2241 results about "Transferase" patented technology

The invention discloses a glycosyl transferase mutant for synthesizing rebaudioside M and a synthesis method of rebaudioside M, and belongs to the technical field of gene engineering. The glycosyl transferase mutant for synthesizing the rebaudioside M is a glycosyl transferase UGT76G4 mutant and / or a glycosyl transferase UGT91C1 mutant, and the glycosyl transferase mutant is a glycosyl transferase UGT76G4 mutant. The glycosyl transferase UGT76G4 mutant is obtained by mutation of an amino acid sequence as shown in SEQ ID NO: 1: 85th amino acid is mutated into R from L, and 90th amino acid is mutated into G from I; the glycosyltransferase UGT91C1 mutant is obtained by mutating an amino acid sequence shown in SEQ ID NO: 3 as follows: the 204th amino acid is mutated into A from L, the 208th amino acid is mutated into A from F, and the 380th amino acid is mutated into D from G. The mutant can realize efficient catalytic synthesis of rebaudioside M, and the yield of rebaudioside M synthesized by an enzymic method is improved.

The present disclosure provides certain piperidinylpyridinylcarbonitrile derivatives, and pharmaceutically acceptable salts thereof, that are inhibitors of Glutaminyl-peptide cyclotransferase (QPCT) and glutaminyl-peptide cyclotransferase-like protein (QPCTL), and are therefore useful for the treatment of diseases treatable by inhibition of QPCT / L. Also provided are pharmaceutical compositions containing the same, and processes for preparing said compounds.

The present invention relates to compounds used as MTA-cooperative inhibitors of protein arginine N-methyl transferase 5 (PRMT5). In particular, the present invention relates to MTA-cooperative PRMT5 inhibitors, including their pharmaceutical compositions comprising the compounds and the methods of use thereof.

According to the glycosyl transferase mutant provided by the invention, the region selectivity of tyrosol is remarkably improved from 51% of a wild type to 99% or above, and when the glycosyl transferase mutant is applied to catalytic synthesis of salidroside, a single glycosylation product salidroside can be generated; besides, the catalytic activity of the microbial glycosyl transferase mutant to tyrosol is remarkably improved, the specific enzyme activity is improved to 97 mU / mg from 14 mU / mg of a wild type, and the substrate conversion rate is higher than 99%; according to the invention, the key problems of difficult glycosyl transferase expression, low regioselectivity and low activity in the salidroside preparation process are successfully solved, and the salidroside has a very great industrial application prospect.

The invention discloses an acyltransferase mutant and application of the acyltransferase mutant in synthesis of N-acetyl-trans-4-hydroxyproline, and belongs to the technical field of enzyme engineering. The acyltransferase mutant provided by the invention contains 10th and 95th mutations on the basis of a corresponding amino acid sequence, such as a sequence as shown in SEQ ID NO.1, or contains 62nd or 64th mutations while containing the 10th and 95th mutations. Compared with an original enzyme before mutation, the acyltransferase mutant obtained by the invention has higher acyltransferase activity, and the conversion rate of trans-4-hydroxy-L-proline is greatly improved, so that the yield of N-acetyl-trans-4-hydroxy proline is further improved, and the yield of N-acetyl-trans-4-hydroxy proline is improved. After the surfactant and the metal ions are added, the yield of the N-acetyl-trans-4-hydroxyproline synthesized by the mutant D62K / D10V / D95I under the catalysis is up to 18.7 g / L.

The invention discloses a glycosyl transferase UGT76G1 mutant and a method for catalytically synthesizing rebaudioside M by using the glycosyl transferase UGT76G1 mutant, and relates to the technical field of biological catalytic synthesis. The glycosyl transferase UGT76G1 mutant is any one of the following components (A)-(C): (A) a protein obtained by performing any one or more of the following mutations on the basis of an amino acid sequence as shown in SEQ ID NO.1: the 89th amino acid is mutated into H from M; the 380th amino acid is mutated from L to M; the 411th amino acid is mutated from A to Y; (B) a protein which has 95% or 98% or more of identity with the amino acid sequence defined by (A) and has the same function as the amino acid sequence defined by (A); and (C) a fusion protein obtained by connecting a tag to the terminal of the protein defined in (A) or (B). The glycosyl transferase UGT76G1 mutant disclosed by the invention is obtained by mutating and screening glycosyl transferase UGT76G1, has higher catalytic activity, and can be used for efficiently synthesizing rebaudioside M by taking rebaudioside D and UDPG as substrates.

The invention discloses a glycosyl transferase UGTSL2 mutant. The UGTSL2 mutant is subjected to any one or more of the following mutations on the basis of an amino acid sequence as shown in SEQ ID NO.1: the 23rd amino acid is mutated from N to E; the 41st amino acid is mutated from R to P; the 91st amino acid is mutated from H to K; the 95th amino acid is mutated from K to D; the 123 amino acid is mutated from E to P; the 136th amino acid is mutated from L to F; the 151st amino acid is mutated from R to F; the 124 amino acid is mutated from H to E; the 168th amino acid is mutated from V to Y; the 198 amino acid is mutated from C to K; the 202nd amino acid is mutated from T to E; the 217th amino acid is mutated from W to K; the 225th amino acid is mutated from P to L; the 226 amino acid is mutated from F to V; the 285th amino acid is mutated from A to V; and the 333rd amino acid is mutated from I to V and the like.

The invention relates to the technical field of microbial genetic engineering, in particular to a sialyltransferase mutant with high yield of 3 '-sialyllactose as well as a construction method and application of the sialyltransferase mutant. The sialyltransferase mutant is selected from at least one of an NST protein or a mutant and a 406 NST protein or a mutant, the NST protein comprises an amino acid residue site in a sequence as shown in SEQ ID NO.1, and the 406 NST protein comprises an amino acid sequence in a sequence as shown in SEQ ID NO.2. The sialyltransferase mutant has the advantages that the sialyltransferase mutant can be used for preparing sialyltransferase, and the sialyltransferase mutant can be used for preparing sialyltransferase. The invention also provides a nucleic acid molecule containing the sialyltransferase mutant, an expression cassette, a recombinant vector, a recombinant microorganism and a construction method and application thereof, and has great significance in large-scale industrial production of 3-sialyllactose.

The invention discloses a glycosyl transferase mutant and an application of the glycosyl transferase mutant in synthesis of rebaudioside. According to the invention, single-point mutation and multi-point mutation are carried out on the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, and a mutant with improved catalytic activity is obtained. The catalytic activity, the substrate specificity and / or the substrate specificity of the glycosyl transferase mutant are / is changed, and the catalytic activity of enzyme to a specific substrate can be remarkably improved by mutation at a specific site. And carrying out induced expression and protein purification on the obtained mutation sequence to obtain the mutant enzyme. The mutant enzyme is used as a catalyst, and UDPG is used as a glycosyl donor, so that the catalytic reaction efficiency of substrates such as stevioside ST, rebaudioside A (RebA) and rebaudioside D (RebD) can be obviously improved. According to the glycosyl transferase UGT76G1 mutant constructed by the research, the catalytic activity of the glycosyl transferase UGT76G1 mutant is improved, and efficient production of rebaudioside M is realized by optimizing a reaction system.

The invention discloses N-acetylglucosamine transferase AcetRL-R349H and an application thereof, and belongs to the technical field of functional enzymes. The amino acid sequence of the N-acetylglucosamine transferase AcetRL-R349H is as shown in SEQ ID NO. 5 (sequence identifier number 5). The invention further discloses the application of the N-acetylglucosamine transferase AcetRL-R349H in the preparation of the chitosan oligosaccharide. The N-acetylglucosamine transferase AcetRL-R349H disclosed by the invention is obtained by carrying out mutation modification on the N-acetylglucosamine transferase AcetRL, and can be used for producing the chitosan oligosaccharide with the polymerization degree of 4 to 6, especially (GlcNAc) 6. The product spectrum of the N-acetylglucosamine transferase is widened, and the N-acetylglucosamine transferase can be used for preparing high-polymerization-degree chitosan oligosaccharide and has a wide application prospect.

The invention discloses an N-acetylglucosamine transferase AcetRS-R346D and an application thereof, and belongs to the technical field of functional enzymes. And the amino acid sequence of the N-acetylglucosamine transferase AcetRS-R346D is as shown in SEQ ID NO. 3. The invention further discloses the application of the N-acetylglucosamine transferase AcetRS-R346D in the preparation of the chitosan oligosaccharide. The N-acetylglucosamine transferase AcetRS-R346D disclosed by the invention is obtained by carrying out mutation modification on the N-acetylglucosamine transferase AcetRS, and can be used for producing the chitosan oligosaccharide with the polymerization degree of 4 to 7, especially (GlcNAc) 7. The product spectrum of the N-acetylglucosamine transferase is widened, and the N-acetylglucosamine transferase can be used for preparing high-polymerization-degree chitosan oligosaccharide and has a wide application prospect.

The invention relates to the technical field of biology, in particular to a beta-1, 4-galactosyl transferase mutant and a method for biologically synthesizing lactose-N-neotetraose by using the beta-1, 4-galactosyl transferase mutant. The invention discloses a protein, which is a protein HpgalT-186 or a protein HpgalT-146-186, a protein HpgalT-146 and a protein HpgalT-146. The protein HpgalT-186 is a protein obtained by mutating an amino acid residue at the 186th site of a wild type HpgalT; the protein HpgalT-146-186 is a protein obtained by carrying out mutation on an amino acid residue at the 186 site and an amino acid residue at the 146 site of wild type HpgalT. On the basis, a series of escherichia coli is constructed and optimized, the yield of the LNnT synthesized through shake flask fermentation of the finally obtained strain reaches 1.73-4.1 g / L, the highest yield in a 5L fermentation tank reaches 20-45.2 g / L, and efficient synthesis of the LNnT is achieved.

The invention belongs to the technical field of chondroitin sulfate biosynthesis, and discloses a recombinant strain for high-yield chondroitin sulfate, application of the recombinant strain and a preparation method of the chondroitin sulfate. The recombinant strain provided by the invention can be used for simultaneously expressing UDP-glucosamine isomerase, UDP-glucose dehydrogenase, chondroitin polymerase, glucosamine synthetase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, glucose-6-phosphate isomerase and glucose phosphate mutase; according to the present invention, glucose-1-phosphate uridyl transferase, ATP sulfonase, sulfotransferase, pyruvate kinase and APS kinase are adopted, and the enzyme molecules are matched with the original carbon metabolism pathway in the recombinant strain cell to jointly form the enzyme-linked pathway having the efficient CSA synthesis ability, such that the high-yield and high-purity preparation of the CSA can be achieved; the method has the advantages of simple operation, high biological safety of the obtained product, easy realization of large-scale production and the like, and has a good application prospect.

The invention discloses acyltransferase for synthesizing a polyester intermediate and application of the acyltransferase. The acyltransferase is specifically a wild-type enzyme with an amino acid sequence as shown in SEQ ID NO: 1 or an acyltransferase variant which is obtained by mutation of the wild-type enzyme and has acyl transfer reaction catalysis activity, and the acyltransferase variant is specifically obtained by replacing at least one amino acid residue in the 209th site, the 166th site and the 119th site in the amino acid sequence of the wild-type enzyme. And the protein with improved acyltransferase activity is obtained. The wild type enzyme and the acyltransferase variant provided by the invention both have acyltransferase activity. The method has an important application value.

The invention relates to methods of amplifying methylated DNA, particularly to methods using a methyl transferase which has activity on hemimethylated DNA, but which essentially lacks de novo methylation activity. The invention further relates to methyl transferases having such activity, and products comprising such methyl transferases, as well as applications of said methods and methyl transferase.

The invention provides a fucosyl transferase polypeptide, which can be applied to the synthesis of human milk oligosaccharides (HMOs), and belongs to the technical field of enzyme engineering. The fucosyltransferase polypeptide provided by the invention comprises polypeptides with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% and 100% same amino acid sequences as polypeptides shown in amino acid sequences such as SEQ ID NO: 1-25. According to the transferase polypeptide disclosed by the invention, the specificity of a polypeptide catalytic substrate is also improved while the ability of the polypeptide as shown in SEQ ID NO: 1 to catalyze and synthesize 2 '-FL is improved.

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

The invention relates to the technical field of biology, in particular to application of N-terminal truncated 8-isopentenyl transferase in production of 8-isopentene kaempferol. The invention provides the N-terminal truncated 8-isopentenyl transferase, and the N-terminal truncated 8-isopentenyl transferase can be used for carrying out isopentenylation on kaempferol to generate 8-isopentene kaempferol. Specifically, the saccharomyces cerevisiae is used as a chassis, under the condition that kaempferol is exogenously added, through overexpression of 8-isopentenyl transferase, biosynthesis of 8-isopentenyl kaempferol in the saccharomyces cerevisiae is realized, and meanwhile, a foundation is laid for subsequent biosynthesis of icaritin. The invention provides a novel reference scheme for biosynthesis of flavonoid compounds.

The invention provides recombinant yeast for producing bakuchiol as well as a construction method and application of the recombinant yeast. Wherein the recombinant yeast is Yarrowia lipolytica containing isopentenyl transferase, and the recombinant yeast is a Yarrowia lipolytica containing isopentenyl transferase. Wherein a gene for coding the isopentenyl transferase is derived from Psoralea coryllifelia Linn (Psoralea coryllifelia Linn), and the isopentenyl transferase is used for coding the isopentenyl transferase. By utilizing the recombinant yeast provided by the invention, the bakuchiol with high yield can be efficiently and quickly prepared through a biosynthesis method, and a technical support is provided for subsequent industrialization of the bakuchiol.

The invention provides an application of citrus flavonoid glycosyl transferase CitUGT73A78 in biosynthesis of flavonoid glycoside, and belongs to the technical field of gene engineering and enzyme engineering. According to the present invention, the function of the CitUGT73A78 gene is cloned and verified, and the in vitro enzyme activity test proves that the CitUGT73A78 recombinant protein has broad-spectrum catalytic activity, and can efficiently catalyze the glucosylation of a variety of flavonoids so as to generate the corresponding flavonoids 7-O-glucoside and / or flavonoids 4 '-O-glucoside. An in-vivo transient overexpression test also proves that the CitUGT73A78 gene has a promoting effect on the accumulation of the content of the citrus flavonoid 7-O-glucoside. The invention not only provides a preparation method for the production of flavonoid 4 '-O-glucoside, has the advantages of simplicity, convenience, high catalytic efficiency, low cost, greenness and safety, but also can be applied to nutritional quality improvement and germplasm innovation of citrus fruits.

The invention relates to a compound enzyme for efficiently converting high-purity rare ginsenoside CK as well as a preparation method and application of the compound enzyme, and belongs to the technical field of microorganisms. The compound enzyme disclosed by the invention comprises beta-glucosidase, fructosyl transferase, lactase, cellulase and beta-glucanase. The beta-glucosidase, the fructosyl transferase, the lactase, the cellulase and the beta-glucanase are selected and matched to obtain the compound enzyme, the ginsenoside CK can be prepared in an enzyme engineering process, and the compound enzyme has the effect of improving the conversion rate of converting the ginsenoside Rb1 into the ginsenoside CK through synergistic interaction.

The invention provides an enzyme modification method based on artificial intelligence. The enzyme modification method comprises the following steps: training an AlphaFold2 model to obtain a glycosyltransferase tertiary structure prediction model; performing multi-sequence comparison on the protein and nucleic acid sequence database and the non-redundant protein feature sequence database based on the active point location recognition model to obtain a comparison result; initializing a matrix of which the length is equal to that of a comparison result, so as to construct a PSSM matrix and screen out potential mutation sites; according to the screened potential mutation sites, constructing an initial mutation library to form a virtual screening mutation library; performing structure reconstruction on the sequence in the virtual screening mutation library based on a glycosyl transferase tertiary structure prediction model so as to predict the glycosyl transferase tertiary structure and perform molecular docking and dynamic simulation on the glycosyl transferase tertiary structure; the method is used for analyzing binding pockets of proteins and ligands and determining dynamic interaction of amino acid residues and ligands in the binding pockets, and the contact number of each amino acid residue and the ligands is used for modifying the tertiary structure of the glycosyltransferase.

The invention discloses a protein palmitoyl transferase prediction method and system based on a multi-branch deep convolutional neural network, and belongs to the technical field of bioinformatics and artificial intelligence. The method comprises the following steps: S1, obtaining a to-be-detected protein sequence; s2, inputting the protein sequence into a pre-trained iPalmT model; and S3, judging whether the target protein is palmitoyl transferase or not according to a model output result. The iPalmT model comprises a coding module, two paths of parallel convolution branches, a feature fusion module and a classification module; and after the convolution layers of each convolution branch are stacked, an SE module is arranged and is used for channel weighting and feature re-calibration. The model extracts multi-level sequence features through convolution kernels of different scales, realizes high-precision prediction through feature fusion and a residual structure, can automatically learn multi-scale features from large-scale data, realizes end-to-end palmitoyl transferase recognition, and has high accuracy and good universality.

Glycosyltransferase UGT76G1 and UGT91C1 mutants and a method for catalytic synthesis of rebaudioside A, D, and M by using same, pertaining to the technical field of biocatalytic synthesis. The glycosyltransferase UGT76G1 mutant can effectively synthesize rebaudioside A using stevioside STV as a substrate. The glycosyltransferase UGT91C1 mutant is obtained by mutation screening of wild-type glycosyltransferase UGT91C1, has higher enzyme activity and catalytic rate, and can effectively synthesize rebaudioside D using rebaudioside A as a substrate. The glycosyltransferase UGT76G1 mutant can also be obtained by mutation and subsequent screening of glycosyltransferase UGT76G1, has higher catalytic activity, and can effectively synthesize rebaudioside M using rebaudioside D and UDPG as substrates.

The invention relates to a diacylglycerol acyltransferase mutant and application thereof in synthesis of triacylglycerol from saccharomyces cerevisiae, and belongs to the technical field of enzyme engineering. Diacylglycerol acyltransferase is a rate-limiting step of a synthetic route of saccharomyces cerevisiae triacylglycerol (TAG), and at present, a research on a DGA1 mutant for efficiently synthesizing TAG is lacked, and a research on the aspect of a DGA1 catalytic mechanism is also lacked. The DGA1 mutant capable of effectively improving TAG synthesis is obtained by screening in combination with a directed evolution technology, and a result shows that 282-site mutation of diacylglycerol acyltransferase can significantly increase the accumulation amount of TAG. Meanwhile, the catalytic mechanism of the DGA1 is preliminarily explored by combining protein structure prediction, molecular docking and molecular dynamics simulation, and a foundation is laid for deeply analyzing the catalytic mechanism of the DGA1. The recombinant saccharomyces cerevisiae is also constructed based on the mutant, so that the TAG synthesis is improved, and meanwhile, the relative proportion of C18: 1 in total fatty acids is remarkably increased.

The invention relates to the technical field of microbial metabolism engineering, in particular to a construction method and application of genetically engineered bacteria for efficiently producing O-acetyl-L-homoserine. The construction method specifically comprises the following steps: (1) taking a strain Escherichia coli W3110 as a chassis, and knocking out a homoserine O-succinyl transferase coding gene metA; (2) knocking out a gamma-cystathionine synthetase encoding gene metB; (3) knocking out a cysteine lyase coding gene metC; (4) carrying out weakening expression on an O-acetyl-L-homoserine thiolase coding gene metYcm from the marine cyclobacterium; (5) overexpressing a ribulose diphosphate carboxylase coding gene rbcLSse from the synechococcus elongate; (6) overexpressing a ribulose phosphate kinase coding gene prkse from the synechococcus slender; and (7) weakening the expression of the citrate synthase coding gene gltA to obtain the genetically engineered bacterium for efficiently producing the O-acetyl-L-homoserine. The strain provided by the invention is used for producing O-acetyl-L-homoserine, and has the advantages of high yield, high yield and high sugar-acid conversion rate.

The invention discloses bacillus subtilis with high yield of surfactin, a related enzyme mutant and a preparation method of the bacillus subtilis, the bacillus subtilis is obtained by a gene modification method and can produce the surfactin with high yield, and the highest yield can reach 9.4 g / L; the invention also provides key enzyme mutants participating in surfactin synthesis in the bacillus subtilis, and the key enzyme mutants comprise a biotin carboxylase IdeHA mutant, a biotin carboxylase accBC mutant, an enoyl reductase FabI mutant, a malonyl transferase FabD mutant and a dihydrolipoyl dehydrogenase IPdU mutant. The key enzyme mutants comprise a biotin carboxylase IdeHA mutant, a biotin carboxylase accBC mutant, an enoyl reductase FabI mutant, a malonyl transferase FabD mutant and a dihydrolipoyl dehydrogenase IPdU mutant. The bacillus subtilis for high-yield surfactin and the preparation method thereof disclosed by the invention are beneficial to realizing industrial production and commercial application of the surfactin.

Provided is a glycosyltransferase UGTSL2 mutant, which is produced through any one or more selected from the group consisting of mutations based on an amino acid sequence shown in SEQ ID NO: 1. Also provided is a glycosyltransferase mutant, which is the following A1) or A2): A1) a protein produced through an amino acid residue substitution in an amino acid sequence shown in SEQ ID NO: 66, where the protein has an identity of 90% or more with and the same function as the amino acid sequence shown in SEQ ID NO: 66; and A2) a fusion protein produced by linking a tag to an N-terminus and / or a C-terminus of the protein in the A1).

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to an alpha-2, 3-sialyltransferase mutant and application thereof. The a-2, 3-sialyltransferase mutants V175N, P198A, G173D, D174Q, F196K and A315H are obtained by performing site-specific mutagenesis on six amino acids in an a-2, 3-sialyltransferase amino acid sequence coded by a gene derived from Campylobacter jejuni (OH4384), the yield of 3 '-sialyllactose is remarkably increased by single-point mutagenesis of the V175N, the yield of 3'-sialyllactose is remarkably increased by single-point mutagenesis of the V175N, the yield of 3 '-sialyllactose is remarkably increased by single-point mutagenesis of the P198A, the G173D, the D174Q, the F196K and the A315H, the optimal recombinant microbial strain 3 '-SL-09 (V175N / P198A / D174Q) is obtained through combination of multiple mutation sites, the yield can reach 5.91 g / L, the bottleneck problem of low catalytic efficiency of sialyltransferase is effectively solved, and an enzyme element with excellent performance is provided for high-efficiency and low-cost biological manufacturing of 3'-SL.

The invention provides a preparation method of antitoxin serum containing an horse F (ab ') 2 fragment, and the specific steps are as follows: a recombinant streptococcus immunoglobulin G degrading enzyme is immobilized to obtain an immobilized enzyme chromatography column, the recombinant streptococcus immunoglobulin G degrading enzyme comprises a glutathione-S-transferase tag and a histidine tag; performing primary treatment on the immune plasma to obtain a pretreated antibody solution; the pretreatment antibody solution is added into an immobilized enzyme chromatography column for digestion, after digestion is completed, ultrafiltration, purification and solution preparation are sequentially conducted through an immobilized glutathione affinity column or a nickel affinity column, an antitoxin serum stock solution is obtained, and the antitoxin serum stock solution contains F (ab ') 2 fragments and does not contain Fc fragments, impure protein and small molecule impurities. Through the process steps, the Fc fragment and impure protein are effectively removed, and only the horse F (ab ') 2 fragment is reserved, so that the immunogenicity of antitoxin serum and the risk of causing anaphylactic reaction are remarkably reduced.