35 results about "Wild type enzyme" patented technology

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 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 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 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.

This invention discloses a desmethylmenoprenone methyltransferase mutant, its construction method, and its applications, belonging to the field of biotechnology. Desmethylmenoprenone methyltransferase is a key enzyme in the MK-7 biosynthetic pathway, responsible for catalyzing the methylation reaction of desmethylmenoprenone to generate a series of methylnaphthoquinone (MK) products, ultimately forming MK-7. This invention, through metabolic network regulation and rational design, successfully obtained 19 single-point mutants, among which 5 mutants showed significantly higher catalytic efficiency than the wild-type enzyme. Experimental results show that the recombinant engineered bacteria constructed based on these mutants exhibit significant advantages in the whole-cell catalytic production of vitamin K2 (MK-7), with MK-7 yield increased by more than 20% compared to the wild-type strain. The excellent catalytic performance of the menG mutant provided by this invention provides strong technical support for the industrial production of vitamin K2, and has broad application prospects and market value.

This invention discloses a highly efficient method for the biosynthesis of butanediamine, belonging to the field of bioengineering. The arginine decarboxylase mutant provided by this invention exhibits a specific enzyme activity of 25.1 U / g under alkaline conditions, approximately 8 times higher than the wild-type enzyme, significantly enhancing the enzyme's catalytic efficiency and providing a highly efficient enzyme catalyst for the biosynthesis of butanediamine. Recombinant strain E. coli BL21(DE3) speA ‑ E482R ‑ E467K ‑ H736E ‑ speB In the catalytic synthesis of butanediamine, there is no need to collect the bacterial cells by centrifugation. The bacterial cells will self-sediment in the culture medium, and complete sedimentation can be achieved within 20-30 minutes. Moreover, the bacterial cell content in the supernatant is only 1% (OD600). By reusing the bacterial cells in the fermentation broth, continuous catalytic reaction can be achieved, significantly increasing the yield of butanediamine.

The application discloses a trehalose-6-phosphate phosphatase and a mutant and application thereof, and belongs to the technical field of biology. The amino acid sequence of the wild-type LcTre6PPase is shown as SEQ ID NO:1, and the encoding nucleotide sequence is shown as SEQ ID NO:2. The mutant is obtained by mutating the isoleucine at the 571th position of the wild-type enzyme into leucine (I571L), the amino acid sequence is shown as SEQ ID NO:3, and the corresponding encoding nucleotide sequence is shown as SEQ ID NO:4. Bidirectional enzyme activity analysis shows that, compared with the wild type, the I571L mutant is improved by about 1.2 times in the direction of catalyzing glucose-6-phosphate and beta-D-glucose-1-phosphate to synthesize trehalose-6-phosphate, and the activity in the direction of catalyzing trehalose-6-phosphate to phosphorolyze is reduced to 0.8 times of the wild type. The mutant shows good application potential in the efficient synthesis of trehalose-6-phosphate, and provides a new enzyme tool for preparing trehalose-6-phosphate.

This invention relates to the field of biotechnology, specifically to an inositol oxidase mutant and its application in the production of glucuronic acid. The inositol oxidase mutant provided by this invention is obtained by point mutation of the amino acid sequence shown in the wild-type inositol oxidase. Compared with the wild-type enzyme, the inositol oxidase mutant constructed by this invention exhibits a significant improvement in catalytic efficiency. Furthermore, this invention also provides an engineered strain with site-directed mutagenesis and knockout of the UxaC gene in the chassis cell genome. This strain exhibits higher catalytic activity towards the substrate, with a catalytic efficiency increased by 74.44% compared to the wild-type enzyme. Even at high substrate concentrations, the conversion rate can still reach over 92.09%, with a production intensity as high as 13.23 g / L / h. It possesses extremely strong substrate tolerance and production intensity, significantly reducing the production cost of glucuronic acid and demonstrating significant industrial application potential.

This invention belongs to the field of molecular biology technology and discloses an improved PCR amplification enzyme, amplification enzyme antibody, and buffer composition for full-component lyophilization and direct amplification, as well as their applications. This invention provides an enzyme combination comprising Taq enzyme and reverse transcriptase. The Taq enzyme is obtained by site-directed mutagenesis based on wild-type Taq enzyme, exhibiting superior lyophilization tolerance, interference resistance, thermostability, and amplification specificity. The reverse transcriptase is obtained by site-directed mutagenesis based on wild-type reverse transcriptase, exhibiting good lyophilization-reconstitution activity, thermostability, reverse transcription efficiency, and specificity. Using the above enzyme combination to prepare full-component lyophilized and direct PCR amplification reagents can simultaneously solve the pain points of traditional reagents, such as reliance on low-temperature transportation and storage, the need for extraction before amplification, cumbersome operation, and weak interference resistance.

This application provides a class of RNA ligase mutants, their preparation methods, and their applications in the preparation of nucleic acid products, belonging to the field of biomedical technology. Compared with wild-type enzymes, the RNA ligase mutants with mutations of Y206A, V207A, I224A, or C226Y provided in this application significantly reduce non-specific ligation impurities generated in the ligation reaction, showing good prospects for industrial application.

The present application relates to a kind of specific synthesis plant inulin of inulin sucrose enzyme mutant and its application, the mutant is the 300th aspartic acid of wild type inulin sucrose enzyme from Lactobacillus reuteri (Lactobacillus reuteri) Limosilactobacillus reuteri ) Mutated into alanine.It precisely cuts off the ability of wild type enzyme to synthesize million Dalton high molecular weight microbial inulin at molecular level, while perfectly retaining the synthesis ability of medium-low degree of polymerization inulin.This mutant fundamentally solves the problem of wild type enzyme product molecular weight distribution extremely wide, component complex, realizes the efficient biosynthesis of specific inulin.At the same time, it can greatly reduce the difficulty and energy consumption of downstream separation and purification process, significantly reduce production cost.In the high-quality inulin large-scale green manufacturing of food and health product industry, it has great application value.

Disclosed herein are mutants of bacteriophage Phi29 DNA polymerase with improved primer recognition, compared to the wild-type enzyme. Certain mutants comprise one or both of the mutations K64R or M97K. The provided mutants are capable of using more efficiently shorter and longer random synthetic DNA primers than wild-type Phi29 DNA polymerase, generating more amplification product in Multiple Displacement Amplification (MDA) reactions. The inventive mutants amplify human genomic DNA with less bias and better coverage in comparison to reactions carried out with wild-type Phi29 DNA polymerase.

The application discloses an enzyme catalytic constant prediction method and system based on deep learning, and belongs to the technical field of bioinformatics and deep learning. In view of the defects of low prediction accuracy and weak generalization ability of the prior art, the application adopts a three-level progressive feature fusion architecture: a 960-dimensional protein semantic feature vector is extracted through an ESMC-300M pre-training model, a 1024-dimensional chemical feature vector is generated by using a SMILES molecular converter based on a Transformer architecture, and the two feature vectors are integrated into a 1984-dimensional combined feature vector through a heterogeneous feature fusion network; and then the logarithmic value of the enzyme catalytic constant is predicted by using an ExtraTreesRegressor ensemble learning model combined with grid search optimization of hyperparameters. The application can achieve an MAE of 0.33 in wild-type enzyme prediction, and still has good generalization performance on unfamiliar enzyme systems with a sequence similarity of less than 40%, which is significantly better than the prior art, and can be widely applied to the fields of industrial enzyme screening, metabolic engineering optimization, drug design and the like, and provides efficient technical support for biological manufacturing and green chemistry.

This invention relates to the field of enzyme engineering, and discloses a mutant enzyme with halogenation activity, its enzyme preparation, encoding gene, recombinant vector, recombinant strain, and applications. The mutant enzyme is an S-adenosyl-L-methionine hydrolase with an amino acid sequence as shown in SEQ ID NO.1, comprising at least one mutation at the F19, P83, and R89 sites; wherein the mutation at the F19 site is selected from at least one of F19P, F19C, and F19L; the mutation at the P83 site is selected from at least one of P83F, P83H, P83E, P83D, P83C, P83L, P83K, and P83I; and the mutation at the R89 site is selected from at least one of R89A, R89C, R89E, R89F, R89S, R89T, R89Q, R89H, and R89D. All of the above mutations enable the enzyme to acquire the new function of catalyzing the production of 5'-halodeoxyadenosine from S-adenosine-L-methionine, exhibiting excellent iodination catalytic efficiency. Its activity exceeds that of wild-type enzymes and is superior to known natural halogenases, thus solving the technical problems of scarce natural halogenase resources and severe chemical halogenation pollution.

The application discloses a D-tagatose 4-epimerase mutant for efficiently synthesizing D-tagatose and application thereof. The wild-type D-tagatose 4-epimerase is screened first, and the wild-type enzyme capable of being used for mutation to further improve activity is obtained. The wild-type enzyme is then subjected to mutation and modification, and a plurality of high-temperature-resistant D-tagatose 4-epimerase mutants are screened, so that the conversion efficiency of the mutants in catalyzing D-fructose to synthesize D-tagatose is improved. The conversion rate of the obtained mutants at 70 DEG C is more than 3 times of that of the wild type, and the mutants can catalyze higher-concentration substrates to be converted compared with the wild type.

The application discloses a glycosyltransferase, which is a parent of wild-type glycosyltransferase described in SEQ ID NO. 1, and comprises the following mutation points: the 125th lysine is mutated into arginine (K125R), the 172nd alanine is mutated into phenylalanine (A172F), the 294th glycine is mutated into cysteine (G294C), and the 311th valine is mutated into cysteine (V311C), and application of the glycosyltransferase in preparation of non-natural proto-ginsenoside. The application rationally designs and constructs multiple groups of mutant UGT109A1 of UGT109A1 by analyzing the structure and evolutionary conservation of the enzyme molecule UGT109A1, and obtains the mutant with improved catalytic performance under the concentration of 30% DMSO through screening, and the glycosylation efficiency of the mutant is relatively improved by 72% compared with the wild-type enzyme. Based on the wide substrate spectrum of UGT109A1, the obtained mutant enzyme with improved glycosylation efficiency under high DMSO concentration provides a new enzyme for green and efficient preparation of functional glycosides.

The application provides a Bst DNA polymerase domain, a mutant and application thereof, and belongs to the technical field of molecular biology. The amino acid sequence of the Bst DNA polymerase domain is shown in SEQ ID NO:1. The Bst DNA polymerase mutant is obtained by replacing the natural DNA binding domain of the wild-type Bst DNA polymerase with the amino acid sequence shown in SEQ ID NO:1. The Bst DNA polymerase mutant exhibits extremely high catalytic efficiency on dUTP, and also has excellent hot-start characteristics, and can reduce the false positive rate of LAMP technology to close to zero. The Bst DNA polymerase mutant also has extremely strong complex sample tolerance, and the detection sensitivity of the pathogen is 20 times higher than that of the wild-type Bst enzyme, and can meet the early and accurate diagnosis requirements of trace infection.

The application discloses a 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutant, a construction method and application thereof, and belongs to the technical field of biology. Eight 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase mutants which are significantly superior to wild-type enzymes in catalytic efficiency are obtained through rational design and screening, and a recombinant Escherichia coli based on the mutants is constructed, so that the yield of vitamin K2 produced by the whole cell catalysis of the constructed recombinant bacteria is more than 30% higher than that of a wild-type strain. The excellent catalytic performance of the menA mutant provided by the application provides strong technical support for the industrialized production of vitamin K2, and has wide application prospect and market value.

The application belongs to the technical field of enzyme engineering, and particularly relates to a beta-1,3-glycosyltransferase mutant and application thereof in catalyzing synthesis of rebaudioside M. The beta-1,3-glycosyltransferase mutant is obtained by mutating the beta-1,3-glycosyltransferase with an amino acid sequence shown in SEQ ID NO. 1 by at least one mutation, i.e. mutating isoleucine at the 14th position into alanine; and / or mutating asparagine at the 50th position into alanine; and / or mutating isoleucine at the 92nd position into leucine; and / or mutating leucine at the 141st position into cysteine or isoleucine. The mutant is applied to catalyzing synthesis of rebaudioside M, and is significantly superior to the wild-type enzyme in catalytic efficiency and thermal stability.

The application discloses a glycosyltransferase Ml CGT and mutants thereof and application thereof in preparing luteolin-3'-O- O glucoside, and belongs to the technical field of biological catalysis and natural product synthesis. Ml The application constructs a glycosyltransferase CGT mutant, which shows high regioselectivity for luteolin and can be used for preparing luteolin-3'-O- O glucoside in a directional conversion mode, the conversion rate reaches 99%, and the catalytic efficiency is 5203.2 L·mol ‑1 ·s ‑1 , which is significantly higher than that of the wild-type enzyme. At The application further constructs a cascade catalysis system comprising the mutant and sucrose synthase SUS1, and realizes in-situ regeneration of UDP-glucose. O By optimizing the UDP concentration, sucrose concentration, temperature and pH conditions, the conversion rate of luteolin-3'-O- O glucoside is further improved, and the yield can reach 90%, so that the application has the application potential in large-scale biological preparation of luteolin-3'-O- glucoside.

This application provides a Phi29 DNA polymerase variant and its application. Compared with the wild-type enzyme, the Phi29 DNA polymerase variant described in this application, which contains substitutions of A484N+S395T, A484Q+S395T, A484Q+T534C, Y281H+S395T, D186E+G401K, or S395T+K402R, exhibits higher DNA synthesis fidelity, effectively reduces errors during amplification, thereby reducing the risk of non-specific amplification and showing good prospects for industrial application.

This invention discloses an NADPH-dependent alcohol dehydrogenase EcYjgB mutant and its applications. The mutant is based on the wild-type alcohol dehydrogenase EcYjgB with mutations occurring at one or more of the following sites: S46, E51, W52, F54, T92, I108, A114, P116, I118, N240, G262, V264, L265, and A286. The amino acid sequence of the wild-type alcohol dehydrogenase EcYjgB is shown in SEQ ID. 1. This mutant is used to catalyze the selective reduction of high concentrations of 5-hydroxymethylfurfural or furfural to synthesize 2,5-furandiethanol or furfuryl alcohol, respectively. Compared to the wild-type enzyme, this mutant exhibits significantly improved catalytic efficiency and significantly reduced substrate inhibition at high substrate concentrations. It is green and efficient, requires no organic reagents, shortens reaction time, and significantly reduces the difficulty and cost of subsequent separation and purification.

This invention discloses an epoxide hydrolase mutant, biomaterials, products, and applications. Compared to the wild-type epoxide hydrolase, this mutant possesses one or more amino acid residue mutations at positions 108, 131, and 256. The catalytic activity of this series of mutants is significantly improved compared to the wild-type enzyme, with the most preferred mutant being F108V / D131N / E256V. (S) - Epoxyphenylethane formation (S) When synthesizing 1,2-ethylene glycol, the yield can reach 93% under optimal reaction conditions. Furthermore, the reaction conditions for the catalytic synthesis of chiral 1,2-ethylene glycol using this series of mutants are mild, and the production process is environmentally friendly, demonstrating excellent industrial application value.

The application provides a 3-steroid ketone-delta 1 -dehydrogenase, mutants and applications thereof. The 3-steroid ketone-delta 1 -dehydrogenase mutant provided by the application is a mutant of one or more core amino acids of a wild-type 3-steroid ketone-delta 1 -dehydrogenase which are related to catalytic activity of the enzyme. Compared with the wild-type enzyme, the 3-steroid ketone-delta 1 -dehydrogenase mutant of the application significantly improves catalytic efficiency of a C1,2 dehydrogenation reaction. By using a recombinant bacterium expressing the mutant, an intermediate with a methyl substitution at a C6 position of a steroid nucleus can be efficiently converted at a high substrate concentration, a yield of a target product is high, no by-product is generated, and a conversion rate is not less than 95%. The process has a short conversion time, a small amount of biological catalysts, and a simple, mild, and environmentally friendly preparation method, and shows a good industrial application prospect.

This invention relates to a BsNTR mutant nitroreductase and its applications, belonging to the field of biotechnology. The BsNTR mutant is BsNTR-M, and its amino acid sequence is shown in SEQ ID NO.2. This invention designs and modifies wild-type BsNTR-WT from Bacillus subtilis. The resulting mutant BsNTR-M not only exhibits significantly improved thermostability (Tm value 14°C higher than wild-type BsNTR-WT) but also demonstrates 1.6 times enhanced enzyme catalytic activity. This effectively overcomes the technical bottlenecks of poor thermostability, weak environmental tolerance, and limited activity inherent in wild-type enzymes. Furthermore, it possesses excellent expression and purification performance, allowing for efficient expression and purification in prokaryotic expression systems while retaining high specificity for nitro compounds and good biocompatibility and environmental safety. Large-scale production can be achieved without complex equipment.