29 results about "Single mutation" patented technology

The invention discloses a 3-sterone-delta1-dehydrogenase mutant and application of the 3-sterone-delta1-dehydrogenase mutant in preparation of a steroid drug intermediate. The mutant is obtained by performing single mutation or multiple mutation on 551, 468, 48 and 157 sites of an amino acid sequence of 3-sterone-delta1-dehydrogenase as shown in SEQ ID NO: 1. The 3-ketosterone-delta1-dehydrogenase is subjected to molecular modification through directed evolution and semi-rational design, the mutant with improved enzyme activity is screened in a high-throughput mode, the mutant can effectively improve the conversion rate of a substrate 11alpha, 17alpha-dihydroxyprogesterone, and the substrate conversion rate of the mutant ISM-2 is improved to 92.4% compared with 36.7% before mutation.

The present invention relates to the technical field of enzyme engineering. Disclosed are an artificial intelligence-based enzyme engineering method and system, a device, and a medium. The method comprises: on the basis of structural information of an engineered enzyme to be modified, using an inverse protein folding model to obtain a plurality of structurally compatible protein sequences on the basis of a given scaffold structure; on the basis of the plurality of structurally compatible protein sequences, obtaining a mutant amino acid frequency and a wild-type amino acid frequency at each sequence position; and on the basis of the mutant amino acid frequency and the wild-type amino acid frequency at each sequence position and the structural information of said engineered enzyme, as well as a first creation strategy, a second creation strategy, and a third creation strategy, obtaining a single-mutation variant set and a combinatorial-mutation variant set of said engineered enzyme. The present invention enables efficient and low-cost generation of single-mutation and combinatorial-mutation variant sets of an engineered enzyme to be modified, thereby achieving precise and efficient gene editing and effectively expanding the applicability and safety of gene editing.

The invention provides a glycosidase mutant and application thereof, the amino acid sequence of the mutant is compared with the amino acid sequence SEQ ID NO.1 of a wild enzyme, in the amino acid sequence SEQ ID NO.1, five sites of M102K, P171L, V203G, T269Y and K299D are respectively subjected to single mutation, pairwise combined mutation, three combined mutation, four combined mutation or one of five combined mutation; the novel glycosidase mutant industrial enzyme is used for synthesis and preparation of hesperetin. The glycosidase mutant enzyme constructed by the invention has the characteristics of low enzyme cost, short conversion time, simple process operation and the like, and has a wide prospect of large-scale industrial application.

The invention relates to the technical field of gene engineering and protein modification, in particular to a high-specific-activity cellulase mutant and application thereof. Compared with the wild type cellulase, the specific activity of the mutants respectively containing single mutation sites of P14A, P19T, S75A, S109N, H118P, M123I, M123L, I130L, I130Q, Y167E, Y167F, Y167W, A176S, A176F, D177S and L192I, provided by the invention, is generally improved by 10.4%-76.1% under a neutral condition. Wherein the specific activity of a single-site mutant containing I130L is the highest, and reaches 183.64 U / mg. The production cost of the cellulase mutant is remarkably reduced, so that wide application of the cellulase mutant is promoted.

Provided are high-specific-activity cellulase mutants and use thereof. Compared to the wild-type cellulase, the specific activity of the provided mutants that comprise single mutation sites P14A, P19T, S75A, S109N, H118P, M123I, M123L, I130L, I130Q, Y167E, Y167F, Y167W, A176S, A176F, D177S, and L192I, respectively, under neutral conditions is generally increased by 10.4% to 76.1%. The single-point mutant comprising I130L has the highest specific activity, which reaches 183.64 U / mg. The production costs of the cellulase mutants are significantly lower, which facilitates the wide use thereof.

The application provides a tyrosine deaminase mutant and application thereof in production of p-coumaric acid. The tyrosine deaminase mutant is obtained by mutating a wild-type tyrosine deaminase with an amino acid sequence shown in SEQ ID No. 1, and the mutation site is selected from at least one of Q108S, Y340Q and P375H. The application analyzes a catalytic active center of the wild-type tyrosine deaminase, and finds that different mutation sites, same mutation sites but different mutation targets can make the mutant show completely different enzyme catalytic activities, and the single mutation of Q108S, Y340Q and P375H can significantly improve the enzyme catalytic activity of the tyrosine deaminase mutant, and the tyrosine deaminase mutant with any two mutation sites, especially with three mutation sites also has excellent enzyme catalytic activity than the wild type, and has great application potential.

The application discloses a 7beta-hydroxysteroid dehydrogenase mutant and application thereof, relates to the field of bioengineering technology, and is designed and reformed according to the amino acid sequence of wild-type 7beta-hydroxysteroid dehydrogenase derived from Collinsella aerofaciens, that is, 17 single-mutation sites or 7 combined-mutation sites of 7beta-hydroxysteroid dehydrogenase are provided, the obtained 7beta-hydroxysteroid dehydrogenase mutant has the thermal stability increased by 2-21.5 DEG C and the activity increased by nearly 1.2-2.24 times compared with the wild-type 7beta-hydroxysteroid dehydrogenase. According to the mutation sites with obvious increase in thermal stability and activity, three preferred mutant proteins are further provided, the three preferred mutant proteins have higher yield, activity and thermal stability compared with the wild-type 7beta-hydroxysteroid dehydrogenase, have wider application conditions, are more suitable for the efficient production of ursodeoxycholic acid by the bioconversion method, and are favorable for large-scale production and industrial application.

The invention provides a Perakine reductase mutant and application thereof in double bond reduction, the mutant is obtained by performing the following single mutation on the 52 site, 57 site or 84 site of a wild Perakine reductase amino acid sequence: D52V / F / Y / N, Y57G / F / N / K and K84D, and ' / ' represents' or '. According to the invention, the selective reduction ability of the Perakine reductase mutant to carbon-carbon double bonds of alpha, beta-unsaturated carbonyl compounds is evaluated, and the high-activity Perakine reductase mutant is obtained through screening. The Perakine reductase is used as a biocatalyst, can realize efficient conversion of alpha, beta-unsaturated carbonyl compounds into saturated ketones, and has the advantages of simple reaction steps, mild conditions, high catalytic efficiency and the like. According to the invention, the application potential of the Perakine reductase in selective reduction of carbon-carbon double bonds of alpha, beta-unsaturated carbonyl compounds is improved.

The invention discloses a cancer genome variation prediction system based on multiple genetic operators, relates to the technical field of genetic variation detection, and aims to solve the problems that the traditional cancer genome variation prediction is mainly based on a static mutation rate and a single mutation type, the dynamic balance and nonlinear evolution of a tumor in mutation, selection and clone expansion processes are difficult to accurately reproduce, and the accuracy is poor. According to the cancer genome variation prediction method and the cancer genome variation prediction system, high-fidelity simulation and evolution reconstruction of the cancer genome are realized by fusing a multi-type mutation mechanism, fitness feedback and a polyclonal competition strategy. According to the method, through the synergistic effect of multiple genetic operators and dynamic parameter adjustment, the dynamic balance and nonlinear evolution of the tumor in the mutation, selection and clone expansion process can be accurately reproduced, and then the accuracy of cancer genome variation prediction is improved. The application provides a brand new technical approach for early screening of cancers, variation detection and precise medical treatment.

This invention discloses a nitrile hydrolase mutant, an engineered bacterium, and its application in the synthesis of (R)-3-cyanohexanoic acid. The nitrile hydrolase mutant is obtained by single or multiple mutations at positions 135, 199, and 59 of the amino acid sequence shown in SEQ ID NO.2. This invention utilizes directed evolution technology to enhance the activity and stereoselectivity of the nitrile hydrolase by constructing a nitrile hydrolase mutant. Compared to the parental nitrile hydrolase PgNITmut, the enantiomeric selectivity (E value) of the mutant PgNITmut / F135L for the substrate 3-cyanohexanonilonitrile is increased from 332 to 419; the activity of the mutant PgNITmut / F135L / R199W is increased by 1.36 times, and the E value is increased to 461; the activity of the mutant PgNITmut / F135L / R199W / T59D is increased by 2.05 times, and the E value is increased to 569. The nitrile hydrolase mutant of this invention has high activity and stereoselectivity. It can be used to synthesize the chiral intermediate (R)-3-cyanohexanoic acid of buvasidan by a highly efficient nitrile hydrolase method, which greatly reduces the cost of industrial production and has good application prospects in the industrial production of buvasidan.

This invention belongs to the field of biodetection technology, specifically disclosing a primer-probe composition and kit for detecting drug-resistant gene mutations in Helicobacter pylori. The composition comprises two sets of primers and probes, using the probe melting curve method as its principle. The composition and universal PCR reagents are used to detect multiple drug resistance mutations in the quinolone resistance gene gyrA and the clarithromycin resistance gene 23S rRNA of Helicobacter pylori in biological samples. This overcomes the limitation of existing methods that only detect single mutation sites in a single tube for Helicobacter pylori drug resistance genes, providing a new, rapid, and efficient method for detecting Helicobacter pylori drug resistance genes.

The application discloses a glycosyltransferase mutant and a method for catalyzing synthesis of sweet tea glycoside derivative Rub2G, the mutant is a single mutation or a combined mutation based on an amino acid sequence of original glycosyltransferase being SEQ ID NO:1, and finally a better catalytic effect is screened out Lb UGT mutant strain _ The SuSy system realizes efficient catalytic synthesis of sweet tea glycoside derivative Rub2G. The application improves the enzyme activity of the glycosyltransferase Lb UGT by site-directed mutagenesis, prolongs the half-life, realizes efficient catalytic synthesis of Rub2G by using the mutant, and has the advantages of mild synthesis method condition, simple operation, short time, high catalytic efficiency, high yield and good application prospect.

The invention discloses a bacillus subtilis alcohol dehydrogenase mutant with improved catalytic activity, and belongs to the field of gene engineering and protein expression. The mutant BsBDHAH42A / V266C / G292A is obtained by performing single mutation and combined mutation on 42,266 and 292 amino acids of (2, 3)-butanediol dehydrogenase (BsBDHA) of bacillus subtilis, the specific activity and catalytic efficiency of the mutant BsBDHAH42A / V266C / G292A are remarkably improved compared with those of wild type BsBDHA for catalyzing (R)-PED, the specific activity reaches 11.6 U / mg, and the catalytic efficiency reaches 2.52 mM <-1 > s <-1 > or above; compared with BsBDHAI49L / V266L in the prior art, the specific activity of the BsBDHAH42A / V266C / G292A mutant disclosed by the invention is improved by 21 times; compared with the mutant BsBDHAI49L / V266L in the prior art, the catalytic efficiency of the mutant BsBDHAI49L / V266L is improved by 31 times; and the conversion rate of catalyzing 2-hydroxyacetophenone in 6 hours reaches 92.1%.

The application discloses a glycosyltransferase mutant and application thereof, and the mutant is obtained by single mutation or multiple mutations of amino acids at positions 58, 181 or 371 of the amino acid sequence shown in SEQ ID NO. 4 or single mutation or multiple mutations of amino acids at positions 54, 152, 241 or 272 of the amino acid sequence shown in SEQ ID NO. 6. The glycosyltransferase is rationally modified by homologous modeling and sequence homology analysis, and a glycosyltransferase mutant with obviously improved enzyme activity is screened. Compared with the wild type, the relative enzyme activity of the mutants UGTM1-3 and UGTM2-4 is respectively improved by 2.88 times and 3.60 times compared with that before modification, and the mutants have great industrial application potential.

The invention relates to the technical field of enzyme engineering, and discloses an enzyme engineering method, system, equipment and medium based on artificial intelligence, and the method comprises the following steps: obtaining a plurality of structurally compatible protein sequences based on a given skeleton structure through a protein inverse folding model according to the structure information of an engineering enzyme to be modified; the method comprises the following steps: obtaining a mutant amino acid frequency and a wild amino acid frequency at each sequence position according to a plurality of structurally compatible protein sequences; and obtaining a single mutation variant set and a mutation combination variant set of the to-be-modified engineering enzyme according to the mutation amino acid frequency and the wild amino acid frequency at each sequence position and the structure information of the to-be-modified engineering enzyme in combination with the first creation strategy, the second creation strategy and the third creation strategy. According to the method, the single mutation variant set and the mutation combination variant set of the engineering enzyme to be modified can be efficiently obtained at low cost, accurate and efficient gene editing is realized, and the application range and the safety of gene editing are effectively expanded.

The invention provides a glycosyl hydrolase mutant and application thereof, the amino acid sequence of the mutant is compared with the amino acid sequence SEQ ID NO.1 of an original wild enzyme, in the amino acid sequence SEQ ID NO.1, six sites of E88K, G167Q, S252Y, G275V, L290P and M328L are respectively subjected to single mutation, pairwise combined mutation, three combined mutation, four combined mutation, five combined mutation or one of six combined mutation; the novel glycosyl hydrolase mutant industrial enzyme is used for synthesizing and preparing naringenin. The glycosyl hydrolase mutant enzyme constructed by the invention has the characteristics of low enzyme cost, short conversion time, simple process operation and the like, and has a wide prospect of large-scale industrial application.

The invention provides a high-activity carbonyl reductase mutant and application of the high-activity carbonyl reductase mutant in synthesis of a series of duloxetine intermediates. The mutant is obtained by performing single mutation or multiple mutation on the 138 site and / or the 195 site of an amino acid sequence as shown in SEQ ID NO. 2. The Mut-A138V-D195A recombinant reductase mutant is constructed and applied to synthesis of a duloxetine chiral key intermediate, an efficient biological preparation way is provided for synthesis of duloxetine key intermediate chiral alcohol, and the method has the advantages of being environmentally friendly, safe, easy and convenient to operate, easy to industrially amplify and the like and has a wide application prospect. And the mutant has the advantages of high catalytic activity, high product selectivity, wide substrate universality, high substrate concentration tolerance and the like, and has a huge industrial application prospect.

The invention discloses a biotin ligase BirA mutant and application thereof, a wild BirA enzyme from Escherichia coli is taken as a research object, single mutation is performed on a protein sequence of the wild BirA enzyme to obtain two mutant proteins BirA-M1 and BirA-M2, the Tm value of the wild BirA enzyme is 46.61 DEG C, and the Tm values of the BirA-M1 and BirA-M2 are respectively 53.50 DEG C and 51.36 DEG C, which are respectively increased by 6.89 DEG C and 4.75 DEG C compared with those of the wild BirA enzyme, so that the biotin ligase BirA mutant can be used for preparing a biotin ligase BirA mutant. Compared with a wild type BirA enzyme, the BirA-M1 and the BirA-M2 have higher thermal stability, the BirA-M1 and the BirA-M2 still keep better activity, and the labeling rates are 1.1 times and 1.2 times of those of the wild type BirA, so that the BirA enzyme mutant protein provided by the invention has remarkably improved thermal stability, and the application range of the BirA enzyme mutant protein in the fields of high-temperature biological research, industrial biological catalysis and the like is widened.

The application discloses a kind of Lb2Cas12a protein mutant activity prediction method and system, the method includes: obtaining the amino acid sequence of Lb2Cas12a protein mutant and the original data of corresponding enzyme activity;Using AAindex indexed as KARS160105 to carry out digital coding to amino acid sequence, obtain digitized amino acid sequence;The digitized amino acid sequence is processed, and the original data set consisting of the mean of digitized amino acid sequence and the number of mutated positions is obtained, and is randomly divided into training set, verification set and test set;Data in training set is used for training, and preliminary model is obtained;The data in verification set is used to adjust preliminary model, obtain the best model, and test the generalization of model on test set;Using the model constructed to process each piece of all Lb2Cas12a protein single mutants, obtain the prediction result of Lb2Cas12a protein mutant activity.The method is convenient to operate.

This invention discloses a highly active T4 DNA ligase mutant, its preparation method, and its applications. In this invention, through crystal structure and bioinformatics analysis combined with site-directed mutagenesis, saturation mutagenesis, and combinatorial mutagenesis, as well as rigorous experimental analysis, several T4 DNA ligase mutants with significantly improved performance were screened, including single mutants and combinatorial mutants. The improved performance includes: enzyme activity, thermal stability, and / or ligation efficiency.

Genetic samples are obtained from separate people, and at least a portion of each are purposefully combined before testing to form a pooled genetic sample. The pooled genetic sample is tested for the presence of a signature for a given known ailment. DNA identification uses discovered InDels in a region of InDel variation in a genetic sample. A pair-wise comparison is performed to reference InDels, and a distance is measured between the first InDel and the reference Indel. Reference kmers are identified in a reference genome, and in a test sample. The plurality of sample kmers are filtered to those which have a 1 edit distance from a corresponding one of the plurality of reference kmers. Reads that have kmers that do not have a 1 edit distance from the corresponding one of the plurality of reference kmers are identified, and multiple single-mutations are eliminated from candidate InDel reads.

The invention discloses a firefly luciferase Fluc mutant and application of the firefly luciferase Fluc mutant, wild firefly luciferase Fluc from North America fireflies is taken as a research object, single mutation or combined mutation is carried out on a protein sequence of the firefly luciferase Fluc to obtain a mutant protein, and the mutant protein is used for preparing the firefly luciferase Fluc mutant. Compared with wild type Fluc, the heat stability and activity of the Fluc mutant protein are improved to a certain degree, compared with the wild type Fluc, the heat stability of the preferable mutant protein is improved by 13.2-14.5 DEG C, the activity is improved by 1.4-2.5 times, the yield is improved by 2.4-3.9 times, the purity of the obtained preferable mutant protein is higher than 99%, and it can be seen that the preferable mutant protein can be applied to the field of industrial production. The luciferase Fluc mutant protein provided by the invention has higher enzyme activity, better thermal stability and higher yield, widens the application conditions of luciferase Fluc, has higher practical application value, and is more suitable for large-scale production and industrial use.

The invention discloses application of a novel methylation marker in differential diagnosis of benign and malignant tumors of mammary glands, and particularly discovers that ANO1 methylation can be used as an accompanying diagnosis marker of an HR < + > / HER2 <-> / PIK3CA mutant subgroup for the first time, an epigenetic basis is provided for precise medication of Alpelisib, and the prediction limitation of a single mutation marker is avoided. According to the present invention, the discrimination efficiency of the methylation level of the specific CpG site of the ANO1 gene in a variety of clinical scenes is verified; the marker is proved to be capable of systematically assisting identification of benign and malignant tumors of mammary glands, typing and distinguishing of different pathological subtypes of breast cancer (such as intraductal carcinoma, invasive ductal carcinoma, invasive lobular carcinoma and the like) and molecular layering guidance (such as PIK3CA mutation state identification in HR < + > / HER2-subtype breast cancer). The invention has important scientific significance and clinical application prospect for identifying breast benign and malignant tumors and evaluating treatment schemes.

The invention discloses a bacillus subtilis alcohol dehydrogenase mutant with improved specific activity and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. Amino acids at 49th, 266th and 292th sites of (2, 3)-butanediol dehydrogenase (BsBDHA) of bacillus subtilis are subjected to single mutation and combined mutation to obtain a mutant of which the specific activity and the catalytic efficiency for catalyzing (R)-1-phenyl-1, 2-glycol are remarkably improved, the specific activity reaches 2.44 U / mg or above, and the catalytic efficiency reaches 0.96 mM <-1 > s <-1 > or above; compared with BsBDHAI49L / V266L in the prior art, the (2, 3)-butanediol dehydrogenase mutant disclosed by the invention is improved by 23% or more; the catalytic efficiency of the mutant BsBDHAI49L / V266L is improved by 2.46 times or more compared with that of the mutant BsBDHAI49L / V266L in the prior art; and the conversion rate of 2-hydroxyacetophenone in 12 hours reaches 95.3%.

The application belongs to the technical field of genetic engineering and enzyme engineering, and discloses a nitrile hydratase mutant and application thereof, wherein the mutant is obtained by single mutation or multiple mutations of positions 22, 42, 63, 73, 105, 131, 145 and 163 of the amino acid sequence shown in SEQ ID NO. 1. The nitrile hydratase mutant of the application can be expressed in a high-density fermentation host bacterium in a heterologous manner, can improve the substrate catalytic concentration to 1000 g / L, is applied to catalytic synthesis of 4-acetyl-2-methylbenzamide, is green, environmentally friendly, non-polluting, has high catalytic efficiency and high product conversion rate.

This application relates to a drug lead compound with ALK G1202R / L1196M double mutation inhibitory activity, a virtual screening method, and its application. The method includes: obtaining resolved single-mutant complex structures from a database; constructing screening models based on the complex structures and performing standardized preprocessing on the crystal structures in each model; optimizing the structure and preparing the conformation of the compound library to be screened; determining the docking method, screening the optimal screening model, clustering the optimal screening model, docking it with the compound library to be screened, and obtaining drug lead compounds with ALK G1202R / L1196M double mutation inhibitory activity through drug-like property filtering, ligand structure clustering, and ADMET property prediction. The technical solution of this application can accurately screen ALK G1202R / L1196M double mutation inhibitory lead compounds, shortening the research and development cycle, reducing costs, and has clinical translational value.

We describe novel Staphylococcal Protein A ligands that enable milder elution pH for use in affinity chromatography. The change in elution pH is the result of point mutations to the protein sequence. Two novel ligands are investigated in this study. The first, designated Z(H18S)4, represents a histidine to serine substitution single mutation. The second, designated Z(H18S, N28A)4, is a double mutant comprising histidine to serine and asparagine to alanine mutations. Both are compared against the unmutated sequence, designated Z4, which is currently utilized in a commercially available Protein A stationary phase for the purification of molecules containing Fc domains. The ligands are coupled to a chromatography support matrix and tested against a panel of antibodies and an Fc fusion protein for elution pH, dynamic binding capacity, step-wise elution, and capture from clarified culture media. Results demonstrate that the novel ligands result in milder elution pH, on average >0.5 pH units, when tested in a pH gradient. For step-wise elution at pH 4.0, the Z(H18S, N28A)4 ligand showed on average a greater than 30% increase in yield compared to Z4. Importantly, for the antibodies tested the mutations did not result in a decrease in dynamic binding capacity or other desirable attributes such as selectivity. A potential application of the novel ligands is shown with a pH sensitive molecule prone to aggregation under acidic conditions.

The invention discloses a bile salt hydrolase BSH mutant protein and application thereof. The mutant protein is obtained by single mutation of an amino acid sequence of wild type bile salt hydrolase as shown in SEQ ID NO.1, and a single mutation site at least comprises any one of H91R, T122G, T122R, M161L, D166S, D167S, C189K, H215F, H215Y, K265P, G279A, A301R, Y305F or S314Q. Wild type bile salt hydrolase BSH from Bifidobacterium longum is used as a research object, an amino acid sequence of the wild type bile salt hydrolase BSH is designed and modified to obtain a series of mutant proteins with improved thermal stability and catalytic activity, and the thermal stability of the mutant proteins is 1.8-3.9 DEG C higher than that of the wild type bile salt hydrolase BSH. And moreover, the catalytic activity is improved by 1.1-4.3 times compared with that of a wild type, and the catalytic activity is obviously improved on the basis of keeping the stability of a protein structure. And a key mutation target set which can be directly used is provided for subsequent protein engineering modification of the bile salt hydrolase BSH.

This invention belongs to the field of bioenzyme technology, specifically relating to a 7α-HSDH enzyme mutant and its applications. The 7α-HSDH enzyme mutant is obtained by single or combined mutations of the protein sequence of the wild-type 7α-HSDH enzyme as shown in SEQ ID NO:1; the single mutations include V50A, I56R, A126I, A129C, or A203P; the combined mutations include V50A / I56R, V50A / A203P, V50A / A126I, V50A / A129C, V50A / I56R / A126I, V50A / I56R / A203P, V50A / I56R / A129C, or V50A / I56R / A203P / A126I. The 7α-HSDH enzyme mutant of this invention exhibits significantly improved activity and stability, and can convert CDCA to UDCA, showing broad application prospects.