379 results about "Enzyme method" patented technology

The invention provides a carbonyl reductase mutant. The carbonyl reductase mutant is obtained by performing single-point mutation or multi-point combined mutation on the 17th site, the 40th site and the 64th site of an amino acid sequence as shown in SEQ ID NO.1. The invention also provides a coding gene, a recombinant vector containing the coding gene, a co-expression engineering bacterium and an application of the co-expression engineering bacterium. Compared with a wild type enzyme, the carbonyl reductase mutant has high activity and high stereoselectivity, the enzyme activity can reach more than two times that of the wild type enzyme, the catalytic efficiency on a substrate precursor ketone is remarkably improved, the yield of a statin drug intermediate and chiral alcohol synthesized by an enzyme method of the carbonyl reductase mutant is remarkably improved, and the yield of a rosuvastatin intermediate (3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R, 3R) is remarkably improved. The highest yields of the (3R, 5R)-6-chloro-3, 5-dihydroxyhexanoic acid tert-butyl ester and the atorvastatin intermediate (3R, 5R)-6-cyano-3, 5-dihydroxyhexanoic acid tert-butyl ester reach 99% and 97.6% respectively, e.e. Is larger than 99%, and the method has high industrial application value.

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 field of bioengineering, in particular to an enzyme composition, a product and application of the enzyme composition in preparation of Ectoine. The invention provides an enzyme composition. The enzyme composition comprises a mutant of 2-hydroxy-4-aminobutyric acid oxidase, a mutant of keto transaminase, a mutant of hydroperoxide hydrolase, a mutant of Ectoine synthetase, a mutant of pyruvic acid-CoA oxidase and lactobacillus helveticus lactic dehydrogenase. According to the method disclosed by the invention, a plurality of mutant enzymes are utilized for combined transformation for the first time, and cheap 2-hydroxy-4-aminobutyric acid is taken as a starting raw material to quickly and efficiently produce Ectoine; the enzyme method has the characteristics of easiness in control, short conversion time, few generated impurities and the like, so that the method has unique advantages in the aspects of low-cost and large-scale mass production of Ectoin.

The invention belongs to the field of gene engineering, and particularly relates to a PET hydrolase mutant with high catalytic activity and application thereof. According to the invention, amino acids at the 62nd site, the 65th site, the 66th site, the 71th site, the 154th site, the 156th site, the 177th site, the 183th site, the 184th site, the 209th site, the 213th site, the 214th site and the 215th site in the PES-H1L92F / Q94Y amino acid sequence of the PET hydrolase shown as SEQ ID NO.2 are mutated by utilizing a structural analysis and site-specific mutagenesis technology to obtain various mutants, and the mutants with improved catalytic activity are obtained. The PET hydrolase mutants promote the hydrolysis efficiency of ester bonds in PET so as to improve the degradation capacity of PET plastics, and can be applied to enzymatic depolymerization of PET plastics in life in the future.

The invention provides a high-thermal-stability deoxyadenosine kinase mutant and application thereof, and belongs to the field of enzyme engineering. Compared with an amino acid sequence as shown in SEQ ID NO.1, the high-thermal-stability deoxyadenosine kinase mutant provided by the invention has mutation of at least one amino acid site, and the mutation comprises V40G, D121T and S154L. In the high-thermal-stability deoxyadenosine kinase mutant provided by the invention, the half-life period of V40G at 50 DEG C is 1.02 h, the half-life period of D121T at 50 DEG C is 9.51 h, and the half-life period of S154L at 50 DEG C is 5.23 h, which are significantly higher than those of a wild type. The high-thermal-stability deoxyadenosine kinase provided by the invention can be used for enzymatic preparation of 2 '-deoxyadenosine monophosphate, deoxyadenosine and deoxyadenosine triphosphate react to obtain deoxyadenosine monophosphate by adopting a one-pot enzymatic method, and the high-thermal-stability deoxyadenosine kinase is simple to operate, can tolerate higher reaction temperature and has the advantage of industrial amplification.

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 relates to heparinase III with high activity on 6-sulfated heparin and application of the heparinase III. The amino acid sequence of the heparinase III (BeHepIII) is as shown in SEQ ID NO. 2, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 1. The invention discovers and discloses a novel heparinase III (BeHepIII) for the first time, the heparinase III is derived from Bacteroides eggerthii, has a higher recombinant expression level in escherichia coli, has very high degradation activity on heparin containing a 6-site sulfated structure, and particularly has the enzyme activity which is 93.5 times of that of commercialized heparinase FhHepIII at present, and the heparinase III can be used for degrading heparin containing 6-site sulfated structure. Therefore, the heparinase III (BeHepIII) provided by the invention has a wide application potential in heparin degradation. In addition, by using heparinase III (BeHepIII), directional excision of a reducing end of heparin oligosaccharide synthesized by a chemical enzyme method is realized, and complete chemical enzyme method synthesis of a pentasaccharide structure with anticoagulant factor Xa activity is successfully realized.

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 discloses a tryptophan synthetase trp B modified gene and a preparation method of 6-fluoro-L-tryptophan, belongs to the technical field of gene modification and amino acid synthesis, and provides a tryptophan synthetase trp B gene subjected to mutation modification, and the nucleotide sequence of the tryptophan synthetase trp B gene is shown as SEQ ID NO.1. After gene modification, substrate molecules can enter the channel more easily. After gene modification, a channel of an active center of tryptophan synthetase is more beneficial to entering of substrates 6-fluoroindole and L-serine, and the yield of 6-fluoro-L-tryptophan is increased. According to the method, a specific strain containing tryptophan synthetase is adopted, tryptophan synthetase can be efficiently expressed by culturing in an optimized culture medium, so that the 6-fluoro-L-tryptophan synthesized by an enzymic method has relatively high catalytic rate and conversion rate, the molar conversion rate of L-serine reaches 86.5% or above, the reaction conditions of the enzymic method synthesis are mild, the enzyme stereoselectivity is strong, the catalytic efficiency is high, and the method is suitable for industrial production. The method is suitable for industrial production.

The invention relates to a method for synthesizing deoxynucleoside triphosphate by an enzymic method, which comprises the following steps: by taking deoxynucleoside monophosphate dNMP as a raw material and taking ATP (adenosine triphosphate) and acetyl phosphoric acid as phosphoric acid donors, synthesizing dNTP (deoxynucleoside triphosphate) by using nucleotide kinase and acetokinase through a one-pot enzymic method. The one-pot enzyme method is used for preparing deoxynucleoside triphosphate, and the method has the advantages of being simple in operation, short in production period, low in production cost, high in yield, small in three-waste discharge, suitable for large-scale industrial production and the like.

The invention discloses a high-efficiency green extraction method of semen plantaginis chitin, and belongs to the technical field of natural product extraction. The method comprises the steps of raw material pretreatment, ultrasonic-assisted enzymolysis, enzyme deactivation, centrifugal separation, ultrafiltration concentration, alcohol precipitation and drying. The core innovation lies in that an ultrasonic-assisted composite enzymolysis synergistic technology is adopted, the dissolution rate of the polysaccharide is remarkably increased, the enzyme activity is protected, and finally the high-purity polysaccharide with the molecular weight of 9.2 kDa and the extraction rate of 13.82% is obtained. Compared with a traditional method, intermittent ultrasound and low-temperature enzymolysis are cooperated, so that polysaccharide chain breakage is effectively reduced; the compound enzyme degrades cell wall components in a targeted manner and breaks through the efficiency bottleneck of a single enzyme; toxic solvents are avoided in the whole process, and the process is environment-friendly. The data of the embodiment shows that the extraction rate is increased by 30% or above compared with that of a single enzyme method, the molecular weight distribution of the product is uniform, and the method is suitable for large-scale production of functional polysaccharides in the fields of food, medicine and the like.

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.

Provided herein is a mutant enzyme comprising a variant of the amino acid sequence shown in SEQ ID NO: 1, as well as compositions comprising said enzyme; and applications of said enzyme and compositions in both therapeutic and non-therapeutic applications.

The invention discloses an alpha-1, 4 glucosidic bond hydrolytic ability enhanced oligomerized 1, 6-glucosidase mutant and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. The oligomerized 1, 6-glucosidase mutant V219A / P227A disclosed by the invention also has the alpha-1, 4 glucosidic bond hydrolytic activity which is not possessed by a wild type enzyme on the basis of retaining the original alpha-1, 6 glucosidic bond hydrolytic activity, and the alpha-1, 4 glucosidic bond hydrolytic activity is improved by 267% compared with that of a single mutant V219A. The mutant V219A / P227A is applied to an enzymatic production process of glucose, maltodextrin is taken as a substrate, glucamylase and the mutant V219A / P227A are added for enzymatic conversion, and the final glucose yield reaches 98.0%; the mutant V219A / P227A is applied to regeneration of glucose from crystalline glucose mother liquor, and the final glucose yield reaches 95.9%, which is 9.35% and 5.15% higher than those of a wild type V219A and a single mutant V219A respectively. Therefore, the mutant is helpful for improving the substrate conversion rate and the product purity in the glucose production process, can be used for regenerating glucose from crystalline glucose mother liquor, and has higher industrial application value.

The invention discloses a novel method for synthesizing an intermediate (S)-3-aminobutyronitrile of a GLP-1 receptor stimulant. According to the synthesis method, a chemical method and an enzyme method are combined for the first time to realize green and environment-friendly high-selectivity synthesis of the (S)-3-aminobutyronitrile. The catalytic reaction system disclosed by the invention is simple, mild in condition and high in selectivity.

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 discloses a new enzyme with acylsulfonate transferase activity or a mutant thereof and application thereof, and belongs to the field of enzyme engineering. On the basis of an NCBI (National Center of Biotechnology Information) database, a new enzyme sequence is mined through a bioinformatics means, a new enzyme is obtained through codon optimization, gene synthesis and cell expression, the activity of acylsulfonate transferase is measured, and a corresponding mutant is obtained by using a directed evolution method. Compared with reported mouse acyl sulfonate transferase (ASTIV), the enzyme sequence obtained in the invention has better catalytic activity in a regeneration system of PAPS, and provides a new choice for enzymatic synthesis of PAPS.

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 invention discloses a preparation method of nebivolol, which combines an enzyme method and a chemical method, and integrates enzyme selectivity and operability of the chemical method. Meanwhile, nebivolol is constructed by adopting a brand new intermediate, chiral center construction is efficient and convenient, the chemical purity and optical purity of the product meet the quality requirements of raw material medicines, atoms are more economical, preparation is more efficient, and industrialization is facilitated.

The invention discloses acyl sulfonic acid transferase and application thereof, and belongs to the field of enzyme engineering. On the basis of an NCBI (National Center of Biotechnology Information) database, a new enzyme sequence is mined through a bioinformatics means to obtain the acyl sulfonate transferase derived from Podarcismuris, a new enzyme is obtained through codon optimization, gene synthesis and cell expression, and the activity of the acyl sulfonate transferase is measured. Compared with reported mouse acyl sulfonate transferase, the acyl sulfonate transferase provided by the invention has better catalytic activity in a regeneration system of PAPS, and a new choice is provided for enzymatic synthesis of PAPS.