89 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 invention belongs to the field of biological pharmacy and biotransformation, and particularly relates to a tagatose-4-epimerase mutant as well as a coding gene and application thereof. The tagatose-4-epimerase mutant disclosed by the invention is obtained by carrying out single mutation or combined mutation on an amino acid residue at a specific position of an amino acid sequence as shown in SEQ ID NO.1. Compared with wild type tagatose-4-epimerase, the mutant has the advantage that the catalytic activity is improved when the D-tagatose is prepared through conversion. The obtained tagatose-4-epimerase mutant has the advantages of being mild in reaction condition, green, environmentally friendly, single in product and easy to separate and purify, and the production cost is greatly reduced.

The invention provides a genome editor enDelIscB and a genome editor enDelIscB-T5E based on an IscB system, and further provides a base editor ICBE and a base editor IABE based on the enDelIscBD60A. According to the invention, a genome editor and a base editor which are more compact in size and higher in cleavage activity and insertion / deletion efficiency are realized by optimizing a single mutation site of the activity of a DelIscB protein cleavage plasmid, an optimized variant of the DelIscB protein and an optimized sgRNA variant of the DelIscB. According to the present invention, it is verified that the albinism model is efficiently constructed by using the enDelIscB editor and the enDelIscB-T5E editor through micro-injection of the mouse embryo, such that the enDelIscB editor and the enDelIscB-T5E editor have great potential in the field of genome editing.

The invention discloses a dsPETase01 plastic degrading enzyme mutant and application thereof, and relates to the technical field of bioengineering, the amino acid sequence of a wild type plastic degrading enzyme (dsPETase01) is designed and modified, 8 dsPETase01 single mutation sites or 19 combined mutation sites are provided, the thermal stability of the obtained dsPETase01 mutant is improved by about 4-21 DEG C compared with that of the wild type dsPETase01, and the dsPETase01 mutant can be used for preparing a plastic degrading enzyme. And the activity is improved by nearly 1.2-3 times. The invention also provides four preferable mutant proteins which have higher yield, activity and thermal stability compared with wild dsPETase01, have wider application conditions, are more suitable for degrading PET (Polyethylene Terephthalate) plastics, and are beneficial to large-scale production and industrial application.

The invention discloses a glucose dehydrogenase GOX-2015 mutant and application thereof, and relates to the technical field of bioengineering, the amino acid sequence of wild type glucose dehydrogenase GOX-2015 is designed and modified, that is, 18 single mutation sites and 16 combined mutation sites are provided, and the glucose dehydrogenase GOX-2015 mutant is obtained. Compared with the wild type GOX-2015, the thermal stability of the obtained GOX-2015 mutant is improved by about 2 to 13 DEG C, and the activity of the obtained GOX-2015 mutant is improved by about 1.2 to 4.5 times. The invention further provides a preferable mutant protein which has higher yield, activity and thermal stability compared with wild type GOX-2015, has wider application conditions, is more suitable for efficient production of NADPH coenzyme by a biological conversion method, and is beneficial to large-scale production and industrial application.

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 discloses an L-threonine aldolase mutant and application of the L-threonine aldolase mutant in synthesis of L-serine. The mutant is obtained by performing single mutation on the 88th amino acid of a wild type L-threonine aldolase amino acid sequence shown as SEQ ID No.2. The invention further discloses a preparation method of the L-threonine aldolase mutant. The invention provides the L-threonine aldolase mutant with better catalytic performance, aldol condensation reaction of formaldehyde and glycine can be efficiently catalyzed to prepare L-serine under the condition of not adding a tetrahydrofolic acid precursor, the yield of the L-serine is increased to 72.3% from 56.7% before mutation, the yield is remarkably increased, and the L-threonine aldolase mutant has the advantages that the L-threonine aldolase mutant has a good application prospect. Good application prospects are shown in the synthesis of the L-serine.

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 RthB protein mutant and its application in isoleucine production. The RthB protein mutant is obtained by performing single-site mutations or multi-site mutations at the 16th, 21st, 117th, and 156th positions of the amino acid sequence shown in SEQ ID NO:1. By mutating and modifying the RthB protein, the L-isoleucine fermentation yield of recombinant microorganisms expressing the mutant protein is increased, and the threonine content in the fermentation broth is decreased, thereby improving the yield and purity of isoleucine.

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 present invention relates to the field of Biotechnology, particularly to a method based on the introduction of a single mutation in the genes encoding the human IL-2 and the muteins derived thereof that results in increased secretion levels in different hosts without affecting their biological functions. In particular, these mutations are based on a non-conservative change in the amino acid located in position 35 in the primary sequence of human IL-2, preferably the substitutions are K35E, K35D and K35Q. Another object of the present invention are the expression systems used to obtain both the recombinant human IL-2 and the muteins derived thereof using the method described in this invention. The above-mentioned method is useful to improve the production efficiency of the recombinant human IL-2 and the muteins derived thereof both at laboratory and industrial scales. The proteins obtained using this method can be used for therapeutic purposes as well as in the in vitro expansion of T cells for adoptive transfer therapies.

The present invention relates to the technical field of genetic engineering and protein engineering modification, and specifically provides a thermostable acidic pectinase mutant. The acidic pectinase mutant contains a single mutation site S98E. After being treated at 80 °C for 3 minutes, the residual enzyme activity rate is increased by 54.48% compared with the wild type, achieving unexpected technical effects. The thermostability of the acidic pectinase mutant provided by the present invention is significantly enhanced and can be widely applied in fields such as feed and food processing.

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 present invention relates to the technical field of genetic engineering and protein engineering modification, and specifically provides an acidic pectinase mutant and its application. The acidic pectinase mutant contains a single mutation site Y219W. After being treated at 80 °C for 3 minutes, the residual enzyme activity rate is increased by 46.77% compared with the wild type, achieving an unexpected technical effect. The heat resistance of the acidic pectinase mutant provided by the present invention is significantly enhanced and can be widely applied in the fields of feed, food processing, etc.

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 present invention discloses a method and device for recommending mutatable sites from small-sample experimental data, comprising the following steps: obtaining small-sample experimental data, including the sequences and data of wild enzymes and mutants, and obtaining the optimal mutant sequence according to the data type of the mutants; predicting the mutant structure based on the optimal mutant sequence; predicting the substrate-binding pocket based on the mutant structure; predicting single mutation sites based on the protein structure model, and selecting residues with a distance from the center of the substrate-binding pocket less than a first threshold as the recommended single mutation sites; selecting sites from the small-sample experimental data, mutating each site in the site set into 19 other amino acids, and pairwise combining them to construct a double mutation set, predicting the mutation results, and obtaining the recommended double mutation sites according to the sorting results; selecting sites from the small-sample experimental data, obtaining the coordinates of the sites in the variant structure for clustering analysis, and selecting 1 site from each cluster to combine with other clusters to construct multi-mutations; predicting the mutation results of the multi-mutations, and obtaining the recommended multi-mutation sites according to the sorting results; the method of the present invention operates effectively under the condition of small-sample data; through the powerful generalization ability of the large language model, the structural and functional information in biomolecules can be captured, so as to effectively encode the sequence of the enzyme for more accurate inference.

The invention discloses construction and application of a novel nitrilase with excellent amide generation capability and a mutant thereof. The amino acid sequence of the nitrilase disclosed by the invention is as shown in SEQ ID NO: 1, and a mutant of the nitrilase is obtained by performing single mutation on the 166th site, the 168th site and the 191th site of the sequence as shown in SEQ ID NO: 1. The nitrilase provided by the invention has excellent amide generation capability, and has important industrial application potential for green and efficient preparation of amide compounds.

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.

The invention discloses a sucrose isomerase mutant with improved thermal stability and a construction method thereof, and belongs to the technical field of gene engineering and enzyme engineering. According to the sucrose isomerase mutant, the thermal stability and the expression quantity are simultaneously improved, the mutant is used for preparing isomaltulose, the half-life period of the single mutant V447E at the temperature of 40 DEG C is prolonged by 1.38 times, the optimum temperature is increased to 35 DEG C from 30 DEG C, and the expression quantity is increased to 33.06 U * mL <-1 > from 18.89 U * mL <-1 >; other mutations are superposed on the basis of the mutant V447E, and the thermal stability of the constructed double mutant V447E / D232P is further improved. Compared with a wild type, the V447E / D232P has the advantages that the optimal temperature is increased by 5 DEG C, the half-life period at 40 DEG C is prolonged by 1.52 times, and the expression quantity is increased from 18.89 U.mL <-1 > to 30.73 U.mL <-1 >.

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%.