40 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 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 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.

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.

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 belongs to the technical field of nucleoside medicine preparation, and particularly discloses a method for efficiently preparing 2-deoxy-D-ribose. According to the method, reactants are added into Escherichia coli fermentation liquor for recombinant expression of thymidine phosphorylase, and acid hydrolysis treatment is carried out after one-step catalytic reaction to directly generate 2-deoxy-D-ribose. The method can be used for catalytically preparing the high-concentration 2-deoxy-D-ribose, so that the production of the 2-deoxy-D-ribose reaches a kilogram level, and large-scale industrialization is realized. The preparation process disclosed by the invention does not need to purify recombinase, is simple, saves cost, saves energy, reduces emission, reduces equipment investment, and further reduces production cost.

The invention discloses a beta-glucosidase gene and an application thereof. Relates to the technical field of enzyme engineering. Comprising a gene of beta-glucosidase, an amino acid sequence and an application of the beta-glucosidase. The optimum temperature of the obtained recombinase B0-BG30 is 40 DEG C, the optimum pH value is 5.6, and the activity of the recombinase B0-BG30 is kept 100% after incubation at 35 DEG C for 2 hours; the activity still exceeds 80% after incubation for 24 h at the pH of 5.0-10.0; the enzyme activity is activated in a 0.5-3.5 M salt solution, and the activity is still kept at 45% or above when the salt concentration is 5 M. Enzyme resources are provided for the aspects of industry, medicine, environmental protection and the like, meanwhile, the research method is widened, and a new thought and a new technical means are provided for detection and application of glycoside compounds.

The invention belongs to the technical field of gene engineering and enzyme engineering, and particularly relates to a method for efficient secretory expression of pichia pastoris for recombinant bacillus lipase LIPBST and BSLA. The method comprises the following steps: respectively constructing recombinant secretory expression vectors containing LIPBST and BSLA mature peptide coding sequences, transforming the recombinant secretory expression vectors into a pichia pastoris host, and realizing efficient secretory expression of the two lipases by optimizing respective inducible expression conditions (the LIPBST is 2% v / v methanol concentration and induction start OD600 is equal to 3, and the BSLA is 1% v / v methanol concentration and induction start OD600 is equal to 1). Through fermentation culture and affinity chromatography purification, the high-purity recombinase is obtained, the LIPBST yield reaches 230 mu g / mL, and the enzyme activity is 195 U / mL; the yield of the BSLA reaches 125 mu g / mL, and the enzyme activity is 126 U / mL. The high-yield expression of the LIPBST and the BSLA is realized in the pichia pastoris for the first time, and the obtained enzyme preparation has excellent alkaline tolerance and medium-temperature activity and is suitable for the fields of detergents, food processing, biological catalysis and the like.

This invention discloses the application of a glutamine-depleting engineered bacterium and its pharmaceutical composition. The engineered bacteria use attenuated bacteria with tumor-targeting capabilities as vector strains. The genome or plasmid of the vector strain integrates a therapeutic gene encoding glutaminase. This method achieves deep and continuous depletion of glutamine in the tumor, avoiding drug resistance caused by tumor metabolic compensation. The inhibition rate against c-Myc-overexpressing soft tissue sarcomas is significantly higher than that of small molecule inhibitors. Enzyme production is only carried out locally in the tumor, with no effect on normal tissues and no systemic toxicity. The engineered bacteria can penetrate the dense tumor matrix and colonize the tumor, solving the problem of poor tissue penetration of small molecule inhibitors. The engineered bacteria continuously multiply and produce enzymes in the tumor, achieving continuous release of glutaminase. The half-life is significantly longer than that of recombinant enzymes, eliminating the need for frequent dosing. The glutaminase optimized by protein engineering has significantly reduced immunogenicity.

The present application relates to the technical field of enzyme engineering, and particularly relates to a recombinant enzyme UvsX and preparation and application thereof.The amino acid sequence of the recombinant enzyme UvsX provided by the present application is shown as SEQ ID NO.1, a recombinant expression strain with a higher expression amount is obtained through optimization of Escherichia coli codon bias and induction conditions, and the recombinant enzyme UvsX with a purity greater than 95% is purified; the recombinant expression vector and strain obtained by the present application can be used for efficient expression of the recombinant enzyme UvsX and preparation of an RPA kit.Compared with a commercially available ordinary recombinant enzyme UvsX, the recombinant enzyme UvsX obtained by the present application has higher activity and can more efficiently perform a recombinant enzyme polymerase amplification reaction.

To provide methods and means for specifically altering the DNA sequence in a genome, in particular for genome editing by deleting or replacing a sequence of interest.SOLUTION: Disclosed is a method for producing a DNA-recombining enzyme to target sites using two non-identical sequences naturally occurring in a genome as the target sites. The invention is in particular useful for medicine, in particular to repair a mutation in a genome or to delete predefined genetic material derived from cells or tissue and to cure diseases. An advantage of the invention is that it allows precise site directed altering of DNA without engaging host DNA repair pathways and thereby works without inducing random insertions and deletions (indels).SELECTED DRAWING: Figure 1