsG genes, and replacing the promoters of ppc, aspC, and gdhA genes with the inducible strong promoter Ptrc, and by performing site-directed mutagenesis on the thrA and asd genes, and simultaneously overexpressing the thrA*, pnhsd, asd, asd*, and rhtA genes using free plasmids, a recombinant strain HOM20 was constructed. This strain, fermented in a 5 L fermenter for 48 h, achieved an L-homoserine yield of 135.4 g / L and a sugar-acid conversion rate of 44%, significantly superior to existing technologies. This invention achieves directional carbon flow allocation, cofactor balance, feedback inhibition removal, and enhanced product efflux through multi-gene synergistic modification, and has broad prospects for industrial application.

The present application relates to a kind of chitosanase mutant WP-E80A, plasmid, recombinant bacteria and its application, belong to enzyme engineering technical field, the amino acid sequence of the chitosanase mutant as shown in SEQ ID NO:1, the amino acid sequence of wild-type chitosanase CsnWP as shown in SEQ ID NO:2.Compared with wild-type chitosanase CsnWP, the 80th amino acid is subjected to site-directed mutagenesis.The present application also provides plasmid and recombinant engineering bacteria comprising the chitosanase mutant WP-E80A gene, and the chitosanase mutant WP-E80A can be prepared to generate chitotriose with chitobiose as substrate.

The application discloses a kind of MS2 virus-like particles based on capsid protein site-directed mutation and its preparation method and application, belong to molecular biological technique field.The amino acid of the beta sheet interface of capsid protein sequence, the amino acid of the interface when two capsid proteins molecules assemble, the carboxyl end site of capsid protein is subjected to site-directed mutation, obtain the mutant that can significantly improve the assembly efficiency and stability of capsid protein dimer.Utilize the mutant to construct MS2 virus-like particles, further by the design of mature enzyme and capsid protein expression element, control mature enzyme weak expression, capsid protein strong expression, to improve assembly efficiency.The application improves the assembly efficiency of MS2 virus by the above strategy, improves the proportion of wrapped RNA, enhances the stability of virus-like particle, greatly prolongs the stable storage time of RNA, improves the purity of wrapped RNA.

The application provides a beta-glucosidase mutant and application thereof in production of ginsenoside Rg3, and belongs to the technical field of genetic engineering.The application provides a beta-glucosidase mutant, and the amino acid sequence is shown as SEQ ID NO:1.The application screens a mutant capable of catalyzing the directional conversion of ginsenoside Rb1 into ginsenoside 20(S)-Rg3 through site-directed mutagenesis.Compared with a wild type, the mutant has a mutation site K715G, and the yield of Rg3 produced by enzymolysis of Rb1 is 585.95 muM, while the yield of the wild type is only 441.22 muM, and the efficiency of the directional conversion of ginsenoside Rb1 into ginsenoside 20(S)-Rg3 is significantly improved.

This invention belongs to the fields of genetic engineering and enzyme engineering, specifically relating to a chitinase mutant ChiTgM and its applications. This invention obtains the chitinase ChiTg through gene cloning, achieves heterologous expression of it in Pichia pastoris through codon optimization, and obtains the chitinase mutant ChiTgM through site-directed mutagenesis and combinatorial mutagenesis. Analysis of the enzymatic characteristics of chitinase ChiTg and the mutant ChiTgM shows that, compared to the starting template ChiTg, the chitinase mutant ChiTgM provided by this invention exhibits a 1.81-fold increase in specific activity and demonstrates excellent hydrolytic properties. Its application in the enzymatic preparation of chitin oligosaccharides can efficiently hydrolyze colloidal chitin, laying the foundation for its further industrial application.

The application discloses a alginate lyase mutant E87Q, a coding gene, a recombinant expression vector and a genetically engineered bacterium, and belongs to the technical field of genetic engineering. The application takes alginate lyase PpAly7A as a starting template, and a mutant with improved relative specific activity is obtained through site-directed mutagenesis. Compared with the wild enzyme PpAly7A, the mutant E87Q has a relative specific activity increased to 133.82%, and the corresponding site can be used as a new target for subsequent rational design.

This invention discloses a specific antibody for identifying K27 ubiquitin chains, belonging to the field of biomedical technology. The antibody is obtained through structural simulation, molecular docking, and site-directed mutagenesis, using the initial screening antibody zl1901 as a parent material. The optimal core strain is zl1901-R3-S8, whose light chain CDR1 and CDR2 regions can bind tightly to the receptor and donor ubiquitin of K27-diUb, while the heavy chain CDR3 region forms a specific hydrogen bond with the P19 residue of the K27-diUb donor ubiquitin. The antibody of this invention exhibits an affinity (KD) of 16.5 nM for K27-linked ubiquitin chains, shows no cross-binding with M1, K6, K11, K48, and K63-linked ubiquitin chains, and only exhibits a very weak binding with K29-diUb. This allows for precise identification of endogenous and exogenous K27-linked ubiquitination modifications, providing a core tool for studying the regulatory mechanism of K27 ubiquitination.

The application provides a formic acid dehydrogenase mutant, which is mutant I or mutant II; the mutant I is a single-point mutant based on wild-type formic acid dehydrogenase and is selected from one of R69H, K89E, R213H, R323H and K326E; the mutant II is a double-point mutant based on R69H and is selected from one of R69H / K89E, R69H / R213H, R69H / R323H and R69H / K326E. The application also provides a coding gene of the mutant and a recombinant expression vector and an engineering bacterium containing the gene. Meanwhile, the application also provides a preparation method of the mutant and an application of formic acid hydrogen production. The application obtains a mutant with high activity and substrate resistance by rational site-directed mutagenesis on wild-type formic acid dehydrogenase, significantly improves the catalytic activity and substrate resistance of the enzyme, improves the formic acid biological hydrogen production efficiency, reduces the production cost and is beneficial to industrial application.

This invention relates to the field of clinical laboratory technology and discloses an aptamer that specifically binds to AZGP1 and its applications. The invention uses magnetic bead SELEX technology to screen for the AZGP1-specific aptamer Seq7. Guided by Mfold secondary structure prediction, the core binding region Seq7T is truncated. Further, through site-directed mutagenesis, insertion, and substitution modifications, an optimized version, Seq7TM, containing specific motifs and capable of forming a compact mini hairpin structure, is obtained, further improving the performance of the nucleic acid aptamer. The aptamer of this invention can be widely used in the preparation of AZGP1 detection reagents, kits, or sensors; the preparation of AZGP1 capture / separation / purification formulations; the development of new methods for rapid early diagnosis of AMD; and the construction of AMD targeted drug delivery systems. It provides a novel and practical tool for early screening, early diagnosis, and early intervention of AMD, and has significant application value in the field of clinical laboratory testing.

This invention relates to a phthalate hydrolase mutant and its application in phthalate degradation, belonging to the field of microbial enzyme technology. The amino acid sequence of the phthalate hydrolase mutant HylD1 is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2. This enzyme can catalyze the reaction to generate MMP using DMP as a substrate; or catalyze the reaction to generate MEP and EMP using DEP as a substrate. In this invention, the encoding gene of the phthalate hydrolase HylD1 from *Paracoccus koningii* BJQ0001 was artificially modified by site-directed mutagenesis at amino acid position 265, resulting in a mutant HylD1 (R265A) with higher activity. Compared with the wild-type phthalate hydrolase HylD1, the absolute enzyme activity of this phthalate hydrolase mutant HylD1 (R265A) in degrading DMP and DEP is increased by 2-fold and 3.6-fold, respectively, and a special product EMP is also produced during the degradation of DEP.

The application discloses a S288C riboflavin kinase mutant and application thereof, and belongs to the technical field of riboflavin kinase.The amino acid sequence of the S288C riboflavin kinase mutant is shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.The S288C riboflavin kinase mutant with high activity, high alkali resistance, high selectivity and suitable for industrial application is obtained by site-directed mutagenesis and screening of the original riboflavin kinase, and the S288C riboflavin kinase mutant is used for preparing FMN, which has wide application prospects.

This invention belongs to the field of molecular biology technology and discloses an improved PCR amplification enzyme, amplification enzyme antibody, and buffer composition for full-component lyophilization and direct amplification, as well as their applications. This invention provides an enzyme combination comprising Taq enzyme and reverse transcriptase. The Taq enzyme is obtained by site-directed mutagenesis based on wild-type Taq enzyme, exhibiting superior lyophilization tolerance, interference resistance, thermostability, and amplification specificity. The reverse transcriptase is obtained by site-directed mutagenesis based on wild-type reverse transcriptase, exhibiting good lyophilization-reconstitution activity, thermostability, reverse transcription efficiency, and specificity. Using the above enzyme combination to prepare full-component lyophilized and direct PCR amplification reagents can simultaneously solve the pain points of traditional reagents, such as reliance on low-temperature transportation and storage, the need for extraction before amplification, cumbersome operation, and weak interference resistance.

This invention discloses a chondroitin 4-O-sulfate transferase with enhanced enzyme activity and stability, and its production method, belonging to the field of biotechnology. This invention truncates and performs site-directed mutagenesis on mouse-derived chondroitin 4-O-sulfate transferase, increasing the enzyme activity to 6062.36 U / L, 2.4 times that of the original strain; and extending the half-life of the final mutant to 228 h at 37°C, 28.5 times that of the original strain. Further optimization of the culture medium reduces production costs while increasing the enzyme activity to 10791.08 U / L, 4.3 times that of the original strain, thus promoting the industrial production of non-animal-derived chondroitin sulfate.

The application discloses a thermophilic phospholipase C mutant and a preparation method and application thereof. Based on a wild-type phospholipase TdPLC shown in SEQ ID NO. 1, a site-directed mutation is carried out, and a mutant T3A is formed by mutating threonine at the 3rd position. The mutant T3A has a significantly improved PA conversion rate, which is increased from 43.69% of the wild type to 73.54%, and has a good industrial potential. The application provides a theoretical basis for further improving the performance of phospholipase C such as TdPLC through protein engineering. The application also comprises nucleotides and construction, vectors and host cells for coding the mutant enzyme, as well as a method for producing the mutant and using the mutant.

This invention discloses a high-yield canthaxanthin genetically engineered bacterium, its construction method, and its applications, relating to the fields of microbial metabolic engineering and synthetic biology. The construction method includes the following steps: introducing key genes into a yeast chassis strain for overexpression to obtain a basic canthaxanthin-producing bacterium; the key genes include... carB Gene, carRP Gene, crBKT Gene, HMG1 Genes and GGS1 Genes; through repeated gene introduction optimization, the quality of the cantharidin-producing strain was improved. crBKT The copy number of the gene was determined to obtain a copy number-optimized strain; the copy number-optimized strain was then... ERG7 The high-yield canthaxanthin genetically engineered bacterium was obtained by performing site-directed mutagenesis to shrink the substrate binding pocket of the gene. The high-yield canthaxanthin genetically engineered bacterium provided by this invention can significantly improve the synthesis efficiency and production yield of canthaxanthin, and has great application prospects in the field of canthaxanthin biosynthesis.

The application relates to the technical field of biological medicine, in particular to a medicine for combined treatment of tumors by using a recombinant oncolytic virus and a small-molecule anticancer drug. The method comprises the following steps: combined treatment of tumors by using a recombinant oncolytic virus and a small-molecule anticancer drug; the small-molecule anticancer drug is a small-molecule anticancer drug targeting EGFR; the recombinant oncolytic virus comprises M protein, G protein, N protein, P protein and L protein after site-directed mutagenesis. The application can kill tumor cells by using a recombinant oncolytic virus and a small-molecule anticancer drug, and the curative effect is 1+1>2.

This invention proposes a UDP-glucosyltransferase mutant and a method for preparing rebaudioside D. Site-directed mutagenesis of UDP-glucosyltransferase is performed, and the mutant plasmid is transferred to *E. coli* to obtain recombinant engineered bacteria. The recombinant engineered bacteria express the protein through fermentation to obtain a crude enzyme solution of the UDP-glucosyltransferase mutant. ADP-dependent enzyme activity and temperature-dependent enzyme activity of the UDP-glucosyltransferase mutant were measured. The mutant ADP-dependent enzyme activity was significantly increased, and it exhibited thermostability. Rebaudioside D was prepared using rebaudioside A, sucrose, ADP, the UDP-glucosyltransferase mutant, and AtSUS sucrose synthase hypercatalytic synthase.

The application discloses a cytochrome P450 enzyme mutant and a method for catalytically synthesizing betulinic acid, and relates to the technical fields of genetic engineering and biological enzyme catalysis.The reported RoCYP01 from Litsea cubeba is used as a mutation template, site-directed mutation is carried out, the substrate channel for the entry and exit of the enzyme catalytic center of a substrate and a product is changed, and a mutant with improved catalytic efficiency for the synthesis of betulinic acid from betulonic acid is obtained.Under the same catalytic conditions, the catalytic activity of the mutant is increased by 198% compared with the wild type, which has important guiding significance for the construction of a betulinic acid yeast engineering strain.