80 results about "Mutant enzyme" patented technology

The invention relates to the technical field of biological catalysis, and discloses an L379A mutant enzyme for preparing rebaudioside I. The enzyme is obtained by the following mutations generated by UGT76G1: leucine of the 379th amino acid sequence is mutated into alanine; the enzyme can be applied to RA in-vitro conversion preparation of RI with higher utilization value, the conversion rate is higher than 50%, and the enzyme activity is remarkably improved by 7 times compared with the original enzyme catalytic conversion enzyme activity. The UGT76G1 mutant disclosed by the invention has the advantages that (1) the blank is filled, and a special enzyme catalyst capable of realizing efficient in-vitro synthesis of rebaudioside I (RI) is provided for the first time; 2) efficiency jump: the catalytic conversion rate is greatly increased from original about 7% to more than 50% (increase gt; and 3) stability and reliability: in the cross-scale reaction of 10mL to 5L, the catalyst has stable catalytic performance, shows excellent industrial application potential, and is suitable for popularization and application.

The invention relates to the technical field of biological catalysis, and discloses an M88V mutant enzyme for preparing rebaudioside I. The M88V mutant enzyme is obtained by the following mutations generated by UGT76G1: M88V: methionine of the 88th amino acid sequence of UGT76G1 is mutated into valine. Aiming at the technical bottlenecks of low RI yield and poor selectivity in the existing RA biotransformation process, an efficient solution is provided for the UGT76G1 mutant developed by the invention. The mutant can specifically catalyze conversion from RA to RI, and the conversion rate of a target product is remarkably increased to 65% or above from general less than 7%. The excellent catalytic performance of the catalyst is verified in milliliter-level to upgraded reaction systems, the key problems of conversion efficiency and process amplification are solved, and a foundation is laid for large-scale green manufacturing of a high-added-value product RI.

The invention relates to the technical field of biological catalysis, and discloses an M88A mutant enzyme for preparing rebaudioside I. The enzyme is obtained by the following mutations generated by UGT76G1: M88A: methionine of the 88th amino acid sequence of UGT76G1 is mutated into alanine; the key conversion rate of RI is greatly increased from 7% to 85% (increased by more than 12 times), and the utilization rate and economical efficiency of raw materials are remarkably improved; the method can be perfectly reproduced under any test scale (10mL, 100mL and 5L), the conversion rate is always anchored to be more than 85%, and incomparable stability and amplification potential are shown.

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 relates to the technical field of biological catalysis, and discloses an M88F mutant enzyme for preparing rebaudioside I. The enzyme is obtained by the following mutations generated by UGT76G1: M88F: methionine of the 88th amino acid sequence of UGT76G1 is mutated into phenylalanine; and the substrate rebaudioside A (RA) can be efficiently and directionally converted into rebaudioside I (RI) with higher value. In an optimized reaction system, the conversion rate stably reaches 40%, and the catalytic efficiency is improved by more than 5-8 times compared with the common reference enzyme. The enzyme preparation has the characteristics of high catalytic activity, mild reaction conditions, simplicity and convenience in operation and the like, shows good stability and reproducibility in laboratory research and large-scale production, and has a wide industrial application prospect.

This invention discloses a laccase mutant, Lcc5-I479T, its expression strain, and its applications. Using laccase from *Coprinopsis cinerea* as the starting enzyme, this invention obtained the mutant gene through molecular docking and positional analysis. After induction expression in engineered bacteria containing the mutant gene, the stable laccase mutant enzyme Lcc5-I479T was obtained. Using ABTS as a substrate, the mutant enzyme Lcc5-I479T showed a stable increase in activity to 8-20 times that of the starting enzyme at 35-50℃. This mutant enzyme has potential application value in the oxidative transformation of aflatoxin.

This invention belongs to the fields of genetic engineering and enzyme engineering, specifically relating to proline hydroxylase mutants and their application and methods in hydroxyproline production. This invention provides p4h... G141A p4h S85A p4h V126A p4h H125A p4h S205A p4h D122A p4h S211A Mutations at specific sites significantly increased the production of hydroxyproline and decreased the proportion of proline in the mutant enzyme. Further saturation mutations at these sites, combined with the dominant mutations, yielded strains with even better performance, such as p4h. S85Q‑V126A p4h S85A‑V126T p4h G141A‑H125S p4h G141A‑H125T Compared with traditional chemical methods, this process has lower production costs, is more environmentally friendly, and allows for large-scale continuous production.

Disclosed herein, inter alia, are mutant enzymes, kits, and methods of use thereof.

The invention discloses a phytase mutant AppA as well as a coding gene, a preparation method and application thereof. The phytase mutant AppA is obtained by mutating amino acids at the 137th site, the 185th site and the 255th site of wild type phytase AppA from asparagine, aspartic acid and tyrosine into valine, asparagine and aspartic acid respectively; the amino acid sequence and the nucleotide sequence of the gene are SEQ ID NO.1 and SEQ ID NO.2 respectively. The catalytic efficiency of the mutant enzyme AppA provided by the invention on sodium phytate is improved by 2.5 times compared with that of a wild enzyme AppA; under the condition of 70 DEG C, the half-life period of the mutant enzyme AppA is 3.4 times that of the wild enzyme AppA. The mutant enzyme AppA has the characteristic of high enzyme activity under a high-temperature condition, which indicates that the mutant enzyme AppA has an important application prospect in industrial production of a feed in which phytic acid needs to be degraded under the high-temperature condition.

The invention discloses a beta 1, 3-galactosyl transferase Pm beta 3GalT-S207H and an application thereof, and belongs to the technical field of functional enzymes. And the amino acid sequence of the beta 1, 3-galactosyl transferase Pm beta 3GalT-S207H is as shown in SEQ ID NO. 3. The invention further discloses the application of the beta 1, 3-galactosyl transferase Pm beta 3GalT-S207H in the preparation of lactose-N-tetrasaccharide. According to the invention, 1, 3-galactosyl transferase Pm beta 3GalT is subjected to mutation modification to obtain a mutant enzyme, and compared with a wild enzyme, the enzyme activity of the mutant enzyme is improved by 157.21% and is obviously improved, and the thermal stability and alkali resistance are also obviously improved. The beta 1, 3-galactosyl transferase Pm beta 3GalT-S207H disclosed by the invention has important theoretical value and application value on the industrial production of LNT (Lipoxystrobin).

The present invention relates to mutants of the bumblebee Yarrowia SBLE enzyme having modulated transesterification and / or esterification activity compared to the bumblebee Yarrowia SBLE enzyme, and to homologues of the bumblebee Yarrowia SBLE enzyme, such as the C. bogoriensis LE enzyme, the B. batistae LE enzyme and the R. riograndensis LE enzyme, having transesterification and esterification activity on bolaform glycolipids. The present invention also provides genetically modified yeast strains comprising a mutant LE enzyme or a bumblebee Yarrowia SBLE enzyme homologue of the present invention. The present invention further provides the use and methods of the mutant LE enzymes, the bumblebee Yarrowia SBLE enzyme homologues and the modified yeast strains of the present invention for the production of acidic glycolipids, lactone-form glycolipids and / or glycolipid oligomers / polymers / esters. The present invention also provides the three-dimensional structure of the bumblebee Yarrowia SBLE enzyme and its use for the design of mutants having modulated hydrolysis and / or transesterification activity.

The present invention relates to mutant enzymes with enhanced properties and methods of using such enzymes to oxidize organic compound substrates. More specifically, provided herein are mutations in cytochrome P450 enzymes that enhance their peroxygenase activity and / or alter product selectivity as compared to wild-type cytochrome P450 enzymes. Also provided are uses of such mutant cytochrome P450 enzymes, e.g., for biocatalytic oxidation of carbon-hydrogen bonds by a peroxygenase pathway, or for oxidation of organic compound substrates.

The invention discloses a beta-glucosidase mutant, an immobilized enzyme of the beta-glucosidase mutant and application of the beta-glucosidase mutant in efficient synthesis of kinsenoside, and belongs to the field of enzyme engineering and natural product biosynthesis. According to the mutant, one or more mutations of W177Y, W179F, Q242A, F245G, R298S and R313T are introduced at key sites of an amino acid sequence of wild type beta-glucosidase, so that the yield of catalytic synthesis of kinsenoside is remarkably improved. Furthermore, a metal organic framework material UiO-66-NH2 is adopted as an immobilization carrier, enzyme immobilization is achieved in a physical adsorption mode, and the obtained immobilized enzyme is excellent in reusability. Under the same reaction conditions, the immobilized mutant enzyme is used for catalyzing synthesis of kinsenoside, and the yield of the immobilized mutant enzyme is 3 times that of a wild enzyme. The invention provides an efficient, green and sustainable kinsenoside biocatalytic synthesis scheme, and the kinsenoside biocatalytic synthesis method has a good industrial application prospect.

The application belongs to the technical field of bioengineering, and particularly relates to a high-temperature-resistant laccase mutant obtained by site-directed mutation through an overlapping PCR technique, and preparation and application thereof. The laccase mutant is obtained on the basis of a wild-type laccase shown in SEQ ID NO. 1, and at least one of D221Y, E231D, Y441H or Y441F mutations occurs, and the mutant enzyme activity and the thermal stability at 80 DEG C are both improved to different degrees relative to the wild type, which is beneficial to expand the application in the industrial fields of degrading lignocellulose, paper pulp bleaching and the like.

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 present application relates to the technical field of biological catalysis, and discloses an M88W mutant enzyme for preparing rebaudioside I and application thereof, wherein the enzyme is obtained by generating the following mutation on UGT76G1: M88W: the methionine in the 88th amino acid sequence of UGT76G1 is mutated into tryptophan; the enzyme can be applied to the preparation of RI with higher utilization value through RA in vitro conversion, and the conversion rate is higher than 90%, and the enzyme has significant catalytic activity. The mutant enzyme of UGT76G1 discovered in the present application has high activity and high catalytic efficiency, is simple to use, and is suitable for wide application.

The invention relates to the field of enzyme engineering, in particular to a mutant of nylon degrading enzyme and application of the mutant. The mutant of the nylon degrading enzyme is obtained by mutation at one or more key sites through stability modification guided by machine learning, substrate channel inlet loop region modification and tetramer interface modification on the basis of a wild type nylon degrading enzyme NylCp2-TS as shown in SEQ ID NO: 1; the mutant enzyme can efficiently degrade nylon materials in various forms, including films, particles and industrial fibers of nylon 6 and nylon 66, and has a wide application prospect in the field of nylon waste biodegradation.

The application discloses a methionine adenosyltransferase mutant and application of the mutant in immobilized reaction, the mutant is obtained by mutating an amino acid sequence of wild-type Escherichia coli methionine adenosyltransferase, the mutant enhances the interaction force between subunits by introducing a disulfide bond at a specific position; and / or, the protein stability is enhanced by constructing a connecting peptide between adjacent subunits. The mutant provided in the application enhances the thermal stability and catalytic efficiency of the enzyme, reduces the optimum reaction pH value, reduces the generation of by-products and the precipitation of a reaction solution. The mutant enzyme can maintain high activity at a temperature of about 40 DEG C, and is particularly suitable for industrial production. In addition, the immobilized form of the mutant enzyme exhibits good stability and reusability in continuous use, significantly reduces production cost, simultaneously reduces environmental pollution, improves the yield of SAM, is more suitable for large-scale industrial production, and has more market competitiveness.

This invention discloses a glycosyltransferase mutant and its application in the synthesis of rebaudioside. Based on the amino acid sequence of the glycosyltransferase shown in SEQ ID NO:1, this invention performs single-point and multi-point mutations to obtain mutants with enhanced catalytic activity. The catalytic activity, substrate specificity, and / or substrate specificity of the glycosyltransferase mutant are altered; mutations at specific sites can significantly improve the enzyme's catalytic activity for specific substrates. The mutant enzyme is obtained by inducing expression and purifying the protein from the obtained mutant sequence. Using the mutant enzyme as a catalyst and UDPG as a glycosyl donor, the catalytic reaction efficiency for the substrates steviol glycoside ST, rebaudioside A (RebA), and rebaudioside D (RebD) can be significantly improved. The glycosyltransferase UGT76G1 mutant constructed in this study improves its catalytic activity, and the efficient production of rebaudioside M is achieved through optimization of the reaction system.

The invention relates to the technical field of gene engineering, in particular to a lipase mutant, a coding gene, a recombinant vector, engineering bacteria and application. According to the lipase mutant, amino acid V at the 142 site of lipase Lip with the amino acid sequence shown as SEQ ID No.1 is mutated into I, amino acid N at the 169 site of the lipase Lip is mutated into D, amino acid K at the 212 site of the lipase Lip is mutated into E, and the amino acid sequence of the lipase mutant is shown as SEQ ID No.2. The lipase activity induced by the engineering bacterium pPICZ alpha A-lip / X33 of the mutant gene obtained by the invention is 6923 U / mL under the condition that the pH value is 9.0, and is 7.1 times that of the original gene engineering bacterium; the residual enzyme activity of the mutant lipase is 90% and is far higher than the residual enzyme activity of the original lipase under the same treatment condition when the temperature is kept at 70 DEG C for 1 h. The mutant enzyme has the characteristics of high temperature resistance and high enzyme activity under an alkaline condition, which indicates that the mutant enzyme has important application prospects in the industries of cotton and linen processing, leather degreasing, sewage treatment and the like.

The invention provides a recombinant mutant dextranase from chaetomium graciliforme as well as a construction method and application thereof, and belongs to the technical field of mutant enzymes. The recombinant mutant dextranase provided by the invention is CgDEXA289V, and the amino acid sequence of the recombinant mutant dextranase is as shown in SEQ ID NO: 21. Non-conservative site mutation is carried out on the basis of a Chaetomium gracile-sourced dextranase coding gene, a high-enzyme-activity mutant is preliminarily screened out through escherichia coli and is expressed in a pichia pastoris expression system, the high-performance mutant enzyme CgDEXA289V is obtained, the enzyme is endo-dextranase and has good substrate affinity for a substrate, and the mutant enzyme CgDEXA289V can be used for preparing a high-performance mutant enzyme CgDEXA289V. Due to higher enzymatic reaction rate and better stability, the enzymolysis preparation efficiency can be improved, the production cost is reduced, and the method has a better application prospect.

The invention discloses a sucrase mutant, an expression strain of the sucrase mutant and application of the sucrase mutant in hydrolysis of carbohydrates in soybean milk, and belongs to the technical field of biology. The sucrase mutant disclosed by the invention is simply recorded as InvDz13-TBT-01, and the amino acid sequence of the sucrase mutant is shown as SEQ ID NO: 2. The sucrase mutant InvDz13-TBT-01 is subjected to heterologous expression in bacillus subtilis, a recombinant strain is obtained, and the specific enzyme activity and the stability of protein produced by fermentation of the recombinant strain are remarkably improved. When sucrose is used as a substrate, the specific enzyme activity of the mutant enzyme is improved by 1.94 times, and when raffinose is used as a substrate, the specific enzyme activity of the mutant enzyme is improved by 1.7 times. Under the condition of 35 DEG C, the stability of the mutant enzyme is improved by 4 times. Compared with the starting enzyme, the thermal stability of the mutant enzyme is greatly improved while the specific enzyme activity is maintained. The sucrase mutant disclosed by the invention has a potential value in the application of hydrolyzing carbohydrates such as raffinose in soybean milk.

The present application relates to the field of enzyme engineering, and in particular to a mutanase and application thereof. At present, mutan is mostly prepared by bacterial extraction, and there are problems such as low product yield and many impurities. The present application provides a mutanase, a MutI gene fragment of the mutanase is obtained by cloning a genome of Leuconostoc pseudomesenteroides G496, and a genetically engineered bacterium is constructed. The mutanase provided by the present application is subjected to enzymatic property analysis, and it is found that the mutanase has good stability under acidic conditions, and the optimal induction expression temperature and reaction temperature are close to room temperature, and the production energy consumption is low and the raw material conversion rate is high. The catalytic product of the mutant enzyme is linear mutan with a high polymerization degree, the molecular weight of the linear mutan is as high as 650 kDa, the linear mutan contains 90% of alpha-1,3 glycosidic bonds, and the structure is relatively dense, and the linear mutan has a good application prospect in the field of biological base materials.

The invention belongs to the technical field of gene engineering, and discloses a D-psicose 3-epimerase mutant enzyme with high temperature stability and application of the D-psicose 3-epimerase mutant enzyme. The mutant enzyme of the D-psicose 3-epimerase is obtained by replacing the 87th site of threonine in the amino acid sequence of the D-psicose 3-epimerase derived from Cabalronia insemination with isoleucine and / or replacing the 242nd site of threonine in the amino acid sequence of the D-psicose 3-epimerase with proline. The mutant enzyme of the D-psicose 3-epimerase is a mutant enzyme of the D-psicose 3-epimerase derived from Cabalronia insemination. The catalytic activity of the mutant enzyme can be improved by 100% or above compared with that of wild DAEase, meanwhile, the thermal stability is also remarkably improved, and the mutant enzyme has good industrial application potential.

The application discloses an in-vitro detection method and kit for O-GlcNAc glycosylation modification of alpha-synuclein, and relates to the field of biochemical detection. Y289L The method comprises the following steps: extracting protein in a sample to be detected, adding UDP-GalNAz substrate beta4GalT Y289L The mutant enzyme and the reaction are carried out to obtain a protein system; DBCO-mPEG is added to the protein system, and the reaction product is subjected to electrophoretic separation; and according to an electrophoretogram, the proportion of the protein subjected to glycosylation modification and the molecular weight distribution thereof are calculated. The application marks specific molecular weight "tags" for O-GlcNAc glycosylation proteins, and the glycosylation-modified proteins are recognized by alpha-synuclein specific antibodies to show specific molecular weight sizes; and the alpha-synuclein polymerization states, such as dimers and polymers, subjected to O-GlcNAc glycosylation modification can be determined and quantified according to protein standards.

The application discloses an acetyl lactic acid synthase mutant and application thereof, and belongs to the technical field of bioactive enzymes. The acetyl lactic acid synthase mutant is acetyl lactic acid synthase-PCC6803-L255I, acetyl lactic acid synthase-PCC6803-V398I or acetyl lactic acid synthase-PCC6803-T488V. The application of the acetyl lactic acid synthase mutant in catalyzing preparation of acetyl lactic acid. The acetyl lactic acid synthase derived from PCC6803 is reformed by a site-directed mutation method, and mutant enzymes, acetyl lactic acid synthase-PCC6803-L255I, acetyl lactic acid synthase-PCC6803-V398I and acetyl lactic acid synthase-PCC6803-T488V, are screened, and the catalytic efficiency of the mutant enzymes is 120%, 130% and 110% of that of the wild enzyme respectively. The application lays a technical foundation for metabolic engineering of a blue-green algae photosynthetic cell factory.

The application discloses a cellobiose epimerase mutant with high lactulose yield and application thereof, and belongs to the technical field of enzyme engineering. Dictyoglomus The cellobiose epimerase (referred to as Disp-WT enzyme) derived from microorganism Bifidobacterium longum sp. is used as a parent, a gene mutation technology is used, a histidine His at the 245th position is replaced by glutamic acid Glu, and a mutant H245E is obtained.The optimal catalytic condition of the mutant enzyme H245E does not change, but the isomerase activity of the enzyme at 85 DEG C is increased to 254 % of the original, and the generation amount of epi-lactose is greatly reduced.This finding has important research value for researching more cellobiose epimerases with low isomerase activity.

The present disclosure provides novel PH20 mutant enzymes that exhibit significantly enhanced aggregation stability, are useful for the development of pharmaceutical subcutaneous injection formulations, facilitate diffusion of pharmaceuticals in subcutaneous tissue, reduce the volume limitations for subcutaneous administration and alleviate swelling or pain from large volume subcutaneous injections.

This invention discloses a mutant acylase and its application in the synthesis of sodium cocoyl glycinate. The mutant acylase is a mutant containing one, two, three, or more substitution mutations of amino acid residues selected from the following sites, based on the amino acid sequence of the acylase shown in SEQ ID NO.2: S3T, P70A, L177C, G207V, R209C, K218N, P244L, S249T, S249N, C271V, Q290K, M309T, C342A, and L394I. The acylase mutant disclosed in this invention exhibits significantly enhanced cocoyl glycinate synthase activity. Furthermore, the mutant enzyme can tolerate higher concentrations of coconut oil acid and cocoyl glycin, which is beneficial for increasing the concentration of the reaction system and thus reducing production costs.

The invention discloses a method for synthesizing spermidine through a double-bacterium co-culture system, and relates to the technical field of bioengineering. The method comprises the following steps: constructing a first strain for synthesizing and secreting ornithine; a second strain is constructed and used for synthesizing aspartic semialdehyde and butanediamine, the second strain comprises carboxyl spermidine dehydrogenase and carboxyl spermidine decarboxylase, the carboxyl spermidine dehydrogenase is a mutant enzyme obtained by performing G13A and / or K159R mutation on an amino acid sequence as shown in SEQ ID NO: 1, and the carboxyl spermidine decarboxylase is a mutant enzyme obtained by performing G13A and / or K159R mutation on an amino acid sequence as shown in SEQ ID NO: 2. The carboxyl spermidine decarboxylase is a mutant enzyme obtained by carrying out D82E single-point mutation on an amino acid sequence as shown in SEQ ID NO: 2; and co-culturing and fermenting the first strain and the second strain to synthesize spermidine. According to the present invention, the metabolism division and the efficient synergy can be achieved, the metabolism load and the butanediamine toxicity accumulation can be reduced, and the catalytic efficiency of the key reaction and the cofactor utilization efficiency can be improved so as to significantly improve the spermidine yield and the conversion efficiency.