25 results about "Ester hydrolase" patented technology

The invention discloses a BHET hydrolase designed based on an inverse folding algorithm and a preparation method of the BHET hydrolase, and relates to the technical field of biology. The preparation method comprises the following steps: selecting polyester hydrolase PHL7, learning a protein crystal structure by using a deep learning algorithm Protein MPNN, carrying out three-dimensional structure modeling by using Esmfold, carrying out molecular docking on candidate protein and BHET, carrying out molecular dynamics simulation, and screening out protein which is well combined with a substrate; performing expression and purification; the invention further discloses the application of the BHET hydrolase in hydrolysis of BHET. Through the screening process, the BHET hydrolase can be simply, rapidly and efficiently screened from the candidate sequences generated in a large scale, and the source of the BHET hydrolase is expanded.

The invention relates to a preparation method of D-panthenol. The preparation method comprises the following steps: (1) carrying out mixed reaction on DL-pantoic acid lactone and D-pantoic acid lactone hydrolase, extracting, and separating a water phase to obtain D-pantoic acid; (2) mixing the D-pantoic acid with a compound enzyme auxiliary agent for reaction to obtain D-pantoic acid lactone; (3) mixing the D-pantoic acid lactone with beta aminopropanol for reaction to obtain D-panthenol crude product slurry; (4) purifying the D-panthenol crude product slurry to obtain D-panthenol; the compound enzyme auxiliary agent comprises esterase, carbonyl reductase, alcohol dehydrogenase and alcohol nitrile enzyme. According to the method disclosed by the invention, the esterase, the carbonyl reductase, the alcohol dehydrogenase and the alcohol nitrile enzyme act synergistically to catalyze an esterification reaction in D-pantoic acid molecules and promote the D-pantoic acid molecules to form a lactone structure, so that side reactions are reduced, the efficiency of the esterification reaction of the D-pantoic acid is improved, and finally, the yield and the purity of the D-panthenol are improved.

The invention discloses a producing strain of D-pantoic acid lactone hydrolase and application of the producing strain. The producing strain is fusarium moniliforme DPLH6, and the preservation number of the producing strain is CGMCC (China General Microbiological Culture Collection Center) No. 42135. The fusarium moniliforme strain disclosed by the invention has the capability of producing the D-pantoic acid lactone hydrolase at high yield, the D-pantoic acid lactone is prepared after the D-pantoic acid lactone hydrolase fermented by the strain is immobilized by glutaraldehyde, less enzyme dosage and shorter catalysis time can be realized, and the ee value of the target product D-pantoic acid lactone is greater than 99%.

The application relates to the field of genetic engineering, and discloses a 2-hydroxyethyl terephthalate diester hydrolase mutant, a coding gene, a recombination carrier, a recombination strain, an enzyme preparation and application. The 2-hydroxyethyl terephthalate diester hydrolase mutant is obtained by mutating at least one of positions 109, 110, 138, 225, 276, 287, 312, 313, 333, 335, 351, 356, 387, 394, 395, 401 and 413 of an enzyme with the amino acid sequence shown in SEQ ID NO:1. The 2-hydroxyethyl terephthalate diester hydrolase mutant has excellent thermal stability, and has better catalytic efficiency at high temperature.

The preparation method comprises the following steps: detecting the concentration of cytoplasmic acyl-coenzyme A thioester hydrolase in a sample by adopting an ELISA (Enzyme-Linked Immunosorbent Assay) method, and carrying out regression analysis through Logistic, the correlation between the serum level of the cytoplasm acyl-coenzyme A thioester hydrolase in an AD (Alzheimer's disease) patient and a vascular dementia (VD) patient and the cognitive impairment degree and the A beta 40 content is evaluated, and whether the expression of the cytoplasm acyl-coenzyme A thioester hydrolase is closely related to the AD progress or not is judged. An effective molecular marker is provided for differential diagnosis of an AD patient and a VD patient, a large number of tests prove that the cytoplasm acyl-coenzyme A thioester hydrolase is highly expressed in the AD patient, the cytoplasm acyl-coenzyme A thioester hydrolase serving as an AD serum molecular marker has high diagnostic value, and when the serum content of the cytoplasm acyl-coenzyme A thioester hydrolase is higher than 62.5 pg / ml, the cytoplasm acyl-coenzyme A thioester hydrolase can assist AD diagnosis and can also serve as an auxiliary index for distinguishing AD and VD. The kit is applied to preparation of a kit for differential diagnosis of AD and VD.

The invention relates to the technical field of bioengineering, in particular to an ester hydrolase mutant and application thereof. According to the present invention, the screened wild-type ester hydrolase derived from Pseudomonas fluorescens is subjected to mutation to obtain the ester hydrolase mutant, the ester hydrolase mutant is applied to the synthesis of the sitagliptin intermediate, and compared with the wild-type ester hydrolase, the ester hydrolase mutant has characteristics of high enzyme activity and high stereoselectivity on the substrate (compound II), the enzyme consumption is significantly reduced, and the yield of the sitagliptin intermediate is significantly improved; the chiral purity of the reaction product (compound I) is greatly improved, and the method has a good industrial application prospect.

The invention provides a method for preparing L-penicillamine, comprising the following steps: subjecting a compound shown in formula I-1 to a nitration reaction under concentrated nitric acid conditions; subjecting a nitrated product shown in formula II-1 to a thio reagent R2-SH under the catalytic action of an alkaline catalyst in a third organic solvent to a Michael addition reaction under an inert gas atmosphere; reducing the nitro group in the thio product shown in formula III-1 to an amino group to generate a racemic penicillamine shown in formula IV-1; subjecting the racemic penicillamine shown in formula IV-1 to ester hydrolase decomposition to generate a thiol-protected L-penicillamine shown in formula V-1; removing the thiol protecting group from the thiol-protected L-penicillamine shown in formula V-1 under strong acid conditions to generate an L-penicillamine salt, and performing alkaline hydrolysis to generate the L-penicillamine; the method of the invention solves the problems of high raw material cost, complicated synthesis steps and low synthesis efficiency in the prior art.

The invention belongs to the field of biotechnology and biocatalysis, and discloses application of ester hydrolase in preparation of beta-keto acid compounds. A new esterase gene is subjected to heterologous overexpression in escherichia coli through a genetic engineering means to obtain a genetically engineered bacterium for efficiently expressing esterase, and the genetically engineered bacterium is obtained by taking escherichia coli as a host and transferring a recombinant plasmid carrying an esterase gene. The engineering bacterium can efficiently express and produce ester hydrolase, has the advantages of strong substrate tolerance, high concentration feeding, high catalytic efficiency, conversion rate of 99% or more, no by-product generation, short reaction period, less biocatalyst dosage, simple and convenient preparation method, mild conditions and environmental friendliness, and has good industrialization prospects.

The invention belongs to the field of biotechnology and relates to a method for preparing D-pantoic acid by enzymatic conversion. The method utilizes a D-pantoic acid lactone hydrolase (D-Lac) mutant to catalyze the splitting of a DL-pantoic acid lactone substrate to generate D-pantoic acid and L-pantoic acid lactone, and after the reaction is completed, the conversion product is extracted and separated to obtain D-pantoic acid and L-pantoic acid lactone. The method has the advantages of high enzyme catalysis efficiency, short conversion time, simple operation, low production cost, and the like.

The invention discloses a PET hydrolase designed by a deep learning algorithm and a preparation method thereof, and relates to the technical field of biologication.The preparation method comprises the steps that polyester hydrolase PHL7 is selected, a protein crystal structure is learned through the deep learning algorithm InstructPLM, three-dimensional structure modeling is conducted through Esmfold, candidate protein and PET are subjected to molecular docking, and molecular dynamics simulation is conducted. Screening out protein which is well combined with a substrate; performing expression and purification; amino acid sequences of the hydrolase HN and / or the hydrolase JW are respectively shown as SEQ ID NO: 1 and / or SEQ ID NO: 2; the invention also relates to application of the novel PET hydrolase in hydrolysis of PET. Through the screening process, the PET hydrolase can be simply, rapidly and efficiently screened from the candidate sequences generated in a large scale, and the source of the PET hydrolase is expanded.

The present invention discloses an immobilization method and application for effectively improving the acid stability of zearalenone lactone hydrolase, belonging to the field of biotechnology. The present invention uses an injection gel method to encapsulate ZEN hydrolase in hydrogel beads based on potassium carrageenan, and the acid stability of the enzyme is improved by co-encapsulation with an alkaline buffer Mg(OH)2. The Mg(OH)2 buffer obtained by the present invention is insoluble under neutral conditions, but dissolves when hydrogen ions diffuse into the hydrogel beads, thereby ensuring a neutral internal pH value. The use of a hydrogel bead co-encapsulation system containing a Mg(OH)2 buffer improves the stability of ZEN hydrolase in an acidic environment, and is suitable for a food-grade delivery system of ZEN hydrolase for acid-sensitive enzymes, with potential for industrial application.

A transgenic strain for producing 3-hydroxy-3-methylbutyric acid is provided. The transgenic strain comprises a host cell with an anabolic pathway for converting carbon sources into acetyl-CoA and a plurality of exogenous genes. These exogenous genes comprise genes encoding acetyl-CoA acetyltransferase (AtoB) or acetoacetyl-CoA thiolase (NphT7), and genes encoding hydroxymethylglutaryl-CoA synthetase (MvaS), a gene encoding 3-hydroxy-3-methylglutaconyl-CoA dehydratase (LiuC), a gene encoding 3-methylglutaryl-CoA decarboxylase (AibAB), a gene encoding thioester hydrolase (YciA, TesB, MenI or YqiA). A method for producing 3-hydroxy-3-methylbutyric acid using the above genetically modified strain is also provided.

The present application provides a method for preparing a chiral intermediate of Niraparib by means of a chemical-enzymatic method. The method provided by the present application comprises: subjecting compound II, as a starting material, to a Michael addition reaction to obtain compound III; then, subjecting compound III to enzymatic resolution using an ester hydrolase to obtain chiral compound IV, with an ee value of up to 99%; and finally, subjecting compound IV to cyclization and amide reduction reactions to obtain chiral intermediate I of Niraparib. The route features simple operation and low cost, and can be applied to industrial production.

This invention discloses a composite nanozyme, its preparation method, and its application. The composite nanozyme uses Prussian blue nanoparticles as a carrier, with a phospholipid bilayer formed by dimyristoylphosphatidylcholine coated on the surface of the Prussian blue nanoparticles. The phospholipid bilayer contains (2,3-dioleoyl-propyl)-trimethylamine and undecylimidazole. This invention is the first to disclose a composite nanozyme formed by modifying the surface of Prussian blue nanoparticles with DOTAP and imidazole groups. The obtained composite nanozyme can effectively catalyze ester hydrolysis, producing at least two orders of magnitude acceleration, which provides a possibility for the direct hydrolysis of cholesterol esters in atherosclerotic plaques by nanomedicines. This invention expands the enzyme-like activity of the Prussian blue nanozyme through special modification, providing a solution for simultaneously achieving multifunctional composite nanozymes with ester hydrolytic enzyme function, catalase-like properties, and superoxide dismutase-like properties.

This invention relates to a cyclohexenecarbamate hydrolase mutant, its preparation method, and its applications. The cyclohexenecarbamate hydrolase mutant provided by this invention, when used as a catalyst in the asymmetric resolution of chiral cyclohexenecarbamate to prepare chiral cyclohexenecarboxylic acid, exhibits suitable conversion rates, high optical purity (chiral purity exceeding 99%), mild reaction conditions, environmental friendliness, simple operation, high yield, low cost, and ease of industrial scale-up. Therefore, the cyclohexenecarbamate hydrolase mutant and its gene of this invention have promising prospects for industrial application development.

The invention discloses an ester hydrolase detection chemiluminescence probe and a preparation method and application thereof, the ester hydrolase detection chemiluminescence probe based on adamantane-dioxetane derivatives can selectively react with chymotrypsin and generate 540nm chemiluminescence, and meanwhile, the ester hydrolase detection chemiluminescence probe has high selectivity and high sensitivity. The chemiluminescent probe for ester hydrolase detection has a structure as shown in a formula I, which is called CL-CHT-O or adamantane-dioxetane derivative for short; # imgabs0 #

The invention belongs to the technical field of bioengineering, and particularly relates to a carboxylate hydrolase expression system and application thereof. The carboxylate hydrolase expression system comprises: (A) a polynucleotide sequence encoding carboxylate hydrolase EST; and (B) a polynucleotide sequence for coding a signal peptide, the polynucleotide sequence for coding the signal peptide being selected from any one of SEQ ID No: 2-7; and (C) a secretory carrier. Host cells loaded with the carboxylate hydrolase expression system can directly secrete esterase into a culture medium in the fermentation process, and meanwhile the expression quantity and catalytic activity of the esterase are improved. According to the invention, the fermentation process is also optimized, the expression potential of the carboxylate hydrolase EST is further released, and the industrial production requirements of high selectivity and high conversion rate are met.

The invention discloses a PAEs hydrolase gene and application thereof. The nucleotide sequence of the dibutyl phthalate hydrolase gene paeH is as shown in SEQ ID NO. 1. PaeH (SEQ ID NO.2) expressed by an engineering strain constructed by using the gene can be used for remarkably degrading PAEs (Polyacrylamide) compounds such as dibutyl phthalate, butyl benzyl phthalate, diamyl phthalate, diisobutyl phthalate and the like. Therefore, the produced PaeH enzyme preparation can be used for removing PAEs compounds such as dibutyl phthalate, butyl benzyl phthalate, diamyl phthalate, diisobutyl phthalate and the like which are remained in soil or water.

The invention belongs to the field of medicinal chemistry, and provides a synthesis method of an edoxaban key chiral intermediate A. According to the synthesis method, 3-cyclohexene-1-formic acid is taken as a starting raw material, bromine ring esterification is carried out, selective hydrolysis is carried out by using specific biocyclic ester hydrolase, and solid-liquid separation is carried out, so that a target compound A is obtained, post-treatment is simple and convenient, the synthesis route is shortened, the used bio-enzyme mother liquor can be reused, the activity is maintained for 9-10 times, and the whole process is environment-friendly, low in cost and suitable for large-scale production; and the molar yield of the produced key chiral intermediate A is high, the ee value of a crude product after resolution reaches 95% or above, and the ee value exceeds 99.0% after refining.

Provided in the present invention is a method for preparing sucralose using a biological enzyme, which belongs to the technical field of biocatalysis. The method refers to a method for removing the acetyl group at the 6-position in sucralose-6-acetate in an enzymatic reaction system by controlling enzymatic reaction conditions to obtain a sucralose product. According to the present invention, based on a study on the enzymatic hydrolysis mechanism of sucralose-6-acetate, efficient sucralose-6-acetate hydrolases are selected. The enzyme-to-substrate ratio, reaction temperature, and pH are optimized by reducing the inhibitory effect of reaction substrates and products on the enzyme, thereby greatly improving the hydrolysis efficiency of sucralose-6-acetate. In addition, in the present invention, pure water is used to replace a 0.15 mol / L PBS buffer system, and a 5 L tank is used to achieve repeated use of enzyme over 120 batches, greatly reducing the cost of starting materials. The operation is simple, the reaction is complete, and the industrialized and large-scale production can be easily implemented.

The invention relates to the technical field of enzyme engineering, and discloses a preparation process of acid-adaptive zearalenone lactone hydrolase, which comprises the following steps: providing recombinant escherichia coli containing a gene for coding mutant protein, the mutant protein mutates the 59th alanine into proline and / or mutates the 161st asparagine into phenylalanine based on a wild enzyme; carrying out enlarge culture on the strain, then carrying out induced expression, collecting thalli, breaking walls and separating supernate; and carrying out affinity chromatography purification and elution on the supernate to obtain the hydrolase. By introducing rigid and hydrophobic side chain mutation, the thermodynamic stability and catalytic efficiency of the enzyme are improved, so that the enzyme has excellent acid resistance; by combining optimized expression and purification conditions, efficient preparation of high-activity soluble protein is realized, the problems that an existing enzyme preparation is easy to inactivate and low in yield in an acid environment are solved, and the method is suitable for industrial production of feed additives.

The application discloses a method for flame-retardant finishing of polyester by graft modification, and belongs to the technical field of textile dyeing and finishing. Specifically, first, polyester fabric is treated by means of ester hydrolase to hydrolyze ester bonds on the surface of the polyester fabric to generate hydroxyl groups; then, methacrylic anhydride is reacted with the hydroxyl groups on the surface of the polyester fabric to introduce vinyl groups on the surface of the polyester fiber; finally, the polyester fabric is immersed in a mixed solution of vinyl phosphonic acid and N,N'-methylene bisacrylamide, and graft polymerization is catalyzed by horseradish peroxidase to form a polyvinyl phosphonic acid layer on the surface of the polyester, thereby realizing the method for flame-retardant finishing of the polyester. The specific steps include: (1) ester hydrolase pretreatment of the polyester; (2) introduction of vinyl groups on the surface of the polyester; and (3) graft polymerization catalyzed by horseradish peroxidase. Compared with the traditional baking finishing method, the method has good flame-retardant finishing, and avoids the shortcomings of easy strength decrease of the polyester and high production energy consumption in the traditional high-temperature baking finishing method.

The invention provides a preparation method of efficient immobilized D-pantoic acid lactone hydrolase. The method comprises the following steps: mixing wet thalli of D-pantoic acid lactone hydrolase with a glutaraldehyde aqueous solution, and pre-crosslinking to obtain a thalli suspension; dissolving sodium alginate in water to obtain a sodium alginate aqueous solution; mixing the thallus suspension with a sodium alginate aqueous solution, and performing emulsification and vacuum degassing treatment to obtain an emulsion; dropwise adding the emulsion into solidification liquid containing calcium chloride and a synergistic cross-linking agent, solidifying, collecting immobilized enzyme microspheres, and washing to obtain the efficient immobilized D-pantoic acid lactone hydrolase. The immobilized D-pantoic acid lactone hydrolase is high in mechanical strength and good in enzyme activity stability, can be repeatedly used for more than 500 times, is applied to catalysis of hydrolysis of DL-pantoic acid lactone, and can realize efficient and low-cost green synthesis of D-pantoic acid.

The present disclosure relates generally to a polypeptide having mono-(2-hydroxyethyl) terephthalate hydrolase (MHETase) activity, more specifically to a polypeptide comprising an amino acid sequence that (i) has at least 70% sequence identity to SEQ ID NO:1 and (ii) differs from SEQ ID NO:1 by an amino acid substitution at one or more positions selected from the group consisting of positions that correspond to amino acid positions 156 to 396, 398 to 410 and 425 to 603 of SEQ ID NO:1.