238 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 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 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.

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 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 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 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 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.

The application discloses a method for preparing putrescine and spermidine by using an enzyme, and belongs to the field of bioengineering.The application successfully provides a novel arginine decarboxylase mutant, the mutant exhibits excellent catalytic performance under alkaline conditions, and the specific enzyme activity reaches 24.9 U / g, which is 8.3 times higher than that of a wild-type enzyme.Based on the constructed arginine decarboxylase mutant, the application develops a two-step synthesis process for efficiently preparing spermidine.The experimental results show that the process successfully realizes significant improvement of the yield of spermidine, and the final yield reaches 200.4 mg / L.

Disclosed herein are methods and compositions for cleavage of nucleic acids from a surface of a solid support. Further described herein are cleavage methods compatible with enzymatic and chemical nucleic acid synthesis methods.

The application discloses a process for producing diacylglycerol oil by an enzyme method in a solvent-free system, and comprises the following steps: taking low-cost lipase as a catalyst, preparing lipase A by immobilization of the enzyme, preparing lipase B by an ester exchange reaction, and then taking the lipase B as a catalyst to catalyze esterification of free fatty acid and monoglyceride in a solvent-free system to prepare diacylglycerol oil.

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 relates to the technical field of biological catalysis, and discloses a mutant enzyme for preparing rebaudioside I. The enzyme is a UGT76G1 mutant, and the mutation points of the mutant are L379V and L126A. The mutated enzyme realizes efficient in-vitro enzymatic synthesis of RI (the conversion rate is 45-60%), the problem of low efficiency (-7%) of an existing path is solved, and the production process has economic feasibility; secondly, the catalytic efficiency is improved by 6-9 times compared with the original level, and the fundamental improvement means that the raw material utilization rate and the production flux are greatly improved; and meanwhile, the seamless connection capability from a laboratory to large-scale production is realized, and a key technical foundation is laid for constructing a stable and controllable industrial production line.

The application discloses an enzymatic production process of diglyceride, and comprises the following steps: using low-cost lipase as a catalyst, preparing lipase A through immobilization of the enzyme, preparing lipase B through an ester exchange reaction, and then using the lipase B as a catalyst to catalyze esterification of free fatty acid and glycerol to produce diglyceride; the method can realize efficient enzymatic production of diglyceride through a series of methods by using low-cost lipase, and the production cost is reduced. The application provides an economical and efficient method for producing diglyceride by using the enzyme method, greatly reduces the use cost of lipase in the enzymatic esterification reaction, and has certain industrialization application prospect.

The present disclosure provides compositions comprising a glycoside hydrolase and one or both of hesperidin and hesperetin, wherein an active site of the glycoside hydrolase comprises first, second, third, fourth, and fifth regions as described herein. The present disclosure also relates to methods for producing a polyphenol aglycone, comprising contacting a phenolic glycoside with a glycoside hydrolase described herein. Also provided are host cells and heterologous expression systems capable of expressing a heterologous glycoside hydrolase, and nucleic acids encoding the glycoside hydrolase.

The invention relates to the field of comprehensive utilization of biological resources, and discloses a method for extracting grease in camellia oleifera shells based on a biological enzyme method. The invention provides a method for extracting grease in camellia oleifera shells based on a biological enzyme method, and the method comprises the following steps: after the camellia oleifera shells are subjected to crushing and pretreatment extraction, carrying out first-stage enzymolysis and second-stage enzymolysis in sequence to obtain the grease in the camellia oleifera shells. According to the oil and fat extraction method, waste recycling of the camellia oleifera shells is achieved, oil and fat in the camellia oleifera shells are extracted through a biological enzyme method, finally, the oil and fat extraction rate of the camellia oleifera shells reaches 4.22%-4.67%, and a feasible scheme is provided for efficient, environment-friendly and low-cost industrial production.

The invention discloses a glycosyl transferase mutant for synthesizing rebaudioside M as well as a product and application thereof, and belongs to the technical field of gene engineering. The technical problem to be solved is that the existing glycosyl transferase and sucrose synthase are low in enzyme activity and insufficient in enzyme activity stability, so that the synthesis efficiency of rebaudioside M (RebM) is not high, and the industrial application of preparing RebM by an enzyme method is limited. According to the key points of the technical scheme, the glycosyl transferase mutant is provided, the mutant is obtained by mutating UDP-glycosyl transferase UGT76G1, and the amino acid sequence of the UDP-glycosyl transferase UGT76G is as shown in SEQ ID NO. 1; the mutation comprises mutation on the 83rd site of SEQ ID NO.1, and the amino acid sequence of the glycosyl transferase mutant is shown as SEQ ID NO.5. The invention further discloses a preparation method of the glycosyl transferase mutant.

The present application relates to a method for preparing a characteristic flavor enhancer of Xanthoceras sorbifolia Bunge based on an enzymatic reaction, which comprises the following steps: 1) fresh or frozen Xanthoceras sorbifolia Bunge sprouts are added with trifluoroacetic acid for enzyme inactivation treatment, ultrasonic water extraction, and then Sevage reagent, petroleum ether and ethanol are sequentially added for impurity removal; 2) preparation of gamma-glutamyl transpeptidase or gamma-glutamyl transpeptidase crude enzyme solution; 3) different sources of gamma-GTP enzyme are reacted with the sulfur-containing flavor precursor substrate of Xanthoceras sorbifolia Bunge in step 1) at a suitable concentration under sealed conditions at 30-50 DEG C and pH 7-8 for 30-60 min, and the characteristic flavor enhancer of Xanthoceras sorbifolia Bunge is obtained. The present application successfully prepares the characteristic flavor of Xanthoceras sorbifolia Bunge in vitro for the first time through an enzymatic reaction, provides a feasible path for the preparation of typical Xanthoceras sorbifolia Bunge flavor spices or flavor enhancers by the biological enzyme method, and is conducive to solving the problems of difficult storage of fresh Xanthoceras sorbifolia Bunge and loss of flavor of Xanthoceras sorbifolia Bunge processing products.