52 results about "Recombinant enzyme" patented technology

The invention belongs to the technical field of biological medicine, and particularly relates to an engineered cell microvesicle and a delivery system based on the engineered cell microvesicle, the system realizes efficient preparation of 1-5 [mu] m cell microvesicles, and the cell microvesicles have a large space volume and can be used for preparing the cell microvesicles. The carrier can be used for loading and delivery of target protein, polypeptide and recombinase which are specifically expressed in mother cells. The system transfects mother cells through lentivirus transfection or plasmid transfection to further produce cell microvesicles, and the microvesicles can load more goods and inherit membrane proteins of the mother cells, and can also effectively load intracellular proteins to realize effective delivery. By virtue of good structural stability, high immunogenicity and excellent biocompatibility, the cell microvesicle reduces systematic toxic and side effects of a traditional carrier, is expected to become an effective drug delivery system, and has great application potential in the field of gene therapy.

The invention belongs to the technical field of biological engineering, and provides curcumin / dihydrocurcumin reductase MaCurA, a coding gene and application. The reductase can efficiently catalyze 1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-diketone to synthesize tetrahydrocurcumin, an efficient solution is provided for industrial preparation of tetrahydrocurcumin, and experiments prove that the recombinase can catalyze the synthesis of tetrahydrocurcumin in the catalysis of 1, 7-bis (4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-diketone. In a reaction system for preparing tetrahydrocurcumin from 2, 5-diketone, actually measured enzyme activity is stabilized at 3.38 U / mg level, and compared with a traditional chemical synthesis method, the production efficiency is remarkably improved.

The invention discloses an artificially optimized cDNA (complementary deoxyribonucleic acid)-based high-expression trametes versicolor chitin endonuclease gene, a recombinant vector, recombinant bacteria, recombinant enzyme and a preparation method thereof. The optimized trametes versicolor chitin endonuclease gene, such as a sequence as shown in SEQ ID NO.1, is constructed into a pichia pastoris expression vector and then converted into a pichia pastoris expression strain; according to the method, recombinant bacteria expressing active trametes versicolor chitin endonuclease are obtained through high-Zeocin-resistance YPD plate screening, high-expression pichia pastoris transformants are further screened from high-Zeocin-resistance transformants, finally fermentation is carried out in a bioreactor, obtained supernate contains a large amount of recombinant active trametes versicolor chitin endonuclease, and the recombinant active trametes versicolor chitin endonuclease can be obtained under the high-density fermentation condition. The expression level of the recombinant trametes versicolor chitin endonuclease reaches 3.1 g / L, and the trametes versicolor chitin endonuclease is efficiently purified through ammonia sulfate fractional precipitation and ion exchange. The recombinase can hydrolyze colloidal chitin to generate chitin oligosaccharide, and has important application value and wide application prospect in the aspects of preparation of functional chitosan oligosaccharide, antibiosis and anti-tumor, agricultural and biological control, environmental protection, industrial catalysis and the like.

The application discloses a carbonyl reductase mutant and application thereof in preparation of (R)-8-chloro-6-hydroxyoctanoate compounds. The application relates to a mutant of carbonyl reductase with improved catalytic performance and stereoselectivity after rational molecular modification, a recombinant expression plasmid containing the mutant gene of the enzyme, a recombinant expression transformant, a preparation method of the recombinant enzyme, and application of the recombinant enzyme or the recombinant cell as a catalyst in asymmetric reduction of 8-chloro-6-carbonyloctanoate compounds (I) to prepare (R)-8-chloro-6-hydroxyoctanoate compounds (II), in particular, in an intermediate (R)-8-chloro-6-hydroxyoctanoate methyl ester of thioctic acid. The disclosed carbonyl reductase SsCR mutant has the advantages of high reaction efficiency and high catalytic activity, and exhibits the advantages of high substrate concentration, low catalyst consumption and short reaction time in the process of catalytic preparation of the compound (II).

The invention discloses polyethylene glycol terephthalate hydrolase PmPETase and application thereof.The hydrolase PmPETase is protein with the amino acid sequence shown in SEQ ID NO.1. An expression system suitable for the PmPETase is constructed, recombinase protein with catalytic activity is obtained, and the polyethylene glycol terephthalate hydrolase PmPETase can be used for preparing the polyethylene glycol terephthalate hydrolase PmPETase. The substrate specificity, the catalytic property and the potential plastic degradation capacity of the PET degrading enzyme are comprehensively analyzed, and a new theoretical basis and application reference are provided for development and utilization of the PET degrading enzyme and a high polymer material green treatment technology.

The present disclosure relates to production of cis-3-hexenol from unsaturated fatty acid, such as alpha linoleic acid and linoleic acid. The process of the present disclosure involves combinatorial and sequential action of recombinant enzymes, such as LOX, HPL and ADH for the production of cis-3-hexenol.

The invention relates to a bacillus velezensis strain resistant to high temperature and alkali, and belongs to the technical field of microbial detoxification and feed safety. The bacillus velezensis B.26 strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.34475 is provided aiming at the problems that in the prior art, the thermal stability of zearalenone (ZEN) degrading enzyme is insufficient, the efficiency is low in an alkaline environment, and toxic metabolites are easily generated by traditional strains. CotA laccase and peroxide reductase Prx secreted by the strain can efficiently degrade ZEN and derivatives alpha-zearalenol and beta-zearalenol, the products are low-toxicity C17H24O4 and C12H16O4, and harmful by-products such as alpha-ZEL / beta-ZEL and the like do not exist. The strain and recombinase thereof are suitable for a high-temperature alkaline environment for feed processing, and can be applied to biological detoxification of ZEN pollution in food and feed on a large scale through an immobilized carrier.

The invention discloses recombinant poria cocos chitin endonuclease as well as a gene, a preparation method and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1 in a sequence table. According to the nucleotide sequence as shown in SEQ ID NO.1, high-level recombinant secretory expression of target protein in pichia pastoris host bacteria can be realized by taking pichia pastoris inducible expression plasmid pPICZ alpha A as a carrier. Under a shake flask fermentation condition, the average secretory expression quantity can reach 300.3 mg / L. The recombinant enzyme obtained by nickel ion affinity chromatography purification not only can effectively hydrolyze colloidal chitin to generate chitosan oligosaccharide (COS), but also can directly degrade chitin-containing raw materials such as shrimps, crabs and the like to generate COS. In addition, the enzyme also shows the activity of hydrolyzing polysaccharides such as cellulose, pectin, agar and the like, and has important potential values in the fields of functional chitosan oligosaccharide preparation, biological energy production, medicine development, feed, environmental protection, agricultural application and the like.

The construct for site-specific insertion of DNA fragments and transgenes into the genome comprises a recombinant enzyme. This enzyme contains a modified HIV-1 integrase (HIV IN) tetramer with multiple mutations favoring a heterodimer structure in which the first and second outer monomers are bound to the first and second inner monomers, respectively. A transcription activator-like effector (TALE) is bound to the N-terminal region of the outer monomer via a polypeptide linker. The two TALEs confer sequence specificity to the enzyme and prevent non-specific interactions with the target DNA structure. The modified HIV IN monomer ensures that the TALE binds to the outermost monomer and that the TALE is positioned relative to the catalytic region of HIV IN. A polynucleotide is bound to the inner monomer of HIV IN. When the TALE is transported to the nucleus, either alone or within a lentiviral capsid, it interacts with the DNA to localize HIV IN, which then incorporates the polynucleotide.

The application discloses a kind of methyl aspartate lyase EcMAL mutant and its preparation method and application, belong to enzyme engineering technical field.The methyl aspartate lyase EcMAL mutant is any one of mutant EcMAL-M1, mutant EcMAL-M2, mutant EcMAL-M3 and mutant EcMAL-M4.The application is obtained methyl aspartate lyase EcMAL mutant by the mutation of the 329th, 331st, 361st and 365th amino acid sites of methyl aspartate lyase combined with high-throughput screening method.The mutant changes the previous recombinant enzyme only L-aspartic acid production status, so that it is possible to produce D-aspartic acid with methyl aspartate lyase as biological catalyst, and it is expected to be used in pharmaceutical industry.

The invention discloses a kit and a method for rapidly and visually detecting grapefruit moth based on RPA-CRISPR / Cas12a. The kit comprises a recombinase polymerase amplification primer and a targeting crRNA sequence, and the amplification primer can specifically amplify a target nucleotide sequence derived from a grapefruit botryosphaeria maculata mitochondrial genome; the primer comprises an upstream primer and a downstream primer, the sequence of the upstream primer is as shown in SEQ ID NO: 1, and the sequence of the downstream primer is as shown in SEQ ID NO: 2; the target crRNA sequence can be specifically combined with a target nucleotide sequence generated by amplification of the RPA primer and activate the bypass cleavage activity of Cas12a protein, and the sequence of the target crRNA is as shown in SEQ ID NO: 3. The detection kit disclosed by the invention has the characteristics of strong specificity, high speed and high sensitivity, and can be used for detecting grape fruits at the initial stage of being infected by the grapefruit botryosphaeria hemsleyana.

A process for producing three recombinant enzymes capable of hydrolyzing each class of organophosphate, carbamate, and pyrethroid insecticides, as well as a formulation containing these enzymes provides the recombinant plasmid DNA vector, in which the protein secretory system has been modified to enhance the secretion of recombinant protein into the periplasmic space or culture medium. A nucleotide sequence encoding PelB (pectate lyase B) signal sequence for periplasmic localization is deleted and replaced with a nucleotide sequence encoding LamB (maltoporin or phage lambda receptor) signal sequence to enable a more efficient secretory recombinant protein. Three recombinant plasmid DNA vectors for the expression of recombinant proteins that are constructed using the modified plasmid DNA vector described above. The recombinant proteins include a recombinant organophosphate-hydrolyzing enzyme; a recombinant carbamate-hydrolyzing enzyme; and a recombinant pyrethroid-hydrolyzing enzyme.

This invention discloses a recombinant 2-O-sulfonyltransferase mutant and its preparation method, belonging to the field of biotechnology. The recombinant enzyme sequence is formed by fusing a truncated (E28~N356) mutant of hamster-derived 2-O-sulfonyltransferase (L119V-R188E-A240I) with an N-terminal maltose-binding protein (MBP) tag. The recombinant 2-O-sulfonyltransferase mutant exhibits higher activity, 1.9 times that of the recombinant wild type, and greater stability, retaining 86.6% of its activity after 5 days at room temperature. Furthermore, the soluble expression level of this recombinant mutant 2-O-sulfonyltransferase is increased by 5 times. This mutant, as a novel biocatalyst with high efficiency, stability, and controllable production costs, has significant application value and broad industrialization prospects in the industrial enzymatic synthesis of glycosaminoglycans such as heparin and heparan sulfate.

The invention belongs to the technical field of virus detection, and particularly relates to a nucleic acid detection primer, a probe, a detection kit, a detection method and application of an immature skin-adding virus. The primer, the probe and the reaction system designed by the invention are high in sensitivity, the lowest detection limit can reach 3 copies / mL, the accuracy is 100%, the specificity is high, and cross amplification with other 76 pathogens is avoided. The kit adopts a recombinase polymerase isothermal amplification technology, does not need a large-scale PCR instrument, can complete detection only in 25 minutes, saves about 2 hours of time compared with a traditional fluorescent PCR detection method, adopts an M13 bacteriophage probe as internal positive quality control, can prevent false negative conditions, and can prevent pollution by adding UNG enzyme. The kit can be used for laboratory detection, field detection and bedside detection, and is especially suitable for detection of the yearly skin-adding virus at quarantine ports, airports and epidemic disease outbreak sites.

The invention discloses a construction method and application of chassis cells for synthesizing carnosine and derivatives thereof, and belongs to the technical field of bioengineering. Compared with the original host cell, the mutant chassis cell formed by the construction method of the multiple escherichia coli gene mutant strains provided by the invention not only effectively relieves the degradation of carnosine, but also keeps the growth performance consistent with that of the original strain, and ensures that the chassis cell normally grows by utilizing a general culture medium and culture conditions. Compared with existing other chassis cells, culture medium components and culture conditions do not need to be changed; compared with an original host cell, the chassis cell disclosed by the invention has the advantages that the degradation of carnosine is obviously relieved; the chassis cell provided by the invention is used for respectively recombining three recombinases from different sources, and compared with the original recombinant cell, the L-histidine conversion rate of a cell-free extraction liquid catalytic system is maximally increased by 20.6%, and the L-histidine conversion rate of a whole-cell catalytic system is increased by 10.5%.

This invention discloses a recombinant glucuronide C5 epimerase mutant, expression vector, recombinant expression engineered bacteria, and their culture and induction methods, belonging to the field of biotechnology. The recombinant enzyme sequence is formed by fusing a truncated (N29–N617) mutation (V173R–V197K–A239V–N269E) with an N-terminal maltose-binding protein (MBP) tag to a human glucuronide C5 epimerase (C5-epi). The recombinant glucuronide C5 epimerase mutant exhibits higher activity, 2.1 times that of the recombinant wild-type, and greater stability, retaining 72.5% of its activity after 5 days at room temperature. Furthermore, the soluble expression level of this recombinant mutant glucuronide C5 epimerase is increased by 4-fold.

The invention discloses thermoacidophilic beta-glucosidase and application thereof, and relates to the technical field of biological enzymes. The thermoacidophilic beta-glucosidase is BGL74, and the amino acid sequence of the thermoacidophilic beta-glucosidase is as shown in SEQ ID NO: 4. According to the invention, the GH1 family beta-glucosidase gene bgl74 is screened and cloned by adopting a metagenome technology from the bottom mud of a hot spring of Longwang temple from Dali Pu'er source in Yunnan province, heterologous expression is realized in escherichia coli, and the recombinase BGL-74 is purified. Research results show that the BGL-74 has the characteristics of heat resistance, acid resistance, glucose resistance and the like, has good affinity and catalytic efficiency on soybean isoflavone glycoside, and verifies the feasibility and application prospect of obtaining high-performance beta-glucosidase through hot spring metagenome mining to promote soybean isoflavone aglycone. And a high-quality biological catalysis tool is provided for functional food development and biological manufacturing.

The application belongs to the technical field of detecting meat components based on nucleic acid technology, and particularly relates to a primer pair, a kit and a detection method for detecting horse meat components in food. The sequences of the primer pair are shown in SEQ ID NO. 1 and SEQ ID NO. 2. The application further discloses a kit for detecting horse meat components in food and a method for detecting horse meat components. The detection method comprises a pair of primers labeled with 6-FAM fluorescent groups and biotin, can realize high-sensitivity target detection, and can detect as low as 0.1% horse meat in adulterated meat samples. Compared with recombinant enzyme polymerase isothermal amplification real-time fluorescence detection, there is no need to design and screen RPA fluorescent probes. In addition, the application has good application prospect and can be successfully used for detecting horse meat components in donkey meat products, beef products, mutton products and venison products.

This invention discloses a polyethylene terephthalate (PET) hydrolase, PmPETase, and its applications. The hydrolase PmPETase is a protein with the amino acid sequence shown in SEQ ID NO:1. This invention constructs an expression system suitable for PmPETase, obtains a recombinant enzyme protein with catalytic activity, and comprehensively analyzes its substrate specificity, catalytic properties, and potential plastic degradation ability. This provides a new theoretical basis and application reference for the development and utilization of PET degrading enzymes and green treatment technology for polymer materials.

This invention discloses a phospholipase D and its application in the preparation of ω-3 PUFAs-PA, belonging to the field of functional enzyme technology. The amino acid sequence of the phospholipase D is shown in SEQ ID NO. 9. The application of phospholipase D in the preparation of ω-3 PUFAs-PA is also disclosed. This invention also discloses a recombinant engineered bacterium expressing OUC-nPLD. The phospholipase D of this invention—OUC-nPLD—is a recombinant enzyme obtained by functional domain recombination based on the Non-HKD phospholipase D of *Streptomyces aquamarine*, and its PLD hydrolytic activity is 221.73 U / mg. This invention constructs a recombinant engineered bacterium expressing OUC-nPLD. The OUC-nPLD of this invention can be used for the efficient preparation of ω-3 PUFAs-PA, which is of great significance for the large-scale production of ω-3 PUFAs-PA.

The invention discloses alkyl hydroperoxidase AhpC capable of degrading aflatoxin B1 and application of the alkyl hydroperoxidase AhpC. The alkyl hydroperoxidase AhpC capable of degrading aflatoxin B1 is excavated from bacillus paraamycoids, a carrier containing the AhpC gene is transferred into a host through a genetic engineering means to obtain a genetic engineering bacterium, and a crude enzyme liquid containing the enzyme AhpC is obtained through expression. The optimal degradation pH of the recombinase AhpC is measured to be 9.0, the optimal degradation temperature is 80 DEG C, and the 12h degradation rate of the recombinase AhpC on aflatoxin B1 with the concentration of 2 mu g / mL can reach 95.19% under the optimal conditions. The enzyme has high degradation rate, good heat resistance and alkali resistance, can be widely applied to degradation of aflatoxin B1 in the industrial fields of agriculture, feed, food and the like, and has good application potential.

The present application relates to the field of recombinant enzyme catalytic engineering technology, and provides a kind of intracellular expression lipase Ndbn and its related recombinant engineering bacteria.The lipase described in the present application has the characteristics of organic solvent resistance, can use aniline and ester as substrate in organic solvent, and enzyme synthesis amide compounds.Compared with existing lipases, lipase Ndbn shows high catalytic activity to weak nucleophile aniline, has a wide substrate spectrum, the aromatic amide catalyzed by it can be used as an intermediate of drug, avoids the shortcomings of high temperature and use of toxic reagent in the process of chemical synthesis of amide, has high atom economy, and meets the concept of green manufacturing.

The invention relates to the fields of coordination chemistry and metal organic chemistry, and discloses a single iron hydrogenase model compound containing 3, 4 and 5-substituted pyridine ligands, the chemical structural formula is as follows: # imgabs0 #, R1 and R2 are identical or different monodentate ligands or R1 and R2 represent bidentate ligands, and R3 and R5 are identical or different and represent H or C1-C6 alkyl. R4 represents H or hydroxyl. The recombinase obtained by recombining the single iron hydrogenase model compound and decoenzyme has better stability and higher catalytic activity.

This invention relates to the field of biological products, and particularly to the preparation and application of hangover relief products. This invention provides an alcohol dehydrogenase mutant, an aldehyde dehydrogenase mutant, an expression vector, a combination of recombinant yeast strains, and yeast single-cell oral capsules. By cloning the alcohol dehydrogenase and aldehyde dehydrogenase genes into the *Saccharomyces cerevisiae* expression plasmid pYX212, and then transforming the *Saccharomyces cerevisiae* into the *Saccharomyces cerevisiae*, engineered yeast is obtained. Utilizing the protective effect of yeast cells, oral administration is achieved, effectively reducing the destruction of recombinant enzymes by gastric juice. The yeast single-cell oral capsules can accelerate alcohol metabolism after daily drinking and promote hepatocyte regeneration; they are convenient and quick to take and have significant effects.

The application discloses a cannabidiolic acid synthase mutant, a high-yield recombinant enzyme Pichia pastoris strain and application, and relates to the technical field of biology.The amino acid sequence of the cannabidiolic acid synthase mutant is shown as SEQ ID NO.1.The mutant CBDAS G183V‑N482W is used as research material, residues around an active site and a glycosylation site are systematically modified, and a key mutant site capable of significantly improving the enzyme activity of the cannabidiolic acid synthase mutant CBDAS G183V‑N482W is screened by combining domain function analysis and computer-aided calculation.Finally, the multiple mutant CBDAS H114E‑S116A‑C176Y‑G183V‑N328Q‑N482W shows excellent catalytic performance and can effectively improve the conversion efficiency of catalyzing cannabigerolic acid to generate cannabidiolic acid.

Embodiments of the present invention provides recombinant systems, methods, and compositions for the biological production of Testosteronan (Testan), a linear α-(1→4)-linked glucuronic acid–N-acetylglucosamine polysaccharide. Microbial hosts engineered to express full-length CtTS, N-terminal truncation variants (including d64-CtTS), or the Pseudomonas CIPTS homolog synthesize high–molecular-weight Testan (≥800 kDa) with low polydispersity. Structural analysis by ¹H NMR and 2D HSQC demonstrates that all recombinant enzymes produce Testan that is chemically indistinguishable from native polymer. Purification using ultrafiltration and strong-anion exchange chromatography yields highly defined molecular-weight fractions, including monodisperse populations (PDI ~1.02). The invention further provides sulfated derivatives of Testan produced via aqueous sulfation methods, as well as Testan and functionalized Testan compositions useful for chromatographic stationary phases, biomaterial scaffolds, and chemically modifiable polymer platforms. These systems provide the first scalable, fermentation-based production methods for Testan and Testan-derived materials.

This invention discloses a PLA-degrading lipase mutant and its application. First, the lipase PaPlaA gene with the amino acid sequence shown in SEQ ID NO.1 was selected. Highly efficient heterologous expression was achieved by constructing a PgapA-T7 dual promoter system and optimizing the RBS sequence through ribosome binding site design, resulting in a recombinant enzyme activity yield of 6.25 U / mL, approximately 2.5 times higher than the original strain. Then, combining molecular dynamics simulations and rational design, the substrate pocket and flexible region were structurally modified to obtain the PLA-degrading lipase mutant. The obtained mutant exhibited excellent catalytic performance when applied to PLA degradation. This invention also provides a composite enzyme preparation obtained by combining the PLA-degrading lipase mutant with the protease nprT612. This composite enzyme preparation can achieve a degradation rate of up to 78.2% for PLA nonwoven fabrics. This provides a stable enzyme catalyst and a feasible synergistic degradation process for the efficient biodegradation of PLA at high temperatures.

The invention discloses a UGT1A10 fluorescent probe substrate with endoplasmic reticulum targeting characteristic and preparation and application thereof, the substrate is not applied to diagnosis and treatment of diseases, and the substrate is characterized in that the structural formula of the substrate is as shown in formula (I). The preparation method comprises the following steps: taking N, N-dimethyl-ethyl-4-bromo-1, 8-naphthalimide and p-hydroxyphenylboronic acid as raw materials, and carrying out palladium-catalyzed Suzuki coupling reaction to obtain the compound. The probe substrate can be used for quantitatively determining the activity of UGT1A10 in various biological systems such as recombinase, cells, tissues and the like, and can be used for rapidly screening inducers and inhibitors of UGT1A10. The probe provided by the invention has high specificity, high sensitivity, low cytotoxicity and excellent endoplasmic reticulum co-localization capability, and provides an effective tool for functional research of UGT1A10 and development of related drugs.

Disclosed herein are compositions for cleaving antibodies and in vivo methods of using these compositions. The compositions comprise recombinant enzymes having human and non-human primate IgM-specific protease activity and human and non-human primate IgG-specific protease activity, providing a means of directly addressing pathogenic IgM and IgG antibodies, which are key players in the initiation of immune dysfunction. The enzymes may comprise a sequence having at least 50% identity to the sequence set forth in SEQ ID NO:17 or SEQ ID NO:18.

The invention discloses a carbon dioxide fixation pathway and application thereof in microorganisms, and belongs to the technical field of synthetic biology. The invention designs a novel carbon fixation system of a carbon dioxide pathway, and the carbon fixation system can increase the ability of any microbial cell to prepare lactic acid by using carbon dioxide; the carbon immobilization system includes an engineered pathway to immobilize carbon dioxide to lactic acid, the invention relates to a recombinant enzyme preparation which comprises a formate dehydrogenase, a carboxylic acid reductase, a glyoxylate ligase, a carboxyl lyase, a serine-pyruvate aminotransferase, a serine deaminase, a malic enzyme, a maloyl CoA synthetase, a maloyl CoA lyase, a glyoxylate reductase, a glycolaldehyde dehydrogenase, a pyruvate bifunctional enzyme, a lactic dehydrogenase, and an alanine-glyoxylate aminotransferase. The threonine aldolase can utilize various one-carbon substrates such as carbon dioxide, formic acid and formaldehyde, and has multiple values in the aspect of substrate utilization.