1579 results about "Transferase" patented technology

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 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 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 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 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 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 present invention provides a fucosyltransferase polypeptide, which can be applied to the synthesis of human milk oligosaccharides (HMOs) and belongs to the field of enzyme engineering technology. The fucosyltransferase polypeptide described in the present application comprises a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, and 100% identical amino acid sequence to the polypeptide shown in SEQ ID NO:1-25. The transferase polypeptide described in the present application improves the ability of the polypeptide shown in SEQ ID NO:1 to catalyze the synthesis of 2'-FL while also improving the specificity of the polypeptide catalyzing the substrate.

The application discloses a specific palmitoyl transferase inhibitor and application thereof, and an active ingredient of the palmitoyl transferase inhibitor is 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol or a salt thereof. Current palmitoylation inhibitors, 2-bromohexadecanoic acid (2-BP), cerulenin and tunicamycin can all inhibit protein palmitoylation in cells, but all are not selective. And the existing palmitoylation inhibitors are similar to endogenous fatty acids, and inevitably affect lipid metabolism in the body, which limits the clinical application. The application discloses, for the first time, the application of 4-(3-(2-(trifluoromethyl)-9H-thioxanthene-9-ylidene)propyl)-1-piperazine ethanol as a specific palmitoyl transferase inhibitor, and the palmitoyl transferase inhibitor is better in specificity and reversible in inhibition.

The present invention provides an ATP phosphoribosyltransferase mutant and its application in histidine production. The mutant is obtained by performing point mutations at positions 84 and / or 205 on the amino acid sequence of the wild-type ATP phosphoribosyltransferase derived from Acinetobacter baumannii. The mutant, the nucleic acid molecule encoding the mutant, the expression cassette containing the nucleic acid molecule, the recombinant vector or the recombinant microorganism are used for the production of L-histidine, and the yield of L-histidine is significantly increased, laying a good foundation for the large-scale industrial production of L-histidine.

The present invention belongs to the field of biotechnology, and specifically relates to a GmSGT2 gene related to plant height and branch development, and a mutant thereof and the use thereof. A Glycine max mutant having fewer branches and decreased plant height is obtained by means of EMS mutagenesis, and a target gene thereof that is located by means of a map-based cloning technique is a GmSGT2 gene of Hedou 12. It is found through searching that the gene encodes soyasapogenol B glucuronide galactosyltransferase, and can galactosylate soyasapogenol B monoglucuronide, thereby affecting the plant height of Glycine max and reducing the branches of Glycine max. Therefore, the GmSGT2 mutant can be used for cultivating dwarf high-yield Glycine max varieties, and is of great significance in the breeding of ideal plant types of Glycine max and research on important agronomic traits of plants. The mutant has broad application prospects and great research value with regard to understanding the plant height and branch regulation mechanism of Glycine max and improving the process of Glycine max breeding; and can provide an excellent germplasm resource stock for regulating the close planting and high-yield breeding of Glycine max.

The invention relates to the field of biological engineering and technology, in particular to an enzyme for synthesizing glabridin, a biological synthesis method of glabridin and a bacterial strain. The invention discloses a series of glabridin synthesizing enzymes (phenylalanine ammonialyase, cinnamic acid-4-hydrogenase, tyrosine ammonialyase, p-coumaric acid coenzyme A ligase, chalcone synthase, chalcone reductase, chalcone isomerase, isoflavone synthase, 2-hydroxyisoflavone dehydratase, 4 '-oxymethyltransferase, 4'-hydroxymethyltransferase and 4 '-hydroxymethyltransferase) derived from glycyrrhiza glabra for the first time. ) can be used in the preparation of a compound (e.g., isoflavone 2 '-hydrogenase, isoflavone reductase, Vissisoketoreductase, isoprenyltransferase, pterocarpin reductase, oxidative cyclase, demethylase, cytochrome P450 reductase, and the like). The de novo synthesis of the glabridin in a microbial cell factory is realized for the first time, and the application prospect of the glabridin biological manufacturing industry is greatly promoted.

The invention belongs to the field of enzyme engineering, and relates to a glycosyl transferase mutant and application thereof in synthesis of salidroside. On the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, iterative saturated mutation is carried out through a molecular modification technology, and the enzymatic activity and catalytic efficiency of the modified glycosyl transferase mutant are greatly improved. The mutation at a specific site can significantly promote the glycosylation activity of a tyrosol substrate. The mutant enzyme is obtained by carrying out induced expression and protein purification on the obtained mutant strain. The substrate conversion rate of the transformed glycosyl transferase mutant for catalyzing the tyrosol glycosylation reaction can reach 95% or above, the yield reaches 55.0 g / L, and an efficient and green new way is provided for salidroside production.

The invention discloses a methyltransferase mutant, a recombinant vector, a recombinant bacterium and application of the methyltransferase mutant in production of pterostilbene, and relates to the technical field of bioengineering. According to the methyltransferase mutant provided by the invention, resveratrol generates pterostilbene under the catalytic action of methyltransferase, and compared with the unmutated methyltransferase, the mutated methyltransferase has the advantages that the affinity of enzyme and a substrate is improved, and the resveratrol is promoted to be converted into pterostilbene. The coding gene of the methyltransferase mutant is transferred into a chassis cell or a chassis strain and is fermented, so that the yield of the pterostilbene can be greatly increased, the efficient synthesis of the plant-derived antioxidant pterostilbene in the microbial chassis cell is realized, and the ever-increasing market demand of the pterostilbene can be met.

The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful for template-independent polynucleotide synthesis, as well as compositions, methods of utilizing these engineered polypeptides, and polynucleotides encoding engineered terminal deoxynucleotidyl transferases.

The invention belongs to the technical field of gene engineering, and particularly relates to an LgtA mutant and application thereof in production of human milk oligosaccharide. According to the present invention, a gene editing technology is adopted to mutate a key enzyme beta-1, 3-N-acetyl glucosamine transferase for synthesizing an intermediate product LNT II so as to obtain a mutant having at least one of R13H mutation, L24M mutation and R205C mutation on the basis of a wild type LgtA represented by SEQ ID NO.1; further, the mutant is applied to the construction process of production plasmids of the LNTII, the LNnT and the LNT, and the yield of the LNTII, the LNnT and the LNT is further increased.

The invention discloses a production method for biosynthesis of rebaudioside M. Reb A / Reb D has poor solubility at a low temperature, and the reaction efficiency can be obviously accelerated when the Reb A / Reb D is catalytically produced in a high-temperature environment, so that the whole reaction system is carried out in the high-temperature environment, and the production efficiency can be improved. Under the reaction temperature environment of 70 DEG C, the wild type first glycosyltransferase or / and the wild type second glycosyltransferase are / is basically inactivated, and the catalytic enzyme combination cannot meet the requirement of industrial biosynthesis of rebaudioside M. The wild-type catalytic enzyme is mutated, so that the wild-type catalytic enzyme can keep high catalytic activity at 70 DEG C. Therefore, the production cost of the rebaudioside M produced by the production method disclosed by the invention is obviously lower than that of the prior art, and the method is suitable for industrial popularization.

The present application relates to the technical field of biocatalysis and biosynthesis, and specifically discloses a natural blue pigment and a biosynthesis method therefor. The present application uses metabolically engineered bacteria to express indigoidine synthetase and phosphopantetheinyl transferase, to catalyze glutamine and N-acetylglutamine to biosynthesize a natural blue pigment N-acetylindigoidine, and deduces its molecular structure by means of mass spectrometry, nuclear magnetic resonance and other methods. The present application achieves catalytic synthesis of N-acetylindigoidine from glutamine and N-acetylglutamine in Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae and Streptomyces. Compared with indigoidine, N-acetylindigoidine has a maximum absorption wavelength of 584 nm, possesses better color brightness, and has more stable coloration that is not easily faded, with a very broad application range and industrial production application prospects.

The invention provides a whey protein deep hydrolysate with low sensitization and an allergy relieving effect as well as a preparation method and application of the whey protein deep hydrolysate. According to the method, the alkaline protease, the neutral protease and the glutamyltransferase are sequentially used for carrying out first enzymolysis, second enzymolysis and third enzymolysis on the whey protein raw material, and the whey protein hydrolysate which is low in molecular weight, good in taste, good in digestion and absorption performance, low in sensitization and capable of effectively relieving allergic reaction of the organism is obtained.