156 results about "Wild type enzyme" patented technology

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.

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 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 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 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 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 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.

The present invention relates to variants of TEV protease that have—compared to the wildtype enzyme—increased stability and catalytic activity as well as altered substrate specificity. The invention further relates to compositions comprising these variants as well as uses thereof and methods in which these variants are employed.

The invention relates to the technical field of enzyme engineering and biology, and discloses an olefin reductase mutant as well as a coding gene, a recombinant vector, a recombinant strain, an enzyme preparation and application thereof. The olefin reductase mutant provided by the invention is obtained by mutating at least one of F297V, F297V / F124N and F297V / F124N / M40F on an enzyme with an amino acid sequence as shown in SEQ ID NO.1. Compared with a wild enzyme, the olefin reductase mutant has the advantages that the efficiency of catalyzing p-hydroxybenzylideneacetone to generate raspberry ketone is obviously improved, complicated reaction environment control equipment is not needed, the cost is low, and the production cost is low. And an efficient and feasible enzyme catalyst scheme is provided for industrial green production of raspberry ketone.

The present invention discloses a P450 enzyme mutant, a single-plasmid three-enzyme co-expression system, and their application in calcifediol synthesis. The P450 enzyme mutants include: using the wild-type P450 enzyme CYP109E1 shown in SEQ ID NO. 1 as a template, and respectively mutating the T at position 78 or the G at position 81 to form the mutants T78A, T78V, T78L, and T78I, G81V, and G81M; and mutating both the T at position 78 and the G at position 81 to form the mutant T78L / G81M. The present invention also constructs a single-plasmid three-enzyme co-expression system of the P450 enzyme mutant and a redox partner, thereby improving the electron transfer efficiency in the P450 enzyme hydroxylation reaction. This system can synthesize calcifediol with higher efficiency under mild conditions, solving the problems of low catalytic efficiency and activity in the synthesis of calcifediol catalyzed by P450 enzymes, and has important industrial application value.

The present invention relates to an improved cysteine-producing strain, in particular to a microbial strain suitable for the fermentative production of L-cysteine, characterized in that the relative enzymatic activity of the enzymes identified by the number EC 2.7. 9.2 in the KEGG database is inactivated or the specific activity is reduced based on the wild-type enzymes, and the enzyme represented by the number EC 2.7. 9.2 in the KEGG database forms an increased amount of L-cysteine as compared to a microbial strain having a wild-type enzymatic activity in which the gene encoding the enzymatic activity is represented by ppsA. The invention also provides methods of producing L-cysteine using these microbial cells.

The invention provides an improved engineered phenylalanine ammonia lyase which can be used for preparing a chiral amine compound, in particular for asymmetrically synthesizing L-2-chloro-phenylalanine. Compared with a wild-type enzyme corresponding to SEQ ID NO: 2, the engineered phenylalanine ammonia lyase polypeptide has better activity and / or stability, can be used for asymmetrically preparing chiral amine compounds with extremely high stereoselectivity, particularly can be used for more efficiently catalyzing o-chloro-cinnamic acid to prepare L-2-chloro-phenylalanine, can accept various ammonia donors, and can be used for preparing the chiral amine compounds. Such as ammonium carbonate, ammonia water and ammonium chloride.

The invention belongs to the technical field of biochemical engineering, and particularly relates to application of a P450 enzyme mutant and engineering bacteria thereof in synthesis of calcifediol, and the mutant is obtained by mutating one or more sites in an amino acid sequence based on a wild type P450 enzyme Vdh; the amino acid sequence of the wild type P450 enzyme Vdh corresponds to the accession number of GenBank, namely CP069288.1. Through systematic molecular dynamics simulation, conservative analysis and substrate binding pocket engineering on the Vdh enzyme, a plurality of key mutation sites are successfully screened and obtained. The constructed combined mutant (such as I144R / N173M / Q310R, Vdh-M3) shows extremely high hydroxylation activity, the pure enzyme activity of the combined mutant can reach 9.8 times that of a wild type enzyme, and the conversion rate from vitamin D3 to calcifediol is greatly increased.

The invention relates to non-naturally occurring mutated arylsulfotransferases comprising (i) an amino acid substitution in at least one amino acid position selected among positions 6, 7, 8, 9, 11, 17, 20, 33, 62, 97, 138, 195, 236, 239, 244, 263, and combinations thereof, wherein the position is relative to the amino acids sequence of rat arylsulfotransferase IV SEQ ID NO: 1 and (ii) an amino acid sequence having at least 60% sequence identity with amino acids sequence SEQ ID NO: 1. The mutated arylsulfotransferase may have a sulfotransferase activity for converting adenosine 3′,5′-bisphosphate (PAP) into 3′-phosphoadenosine-5′-phosphosulfate (PAPS) enhanced compared to the wild-type enzyme.

The invention discloses short-chain dehydrogenase for synthesis of (S)-1-(2-methoxy-3-bromophenyl) ethanol as well as a mutant, a coding gene, a plasmid, a genetically engineered bacterium and application of the short-chain dehydrogenase, and belongs to the technical field of biocatalysts. The mutant is obtained by mutating methionine at the 97th site into isoleucine on the basis of a wild type enzyme shown in SEQ ID NO.1 (SEQ ID NO.3). The invention further discloses a preparation method of the short-chain dehydrogenase. The invention also provides a coding gene, an expression vector, a genetically engineered bacterium and a double-enzyme catalytic system containing the mutant and formate dehydrogenase. The system can efficiently and highly selectively catalyze and synthesize the target chiral alcohol under mild conditions, and the conversion rate is gt; 95%, eet; 99% of the method is suitable for green preparation of the key intermediate of the medicine lutrombopag.

The application discloses a cutinase mutant and application thereof to efficient degradation of PET. The amino acid sequence of the cutinase mutant is shown in SEQ ID NO. 2, and is obtained by replacing phenylalanine at the 239th amino acid in the amino acid sequence of a wild-type cutinase shown in SEQ ID NO. 1 with alanine. The cutinase mutant is expressed in E. coli, and compared with the wild-type enzyme, the activity of the mutant enzyme is increased in stability, the catalytic efficiency is improved, and the yield of a PET plastic (polyethylene terephthalate, PET) degradation product is increased by 42.6 times. The improved cutinase mutant improves the PET degradation efficiency, reduces the production cost, and is more suitable for industrial application.

The present application relates to the field of genetic engineering and bioengineering technology, in particular to a Candida antarctica lipase B mutant and application thereof. The Candida antarctica lipase B mutant provided by the present application is obtained by joint mutation of the 143th and 284th positions in the amino acid sequence shown in the wild type lipase B. The Candida antarctica lipase B mutant constructed by the present application has 12.5 times higher enzyme activity than the wild type enzyme, and the protein expression amount is as high as 1.8-1.9 g / L under the scale of 5 L fermenter, which can significantly improve the yield and reduce the production cost. In addition, the Candida antarctica lipase B mutant can be used for synthesis of high value-added, non-natural substrates such as vitamin C fatty acid ester and vitamin A oleate, which lays a foundation for the application of lipase in the field of fine chemical industry.