58 results about "Glycosynthase" patented technology

A chemoenzymatic method for the preparation of a core-fucoslyated glycoprotein or glycopeptide, including (a) providing an acceptor selected from the group consisting of a fucosylated GlcNAc-protein and fucosylated GlcNAc-peptide; and (b) reacting the acceptor with a donor substrate including an activated oligosaccharide moiety, in the presence of an endoglycosidase (ENGase) selected from Endo;F1, Endo-F2, Endo-F3, Endo-D and related glycosynthase mutants to transfer the oligosaccharide moiety to the acceptor and yield the structure defined core-fucosylated glycoprotein or glycopeptide. The donor substrate includes, in a specific implementation, a synthetic oligosaccharide oxazoline. A related method of fucosylated glycoprotein or fucosylated glycopeptide remodeling with a predetermined natural N-glycan or a tailor-made oligosaccharide moiety, and a method of remodeling an antibody to include a predetermined sugar chain to replace a heterogeneous sugar chain, are also described.

A method for generating human milk oligosaccharides (HMOs) or precursors thereof, compounds obtainable by the method, and uses and compositions involving such compounds. The method comprising the steps of a) providing at least one donor selected from the group of compounds of any of formulae 5 to 10, b) providing at least one acceptor from a group of lactose, LNT, LNnT and derivatives thereof, c) providing at least one enzyme comprising a transglycosidase activity and/or a glycosynthase activity, d) preparing a mixture of the at least one donor, at least one acceptor and at least one enzyme provided in steps a), b) and c); and e) incubating the mixture prepared according to step d).

The present invention provides for recombinant Endo-D and selected mutants that exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of glycoproteins wherein a desired sugar chain is added to a core fucosylated or nonfucosylated GlcNAc-protein acceptor by transglycosylation. Such recombinant Endo-D and selected mutants are useful for efficient glycosylation remodeling of IgG1-Fc domain.

The object of the present invention is to provide an alpha-isomaltosylglucosaccharide-forming enzyme, process of the same, cyclotetrasaccharide, and saccharide composition comprising the saccharide which are obtainable by using the enzyme; and is solved by establishing an alpha-isomaltosylglucosaccharide-forming enzyme which forms a saccharide, having a glucose polymerization degree of at least three and having both the alpha-1,6 glucosidic linkage as a linkage at the non-reducing end and the alpha-1,4 glucosidic linkage other than the linkage at the non-reducing end, by catalyzing the alpha-glucosyl-transfer from a saccharide having a glucose polymerization degree of at least two and having the alpha-1,4 glucosidic linkage as a linkage at the non-reducing end without substantially increasing the reducing power; alpha-isomaltosyl-transferring method using the enzyme; method for forming alpha-isomaltosylglucosaccharide; process for producing a cyclotetrasaccharide having the structure of cyclo{->6)-alpha-D-glucopyranosyl-(1->3)-alpha-D-glucopyranosyl-(1->6)-alpha-D-glucopyranosyl-(1->3)-alpha-D-glucopyranosyl-(1->} using both the alpha-isomaltosylglucosaccharide-forming enzyme and the alpha-isomaltosyl-transferring enzyme; and the uses of the saccharides obtainable therewith.

The present disclosure relates to novel glycosynthase enzymes for glycoprotein engineering and/or homogeneous antibody remodeling. The enzyme variants, termed EndoSd-D232M and EndoSz-D234M, contain the glycan conjugation and/or modification activity at the conserved N297 glycosylation site of Fc region of an exemplary antibody. It has been demonstrated that the glycosynthase activities of EndoSd-D232M and EndoSz-D234M can be applied to various mAbs targeting different receptors, including, but not limited to, Globo H, SSEA-4, SSEA-3 series of receptors (OBI-888; Globo H ganglioside), Herceptin (Her 2 receptor), Perjeta (Her 2 receptor) and Vectibix (EGFR receptor). It has been found that both mAb-GlcNAc and mAb-GlucNAc(F) were suitable substrates for both EndoSd-D232M and EndoSz-D234M. The ADCC assay of related products, OBI-888-G2S2 and Herceptin-G2S2, showed that the remodeled homogeneous antibody, mAb-G2S2, has an increased relative activity ranging from 3 to 26 folds.

The invention relates to mutant trehalose synthase as well as an expression gene and application thereof. The nucleotide sequence of the expression gene of the mutant trehalose synthase is as shown in SEQ ID NO. 1. The amino acid sequence of the mutant trehalose synthase is as shown in SEQ ID NO. 2. On the basis of a three-dimensional structure predicted by pseudomonas stutzeri Qlu3, the invention conducts key amino acid site-specific mutagenesis on the active center for the first time, so that the mutant protein of the trehalose synthase is obtained; the mutant trehalose synthase is simple and convenient in preparation method, high in yield, high in purity and good in thermal stability; and compared with wild trehalose synthase, the trehalose conversion rate of the mutant is improved by 4.5% and the generation amount of glucose, as a byproduct, is reduced by 69.4%.

An object of the present invention is to provide a means for producing O-fucosylated protein in large quantities, a means for searching for new genes associated with the synthesis system of O-fucosylated protein or the proteins expressed by the genes, and a means for elucidating the functions thereof. According to the present invention, a yeast transformant characterized in that the genes associated with the synthesis system of O-fucosylated protein are a GDP-fucose synthase gene, a GDP-fucose transporter gene, a fucosyltransferase gene, and a fucose receptor gene is provided.

Disclosed are a recombinant Corynebacterium glutamicum (C. glutamicum) for producing fucosyllactose which is transformed to express α-1,2-fucosyltransferase, GDP-D-mannose-4,6-dehydratase (Gmd), GDP-L-fucose synthase (WcaG) and lactose permease (LacY), wherein the Corynebacterium glutamicum has phosphomannomutase and GTP-mannose-1-phosphate guanylyltransferase, and a method for producing fucosyllactose using the same. According to the recombinant Corynebacterium glutamicum and the method for producing fucosyllactose according to the present invention, with use of a GRAS Corynebacterium glutamicum strain, which is safer than conventional Escherichia coli, 2′-fucosyllactose can be produced at a high concentration while overcoming drawbacks of conventional methods associated with industrial inapplicability resulting from low production concentrations.

The invention discloses a glucosamine synthase producing bacterium based on a CRISPR-Cas9 technology and a construction method and application of the glucosamine synthase producing bacterium. The glucosamine synthase producing bacterium is obtained in the modes that a glucosamine acetylase gene gnal and a glucosamine synthase gene glms are connected to a carrier pET-28a, and then a recombinant plasmid is transferred into a double-gene deletion host bacterium obtained by knocking out a glucosamine metabolism gene naggE and a glucosamine metabolism gene mannX of an E.coli BL21(DE3) genome through the CRISPR-Cas9 gene editing technology. The recombinant bacterium is applied to fermentation culture, on a shake flask, 7.51 g/L of glucosamine can be accumulated, 3.73 g/L of acetylglucosamine canbe accumulated, and the total output is 11.24 g/L; and on a fermentor of 5 L, 7.30 g/L of the glucosamine can be accumulated, 10.53 g/L of the acetylglucosamine can be accumulated, and the total output is 17.83 g/L.

The invention provides a heat resistant beta-amylase-trehalose synthase fusion enzyme, an expression gene of the heat resistant beta-amylase-trehalose synthase fusion enzyme, an engineering bacterium secreting the fusion enzyme and application, and belongs to the technical field of genetic engineering. The heat resistant beta-amylase-trehalose synthase fusion enzyme comprises an amino acid sequence with SEQ ID No.1 and another amino acid sequence, and the similarity of the another amino acid sequence to the amino acid sequence with SEQ ID No.1 is 80%-100%. The expression gene of the heat resistant beta-amylase-trehalose synthase fusion enzyme comprises an amino acid sequence with SEQ ID No.2 and another amino acid sequence, and the similarity of the another amino acid sequence to the amino acid sequence with SEQ ID No.2 is 80%-100%. The obtained beta-amylase-trehalose synthase fusion enzyme has a heat resistant property, directly converts amylose into trehalose at the higher temperature, saves the complicated steps of separating and purifying endoenzyme, simplifies the production process of the trehalose, and reduces the production cost.

The invention discloses a method for expressing trehalose synthase by Corynebacterium glutamicum through an EF-Tu promoter and application of the method. The method comprises the following steps: fusing the promoter PEF-Tu of a translation elongation factor EF-Tu in Corynebacterium glutamicum ATCC13032 to the upstream of a trehalose synthase gene of Corynebacterium glutamicum ATCC13032, and cloning to a Corynebacterium glutamicum induced expression plasmid pXMJ19, and the trehalose synthase can be over-expressed in the Corynebacterium glutamicum without induction and used for converting maltose to synthesize trehalose. According to the invention, the corynebacterium glutamicum endogenous strong constitutive expression promoter is utilized to express the target protein, overexpression can be realized without induction, and the production cost can be reduced in actual production.

The invention relates to a beta-amylase-trehalose synthase fused enzyme and an expression gene and application thereof. The amino acid sequence of the beta-amylase-trehalose synthase fused enzyme is as shown in SEQ ID NO.1; the nucleotide sequence of the expression gene of the beta-amylase-trehalose synthase fused enzyme is as shown in SEQ ID NO.2. According to the invention, beta-amylase and trehalose synthase are fused for the first time, and the obtained beta-amylase-trehalose synthase fused enzyme can be used for producing trehalose by utilizing amylose or straight-chain dextrin, and moreover the influence of substrate maltose on the separation of the product trehalose is reduced when the trehalose synthase acts on maltose to generate trehalose.

The invention relates to a method for synthesizing trehalose under catalysis of co-immobilized double enzymes. According to the method, recombinant escherichia coli capable of synthesizing high-temperature-resisting trehalose synthase in a great quantity by fermentation culture is subjected to permeability treatment, and beta-amylase is added; chitosan and sodium alginate are used for co-immobilizing to form micro-spheres; starch with the weight content of 25%-30% is catalyzed by alpha-amylase and then is subjected to a contact reaction with the micro-spheres to generate the trehalose, wherein after the micro-spheres are repeatedly utilized for 8-16 times, the conversion rate of the starch can be up to 60%-65%. By adopting the method provided by the invention, the high-quality trehalose can be provided in a large-scale, low-cost and high-efficiency manner.

The invention discloses fusion protein, as well as an efficient expression method and application thereof and belongs to the technical field of fusion protein. The fusion protein is AtSUS1-RsUGT72B14fusion protein, including core fragments of arabidopsis thaliana sucrosesynthaseAtSUS1 and rhodiola sachalinensis uridine diphosphate glucosyl transferase RsUGT72B14. According to the codon preferenceof escherichia coli, an optimized gene sequence of the AtSUS1-RsUGT72B14 fusion protein is obtained by combining the characteristics of gene sequences of AtSUS1 and RsUGT72B14 and introducing GGSG four amino acid connecting peptide sequences by designing a restriction enzyme cutting site connection method, efficient expression of the AtSUS1-RsUGT72B14 fusion protein is realized, and the fusion protein can be used for catalytically synthesizing salidroside.

A polypeptide includes, in the amino acid sequence of SEQ ID NO: 1 or a similar sequence, at least one specific amino acid substitution on at least one of the 14th, 37th, 209th, 293rd, and 319th amino acid residues from the N-terminal of the amino acid sequence of SEQ ID NO: 1. A polynucleotide, an expression cassette, a vector, and a transformant include a base sequence encoding the amino acid sequence of the polypeptide. A method of producing the polypeptide and a method of producing 2-deoxy-scyllo-inosose are also provided.