152 results about "Glycosyl donor" patented technology

The current invention provides polypeptides and polypeptide conjugates that include an exogenous N-linked glycosylation sequence. The N-linked glycosylation sequence is preferably a substrate for an oligosaccharyltransferase (e.g., bacterial PgIB), which can catalyze the transfer of a glycosyl moiety from a lipid-bound glycosyl donor molecule (e.g., a lipid-pyrophosphate-linked glycosyl moiety) to an asparagine (N) residue of the glycosylation sequence. In one example, the asparagine residue is part of an exogenous N-linked glycosylation sequence of the invention. The invention further provides methods of making the polypeptide conjugates that include contacting a polypeptide having an N-linked glycosylation sequence of the invention and a lipid-pyrophosphate-linked glycosyl moiety (or phospholipid-linked glycosyl moiety) in the presence of an oligosaccharyltransferase under conditions sufficient for the enzyme to transfer the glycosyl moiety to an asparagine residue of the N-linked glycosylation sequence. Exemplary glycosyl moieties that can be conjugated to the glycosylation sequence include GlcNAc, GlcNH, bacillosamine, 6-hydroybacillosamine, GalNAc, GaINH, GlcNAc-GlcNAc, GlcNAc-GlcNH, GlcNAc-Gal, GlcNAc-GlcNAc-Gal-Sia, GlcNAc-Gal-Sia, GlcNAc-GlcNAc-Man, and GlcNAc-GlcNAc-Man(Man)2. The transferred glycosyl moiety is optionally modified with a modifying group, such as a polymer (e.g., PEG). In one example, the modified glycosyl moiety is a GIcNAc or a sialic acid moiety.

Provided are methods for the preparation of glycosylation products, including those represented by formula I:

Sugar-O—R′  I

• • comprising the step of combining R′—OH, a glycosyl sulfide glycosyl donor (“thioglycoside donor”), a hypervalent iodine alkyl-transfer activating reagent, and a base. In an embodiment, the hypervalent iodine alkyl-transfer activating reagent is (phenyl(trifluoroethyl)iodonium triflimide).

The invention provides a method for improving functional property of rice protein by protein-polyoses graft coupling technology, which belongs to the technical field of hydrophobicity vegetable protein modification. The method is to carry out glycosylation modification on a side product of producing rice starch and rice syrup, namely the rice protein as a raw material by different glycosyl donors through a wet method. Dissolvability, emulsibility, foamability and other functional properties of the rice protein are effectively improved and sufficiently utilized through the graft reaction of the rice protein and sugar. Compared with methods chemically modifying the vegetable protein, such as alkylation, phosphorylation, deamidization and the like, the method is safe and environment friendly, a rice protein-polyoses graft coupling product can be used as a natural macromolecular emulsifying agent or a protein nutritional reinforcing agent to sufficiently reflect and exert low sensitivity and high nutritive characteristic of the rice protein, thereby effectively expanding the application field of the rice protein. Meanwhile, the method provides theoretical reference for deep research and development of the hydrophobicity vegetable protein.

The present invention relates to a group of glycosyl transferase and applications thereof, and specifically provides applications of the glycosyl transferase gGT29-7 and a derived polypeptide thereof in terpene compound glycosylation catalysis and new saponin synthesis, wherein the glycosyl transferase can specifically and efficiently transfer the glycosyl from a glycosyl donor to the first glycosyl on C-3 site and/or C-6 site of a tetracyclic triterpene compound so as to extend the sugar chain. The glycosyl transferase of the present invention can further be used for constructing artificial ly-synthesized rare ginsenosides and a variety of new ginsenosides and derivatives thereof.

The invention relates to a group of glycosyl transferases and applications of the glycosyl transferases. Particularly, the invention provides applications of glycosyl transferases gGT25, gGT13, gGT30, gGT25-1, gGT25-3, gGT25-5, gGT29, gGT29-3, gGT29-4, gGT29-5, gGT29-6, gGT29-7, 3GT1, 3GT2, 3GT3 and 3GT4, and derived peptides of the glycosyl transferases in glycosylation catalysis and new saponin synthesis of terpenoids. The glycosyl transferases can specifically and efficiently catalyze hydroxyl glycosylation of C-20 bit and/or C-6 bit and/or C-3 of a tetracyclic triterpenoid compound substrate, and/or transfers the glycosyl groups from glycosyl donors to the first glycosyl groups of the C-3 bit and C-6 bit of the tetracyclic triterpenoid compound to extend a carbohydrate chain. The glycosyl transferases disclosed by the invention can also be applied to building synthetic rare ginsenosides and a plurality of novel ginsenosides and derivatives of the ginsenosides.

The invention discloses 1, 2-anti form glucoside derivative of oxazole compound. During the preparation, 4-(3, 4, 5-trimethoxy phenyl)-5-(3-hydroxyl-4-methoxy phenyl oxazole is used as the acceptor of glycosyl, bromo sugar of D-glucose, D-galactose, L-arabinose, D-xylose, L-fucose or lactose with full acetyl protection, or trichlorine imine ester of L-rhamnose or D-mannose with full acetyl protection is adopted as the donator of glycosyl, glycosylation is performed with the catalysis of alkali and tetrabutyl ammonium bromide or lewis acid, to obtain glucoside derivative containing acetyl protecting group; and then the acetyl protecting group is removed by utilizing methyl alcohol and sodium methoxide, to obtain 1,2 anti-form glucoside derivative of oxazole compound. The preparation method is simple, highly effective and universal, the route is short, the water-solubility of the product is good, the bioavailability is high, thereby the glucoside derivative can be applied as antineoplastic medicine inhibiting microtubule assembly and selectively targeting tumor blood vessels.

The present invention provides a method for preparing a glycoside of a flavonoid compound, which comprises the step of treating flavonoid and a glycosyl donor with an enzymatic agent having glycosylation activity and being derived from the genus Trichoderma (preferably Trichoderma viride or Trichoderma reesei). Such a flavonoid compound includes a catechin compound or a methylated derivative thereof, and the glycosyl donor includes a carbohydrate containing a maltotriose residue (preferably maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, dextrin, γ-cyclodextrin or soluble starch). Glycosides obtained by the present invention have higher water solubility, improved taste, and increased stability. The present invention also provides novel glycosides of catechin compounds, which are obtained by the method of the present invention.

The invention discloses a method for improving the water solubility of dye lignin by utilizing cyclodextrin glucosyltransferase transglycosylation reaction, and belongs to the field of enzyme engineering. A gene (GenBank accession no.JX412224), which is derived from P.macerans strain JFB05-01 (CCTCC NO:M208063) and optimized by a cyclodextrin glucosyltransferase codon, is connected to pET20b(+) plasmid and is transferred to an E.coli BL21(DE3) cell to establish gene engineering bacteria for producing the cyclodextrin glucosyltransferase. The pure cyclodextrin glucosyltransferase, which is obtained through fermentation enzyme production and purification, is applied to catalytic transglycosylation reaction, alpha-cyclodextrin serves as a glycosyl donor, the dye lignin serves as glycosyl receptor, and at least four dye lignin glycosylated derivatives (Glc)n-genistein (n is equal to 1, 2, 3 and 4) are finally obtained, wherein Glc-genistein and (Glc)2-genistein are main products, in particular the water solubility of the (Glc)2-genistein is greatly improved compared with that of the dye lignin.

The invention discloses a genetically engineered bacterium for catalyzing glucose glycosylation of a flavonoids compound and an application thereof. Specifically, a Beta-glycoside hydrolases gene of a chassis biological cell is removed according to the genetically engineered bacterium, and endogenous degradation of a glycosylation product is reduced; the high-expression regulation is performed to two key enzymes of glucophosphomutase (PGM) and uridine diphosphate glucose pyrophosphorylase (UGP1) of the chassis biological cell for participating in the synthesis of glycosylated-donor uridine diphosphate glucose (UDP-Glu), and a UDP-glucuronosyltransferase gene for catalyzing the flavonoid aglycones glucose glycosylation is combined so as to obtain a genetically engineered strain for effectively catalyzing the glucose glycosylation of the flavonoids compound, and finally scutellarein is used as a catalytic substrate for fermenting for 54 h in 10 L of a fermentation tank, wherein the yield of scutellarein-7-O-glucose of the glycosylation product is up to 1200 mg/L. The used strategy provides a technology reference for biologically-catalyzing chemical micromolecular glycosylation by using a microbiological method.

The invention discloses a glycosyltransferase mutant and a method for catalytically synthesizing rebaudioside A by using the same. The amino acid sequence of original glycosyltransferase is SEQ ID NO:1. According to the experiment, Asn196, Asn78, Asn400, Asn69, Gln72, Gln198, Gln178, Gln160 and Thr319 are screened on the basis of prediction obtained by starting from surface residues Q, N and T ofUGT76G1 and combining data analysis results of solvent accessible surface area, B-factor and the like, and finally, a UGT-SuSy system of the better mutant strain 76G1_Q72E-N196D-T319E is screened outby virtue of single-point or multi-point iterative mutation in Thr81, so as to realize efficient catalytic synthesis of the rebaudioside A. The glycosyltransferase UGT76G1 or a mutant gene thereof isconnected with a sucrose synthase gene to obtain a recombinant plasmid, and a double-enzyme co-expressed recombinant strain is constructed. By constructing a double-enzyme system, regeneration of in-vivo UDPG is realized, the problem of sources of expensive glycosyl donors is effectively solved, the cost is reduced, and application of biotechnology industry is promoted. The mutant is simple to prepare, the rebaudioside A is synthesized through efficient catalysis in a short time, and the biological enzyme catalysis method is green, environmentally friendly and pollution-free and is more suitable for current green industrial processing and production.

The invention relates to glycosyltransferases, mutants and application thereof. Specifically, the invention provides an in vitro glycosylation method provided comprisescomprising the following steps:transferring glycosyl groups of a glycosyl donor to C-3 hydroxy groups of tetracyclic triterpenoids in the presence of glycosyltransferase to form glycosylated tetracyclic triterpenoids, wherein the glycosyltransferase is glycosyltransferase or derivated polypeptide derivatives thereof shown as SEQ ID NO.4, or glycosyltransferase or derivated polypeptide derivatives thereof shown as SEQ ID NO.21.

The invention discloses glycosylated and ultrasonic compound modified high-gel soy protein powder as well as a preparation method and application thereof. The preparation method of the glycosylated and ultrasonic compound modified high-gel soy protein powder comprises the following steps: (1), adding distilled water into protein powder and a glycosylated donor to obtain a mixture, wherein the massratio of the protein powder to the glycosylated donor is (1 to 1) to (3 to 1), uniformly stirring the mixture to prevent generation of a large quantity of bubbles so as to prepare a mixed solution with the concentration being 1 percent to 6 percent; (2), deeply inserting an ultrasonic probe into the mixed solution, controlling ultrasound output power to be 200W to 500W, controlling ultrasound treatment time to be 15 minutes to 240 minutes and setting a temperature to be 60 plus/minus 2 DEG C; and (3), taking out the reacted mixed solution after reaction is finished, and instantly putting themixed solution in ice water bath and then performing cooling to a room temperature, and performing lyophilization to obtain a sample. According to the preparation method disclosed by the invention, glycosylation is carried out by adopting a conventional moist heat method, the process is relatively simple, and any chemical reagents are not added, and therefore, green and no pollution can be achieved; the treatment is carried out at the same time by adopting an ultrasonic way, the glycosylation reaction time is shortened, a reaction process is easily controlled, and soy protein products subjected to compound modification has better gelation properties.

The invention discloses cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and a preparation method thereof, and belongs to the field of genetic engineering and enzyme engineering. 195-bit tyrosine (Tyr) of CGTase of P.macerans strain JFB05-01 (CCTCC NO:M208063) is replaced by serine (Ser), 260-bit tyrosine (Tyr) is replaced by arginine (Arg), and 265-bit glutamine (Gln) is replaced by lysine (Lys), so that the AA-2G yield is respectively improved by 23 percent, 44 percent and 40 percent. According to combined mutation of the mutant strains, double mutants Y195S/Y260R, Y195S/Q265K and Y260R/Q265K and A three-point mutant Y195S/Y260R/Q265K are obtained. A Glycosyl donor is produced by the maltodextrin, so that the AA-2G yield is respectively improved by 57 percent, 49 percent, 55 percent and 59 percent; and compared with the mild type CGTase, the mutant strains facilitate the production of the AA-2G for glycosyl donors by using maltodextrin.

The invention provides a preparation method of glycosylated hesperetin. The preparation method comprises the specific steps that raw materials and a glycosyl donor are put into a reaction buffer solution; after the materials and the solution are mixed evenly, glycosidase and glycosyl transferase are added in a reaction system, wherein the raw materials are hesperidin, hesperetin, an extract or a conversion product of neohesperidin and a derivative thereof. According to the prepared glycosylated hesperetin, 1-9 glycosyl groups are added on a flavonoid parent, so that the solubility of the hesperetin is greatly improved, the problem of poor solubility of the hesperetin is solved, the problems about decomposition and absorption of the hesperetin in the human body are also solved, and the bioavailability of the hesperetin in citrus is greatly improved.

The present invention relates to the new process of synthesizing furasteroid saponin and its derivative chemically with C27 helical sterane type sapogenin and various kinds of saccharide as material. The synthesis process includes the following steps: 1. synthesizing completely protected C27 helical steroid saponin through the reaction of C27 helical sterane type sapogenin and acyl radical, alkyl radical or silicon radical protected glycosyl donor; 2. reaction of completely protected helical steroid saponin and oxidant to obtain oxidized product; 3. reaction of oxidized helical steroid saponin and oxyphilic reagent to produce place 26 halogenated saponin derivative; 4. nucleophilic substitution reaction of nucleophilic reagent and the halogenated product to introduce XR3 radical; 5. reducing the place 16 carbonyl radical in saponin selectively with reductant; and 6. eliminating all the protecting radicals to obtain furasteroid saponin and its analog.

The invention discloses a cyclodextrin glycosyl transferase with improved maltodextrin substrate specificity and a preparation method thereof and belongs to the fields of genetic engineering and enzyme engineering. According to the invention, for CGTase derived from peanibacillus macerans, lysine at a 47th site, tyrosine at a 89th site, asparaginate at a 94th site and aspartic acid at a 196th site are respectively mutated into leucine K47L, phenylalanine Y89F, praline N94P and tyrosine D196Y, so that AA-2G yields are respectively increased by 30.7%, 10.9%, 10.0% and 31.7%. Complex mutation is carried out on the mutant strains to obtain double mutants namely K47L/Y89F, K47L/N94P, K47L/D196Y, Y89F/N94P, Y89F/D196Y and N94P/D196Y, three-point mutants namely K47L/Y89F/N94P, K47L/Y89F/D196Y, K47L/N94P/D196Y and Y89F/N94P/D196Y and a four-point mutant namely K47L/Y89F/N94P/D196Y. Yields of AA-2G produced by the mutants while maltodextrin is utilized as a glycosyl donor are respectively increased by 42.6%, 11.0%, 37.6%, 43.6%, 43.6%, 41.6%, 44.5%, 50.5%, 20.8%, 35.6% and 64.4%. Compared with the wild type CGTase, the mutants is more beneficial to production of the AA-2G while the maltodextrin is utilized as the glycosyl donor.

The invention provides flavones-6-hydroxylase and application thereof to scutellarin synthesis and particularly provides flavones-6-hydroxylase which is capable of catalyzing apigenin 6-coordinate hydroxylation reaction to obtain scutellarein. In addition, the invention provides a method for preparing the scutellarein into scutellarin by adoption of glycosylated transferases and glycosyl donors.

The invention provides a convergent stevioside preparing method. The method comprises the following steps: a stevioside receptor, base and a phase transfer catalyst are dissolved in a solvent, and arereacted at room temperature for 5 to 60 minutes, a glycosyl donor 2 is added, a reaction is carried out at 40 to 60 DEG C for 10 to 18 hours, a glycosidation product is obtained with a yield being 80to 85%, and a protecting group is removed to obtain high-purity compounds Red A, Red D and Red M. The method provides a succinctly implementable strategy to facilitate the large acquisition of stevioside, especially stevioside with low content.

The invention discloses a method for improving beta-glucosidic bond stereoselectivity through a bis(trifluoromethane sulfonimide) reagent activation glycosylation reaction. According to the method, under the action of a catalytic amount of bis(trifluoromethane sulfonimide) reagent, a glycosyl donor with a leaving group being the trichloroacetic imidic acid ester group or acetylenic acid ester group is subjected to coupling with different glycosyl receptors, the using amount of the bis(trifluoromethane sulfonimide) reagent is small, the reaction condition is mild, the product yield is high, and a beta-glycosylation product can be obtained in a high-stereoselectivity mode.

The invention discloses maltose substrate specificity improved cyclodextrin glycosyltransferase and a preparation method thereof, and belongs to the field of genetic engineering and enzyme engineering. The 47th lysine (Lys) of CGTase of P.macerans strain HFB05-01 (CTCC NO:M208063) is respectively replaced by phenylalanine (Phe), tyrosine (Tyr) and proline (Pro), so that the output of AA-2G is respectively improved by 17.1%, 22.1% and 32.9%. Compared with the wild CGTase, the mutant strains are more conductive to producing AA-2G for glycosyl donors by utilizing maltose, so that important industrial application prospect is provided.

The invention discloses a N glycosyl transferase AaNGT. An amino acid sequence thereof is shown as SEQ ID NO.1. The invention also discloses an application of the N glycosyl transferase AaNGT in polypeptide galactosylated modification. AaNGT is capable of supplying a simple and convenient method for the forming of glycopeptide; the capacity of AaNGT for utilizing UDP-Glc is obviously higher than that of the reported N glycosyl transferase ApNGT which is sourced from Actinobacillus pleuropneumoniae; the AaNGT also can transfer the special glycosyl donor UDP-GlcNH2 to the polypeptide containing N glycosylation sites, and then the other glycosyl transferases can be utilized to convert the GlcNH2 into the GlcNAc so as to form natural N glycan linking, so that a new method is supplied for the development of the glycoprotein vaccine. The invention is beneficial to the AaNGT becoming a protein modified tool enzyme.

The invention provides a method of industrial quick production and preparation of a glucosyl stevioside mixture. The method comprises the following steps of: step I, performing enzymatic reaction: dissolving oxidized starch and stevioside, adding CGT (cyclodextrin glycosyltransferase) for the enzymatic reaction at 40-55 DEG C for 7-12h, and performing enzyme deactivation to finish the reaction, step II, performing decoloration ad deodorization, and step III, performing filtration, concentration and drying to form a product. The method takes the oxidized starch as a glycosyl donor; technical steps are reduced; a transglycosylation effect is improved; and the method is simple in technology and easy to operate, reduces requirements of industrialization on equipment and energy consumption at the same time, shortens working hours, lowers the cost and increases unit production capacity.