193 results about "Wild type enzyme" 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 discloses a high-performance carbonyl reductase mutant and application thereof in synthesis of a series of chiral alcohols. The high-performance carbonyl reductase mutant is obtained by performing the following mutations on an amino acid sequence as shown in SEQ ID NO.2: the 69th alanine mutates into any one of lysine, leucine, aspartic acid or asparagine, and the 69th alanine mutates into any one of lysine, leucine, aspartic acid or asparagine; and / or the 100th glycine is mutated into any one of glutamic acid, serine, lysine or asparagine. Compared with a wild type enzyme, the thermal stability and the catalytic activity of the obtained mutant are remarkably improved, particularly, single point mutants Mut-A69N, Mut-G100E and Mut-G100N and a combined mutant Mut-A69N-G100N show excellent relative enzyme activity, thermal stability and substrate tolerance. The mutant has high catalytic activity and stability to various chiral alcohols, and the contradiction that the traditional carbonyl reductase is easy to inactivate at high temperature and the catalytic efficiency is difficult to achieve at the same time is solved. The invention provides an efficient and stable enzyme catalysis tool for green biological preparation of chiral drug intermediates.

The invention discloses a high-efficiency lysine decarboxylase mutant and an application of the high-efficiency lysine decarboxylase mutant in synthesis of pentamethylene diamine. A series of mutants including a single mutant (such as N218G, T222V, C244P and the like), a double mutant and a three mutant are obtained by carrying out multi-site mutation on wild type lysine decarboxylase (SEQ ID NO.2) from Klebsiella grimontii. The catalytic efficiency of the mutant is remarkably improved, and the activity of the mutant is improved by 5.0-10.2 times compared with that of a wild type enzyme. According to the method disclosed by the invention, the substrate conversion rate of 99% or above can be realized within 2 hours through an optimized process of constructing an expression vector and a genetically engineered bacterium, inducing expression of lysine decarboxylase and synthesizing 1, 5-pentanediamine by utilizing a whole-cell catalysis technology. Compared with a traditional chemical method and a natural enzyme method, the method disclosed by the invention has the advantages of simplified process, high catalytic efficiency, low production cost and the like, and is suitable for producing bio-based 1, 5-pentamethylene diamine and nylon 56 salt.

The invention discloses a polyphosphate kinase mutant, a biological material, a catalyst and application, and belongs to the field of protein engineering, and compared with a wild enzyme, the mutant has one or more amino acid residue mutations in the 307th site, the 311th site, the 320th site, the 408th site, the 419th site and the 431th site. The mutant disclosed by the invention is obtained by combining a semi-rational design strategy with active site region prediction and mutation library design screening, and the enzyme activity and the relative yield are remarkably improved: the efficiency of catalytic synthesis of GTP by using the preferable mutant Y431P / K311F / W408A with GMP as a substrate is 2.6 times that of a wild type enzyme, and the relative yield is improved by 150%; the efficiency of the optimized mutant Y431P / K311F / R419L for catalytically synthesizing GTP by taking GDP as a substrate is 1.9 times that of a wild enzyme, and the relative yield is increased by 82%. The key mutant obtained by screening can realize efficient synthesis from cheap NDP or NMP to high-value NTP, and has an excellent industrial application prospect.

The invention belongs to the field of gene engineering, and discloses a sucrose isomerase mutant, a high-yield isomaltulose engineering bacterium and a construction method and application thereof. According to the sucrose isomerase mutant obtained through rational design of an enzyme molecular structure, the enzyme activity and the isomaltulose conversion rate of the sucrose isomerase mutant are remarkably improved, compared with a wild enzyme, the specific enzyme activity of the sucrose isomerase mutant is improved by 2.29 times, the isomaltulose conversion rate is improved to 98.9% from 91.1%, the impurity content is lower, and therefore the sucrose isomerase mutant is more suitable for synthesis of isomaltulose. A recombinant engineering bacterium of the high-yield isomaltulose engineering bacterium is further constructed; the recombinant engineering bacterium capable of efficiently expressing the sucrose isomerase is constructed by utilizing ubiquitin-like protein modified molecular genes to be fused with sucrose isomerase mutant coding genes, the enzyme activity of the prepared sucrose isomerase can reach 40.5 U / mg wet cells, and the recombinant engineering bacterium has a great application prospect in industrial production.

Provided are a marine phospholipase C mutant and a use thereof. Compared with wild-type (WT) enzyme protein, at 40°C and under conditions where enzyme protein activity remains essentially unchanged, the enzyme protein half-life of the constructed mutant can be increased to 3.46 and 9.25 times that of the original enzyme protein, and the residual phosphorus content in degummed oil can be reduced from 551 ppm to 72 ppm, which shows a significant improvement over the final residual phosphorus content of 175 ppm for the blank control and the final residual phosphorus content of 131 ppm for the WT. These results demonstrate that the enzyme has improved thermal stability, enables oil degumming under various conditions, and is suitable for a broader range of industrial applications.

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 discloses an alpha-amylase mutant, a biological material, a catalyst and application, and compared with wild type alpha-amylase, the alpha-amylase mutant has mutation of two or more amino acid residues in the 57th site, the 124th site, the 193th site, the 247th site, the 261th site, the 382th site and the 417th site. The mutant has excellent high-temperature resistance and still keeps higher enzyme activity compared with a wild type enzyme after high-temperature treatment, the residual enzyme activity of the preferable mutant is 68% after the preferable mutant is treated for 0.5 hour at 110 DEG C and is improved by 11.3 times compared with the wild type enzyme, and the requirement for extreme high-temperature resistance in industrial application can be met.

The invention discloses an SAM hydrolase mutant and application thereof, the specific activation effect of halogen anions (F <->, Cl <->, Br <-> and I <->) on SAM hydrolase (containing MJ1651 wild type and mutant) is found for the first time, and the active sequence of the SAM hydrolase is F <-> > I <-> > Br <-> = Cl <->. According to the method, 400 mM of halide salt is added into a reaction system, so that the catalytic efficiency is improved in a breakthrough manner; the yield of wild type enzyme adenosine is improved by 20-300% (optimally, an F-system); the yield of the mutant R89C is soaring up by nearly 6 times under the action of KF; the activity of the immobilized enzyme is still more than 90% after the immobilized enzyme is repeatedly used for 5 times. The method is easy and convenient to operate, green production is achieved with water as the solvent (three wastes are reduced by 90%), the comprehensive cost is reduced by 41%-80 / kg, and an efficient solution is provided for industrial biological manufacturing of antiviral drug intermediates and the like.

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 high-activity tyrosine phenol lyase mutant, which is characterized in that glycine is inserted into the 73rd site and the 74th site of an amino acid sequence of TPL (tyrosine phenol lyase) as shown in SEQ ID NO.1, and leucine at the 282nd site is mutated into one or more of glycine; the amino acid sequence of the high-activity TPL mutant is as shown in SEQ ID NO. 3. And a nucleotide sequence for coding the high-activity TPL mutant is as shown in SEQ ID NO. 4. The invention relates to the technical field of biochemical engineering, and provides a TPL mutant with high activity and high phenol tolerance, the enzyme activity of the TPL mutant is improved by two times compared with that of wild TPL, the phenol tolerance of a substrate is improved by one time, and the enzyme activity detection time is shortened to be within 20 min from 3 h.

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 phospholipase D mutant with improved thermal stability and a preparation method thereof. The amino acid sequence of the phospholipase D mutant is as shown in SEQ ID NO.4; according to the phospholipase D mutant A426P with improved thermal stability, the mutant is incubated for 30 min at 55 DEG C and 60 DEG C respectively, the residual enzyme activity is 85% and 58% respectively, the activity of wild-type enzyme is only 56% and 7% under the same treatment condition, the thermal stability of the mutant enzyme is remarkably improved, and the phospholipase D mutant A426P can be used as a phospholipase D mutant. The problem that phospholipase D is not ideal in thermal stability in biological catalytic reaction is solved.

The invention discloses an N-acetyl glucosamine 2-epimerase mutant as well as preparation and application of the N-acetyl glucosamine 2-epimerase mutant. The mutant enzyme comprises an amino acid sequence as shown in SEQ ID No.3. The preparation method comprises the following steps: by taking a recombinant vector containing a Sumo enhancing element and a wild type N-acetyl glucosamine 2-epimerase gene as a template, carrying out PCR (Polymerase Chain Reaction) by adopting a mutation primer to obtain a mutant gene segment; transforming the mutant gene segment into an engineering bacterium to obtain a recombinant bacterium for expressing the N-acetyl glucosamine 2-epimerase mutant protein; culturing recombinant bacteria, collecting and crushing thalli, centrifuging thallus crushing liquid, and taking supernatant, so as to obtain crude enzyme liquid containing the enzyme mutant; the thermal stability of the wild-type enzyme is improved through point mutation of specific amino acid sites, and the substrate affinity and the catalytic efficiency of the mutant are superior to those of the wild-type enzyme. The mutant can be used for efficient catalytic preparation of lactulose, and has the advantages of few byproducts, high product yield and the like.

The present application relates to a deoxyribonuclease mutant and its application in preparing PDRN. The amino acid sequence of the deoxyribonuclease mutant is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2. The present application also provides the application of the deoxyribonuclease mutant and / or the encoding gene of the deoxyribonuclease mutant in preparing PDRN, and the specific application method. The present application is based on the deoxyribonuclease expressed by the DNASE1 gene of bovine pancreas tissue, and the amino acids at positions 69, 132 and 215 are selected for iterative saturation mutation. Compared with the wild-type enzyme, the enzyme activity of the deoxyribonuclease mutant A69H-S132R-Q215C is increased by 2.15 times, reaching 4.1x10 6 U / mL, and the DNA extraction rate of salmon PDRN prepared by using the mutant enzyme prepared by the present application is 15.2%, the DNA content is 99.1%, and the molecular weight is 1.05 million Daltons, which is significantly improved compared with the wild-type enzyme, and the PDRN in this molecular weight range has more significant biological activity.

The invention discloses a lactic dehydrogenase LdLDH mutant and application thereof, a wild type LdLDH enzyme derived from Lactobacillus delbrueckii subsp. Bulgaricus is taken as a research object, the amino acid sequence of the wild type LdLDH enzyme is designed and modified to obtain a mutant protein, the mutant protein is named as LdLDH-M, the amino acid sequence of the mutant protein is shown as SEQ ID NO.2, the Tm value of the LdLDH-M is 77.1 DEG C, the Tm value of the LdLDH-M is 77.1 DEG C, and the Tm value of the LdLDH-M is 77.1 DEG C; compared with the wild type LdLDH enzyme, the Tm value of the LdLDH enzyme is increased by 27.4 DEG C, and the enzyme activity of the LdLDH enzyme is increased by 1.4 times. Therefore, compared with the wild type LdLDH enzyme, the LdLDH-M has remarkably improved thermal stability and enzymatic activity, and is more beneficial to widening the application range of the LdLDH-M in the fields of high-temperature biological research, industrial biological catalysis and the like.

The invention discloses a persistent endo-cellulase mutant with high specific activity and thermal stability and application of the persistent endo-cellulase mutant. A series of persistent endo-cellulase mutants are obtained by taking persistent endo-cellulase EG5C-1 derived from Bacillus subtilis as a template and combining mutation sites with deep learning prediction and a greedy algorithm, and compared with a wild enzyme, the optimal mutant M8 has the advantages that the activity of a substrate CMC (Carboxymethyl Cellulose) is improved by seven times, and the activity of a substrate Avicel is improved by four times. The content of reducing sugar generated by degrading filter paper by the continuous endo-cellulase mutant is greatly increased, and the continuous endo-cellulase mutant has important significance on efficient degradation of a cellulose substrate and reduction of the production cost.

The invention belongs to the technical field of biology, and particularly relates to a T4 DNA ligase mutant with improved connection fidelity. The T4 DNA ligase mutant is obtained by mutating any one or more amino acid sites of lysine at the 365th site and lysine at the 367th site of wild type T4 DNA ligase. Compared with a wild type T4 DNA ligase, the T4 DNA ligase has higher fidelity, and the fidelity of the T4 DNA ligase is maximally improved to more than 10 times of that of the wild type T4 DNA ligase.

The present disclosure discloses a thermophilic recombinant type II pullulanase and the application thereof, and belongs to the technical field of genetic engineering. The present disclosure obtains a thermophilic recombinant type II pullulanase by heterologously expressing type II pullulanase in Escherichia coli. Its optimum pH is 6.6, it has better pH tolerance under the conditions of pH 5.8-8.0, and its optimum temperature is 95° C. After incubating at 95° C. for 10 h, the remaining enzyme activity is greater than 50%. It can exhibit higher specific enzyme activity under strong reducing conditions. For example, adding DTT to the culture environment can increase the specific enzyme activity of Sumo-PulPy by 237.2%. The present disclosure also provides the combined truncation mutant Δ28N+Δ791C of type II pullulanase Sumo-PulPy. The specific enzyme activity of the enzyme mutant is 32.18±0.92 U / mg, which is 5.99 times as high as that of the wild-type enzyme, thereby having important industrial application value and potential.

The application discloses an acyltransferase for synthesizing polyester intermediates and application thereof. The acyltransferase is specifically a wild-type enzyme or a mutant acyltransferase variant with catalytic acyltransferase activity, which is obtained by substituting at least one of amino acid residues at positions 209, 166 and 119 in the amino acid sequence of the wild-type enzyme. The acyltransferase variant is specifically a protein with improved acyltransferase activity. Both the wild-type enzyme and the acyltransferase variant provided by the application have acyltransferase activity. The application has important application value.

The invention discloses a preparation method and application of a lactic dehydrogenase mutant with improved thermal stability. The lactic dehydrogenase mutant is obtained by amplifying a mutation primer by taking a plasmid of wild lactic dehydrogenase as a template; the amino acid sequence of the wild type lactic dehydrogenase is as shown in SEQ ID NO. 1; lactic dehydrogenase mutants M53D and S309P with improved thermal stability are obtained, the residual enzyme activity of the S309P mutant is 76% after the S309P mutant is incubated for 1.5 h at 50 DEG C, and only 50% of the activity of wild type enzyme remains under the same treatment condition.

The invention discloses a construction method of a high-activity mutant of chondroitin 6-O sulfotransferase, and belongs to the field of biological enzyme engineering and synthetic biology. Interaction sites of MusC6 protein and a substrate PAPS are accurately analyzed through a computer-aided molecular docking technology, and key active sites are directionally modified by adopting a strategy of combining rational design and saturated mutation. Experimental results show that the catalytic performance of the obtained mutant S139T / S140L / Y364M / K412V is remarkably improved, and the enzyme activity of the mutant reaches 1182U / L and is improved by 4.4 times compared with the enzyme activity of a wild type 270U / L. According to the invention, the technical bottleneck of low catalytic efficiency of a wild type enzyme on a natural substrate is solved through a multi-site synergistic mutation strategy, and a biocatalyst with industrial application value is provided for efficiently synthesizing chondroitin sulfate C by a microbiological method.

The application belongs to the technical field of bio-chemical industry, and particularly relates to a P450 enzyme mutant and application of an engineering bacterium thereof in synthesis of calcifediol, the mutant is one or more point positions in an amino acid sequence based on a wild type P450 enzyme Vdh being mutated; the amino acid sequence of the wild type P450 enzyme Vdh corresponds to GenBank accession number CP069288.1. Through systematic molecular dynamics simulation, conservation analysis and substrate binding pocket engineering on the Vdh enzyme, multiple key mutation sites are successfully screened and obtained. The constructed combined mutant (such as I114R / N173M / Q310R, Vdh-M3) shows extremely high hydroxylation activity, and the pure enzyme activity can reach 9.8 times of the wild type enzyme, greatly improving the conversion rate from vitamin D3 to calcifediol.

This invention discloses a pullulanase mutant with enhanced catalytic activity against highly branched dextrin substrates, its construction method, and its applications, belonging to the fields of enzyme engineering and biotechnology. This invention obtains two mutants, D139A and D139E, by site-directed mutagenesis of the pullulanase derived from Klebsiella pneumoniae at position 139 aspartic acid residue. Compared with the wild-type enzyme, both mutants maintain high hydrolytic activity against pullulan polysaccharides while significantly improving hydrolytic activity against dextrin substrates with high α-1,6 glycosidic bond content. Specifically, the hydrolytic activity against dextrin substrates with 6% and 10% α-1,6 bond content is increased by up to 242% and 110%, respectively. The pullulanase mutants obtained in this invention exhibit excellent catalytic performance in the enzymatic debranching reaction of highly branched dextrin substrates, showing promising application prospects in starch deep processing and related biomanufacturing fields.

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.

According to the invention, beta-glycosidase which has a catalytic effect on vincoside, loganin and strictosamide is selected, and a foundation is laid for diversity and possibility of an aglycone production process. By researching the three-dimensional structure information of the Gelsemium elegans Beta-glycosidase, a zymoprotein structure information basis is provided for molecular modification of the Gelsemium elegans Beta-glycosidase, enzyme variants capable of improving substrate selectivity, thermal stability and catalytic efficiency are obtained more efficiently, and the requirements of industrial application are met; the invention provides a truncated mutant enzyme with improved enzymatic activity, and through induced expression and activity screening, it is found that the enzyme activity of the obtained mutant enzyme to loganin and strictosamide is about two times higher than that of a wild enzyme.

The present application relates to a kind of persistence endocellulase ancestor and its application.The present application uses the persistence endocellulase of Bacillus subtilis as mother, uses ancestor sequence reconstruction technology, deduces the evolutionary relationship based on systematics analysis, deduces the amino acid sequence of ancestor enzyme from extinct organism using computer calculation.The persistence endocellulase ancestor includes ASR95, ASR106, ASR107, ASR108, ASR109, ASR110, ASR145.Compared with wild-type enzyme, the ancestor enzyme has high catalytic efficiency, good thermal stability and other excellent properties, so that the potential direction of producing reducing sugar by degrading filter paper is realized.

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.

The present application belongs to the technical field of genetic engineering, and relates to a CrtI enzyme mutant, a recombinant plasmid, a recombinant strain and application thereof. The CrtI enzyme mutant provided by the present application is obtained by random mutation of a wild-type CrtI gene through error-prone PCR, and the amino acid sequence thereof is shown as SEQ ID NO. 1. The obtained mutant has multiple site variations in the amino acid sequence, thereby significantly improving the catalytic efficiency and substrate affinity of the enzyme. The present application further constructs a recombinant expression vector containing the CrtI enzyme mutant gene, and transforms the recombinant expression vector into a red Phaffia yeast competent cell, and obtains a high-yield astaxanthin recombinant strain through resistance screening and pigment phenotype screening. Compared with the wild-type CrtI enzyme, the CrtI enzyme mutant of the present application significantly improves the conversion rate of beta-carotene to astaxanthin in the red Phaffia yeast.