66 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 tyrosine ammonia lyase mutant and application thereof in production of p-coumaric acid. The tyrosine ammonia lyase mutant is obtained by mutating wild type tyrosine ammonia lyase with an amino acid sequence as shown in SEQ ID No.1, and a mutation site is selected from at least one of Q108S, Y340Q and P375H. The catalytic activity center of wild type tyrosine ammonia lyase is analyzed, and site-directed mutagenesis is carried out on five catalytic activity center sites, so that the mutant shows different enzyme catalytic activity when the mutation sites are different, the mutation sites are the same but the mutation targets are different; q108S, Y340Q and P375H single mutations can significantly improve the enzyme catalytic activity of the tyrosine ammonia lyase mutant, and the tyrosine ammonia lyase mutant having any two mutation sites, especially having three mutation sites, also has better enzyme catalytic activity than the wild type, and has great application potential.

The invention discloses a lipase mutant, an engineering bacterium and application of the lipase mutant in preparation of (2S, 3R)-p-methylsulfonylphenyl serine ethyl ester. The lipase mutant is obtained by performing single mutation or combined mutation on 67th, 115th or 400th amino acid of an amino acid sequence as shown in SEQ ID No.1. The invention further discloses a preparation method of the lipase mutant, the engineering bacterium and the application of the lipase mutant in preparation of (2S, 3R)-p-methylsulfonylphenyl serine ethyl ester. The lipase mutant provided by the invention has the advantages that the enzyme activity is obviously improved, the selectivity is not reduced, the reaction conversion rate of the lipase mutant serving as a catalyst reaches 50%, the required reaction time is obviously shortened by 1-7 hours, and the lipase mutant has more excellent catalytic activity. Under the optimal system, the conversion rate after 1000 mM of a substrate is split for 4 h reaches 50.5%, e.e.gt; therefore, a certain basis is provided for industrial preparation of optically pure (2S, 3R)-p-methylsulfonylphenyl serine ethyl ester.

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 present application relates to the technical field of bioengineering, and particularly relates to a polyphosphate kinase mutant with high catalytic activity and application thereof in synthesis of glutathione. The present application provides a polyphosphate kinase mutant with high catalytic activity, which is characterized in that the polyphosphate kinase mutant is mutated from an amino acid sequence shown in SEQ ID NO: 2 into one of the following: (1) single mutation is made to the 234th amino acid in the amino acid sequence shown in SEQ ID NO: 2; (2) double mutations are made to the 234th and 166th amino acids in the amino acid sequence shown in SEQ ID NO: 2; wherein a nucleotide sequence of a coding gene coding the amino acid sequence shown in SEQ ID NO: 2 is as shown in SEQ ID NO: 1. ch PPK-N234A and double-site mutation polyphosphate kinase mutant ch PPK-N234A / K166D has high activity, and the enzyme activity is increased by 4.19 times and 6.12 times, respectively, compared with wild-type polyphosphate kinase.

The present invention discloses an L-aspartate aminotransferase mutant and its application. The L-aspartate aminotransferase mutant is obtained by subjecting the amino acid at position 104 of the amino acid sequence of L-aspartate aminotransferase to a single mutation. The L-aspartate aminotransferase mutant is used to synthesize L-phenylglycine derivatives. The L-aspartate aminotransferase mutant is derived from Escherichia coli. After homologous sequence analysis and molecular docking simulation, combined with site-directed mutagenesis verification, a single-point mutation is performed on the amino acid at position 104 to obtain a T104S mutant. The L-aspartate aminotransferase mutant of the present invention can efficiently catalyze the production of L-phenylglycine derivatives. Compared with the wild type, the specific activity of the T104S mutant towards o-chlorophenoxyacetic acid is 3.2 times that of the wild type.

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.

This invention discloses a tyrosine phenol lyase mutant and its application in synthesizing tyrosine derivatives. The mutant is obtained by single or multiple mutations at position 18 or 417 of the amino acid sequence shown in SEQ ID NO.2. Compared with the wild type, the tyrosine phenol lyase mutant provided by this invention exhibits superior enzyme activity and catalytic performance. The TPL mutant synthesizes levodopa at a cumulative concentration of over 160 g / L, L-tyrosine at a cumulative concentration of over 80 g / L, 3,4,5-TOPA at a cumulative concentration of over 16.4 g / L, 3-fluoro-L-tyrosine at a cumulative concentration of over 33.1 g / L, and 2-fluoro-L-tyrosine at a cumulative concentration of over 20.5 g / L, with an optical purity greater than 99.9%.

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%.

Provided in the present invention are a mutant of Burkholderia cepacia lipase, and a recombinant expression vector thereof and the use thereof. The mutant comprises P131S, T132A, T251A, T251S, P131A, P131Q, T132G, Q88S or P113A single mutation, Q88S and P113A double mutation, and combinations of multi-site mutations including combination 1 of Q88S, P113A, T132G and L287S, combination 2 of Q88S, P113A, P131S and L287S, and combination 3 of Q88S, P113A, P131S and T251A.

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 application discloses a cytochrome P450 enzyme mutant derived from Streptomyces and application of the cytochrome P450 enzyme mutant in synthesis of ursodeoxycholic acid, wherein the cytochrome P450 enzyme mutant is obtained by single mutation or multiple mutations of positions 181, 192, 249 or 288 of the amino acid sequence shown in SEQ ID NO. 2. The cytochrome P450 enzyme mutant has higher activity and regional selectivity, and can use cheap lithocholic acid as a substrate, and hydroxylate 7beta of the substrate by using the high-efficiency cytochrome P450 enzyme to synthesize ursodeoxycholic acid in one step, thereby greatly reducing the industrial production cost, and having a good application prospect in industrial production of ursodeoxycholic acid.

The invention belongs to the technical field of biological enzyme engineering, and particularly relates to an L-lactic dehydrogenase mutant and application thereof. The L-lactic dehydrogenase mutant provided by the invention is obtained by performing single mutation, pairwise combined mutation, three combined mutation or four combined mutation on the 105th site, the 122th site and the 85th site on an amino acid sequence shown by wild type L-lactic dehydrogenase. When the L-lactic dehydrogenase mutant provided by the invention is used for producing 2-hydroxy-4-methylthio-butyric acid, compared with a wild enzyme, the enzyme activity of the mutant is greatly improved, the catalytic performance is improved by about 9 times, the enzyme dosage in industrial production can be remarkably reduced, the space time yield of a product is improved, the production cost is reduced, and the L-lactic dehydrogenase mutant is suitable for large-scale popularization and application.

The invention discloses computer equipment, a sequencing data processing method and a storage medium. The method comprises the following steps: acquiring first sequencing data corresponding to cfDNA and leukocyte DNA of a target sample; detecting the first sequencing data corresponding to the cfDNA and the leukocyte DNA according to multiple mutation detection strategies to obtain a mutation detection result of the cfDNA corresponding to each mutation detection strategy and a mutation detection result of the leukocyte DNA corresponding to each mutation detection strategy; combining the multiple mutation detection results of the cfDNA to obtain a first detection result of the cfDNA; combining the plurality of mutation detection results of the leukocyte DNA to obtain a second detection result of the leukocyte DNA; and determining a target detection result of the target sample according to the first detection result, the second detection result and a preset reference sequence set. According to the method and the device, false positive or false negative results possibly caused by a single mutation detection strategy can be avoided, and the accuracy of mutation detection on the target sample is improved.

The present invention discloses a thermostable cellobiose epimerase mutant and its application. The mutant is obtained by a single mutation at position 242 of the amino acid sequence shown in SEQ ID No. 1. The present invention molecularly modifies the cellobiose epimerase (DiCE) from Dictyoglomus thermophilum to screen for mutants with improved thermostability. The resulting mutants have significantly improved lactulose conversion efficiency. Mutant A242L, in particular, can tolerate temperatures of 80°C and has a lactulose conversion rate of 64.41%, showing promising prospects for industrial application.

Methods and oligonucleotide reagents for genotyping Epstein-Barr virus are disclosed. In particular, genetic profiling is used to detect BALF2 variants in the genome of Epstein-Barr virus in an infected individual to predict the risk of an individual developing nasopharyngeal carcinoma. Primers and allele-specific probes are provided for performing nucleic acid-based diagnostic assays to determine which alleles are present at single nucleotide polymorphisms (SNPs) in the BALF2 gene of Epstein-Barr virus in biological samples from potentially infected subjects. These primers and allele-specific probes can be used for amplifying target sequences to allow rapid detection of a single mutation or multiple mutations in the BALF2 gene simultaneously in a single assay. In addition, methods are provided for identifying individuals at high risk of developing nasopharyngeal carcinoma who are in need of further screening and treatment.

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 present invention relates to the field of biological detection technology, and discloses a primer probe set, a kit, and a detection method for detecting biallelic CEBPA gene mutations. The primers are used for full-length amplification of the CEBPA gene coding region, and include an upstream primer and a downstream primer, the upstream primer having a nucleotide sequence as shown in SEQ ID No: 1, and the downstream primer having a nucleotide sequence as shown in SEQ ID No: 2; the probe hybridizes with the CEBPA gene sequence and introduces a single mutation on either side of the N-terminus of the CEBPA gene where a single base mutation occurs. The detection method provided by the present invention has high specificity and accuracy. The primers, probes, and detection method provided by the present invention can effectively detect whether two mutations in the CEBPA gene are biallelic mutations, providing strong technical support and reliable detection means for further determining the prognosis of AML patients.

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 Sortase A transpeptidase mutant and application thereof in the technical field of bioengineering, the Sortase A transpeptidase mutant is obtained through single mutation or double mutation on the basis of a protein sequence shown in SEQ ID NO.2, the protein sequence shown in SEQ ID NO.2 is a truncated mutant at the 60th-206th site made on wild type Sortase A transpeptidase shown in SEQ ID NO.1, and the Sortase A transpeptidase mutant is obtained through single mutation or double mutation on the basis of the protein sequence shown in SEQ ID NO.2. The Sortase A transpeptidase mutant provided by the invention has higher yield, activity and thermal stability, and has wider application conditions. Besides, a cysteine is introduced to the C terminal of the Sortase A transpeptidase mutant protein, and the Sortase A transpeptidase mutant is fixed on the magnetic beads by utilizing the principle of sulfydryl covalent coupling, so that the application value of the Sortase A transpeptidase mutant in protein labeling is realized.