339 results about "Directed mutagenesis" patented technology

Disclosed are methods for the genetic construction and expression of antibody-based fusion proteins with enhanced circulating half-lives. The fusion proteins of the present invention lack the ability to bind to immunoglobulin Fc receptors, either as a consequence of the antibody isotype used for fusion protein construction, or through directed mutagenesis of antibody isotypes that normally bind Fc receptors. The fusion proteins of the present invention may also contain a functional domain capable of binding an immunoglobulin protection receptor.

The invention discloses a tobacco polyphenol oxidase gene NtPPO1 and a site-directed mutagenesis method and application thereof. The nucleotide sequence of the tobacco polyphenol oxidase gene NtPPO1 is shown in SEQ ID:No.1, the coded amino acid sequence is shown in SEQ ID:No.2. The invention further discloses a clone method of the tobacco polyphenol oxidase gene NtPPO1. The clone method comprisesthe specific steps that firstly, cDNAs of tobacco leaves are synthesized; secondly, PCR amplification of the NtPPO1 gene is conducted; thirdly, a CRISPR/Cas9 carrier of the NtPPO1 gene is constructed;fourthly, sequencing detection of the NtPPO1 gene mutation is conducted. The NtPPO1 gene has a wide application prospect in preventing tobacco browning. The tobacco polyphenol oxidase gene and the encoded protein thereof provide the gene and technology support for crops especially for anti-browning breeding of tobaccos.

The invention provides a gene, recombinant plasmid and polypeptide for an anti-tumor binary polypeptide. The gene of the recombinant anti-tumor binary polypeptide is obtained by connecting in an operable way the gene of a coding antibody simulator with a recombinant bacillus anthraci protein antigen gene. The recombinant plasmid of the invention is formed by inserting the gene of the coding antibody simulator by double-chain oligomeric nucleotide directed mutagenesis method into the recombinant bacillus anthraci protein antigen gene. The obtained recombinant plasmid is infected into engineering bacillus coli BL-21 to get engineering bacillus coli cell of anti-tumor binary polypeptide; the anti-tumor binary polypeptide can be obtained by expanding the bacillus coli, settling in centrifugal way the bacillus coli body, crushing in altrasonic way, settling and crushing bacillus coli body by hi-speed centrifuging and treating the upper clean solution. The anti-tumor binary polypeptide is of special targeting characteristic, higher efficiency in killing special physical tumor than prior anti-tumor medicine, and will not attack normal cells, and has much lower toxicity and poor-reaction than prior anti-tumor medicine.

The present invention provides compositions and improved methods for multi-site directed mutagenesis and DNA shuffling. The present compositions and methods provide increased mutation frequency and increased number of transformants which allow one to sequence only a few clones in order to identify the correct mutants and to obtain the desired mutant by screening large number of transformants in a short time. Moreover, the inclusion of FEN-1, PEF and optimized buffer and cycling conditions provided in the present invention should also facilitate random mutagenized library construction and the mutagenesis of large or difficult templates.

The invention discloses an omega-transaminase mutant and a preparation method and application thereof and belongs to the technical field of molecular biology. The amino acid sequence of the omega-transaminase mutant is as shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12. The invention further provides the gene of the omega-transaminase mutant, an expression unit containing the gene, recombinant plasmid and a transformant. The preparation method includes: using an NCBI database and BLAST software to perform comparison screening to obtain the homologous amino acid sequence of omega-transaminase, using a Weblogo program to obtain a sequence consistency result, using the homologous amino acid sequence, the sequence consistency result and the sequence of the omega-transaminase to determine amino acid residue sites which need to be mutated, and using site-directed mutagenesis technology to perform experimental verification. The method has the advantages direct mutation probability can be increased effectively, experiment efficiency and feasibility are increased, and mutant enzymes whose thermodynamic stability is evidently better than that of wild enzymes can be screened out.

The present invention provides methods and compositions for the construction and direct transformation of site-saturation libraries into Bacillus. This method avoids the need for the use of intermediate hosts, such as E. coli for the development of Bacillus strains suitable for the production of proteins.

The invention relates to a mutant of a cyclodextrin glucosyltransferase with the capability of highly yielding beta-cyclodextrin and a mutation method, which belong to the fields of gene engineering and enzyme engineering. The invention improves the capability of the cyclodextrin glucosyltransferase (CGT enzyme for short) for producing the beta-cyclodextrin by a rite-directed mutagenesis method, provides a mutant proposal for improving the capability of CGT enzyme from Peanibacillus macerans JFB05-01 (CCTCC NO: M 208063) for producing the beta-cyclodextrin, and substitutes Lys on the 47 position of the CGT enzyme for Arg, His and Thr respectively; the beta-cyclodextrin production capacity of the obtained three mutant enzyme of K47R, K47H and K47T is improved compared with wild type CGT enzymes, wherein the mutant enzyme K47T is particularly obvious. The mutant enzymes are more favorable for industrial production of the beta-cyclodextrin than the wild type CGT enzymes.

The invention provides feruloyl esterase and a preparing method and application thereof. A feruloyl esterase gene coming from a soil macro gene library have the nucleotide sequence and amino acid sequence shown in SEQ ID NO.1 and SEQ ID NO.2. The gene contains a tetrapeptide SXXK sequence motif which is rarely seen, and after the esterase gene is inserted into plasmid pET28a(+), the gene is transformed into escherichia coli BL21(DE3) to achieve heterogeneous expression. The molecular weight of purified recombinase (DLFae4) is 38.3 kDa. Besides, it is put forward for the first time that novel feruloyl esterase can hydrolyze penbritin, penicillin, cefazolin and other lactam antibiotics. As is shown by site-directed mutagenesis experiments, a catalysis triplet of DLFae4 is composed of serine(S11), histidine (H74) and aspartic acid (D302), and the mutation of any of serine (S11), histidine (H74) and aspartic acid (D302) can cause loss of the catalysis capability of DLFae4. DLFae4 has a high hydrolytic activity on methyl ferulate and has good heat stability. In the presence of cellulase, DLFae4 can obviously increase the amount of ferulic acid released from destarched wheat bran. Due to peculiar activities and enzymatic characteristics of novel feruloyl esterase, novel feruloyl esterase can be applied to feed, paper making, food, pharmacy and other fields.

The invention discloses tetrahydropyridine high-yield Corynebacterium glutamicum and application thereof and belongs to the field of bioengineering. Feedback inhibition is relieved through site-directed mutagenesis of Corynebacterium glutamicum aspartokinase gene lysC, and expression of mutational LysC is intensified through promoter replacement. Further, pentose phosphate pathway of host bacteriais further intensified through promoter replacement to meet needs on reducing power NADPH in efficient synthesis of tetrahydropyridine. Finally, tetrahydropyridine high-yield Corynebacterium glutamicum is obtained by transferring tetrahydropyridine synthetic gene cluster ectABC of P. stutzeri into recombinant bacteria. By recombinant Corynebacterium glutamicum, tetrahydropyridine can be efficiently synthesized by utilizing cheap raw materials like glucose and maize plasm; compared with recombinant Escherichia coli produced strains, tetrahydropyridine high-yield Corynebacterium glutamicum hasbetter bio-safety and is of great significance to industrial production and large-scale application of tetrahydropyridine.

An immunogenic detoxified protein is provided which comprises the amino acid sequence of subunit A of an E. coli heat labile toxin (LT-A) or a fragment thereof in which at least amino acid Ala-72 of the A subunit is mutated, preferably by substitution with Arg. The toxoid is useful as vaccine against an enterotoxigenic strain of E. coli and is produced by recombinant DNA means by site-directed mutagenesis. It is also an effective adjuvant.

The invention relates to a method for modifying an animal genome by directed mutagenesis, in particular relates to a novel directional transformation method of a specific gene of an animal genome by utilizing zinc finger nuclease. The method comprises the following steps: designing the zinc finger nuclease which specifically identifies and cuts a targeting gene according to a sequence in a target animal target gene region; validating the directional cutting capability and efficiency of the zinc finger nuclease to the target gene by using an independent expression system; and carrying out directional genetic modification on the target gene of the embryo of a target animal to obtain a stable genetic character by utilizing the selected zinc finger nuclease. Furthermore, the invention also relates to a main gene mutation mode which is obtained by using the independent expression system, selecting and utilizing the zinc finger nuclease, and a filial generation obtained through efficient screening and oriented genetic modification by utilizing the selected gene mutation mode. On the other hand, the invention relates to the zinc finger nuclease which is obtained by utilizing the method and is capable of specifically identifying the myostatin gene of pelteobagrus fulvidraco and directionally knocking out the myostatin gene of the pelteobagrus fulvidraco.

The invention relates to an organic potassium fertilizer microbial agent. The organic potassium fertilizer microbial agent is characterized by being prepared by the following steps: after salt-tolerance colloid bacilli are activated on a nitrogen-free culture medium, the salt-tolerance colloid bacilli are transferred into a liquid culture medium and cultured, thalluses are collected by centrifugation, then kieselguhr, active carbon or plant ash with equivalent quantity is added as an adsorbent, and the mixture is dried at low temperature to obtain the organic potassium fertilizer strain agent. The invention also relates to a production method of biologic organic fertilizer. The organic potassium fertilizer microbial agent is prepared by combining a culture medium prepared by adopting seawater or bittern and the salt-tolerance colloid bacilli which are obtained by chemomorphosis or radialization mutagenesis and directed mutagenesis, can adapt to the seawater with a certain concentration and contain potassium bittern, the potassium content of the organic potassium fertilizer microbial agent is higher, and the organic potassium fertilizer microbial agent can be used as the biologic organic fertilizer, can be directly applied to common soil and salt alkali soil and can promote the absorption of plants to the soil and potassium in the microbial agent. The organic potassium fertilizer microbial agent also can be used as the organic fertilizer microbial agent and can produce organic potassium fertilizer and composite potassium fertilizer.

The invention discloses a glutamic acid decarboxylase mutant and a preparation method thereof and application. An amino acid sequence of the mutant is as shown in SEQ ID No. 2, and a nucleotide sequence is as shown in SEQ ID No. 1. The invention further discloses an expression unit containing genes for encoding the glutamic acid decarboxylase mutant, a recombinant plasmid and a transformant. According to information of comparison with a thermococcus kodakarensis glutamic acid decarboxylase (GAD) amino acid sequence, a method of site-directed mutagenesis is adopted to introduce proline residue at an amino acid locus corresponding to lactobacillus brevis GAD, and the thermal stability of the GAD is improved through rational design. The mutant has better thermal stability in the process of catalyzing L-glutamic acid or sodium salts thereof to generate gamma-aminobutyric acid and is favorable for industrial production of gamma amino acid butyric acid (GABA).

The invention provides a molecular internal standard quality control method and a kit for biological sample nucleic acid detection. The molecular internal standard quality control method and the kit can realize sample quality control such as sample identification and detection analysis. Quality control molecular internal standards are specific exogenous nucleotide sequences different from nucleotide sequences of tested samples and have homology less than or equal to 99% with the tested samples. The specific exogenous nucleotide sequences are mixed with the tested samples uniformly, and corresponding records are made and simultaneously, follow-up treatment is carried out. Through DNA synthesis, site-directed mutagenesis and gene cloning technologies, the quality control molecular internal standards are prepared. The quality control molecular internal standards are used in an identifier quality control system for biological sample nucleic acid detection. Through common technologies of amplification, fragment length analysis, SNP typing, multiple fluorescent polymerase chain reaction (PCR), capillary electrophoresis and sequencing, quality control molecular internal standard information can be obtained and be used for sample verification so that quality control processes of identification and acceptance inspection of a sample are realized simply and easily.

The invention discloses recombined alkaline pectinase with good pH stability and high specific enzyme activity and a construction method thereof. A Site-directed mutagenesis technique is adopted for mutagenesis of an alkaline pectinase coding gene pel168 of bacillus subtilis 168 and 457-bit guanine G is transformed into thymine T so that the 132-bit valine V of the mature peptide of the coded pectinase is mutated into phenylalanine F, a mutant gene pel168V132F is expressed in colon bacillus Rosset Blue (DE3) and subjected to inducible expression, bacterium breaking and purification, the purified enzyme is subjected to enzyme activity measurement, and the relative activity of the obtained pectinase mutant PEL168V132F is much higher than that of the original pectinase PEL168 when the pH is 3, 5 and 6; and meanwhile, the specific enzyme activity is 2.25 times that of the original pectinase, thus good foundation is laid for wide application of the alkaline pectinase.

The invention relates to a sucrose phosphorylase mutant with improved enzyme activity and a construction method and application of the sucrose phosphorylase mutant, and belongs to the technical fieldof genetic engineering. The amino acid sequence of the mutant is shown in SEQ ID NO: 1. Based on sucrose phosphorylase which is derived from Leuconostoc mesenteroides, site-directed mutagenesis is performed on the mutant so as to improve the enzyme activity of sucrose phosphorylase, the mutant is expressed in Corynebacterium glutamicum, and is used as a whole-cell catalyst for production of 2-O-alpha-D-glycosylglycerol, and a large amount of 2-O-alpha-D-glycosylglycerol can be produced efficiently in a short period of time at the level of a fermenter of 5 L, so that industrial application prospects for 2-O-alpha-D-glycosylglycerol production from sucrose phosphorylase is expanded, and large-scale industrial application is achieved.

The present invention provides a novel vector system and thereby a novel method for the simplified construction of recombinant vectors for directed mutagenesis. Said vector system is used to modify the eukaryotic genome, particularly of embryonic stem cells, at precise and predefined loci by the means of homologous recombination. Furthermore, said system finds its usage in the generation of new strategies for gene therapy and in the generation of genetically modified higher eukaryotic organisms.

The invention relates to the field of molecular genetics, in particular to the application of a molecular marker method in pig breeding for disease resistance, wherein according to the molecular marker method, the molecule at a mutation site in a CYP3A88 gene 5' regulatory region of a pig is marked. The inventor of the method discovers that the CYP3A885' regulatory region of a large Chinese streaky-head pig and the CYP3A885' regulatory region of a Duroc long hybrid pig have a plurality of differences, wherein an A-to-T mutation exists at the -78 site, through a luciferase reporter gene system, the fact that promoter activity of the -78 site is lowered remarkably after an A at the -78 site is mutated into a T in a site-directed mutagenesis mode is found, and the promoter activity of the -78 site is the same as the low level of messenger ribonucleic acid (mRNA) expression of a CYP3A88 gene of the porcine reproductive and respiratory syndrome (PRRS)-resistant large Chinese streaky-head pig. Therefore, through genotype detection of the -78 site of the CYP3A88 regulatory region in a pig genome, the genotype of the -78 site of the CYP3A88 regulatory region can be used as a modular marker associated with traits of the PPRS, the molecular marker method not only is simple, convenient and rapid, but also cannot be affected by the environment, and early selection for breeding can be realized.

The present invention is directed to methods and compositions for use of homologous recombination for directed evolution, gene reassembly, and directed mutagenesis. One aspect of the present invention relates to methods for use of bacterial conjugative transfer and homologous recombination for directed evolution, gene reassembly, and directed mutagenesis. Another aspect of the present invention relates to compositions for use in or produced by the methods of the present invention, including libraries, archives and databases.

The invention relates to the field of gene engineering, particularly a high-specific-activity endo-xylanase NPWXynB, and a gene and application thereof. Orthogenesis and site-directed mutagenesis are utilized to perform molecular modification on endo-xylanase XynB derived from rumen fungus Neocallimastix patriciarum to obtain the high-specific-activity xylanase NPWXynB. In the amino acid sequence, the 58th site Y is substituted by F, the 78th site M is substituted by Y, the 94th site Y is substituted by S, the 143rd site V is substituted by L, the 145th site E is substituted by R, and the 182nd site D is substituted by R. The specific activity of the endo-xylanase mutant NPWXynB provided by the invention is 11500 U/mg under the condition of 37 DEG C, which is enhanced by 82.5% as compared with the specific activity of the original endo-xylanase XynB. The maximum enzyme activity of the NPWXynB-gene-containing recombinant engineering bacterium can reach 153000 U/mL, and the production cost is greatly lowered.

The invention relates to a site-directed mutagenesis genetic engineering batroxobin is capable of clotting animal plasma containing an anti-coagulant agent. The site-directed mutagenesis genetic engineering batroxobin is characterized in that: compared with the amino acid sequence of a natural batroxobin, the amino acid sequence of the site-directed mutagenesis genetic engineering batroxobin has a mutation from Arg to Lys at the 45th digit. The site-directed mutagenesis genetic engineering batroxobin having higher bioactivity can be produced in a large scale through a secreted expression mode.

The invention relates to a high-temperature resistant and high-alkali resistant xylanase as well as a gene, an engineering bacterium and a preparation method of the xylanase. The technical scheme is as follows: a method which comprises the following steps of: implementing high-temperature resistant and high-alkali resistant directed molecular modification on a xylanase gene XynG1-1 derived from paenibacillus campinasensis through an error-prone polymerase chain reaction (PRC) and a site-directed mutagenesis technology, to obtain a high-temperature resistant and high-alkali resistant xylanase mutant gene XynG1-1B43CC16; and through a bacillus megatherium expression system, secreting and expressing the high-temperature resistant and high-alkali resistant xylanase. The invention solves the problem of limited application of the existing xylanase which fails to consider both high-temperature resistance and high-alkali resistance in the paper making industry, and meets the demand of the xylanase to implement biological pulping and biological bleaching under a high-temperature and high-alkali environment; and the whiteness and yield of the paper pulp are improved, dosage of chemical in the paper making industry is reduced, environmental pollution is relieved, and clean production of the paper making industry is promoted.

The invention belongs to the technical field of gene engineering and enzyme engineering, and discloses an application of a phenylpyruvate decarboxylase mutant M538A in the biological fermentation production of phenethyl alcohol. Through a site-directed mutagenesis way, 538th-site methionine which is from Lactococcus lactis subsp. Lactis of which the preservation number is CICC6246 and is near a phenylpyruvate decarboxylase KivD molecular catalytic structural domain is mutated into alanine. The enzyme activity of the phenylpyruvate decarboxylase is obviously improved, the problem that the existing phenylpyruvate decarboxylase has low catalytic efficiency for phenylpyruvic acid can be solved, the phenylpyruvate decarboxylase mutant M538A is used for the biological fermentation of phenethyl alcohol, a new thought is provided for the production of the phenethyl alcohol, and the phenylpyruvate decarboxylase mutant M538A is suitable for large-scale promotion.