63 results about "Carboxylesterase" patented technology

The invention discloses a fusion enzyme capable of efficiently degrading PET (polyethylene terephthalate) plastic and application of the fusion enzyme. The invention provides a recombinant fusion enzyme. The recombinant fusion enzyme comprises a mutant KL-MHETase of thermophilic carboxylesterase Est30 and a PET (Polyethylene Terephthalate) degrading enzyme FAST-PETase, the amino acid sequence of the mutant KL-MHETase of the thermophilic carboxylesterase Est30 is as shown in SEQ ID NO. 3; the amino acid sequence of the FAST-PETase is as shown in the 284th to 546th amino acids of SEQ ID NO.1 (Sequence Identifier Number 1). The fusion enzyme is used as a biocatalyst, the activity of catalyzing hydrolysis of MHET at 50 DEG C is 52.6 times that of FAST-PETase, and the fusion enzyme has high enzyme activity and thermal stability. PET degradation is catalyzed at the temperature of 50 DEG C, the conversion rate of PET is 90% after 48 hours of reaction, and meanwhile, the TPA content in the product is close to 100%.

The invention discloses a high-activity and high-thermal-stability MHET hydrolase mutant and application thereof. The invention provides a mutant protein of thermophilic carboxylesterase, which is shown as 1) or 2): 1) the protein shown as 1) is a protein obtained by mutating amino acid residues at the 130th site and the 171th site of wild thermophilic carboxylesterase shown as SEQ ID NO.2 or a sequence corresponding to the SEQ ID NO.2 and keeping other amino acid residues unchanged; (2) the protein is obtained by mutating amino acid residues at the 127th site, the 130th site and the 171th site of wild thermophilic carboxylesterase and keeping other amino acid residues unchanged. Experiments prove that the activity of the modified mutant for catalyzing MHET hydrolysis at the temperature of 50 DEG C is improved by 95 times compared with that of a wild type, and the modified mutant has higher enzyme activity and thermal stability and is the first enzyme capable of efficiently catalyzing MHET hydrolysis at the temperature of 50 DEG C at present.

The invention discloses a marine-derived carboxylesterase and an application of the marine-derived carboxylesterase in degradation of PET (Polyethylene Terephthalate) plastics. The amino acid sequence of the enzyme is as shown in SEQ ID NO.1, experiments show that Tm of RbCE1 is 79.5 DEG C, 40 mM of depolymerization monomers can be generated after the enzyme treats low-crystallinity PET powder at 60 DEG C for 4 hours, the catalytic activity is equivalent to that of LCC, and good degradation activity is shown. Through escherichia coli heterologous expression, 0.4 g / L high expression quantity can be achieved in a shake flask, and the potential of remarkably reducing the enzyme production cost is achieved. Therefore, the novel carboxylesterase RbCE1 is suitable for degrading PET plastics and composite materials thereof, and is a potential enzyme preparation for efficient biological catalysis of a waste PET plastic waste enzymatic depolymerization recovery treatment process.

The invention relates to a process for producing three recombinases capable of hydrolyzing organophosphorus, carbamate and pyrethroid insecticides, and preparations containing these enzymes. The present invention provides recombinant plasmid DNA vectors in which the protein secretion system has been modified to improve the ability of recombinant proteins to secrete into the space around the plasmid or culture medium. A nucleotide sequence for coding a PelB (butyric acid lyase B) signal sequence is deleted and substituted by a nucleotide sequence for coding a LamB (maltodextrin or phage lambda receptor) signal sequence, so that the secretion efficiency of the recombinant protein is higher. The invention discloses three recombinant plasmid DNA (deoxyribonucleic acid) vectors which are constructed by utilizing the improved plasmid DNA vectors and are used for expressing recombinant proteins. The recombinant protein comprises (a) a recombinant organophosphate hydrolase having at least 80% sequence identity with the amino acid sequence of methyl parathion hydrolase (MPH) (Protein Database (PDB) ID: 1P9E) as shown in SEQ ID NO: 1, wherein the recombinant organophosphate hydrolase has hydrolytic activity on organophosphate insecticides; (b) a recombinant carbamate hydrolase having at least 80% sequence identity with the amino acid sequence of L-aminopeptidase-D-Ala-esterase / amidase (aryl amidase) (MAH) (PDB ID: 1B65), as shown in SEQ ID NO: 2, wherein the recombinant carbamate hydrolase has hydrolytic activity on carbamate insecticides; and (c) a recombinant pyrethroid hydrolase having at least 80% sequence identity with the amino acid sequence of the pyrethroid hydrolytic carboxylesterase (EstA) (PDB ID: 4CCY) as shown in SEQ ID NO: 3, wherein the recombinant pyrethroid hydrolase has hydrolytic activity on a pyrethroid insecticide. The embodiment of the invention further provides three recombinant bacterial strains, an escherichia coli strain BL21 (DE3) is used as a host cell, and each recombinant plasmid DNA vector is used for transformation. The invention also discloses culture conditions of the recombinant strain, wherein the culture conditions comprise culture medium composition, induction conditions and enzyme harvesting and enzyme activity detection methods. In addition, the embodiment of the invention also provides a preparation which contains the three recombinases as functional components.

The invention discloses a double-targeting near-infrared ratiometric fluorescent probe and application thereof in preparation of a carboxylesterase detection reagent. The structure of the double-targeting near-infrared ratiometric fluorescent probe is shown as follows: # imgabs0 #. A quinoline salt part in the fluorescent probe ZZD can target mitochondria, and the ZD structure after response can be positioned on lipid droplets. The fluorescent probe ZZD has the advantages of good response capability, excellent double-organelle targeting capability and near-infrared emission in living cells. The fluorescent probe ZZD is expected to become a useful fluorescent tool for researching lipid droplet and mitochondrial related diseases in the future.

The present invention relates to a fluorescent probe of carboxylesterase 2 and its synthesis method and application. The fluorescent probe uses 1,8-naphthalimide as a fluorescent group, introduces 4-morpholineacetic acid to locate in lysosomes, and uses carboxylate as an enzyme cleavage site to construct a carboxylesterase 2 fluorescent probe. The probe induces the breakage and departure of its carboxylester group through an enzymatic reaction with carboxylesterase 2, and detects the presence and activity of carboxylesterase 2 by changes in the fluorescence intensity of the probe and its reaction product at 560nm. It can be used for qualitative and quantitative detection of carboxylesterase 2 activity in various living cells and subcellular levels.

The present invention relates to fusarium toxin cleavage polypeptide variants of a fusarium toxin carboxylesterase having SEQ ID No.1, 46 wherein said polypeptide variants each have an amino acid sequence shortened by 47 amino acids at the N-terminus and said amino acid sequence has a sequence identity of at least 70%, preferably 80%, particularly preferably 100%, to the amino acid sequence segments 48-540 of SEQ ID No.46, i.e. SEQ ID No.1; and isolated polynucleotides encoding the polypeptide variants; and an additive for cleaving fusarium toxins comprising at least one polypeptide variant and optionally at least one adjuvant; the invention also relates to the use of said polypeptide variants or additives for hydrolytically cleaving at least one fusarium toxin, and to a method for hydrolytically cleaving at least one fusarium toxin.

The present disclosure provides engineered carboxyesterase enzymes that have the ability to catalyze amide bond formation. Also provided are polynucleotides encoding the carboxyesterase enzymes, host cells capable of expressing the engineered carboxyesterase enzymes, and methods of using the engineered carboxyesterase enzymes to make commercially valuable amides. Also provided are amides that are made using the engineered carboxyesterase enzymes.

The invention belongs to the technical field of genetic engineering, and discloses application of an anchoring protein INP-N in a PET degrading enzyme system displayed on the surface. According to the invention, after efficient anchoring protein INP-N is screened, FAST-PETase is mediated by the efficient anchoring protein INP-N to construct recombinant escherichia coli of which the surface displays PET degrading enzyme. Furthermore, hydrophobin HFBII is introduced into the surface display system, so that the relative enzyme activity of the PET degrading enzyme is improved by 1.1 times or more. Meanwhile, FAST-PETase and carboxylesterase Est30KL are synchronously expressed, so that the proportion of TPA in a degradation product can reach 95% or above. In addition, the PET degrading enzyme surface display system provided by the invention is excellent in cycle performance, more than 65% of activity can still be retained when PET is cyclically degraded for the third time, and the application cost can be greatly reduced. The method can be applied to the fields of degradation of PET products, preparation of PET degradation agents or BHET degradation agents and the like.

The invention discloses a molecular marker for interaction resistance of panonychus citri to insecticides as well as application and a detection method of the molecular marker. According to the molecular marker for the cross resistance of the panonychus citri to the multiple medicaments, the molecular marker is carboxylesterase genes PcCCE11 and / or PcCCE7, the nucleotide sequence of the PcCCE11 gene is as shown in SEQ ID NO.1, and the nucleotide sequence of the PcCCE7 gene is as shown in SEQ ID NO.2. The invention further discloses a preparation method of the molecular marker for the cross resistance of the panonychus citri to the multiple medicaments.

The invention discloses a pyridyl-based water-soluble dual-response fluorescent probe as well as a preparation method and application thereof, and relates to the technical field of fluorescent probes. The technical scheme is as follows: the pyridyl-based water-soluble dual-response fluorescent probe has the following structure: # imgabs0 #. According to the invention, acetyl is used as a recognition site of carboxylesterase, terminal benzyl pinacol ester is used as a reaction site of hypochlorite, and the fluorescent probe capable of simultaneously responding to on-off and ratio of hypochlorite and carboxylesterase is prepared; in addition, pyridinium provides an effective mitochondrial targeting site, so that the probe can detect mitochondrial hypochlorite and carboxylesterase in living cells.

The invention provides a method for improving the molecular weight of polyhydroxyalkanoates (polyhydroxyalkanoates, called PHA for short) synthesized by microorganisms, and relates to a method for improving the molecular weight of the polyhydroxyalkanoates (polyhydroxyalkanoates, called PHA for short). By knocking out or knocking down endogenous (intracellular) carboxylesterase, lipase, esterase, thioesterase and PHA degrading enzyme genes of bacteria for synthesizing PHA, the molecular weight of various synthesized PHA is obviously improved. The method has universality for improving the molecular weight of various PHAs synthesized by different species of bacteria, the mechanical property of the PHA can be improved by improving the molecular weight of the PHA, and new performance is provided for expanding the application of the PHA.

The present invention discloses a pyridine-based water-soluble dual-response fluorescent probe and a preparation method and application thereof, and relates to the technical field of fluorescent probes. The technical scheme is as follows: the pyridine-based water-soluble dual-response fluorescent probe has the following structure:. The present invention uses acetyl as the recognition site of carboxylesterase and terminal benzyl pinacol ester as the reaction site of hypochlorite to prepare an on-off and ratio fluorescent probe that can simultaneously respond to hypochlorite and carboxylesterase; in addition, the pyridinium salt provides an effective mitochondrial targeting site, which enables the probe to detect mitochondrial hypochlorite and carboxylesterase in living cells.

The present invention belongs to the field of biomedicine, and specifically relates to a diagnostic and therapeutic integrated probe responsive to carboxylesterase, and a preparation method and application thereof. The present invention discloses a small molecule diagnostic and therapeutic probe represented by the following structural general formula. This small molecule diagnostic and therapeutic probe is obtained by reacting a near-infrared fluorescent dye, chlorambucil, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and 4-dimethylaminopyridine (DMAP) in an organic solvent. The present invention encapsulates the above small molecule diagnostic and therapeutic probe with poly(lactic acid-co-glycolic acid)-polyethylene glycol (PLGA-PEG) to obtain a nano diagnostic and therapeutic integrated probe. The fluorescence wavelength of the obtained nano diagnostic and therapeutic integrated probe is located in the near-infrared region, which can realize near-infrared fluorescence imaging diagnosis of tumors, and has combined therapeutic properties of chemotherapy, photodynamic therapy, and sonodynamic therapy, and can significantly kill tumor cells.

The invention relates to an application of carboxylesterase TfdI in degradation of phenoxy carboxylic acid herbicides. The invention provides application of rhodococcus pyridinivorans Y6 in degrading phenoxy carboxylic acid herbicides, a novel phenoxy carboxylic acid herbicide degrading enzyme TfdI is cloned from the rhodococcus pyridinivorans Y6, the rhodococcus pyridinivorans Y6 has excellent catalytic action on phenoxy carboxylic acid herbicides, the degradation rate on 2, 4-D isooctyl ester with the concentration of 100 mg / L within 1 h is 100%, and the degradation effect is stable. The excellent degrading enzyme provided by the invention has good application potential in bioremediation of the environment polluted by the phenoxy carboxylic acid herbicide, a new scheme is provided for solving the problem of residual pollution of the 2, 4-D isooctyl ester herbicide in agricultural production, and meanwhile, a herbicide degradation gene resource library is enriched.

Methods of treating a metabolic disorder in a subject are provided. Aspects of the method include enhancing extracellular Carboxylesterase 2 (CES2) activity in the subject in order to treat the subject for the metabolic disorder. Also provided are compositions for use in practicing the methods.

The invention discloses a pH / esterase dual-response nano pesticide delivery system as well as a preparation method and application thereof. The nano pesticide delivery system comprises a carboxymethyl chitosan-polycaprolactone graft copolymer (CMCS-g-PCL) carrier, and a pyrethroid pesticide which is loaded on the carrier through physical embedding. The nano pesticide delivery system has good storage stability and foliar affinity, and the photolysis resistance and enzymolysis resistance of the loaded pesticide are remarkably improved. After application, the system shows excellent environmental responsiveness, keeps stable deposition under an acidic condition, and accurately triggers pesticide release in alkaline intestinal tracts of lepidoptera pests and in a carboxylesterase environment. The method is suitable for precise prevention and control of lepidoptera pests, and has the effects of pesticide reduction and environmental risk regulation and control.

The application discloses an esterase whole cell catalyst and preparation and application thereof, the esterase whole cell catalyst can express a carboxylic acid esterase mutant disclosed in the application, the carboxylic acid esterase mutant is subjected to the following mutations on the amino acid sequence shown in SEQ ID NO. 1: the glycine at the 236th position is replaced by valine, the arginine at the 417th position is replaced by phenylalanine, and the threonine at the 545th position is replaced by alanine. The application effectively solves the problems of low catalytic activity of a warming catalyst for resolving methyl chrysanthemate and poor product tolerance in the prior art. The esterase whole cell catalyst of the application significantly improves the activity of selectively resolving methyl chrysanthemate to prepare dextro-trans chrysanthemic acid, and the optical purity can reach more than 99%. Moreover, the carboxylic acid esterase mutant disclosed in the application can be expressed in a soluble form in cell supernatant, can be produced in a large scale through microbial fermentation, has mild reaction conditions, and is simple to operate, and is more suitable for industrial production.

The application discloses a carboxylic esterase lyophilization protective agent, a preparation method and application thereof, and belongs to the technical field of bioengineering. The carboxylic esterase lyophilization protective agent is a single-component polyvinylpyrrolidone; wherein, the polyvinylpyrrolidone has a viscosity-average molecular weight Mv of 10000-15000000; a K value of 17-90; when used for lyophilization protection of carboxylic esterase, the carboxylic esterase lyophilized powder can effectively improve the stability of the carboxylic esterase lyophilized powder at normal temperature, prevent the purity and activity of the carboxylic esterase lyophilized powder from decreasing, and the lyophilized powder cake is complete, easy to be powdered, the enzyme powder resuspension is clear and transparent, the enzyme activity recovery rate is high, and the carboxylic esterase is convenient for being applied to different application scenarios.

The present invention discloses a fluorescence probe for two - photon detection CE, its preparation, screening and application, belonging to the technical field of synthesis and detection. The structural formula of the fluorescence probe is as follows: #imgabs0# wherein, R1 is H, R2 is H; or, R1 is H, R2 is F; or, R1 is F, R2 is H. The fluorescence probe is synthesized in one step by reacting 7 - amino - 4 - methylcoumarin with differently substituted chloroacetyl chloride and triethylamine to obtain the fluorescence probe P. By comparing the fluorescence response multiples, response times and stabilities of the three fluorescence probes, the fluorescence probe P2 with R1 being H and R2 being F is selected for subsequent testing and application. The fluorescence probe P2 can react with CE to break the amide bond, and the ICT effect disappears, resulting in enhanced fluorescence. Therefore, the fluorescence probe P2 can be used to detect CE, and the probe has a simple structure, and has the advantages of high selectivity, high sensitivity and rapid response, etc.

This invention discloses a carboxylesterase-activated mitochondrial-targeting fluorescent probe with photodynamic therapy properties, its preparation method, and its uses. The structure of the mitochondrial-targeting fluorescent probe is shown below. The mitochondrial-targeting fluorescent probe CML of this invention can specifically respond to different concentrations of carboxylesterases (CEs), with a detection limit as low as 0.30 U / L. Simultaneously, this probe can specifically target mitochondria, and through structural optimization (replacing the methyl group on the indole salt with a butyl group), its mitochondrial anchoring ability is significantly superior to that of methyl-substituted derivatives. Cytotoxicity experiments confirmed that the probe has good biocompatibility; confocal fluorescence imaging experiments showed that this probe can effectively monitor the fluorescence changes of endogenous and exogenous CEs in HepG2 cells. Most importantly, after being specifically activated by carboxylesterases, the probe can efficiently generate reactive oxygen species under light conditions, thereby exerting a photodynamic therapeutic effect on cancer cells.

The invention provides a compound for in-vivo detection of carboxylesterase 2, or a salt thereof, or a stereoisomer thereof, and a preparation method and application thereof, and belongs to the technical field of in-vivo luminescence detection of organisms. The invention provides a bioluminescent probe as shown in a formula I which is described in the specification. The probe is high in sensitivity, good in selectivity and suitable for accurate quantitative and qualitative analysis of carboxylesterase 2. Moreover, the preparation process is simple and convenient, the preparation cost is low, the operation condition is mild, the biocompatibility is good, the application prospect is wide, and the method is suitable for medical and biological research on carboxylesterase 2 detection in a living body. # imgabs0 #

The invention belongs to the field of biological enzyme catalysis, and provides a carboxylesterase CarEst3 mutant, the mutant is obtained by mutating phenylalanine at the 66th site of an amino acid sequence as shown in SEQ ID NO.2 into arginine, and the mutant can be used for catalyzing kinetic resolution of racemization 3-cyclohexene-1-methyl formate to prepare (S)-3-cyclohexene-1-formic acid. High stereoselectivity is achieved, and large-scale production can be achieved; the method for preparing (S)-3-cyclohexene-1-formic acid has the advantages of mild reaction conditions, high substrate concentration, good stereoselectivity, simple post-treatment, high product yield and the like.

The application relates to a synthesis method of a compound of formula I, a genetically engineered bacterium for synthesizing terpenes and application of the genetically engineered bacterium, relates to the field of biosynthesis, and a synthesis method of terpenes, and comprises the following method: a compound of formula II is sequentially subjected to the action of cytochrome oxidase, carboxylic acid esterase and cyclase to generate the terpene compound shown in formula I. The application solves the problem of multiple synthesis steps of the compound shown in formula I. The application solves the problem of low enzyme rate of each step through gene optimization.

The invention provides a biosynthesis method of an esdoxaban tosylate chiral intermediate. According to the preparation method, 3-cyclohexene-1-carboxylic acid methyl ester is taken as a starting raw material, and a target intermediate is finally obtained through carboxylesterase resolution, hydrolysis, hydroxylation, dehydrogenation oxidation, transamination reaction, amidation and Boc2O reaction in sequence. According to the method, high-selectivity carboxylesterase is used for realizing chiral resolution; p450 oxidase with excellent regioselectivity and stereoselectivity is utilized to realize specific hydroxylation of a C-H bond under a mild condition, and a traditional process depending on a highly toxic chemical oxidant is overturned; then, clean oxidation is realized through alcohol dehydrogenase, and a way is laid for subsequent reaction; most importantly, the transaminase is adopted to directly construct the chiral amine, so that the use of a high-risk reagent sodium azide is thoroughly avoided, and major potential safety hazards are eliminated from the source; and finally, green amidation is realized by virtue of lipase, and an acyl chloride method for generating corrosive wastewater is replaced.

The invention relates to the technical field of enzyme gene engineering, and provides a carboxylesterase mutant and an application thereof in preparation of (S)-3-cyclohexene-1-formic acid in order to solve the problems of poor substrate selectivity and high raw material cost in preparation of (S)-3-cyclohexene-1-formic acid by a biological method in the prior art. The mutant is D253Y, N329F, V133A / N329F, V133A / D253Y, N329T / D253Y, N329V / D253Y, N329S / D253Y, N329Q / D253Y, N329M / D253Y, N329L / D253Y or N329H / D253Y obtained by mutation of CarEst3 esterase with an amino acid sequence as shown in SEQ ID NO: 1. The carboxylesterase mutant can be used for preparing (S)-3-cyclohexene-1-formic acid, and the selectivity is good.

This invention discloses an alkaloid, its preparation method, and its application in the preparation of human carboxylesterase 2 inhibitors. The residue of Paeonia lactiflora after ultrasonic extraction was extracted with 95% ethanol and then with ethyl acetate to obtain ethyl acetate extract CS-5. Separation was performed by silica gel column chromatography, using a gradient elution with petroleum ether-acetone mixed solvent and dichloromethane-methanol mixed solvent to obtain six eluent fractions, named A-F. Compound D was separated by silica gel column chromatography, Sephadex LH-20 column chromatography, and semi-preparative HPLC to obtain compound I-2. Compound F was separated by Sephadex LH-20 column chromatography and semi-preparative HPLC to obtain compounds I-1 and I-3. Compound I-1 was separated by a chiral HPLC column to obtain enantiomers I-(+)-1 and I-(–)-1. The four alkaloids of this invention have a pH range of 13.65–14.16. m At concentrations of M, it can significantly inhibit the activity of human carboxylesterase 2. Therefore, it can be applied to the preparation of attenuated and protective agents for anticancer drugs such as irinotecan.

The invention belongs to the technical field of medicinal chemistry, and discloses a human carboxylesterase 2A (hCES2A) self-activated covalent inhibitor as well as a preparation method and application thereof. The inhibitor is a novel benzamide-O-carbamate derivative (I) with a structure as shown in a formula (I) and pharmaceutically acceptable salts thereof, and the compound can powerfully and highly selectively inactivate the activity of human intestinal tract hCES2A. Further, the oral bioavailability of the oral prodrug is improved or the intestinal toxicity caused by the carboxylesterase 2A activated prodrug (such as irinotecan) is relieved. The IC50 value of the compound for inhibiting human carboxylesterase 2A can reach a nanomole level and is dose-dependent and time-dependent. The compound provided by the invention also has good safety, and has the advantages of simple preparation process, high yield and the like, which indicates that the compound has good application prospects.

The present disclosure provides engineered carboxylesterase enzymes having improved activity in catalyzing cyclization of short polypeptides, as compared to a naturally occurring wild-type carboxylesterase enzyme. These variants catalyze the cyclization of linear tetrapeptides with high regioselectivity. Also provided are polynucleotides and expression vectors encoding the carboxylesterase enzymes and methods of using the carboxylesterase enzymes to generate cyclic tetrapeptides containing non-canonical amino acids.

The invention discloses a camptothecin-loaded enzyme response self-assembly polypeptide prodrug as well as a preparation method and application of the camptothecin-loaded enzyme response self-assembly polypeptide prodrug. Belongs to the technical field of polypeptide prodrugs and preparation methods and application, the camptothecin-loaded enzyme response polypeptide prodrug is synthesized through a solid-phase organic synthesis reaction, the camptothecin-loaded enzyme response polypeptide prodrug can specifically recognize and shear with fibroblast activated protein alpha and carboxylesterase, in-situ self-assembly is carried out to form nanofibers, and a small-molecule chemotherapeutic drug camptothecin is released; the response type self-assembly polypeptide prodrug disclosed by the invention is simple to synthesize and high in stability; the polypeptide prodrug can be subjected to high-specificity in-situ self-assembly and camptothecin release under the action of fibroblast activated protein alpha and carboxylesterase overexpressed by tumor cells, so that efficient delivery and long retention of the drug to a tumor area are realized; the invention fills the blank in the aspects of design and preparation method of the polypeptide prodrug capable of responding to two enzyme markers to form a nano self-assembly and camptothecin slow release.