296 results about "Enzyme method" patented technology

The invention belongs to the field of gene engineering and enzyme engineering, and particularly relates to a method for producing rebaudioside M by an enzyme method. The method comprises the following steps: S1, adding a mixed solution 1 of recombinant cell disruption liquid into a substrate, adding into a buffer solution to obtain a pre-reaction solution 1 (a mixture of recombinant cell disruption liquid of a recombinant strain UGT11 (S158F) and a recombinant strain AtSUS (P94N)), and collecting precipitate after reaction; and S2, after the precipitate is dissolved, adding a mixed solution 2 (a mixture of recombinant cell disruption solutions of a recombinant strain UGT76G1 (G83Q) and a recombinant strain AtSUS (P94N)) of recombinant cell disruption solutions, adding a buffer solution to obtain a pre-reaction solution 2, and collecting the reaction solution to obtain Reb M. The method provided by the invention can effectively solve the problems of low content and poor specificity of the extracted Reb M, and can realize the conversion rate of 93.33% from Reb A to Reb M.

The invention relates to the technical field of biological catalysis, and discloses an L204A mutant enzyme for preparing rebaudioside I. The enzyme is obtained by the following mutations generated by UGT76G1: L204A: leucine of the 204 amino acid sequence of UGT76G1 mutates into alanine; the high-efficiency in-vitro enzymatic preparation of the rebaudioside I (RI) is realized for the first time, the catalytic efficiency of the rebaudioside I (RI) is improved by more than 7 times (from about 7% to more than or equal to 50%) compared with that of an original enzyme, the high-efficiency performance can be stably maintained under different scales of 10mL to 5L, a solid foundation is laid for industrial application of the RI, and the preparation method is simple, controllable in process condition and suitable for popularization and application.

The invention discloses a method for synthesizing ceramide NP through two-step enzyme catalysis, which comprises the following steps of: firstly, adding lipase into vegetable oil to catalyze and combine fatty acid to decompose into free fatty acid, and centrifuging to remove the lipase; a small amount of phytosphingosine is added into the hydrolyzed vegetable oil, the raw material vegetable oil is used as a solvent or a squalane system solvent for incubation, and then immobilized lipase is added for catalytic synthesis of ceramide. The invention establishes a two-enzyme method for efficiently synthesizing the ceramide NP compound, and the process is natural, environment-friendly and non-toxic. The reaction conditions are optimized through response surface analysis, meanwhile, the immobilized lipase shows excellent cycle performance, and the method is easy to operate, environmentally friendly, green and economical and has wide application prospects in the fields of cosmetics, health care products, biological medicine and the like.

The invention discloses a high-stability carbonyl reductase mutant and application of the high-stability carbonyl reductase mutant in chiral alcohol synthesis. An amino acid sequence as shown in SEQ ID NO.2 is mutated as follows: 20th leucine is replaced by any one of lysine, threonine, aspartic acid or isoleucine, and / or 55th leucine is replaced by any one of methionine, serine, lysine or isoleucine, and / or 55th leucine is replaced by any one of methionine, serine, lysine or isoleucine. Compared with a wild type enzyme, the stability of the obtained mutant is remarkably improved, particularly, single-point mutants Mut-L20I and Mut-L55I and a combined mutant Mut-L20I-L55I show excellent relative enzyme activity, thermal stability and substrate tolerance. The mutant has high catalytic activity and stability to various chiral alcohols, has the advantages of high catalytic efficiency, high thermal stability, strong substrate tolerance, easiness in fermentation production and the like, and is suitable for industrial application of enzymatic synthesis of chiral alcohols.

The invention belongs to the technical field of preparation of food additives, and particularly relates to a method for extracting sa-hao seed gum and oligosaccharide thereof by using a compound enzyme method. According to the method provided by the invention, complete artemisia desertorum seeds are taken as a raw material, enzymolysis is carried out by using a compound system consisting of xylanase, cellulase and pectinase, and after enzyme deactivation and alcohol precipitation, the artemisia desertorum gum or the artemisia desertorum gum oligosaccharide can be obtained. Compared with a traditional water extraction method and an acid-base extraction method, the extraction yield is remarkably increased through the enzymolysis synergistic effect, the process is green and safe, no chemical residue exists, the molecular weight of the product is controllable, and high purity and functional activity are both achieved. The method is easy and convenient to operate, low in cost and suitable for large-scale production, and has wide application prospects in the fields of food, medicine and functional oligosaccharide development.

The invention relates to a diacylglycerol acyltransferase capable of synthesizing triglyceride as well as a coding gene and application of the diacylglycerol acyltransferase, and belongs to the field of enzymology, the nucleotide sequence of the gene for coding the diacylglycerol acyltransferase is as shown in SEQ ID NO.1, and the amino acid sequence of the diacylglycerol acyltransferase is as shown in SEQ ID NO.2. The invention also provides a recombinant plasmid and a recombinant engineering bacterium containing the gene of the enzyme. Diglyceride and an acyl donor are used as substrates, and triglyceride is catalytically synthesized through an acyl transfer reaction by using the enzyme. The invention provides an efficient tool for realizing enzymatic biosynthesis and large-scale production of natural triglyceride.

The invention discloses diglyceride prepared by an enzymic method as well as a preparation method and application thereof, and belongs to the technical field of deep processing of grease. The method comprises the following steps: adding fatty acid into a choline chloride-glycerol-urea ternary eutectic solvent, uniformly mixing, and then adding candida antarctica lipase B for esterification reaction to obtain a reaction mixture; separating the reaction mixture, and recovering an upper-layer oil phase; and separating and purifying the upper-layer oil phase to obtain the diglyceride. According to the method, the reaction rate is high, the selectivity of the diglyceride in the esterification process is high, the yield of the diglyceride can reach 65% or above by reacting for 1 h, the selectivity of the diglyceride is 75% or above, and the purity of the separated and purified diglyceride is 90% or above; in addition, the method is simple to operate, and the reaction product and the eutectic solvent can be separated through simple centrifugation after the reaction is finished; in addition, the monoglyceride content in the product is low, and the subsequent separation and purification difficulty is greatly reduced.

The invention discloses application of double enzymes in one-step preparation of L-selenium methyl selenocysteine, and belongs to the technical field of biochemical engineering. In the method, racemic N-acetyl selenium methyl selenocysteine is used, and the L-selenium methyl selenocysteine with high optical purity is prepared in a green and efficient manner through co-catalysis of stereoselective L amino acid acylase and N-acetyl amino acid racemase. The catalyst is prepared through fermentation by adopting a one-bacterium two-enzyme method. Synchronous racemization and selective hydrolysis are realized by a one-step method, the theoretical conversion rate is 100%, the actual conversion rate is 95%, and the product is e.egt; the concentration of the substrate is 8%.

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 an alcohol oxidase mutant and application thereof. Specifically disclosed are a mutant derived from Gloeophyllum trabeum alcohol oxidase (GtrAOX-M2), a nucleic acid encoding the alcohol oxidase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, a preparation method of a recombinant alcohol oxidase catalyst, and a preparation method of the recombinant alcohol oxidase catalyst. The invention also discloses a method for catalyzing methanol to be oxidized into formaldehyde by using the alcohol oxidase, the alcohol oxidase mutant or the recombinant alcohol oxidase catalyst. The activity of the alcohol oxidase mutant provided by the invention is obviously improved compared with that of a female parent, and the enzyme is high in activity and can be heterologously expressed in escherichia coli, so that the alcohol oxidase mutant can efficiently oxidize methanol into formaldehyde and oxidize the methanol into formaldehyde by a one-carbon unit biological utilization methanol enzyme method; wide application prospects are shown in the aspect of subsequent conversion and generation of polyhydric alcohols.

The invention discloses a method for preparing butanediamine and spermidine by an enzyme method, and belongs to the field of bioengineering. The novel arginine decarboxylase mutant is successfully provided, the mutant shows excellent catalytic performance under the alkaline condition, and the specific enzyme activity of the mutant reaches 24.9 U / g and is improved by 8.3 times compared with that of wild type enzyme. The invention develops a two-step synthesis process based on the constructed arginine decarboxylase mutant, and the two-step synthesis process is used for efficiently preparing spermidine. Experimental results show that the process successfully realizes the remarkable increase of the yield of spermidine, and the final yield reaches 200.4 mg / L.

A method for preparing theasinensin A (TSA) by means of a low-temperature enzyme process. The method comprises using a plant polyphenol oxidase enzyme extract or polyphenol oxidase enzyme formulation as an enzyme source, using epigallocatechin gallate (EGCG) as a substrate, using as a reaction system a buffer solution having a certain pH value, and performing an enzymatic reaction at a low temperature and subsequent steps such as column chromatography, distillation and concentration, liquid phase preparation, and freeze drying, thereby synthesizing TSA in a directional enzymatic manner. The method achieves EGCG enzymatic oxidation at a low temperature and obtains dimeric TSA by means of a conversion reaction, laying a theoretical and practical basis for green, safe, efficient and large-scale industrial production and utilization of TSA.

The invention relates to the technical field of biochemistry, in particular to an enzyme mutant and application thereof in preparation of conopeptide. According to the preparation method, a strategy of combining green chemistry and an enzyme method is adopted, seven tripeptide fragments are taken as raw materials, a conopeptide main chain is prepared through a liquid-phase synthesis method, and then enzymatic precise modification and synthesis are realized by utilizing a multiple directed evolution enzyme concerted catalysis system. Based on the efficient connection characteristic of a liquid-phase synthesis system and the high substrate specificity of an enzymatic system, the complex protection group operation in traditional solid-phase synthesis is effectively avoided, generation of by-products is remarkably reduced, meanwhile, fixed-point modification and precise assembly of a conopeptide main chain are achieved, and the application prospect is wide. And an efficient and green new path is provided for the continuous production of the cosmetic-grade high-purity conopeptide.

The invention belongs to the technical field of natural product extraction and enzyme engineering, and particularly relates to a method for extracting red pomelo polyphenol based on an enzyme method. The method aims at solving the problems of high cost and the like caused by easy inactivation and difficult recovery of enzymes in most red pomelo extraction processes. Waste such as pericarp and pomelo seeds generated by red pomelo processing are used as carbon sources, a biochar material with a hierarchical porous structure is prepared through programmed heating carbonization activation, pectinase, cellulase and naringinase are immobilized by adopting a spatial zoning sequential immobilization strategy and then react with the red pomelo, and the biochar material with the hierarchical porous structure is prepared. The immobilized enzyme is rapidly recovered through magnetic separation to realize repeated utilization of the enzyme, and finally red pomelo polyphenol powder is obtained through multi-step treatment, so that recycling is realized, and the yield of polyphenol extracted by an enzyme method is improved.

The invention relates to the technical field of biology, in particular to a collagen extraction activity monitoring method based on a refined enzyme extraction technology, which comprises the following steps: preparing collagen labeled by quantum dots and modified by Raman tags into a bimodal probe; a microfluidic monitoring system is constructed based on an integrated reaction chamber, a temperature control module, a pH monitoring module and a signal acquisition module; mixing a sample to be detected with the bimodal probe, adding a structure protective agent, and injecting into a reaction chamber of the microfluidic detection system; synchronously acquiring a fluorescence signal and a Raman signal through the signal acquisition module, and determining to return to the step S2 to recalibrate the signal acquisition module based on a signal-to-noise ratio of the signal; and carrying out fusion analysis on the fluorescence signal and the Raman signal to obtain collagenase activity, triple helix structure integrity and enzymolysis kinetic parameters. The method solves the problem that collagenase activity and collagen triple helix structure integrity data cannot be accurately obtained in a refined enzyme extraction scene.

The invention discloses a chemical-enzymatic coupling synthesis method of oseltamivir phosphate, which comprises the following steps: by taking monasic acid OS-01 as a substrate, sequentially carrying out S1 esterification, S2 ketalation, S3 selective ring opening, S4 TEMPO oxidation, S5 primary transaminase reaction, S6 acetylation, S7 secondary transaminase reaction and S8 phosphorylation reaction to obtain the final product oseltamivir phosphate OS-09. According to the method, the step of sodium azide required by a traditional route is avoided, the production safety and the industrialization possibility are enhanced, two different transaminases are used for respectively constructing different chiral centers, the steps of protecting groups and deprotecting groups on the traditional route are not needed, the overall route is short, the used chemical reagents are few, the yield is high, and the method is suitable for industrial production. The industrial requirements of environmental protection and low cost are met.

The application discloses a crystallization equipment applied to an enzyme method for preparing calcium gluconate, belonging to the technical field of crystallization equipment, which comprises a tank body, a liquid adding opening is arranged on the side wall of the tank body, a cylinder body is arranged in the tank body, a cleaning rod is arranged between the cylinder body and the inner wall of the tank body, the cleaning rod is used for cleaning the inner wall of the tank body and the outer wall of the cylinder body, a plurality of plug-in plates are slidably connected to the inner bottom wall of the cylinder body, and the plurality of plug-in plates are arranged in the solution. The plug-in plates, the inner wall of the tank body and the outer wall of the cylinder body can reduce the nucleation barrier of the solution surface, the melt solution can form crystal nuclei at the locations with unevenness, and the solution can form nuclei locally at a very small cooling degree, so that the crystallization efficiency is accelerated, and the nucleation barrier condition is created for the solute to preferentially nucleate, thereby reducing the nucleation time of the solute.

The application belongs to the technical field of organic synthesis, and particularly relates to a preparation method of alpha soludex. The preparation method takes 11-ketoprogesterone as raw material, firstly acetylates the ketone group at the 3 position, then obtains 3beta-hydroxyl through a composite biological enzyme method, and finally obtains alpha soludex through hydrogenation and translocation reaction. The composite biological enzyme method is used to obtain 3beta-hydroxyl, has good specificity, mild reaction conditions, does not need special equipment, and has high conversion efficiency. The preparation method of the application is easy to purify the reaction products of each step, the total mass yield of the final alpha soludex product is greater than 85%, and the HPLC purity is greater than 99.5%. The preparation method of the application has low cost and is suitable for industrial large-scale production.

The application belongs to the technical field of bioengineering, and particularly relates to a xanthan gum cleavage ancestral enzyme AncXLY196 based on ancestral sequence reconstruction, an amino acid sequence of which is shown as SEQ ID NO. 2. Based on the xanthan gum cleavage ancestral enzyme, a mutant AncXLY196-X7 is further obtained, an amino acid sequence of which is shown as SEQ ID NO. 4, xanthan gum degradation activity of which reaches 2.5 times of that of the xanthan gum cleavage ancestral enzyme, and the optimal catalytic temperature of the xanthan gum cleavage ancestral enzyme and the mutant thereof is increased to 45 DEG C, and the enzyme activity of the mutant of the xanthan gum cleavage ancestral enzyme is reserved by 50-80% after being treated at 45-55 DEG C for 12-24 h. The application provides a new enzyme resource for efficient enzymatic degradation of xanthan gum and preparation of functional oligosaccharides.

The application discloses a lipase mutant and a method for biocatalytically preparing dihydrohuperzine by using the same, and relates to the technical field of bioengineering. The application provides a lipase mutant, and the amino acid sequence of the lipase mutant is shown as SEQ ID NO:2. The application also provides a method for synthesizing dihydrohuperzine D by using the lipase mutant, realizes biosynthesis of dihydrohuperzine D, adopts one-step synthesis by using an enzyme, does not need a group protection and deprotection step, is green and environment-friendly in the production process, greatly reduces industrial wastewater discharge, and has important application value.

The invention discloses a key enzyme farnesyl pyrophosphate synthase of a black fungus terpenoid synthetic route, and belongs to the technical field of protein. The invention aims to provide a key enzyme gene of a mevalonic acid pathway synthesis pathway in black fungus. The invention provides an FPPS protein of black fungus. The amino acid sequence of the FPPS protein is as shown in SEQ ID NO. 2. A solid foundation is laid for deeply exploring functions of key genes for triterpene synthesis, and meanwhile important data support is provided for systematically explaining specific effects of the genes in terpene synthesis routes.

The invention provides a beta-glucosidase mutant from Paenibacillus phyllosphaerae, and a method for efficiently preparing rare ginsenoside F2 by using an enzyme method and a whole-cell method, and particularly provides a beta-glucosidase mutant from Paenibacillus phyllosphaerae and a method for efficiently preparing rare ginsenoside F2 by using a beta-glucosidase mutant from Paenibacillus phyllosphaerae. The method comprises the following steps: engineering-modifying beta-glucosidase by utilizing computer-aided design, constructing a mutant library, carrying out primary screening and secondary screening to obtain two high-activity mutants Q7D and G189A, constructing a combined mutant Q7D / G189A, transforming the combined mutant Q7D / G189A into genetically engineered bacterium escherichia coli for expression, and producing F2 by utilizing a whole-cell method. Finally, the highest yield of 18.01 g / L is achieved after the reaction is carried out for 10 h in a 5 L fermentation tank and is 6.82 times that of a shake flask, and the purity of F2 reaches up to 82.20% after the reaction is carried out for 24 h finally. The invention not only provides an efficient solution for large-scale production of rare saponin F2, but also provides a transferable framework for designing and engineering diversified glycoside hydrolase in customized biological catalysis.

The invention provides a method for enzymatic synthesis of sucrose laurate in a deep eutectic solvent. DL-menthol is used as a hydrogen bond donor, lauric acid is used as a hydrogen bond acceptor, and the DL-menthol and the lauric acid are heated and stirred according to the molar ratio of 1: 2, so that the eutectic solvent required by an experiment is generated. After the eutectic solvent prepared by the method is compounded with pyridine, not only can the enzyme activity be maintained, but also the solvent performance is relatively good, and the eutectic solvent can be used for enzymatic synthesis of sucrose ester. The method comprises the following steps: adding sucrose and vinyl laurate into a 15mL reaction tube, then adding Novozymes 435 and a DL-menthol: lauric acid (1: 2)-pyridine solvent, and carrying out a stirring reaction under the conditions of 50 DEG C and 300r / min. After the reaction is carried out for 72 hours, the reaction yield is monitored by a liquid phase, and the yield of the sucrose laurate is 97.65%. The invention provides a new thought for enzymatic synthesis of sucrose ester.

The invention discloses a method for improving yield of anthocyanin extracted by an enzymic method, according to the method, phospholipase is added and used to act on a cell membrane structure, so that the extraction rate of the anthocyanin and the extraction rate of galactoside in the anthocyanin are obviously improved, and the extraction effect of the anthocyanin is greatly improved through a three-stage enzymolysis system. The total enzymolysis extraction time is within 4 h, and compared with 6-7 h of a traditional enzymatic extraction process, the production period is greatly shortened.

The present invention relates to the field of enzyme engineering, and more particularly to enzyme mutants, enzyme compositions or their immobilized enzymes and their applications and methods for preparing L-carnitine. The present invention provides a simple L-carnitine holoenzyme preparation process, the overall process of which utilizes bulk betaine (N,N,N-trimethylglycine) and acetic acid as raw materials. L-carnitine is directly obtained by two-step continuous conversion by carboxyl reductase (CAR) and aldolase (DERA). This route can be completed in one step using a crude enzyme solution, or by utilizing a recyclable immobilized enzyme, and the reduced coenzyme II (NADPH) and adenosine triphosphate (ATP) used in the first step of the reaction can also be recycled using corresponding enzymes to further reduce production costs. Therefore, the patented L-carnitine preparation process has many advantages, such as being fully green, simple in process, inexpensive to produce, and easy to scale up.

The invention relates to a chemoenzymatic synthesis method of a high-glucose-type homogeneity mucoprotein core structural domain. The method comprises the following steps: firstly, catalytically synthesizing a key alpha-linked glycosylated amino acid block Fmoc-GalNacalpha-Ser / Thr-OH at a low temperature (-20 DEG C to 0 DEG C); then assembling the building blocks on a repeated polypeptide skeleton rich in proline, serine and threonine at accurate intervals (3-5 amino acid residues) through a solid-phase peptide synthesis technology to form a'sugar chain growth scaffold '; then, a series of high-specificity glycosyl transferases are used for carrying out sequential and directional sugar chain enzymatic extension on the scaffold, and a uniform core structure (such as a core type 3) is constructed; and finally, performing high-efficiency purification by a three-step series method of size exclusion chromatography, ion exchange chromatography and lectin affinity chromatography. The technical bottleneck of inhomogeneity (lt, 70%) of glycoforms in a traditional method is overcome, the mucoprotein core fragment with glycoform homogeneity larger than or equal to 95% and bioactivity highly similar to that of natural mucoprotein can be prepared on a large scale, and the method has wide application prospects in the biomedical fields of mucous membrane protective agents, drug delivery carriers and the like.

The present application relates to the technical field of biotechnology, and discloses a C26 hydroxylase for improving the biosynthesis efficiency of diosgenin, a coding gene, a preparation method and an application. The protein of the C26 hydroxylase is any one of the following a), b) or c): a) a protein with an amino acid sequence shown in SEQ ID NO. 2; b) a fusion protein obtained by connecting a tag to the N terminal and / or C terminal of the protein shown in SEQ ID NO. 2; and c) a protein with the same function obtained by substitution, deletion and / or addition of one or more amino acid residues to the amino acid sequence shown in SEQ ID NO. 2. The C26 hydroxylase provided by the present application can improve the biosynthesis efficiency of diosgenin, and provides a key gene resource for producing diosgenin by using an enzyme method.

To provide an immobilized enzyme composition for hexose production or an improved process for hexose production. [Solution] The present invention relates to an immobilized enzyme composition for the preparation of hexoses. Examples of hexoses include tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gross, idose, and inositol. The present invention also relates to an enzymatic process for preparing hexoses from sugars by contacting starch derivatives with the immobilized enzyme composition of the present invention.

The invention belongs to the technical field of biochemistry, and particularly relates to an efficient enzymatic synthesis method of holothurian glycosaminoglycan. According to the invention, a high-efficiency enzymatic synthesis approach of the sea cucumber glycosaminoglycan is established, sulfated fucose and chondroitin sulfate are used as raw materials, and the sea cucumber glycosaminoglycan with anticoagulant activity is efficiently prepared by using the alpha 1, 3-fucosyltransferase mutant and L-fucose kinase / GDP-fucose pyrophosphorylase and adopting a one-pot multi-enzyme synthesis strategy. And important technical support is provided for development of novel anticoagulant drugs.

The application uses cheap and easily available hexose acid as a starting material, first obtains two precursors of 2,5-furan dicarboxylic acid synthesis, namely hexahydroxy acid and 4-deoxy-5-dehydro-hexahydroxy acid through enzyme catalytic reaction, and then 4-deoxy-5-dehydro-hexahydroxy acid is dehydrated under acidic conditions to obtain 2,5-furan dicarboxylic acid.