35 results about "Mannosidase" patented technology

The invention features a method of producing a high mannose glucocerebrosidase (hmGCB) which includes: providing a cell which is capable of expressing glucocerebrosidase (GCB), and allowing production of GCB having a precursor oligosaccharide under conditions which prevent the removal of at least one mannose residue distal to the pentasaccharide core of the precursor oligosaccharide of GCB, to thereby produce an hmGCB preparation. Preferably, the condition which prevents the removal of at least one mannose residue distal to the pentasaccharide core is inhibition of a class 1 processing mannosidase and / or a class 2 processing mannosidase. The invention also features an hmGCB preparation and methods of using an hmGCB preparation.

The present invention relates to eukaryotic host cells having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation. Host cells with modified lipid-linked oligosaccharides are created or selected. N-glycans made in the engineered host cells exhibit GnTIII activity, which produce bisected N-glycan structures and may be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

This invention is to provide a process for producing a glycoprotein comprising a mammalian type sugar chain, characterized in that the process comprises introducing an α-1,2-mannosidase gene into a methylotrophic yeast having a mutation of a sugar chain biosynthesizing enzyme gene, so that the α-1,2-mannosidase gene is expressed under the control of a potent promoter in the yeast; culturing in a medium the methylotrophic yeast cells with a heterologous gene transferred thereinto; and obtaining the glycoprotein comprising a mammalian type sugar chain from the culture. Using the newly created methylotrophic yeast having a sugar chain mutation, a neutral sugar chain identical with a high mannose type sugar chain produced by mammalian cells such as human cells, or a glycoprotein comprising such a neutral sugar chain, can be produced in a large amount at a high purity. By introducing a mammalian type sugar chain biosynthesizing gene into the above-described mutant, a mammalian type sugar chain, such as a hybrid or complex, or a protein comprising a mammalian type sugar chain can be efficiently produced.

A method for producing human-like glycoproteins by expressing a Class 2 α-mannosidase having a substrate specificity for Manα1,3 and Manα1,6 glycosidic linkages in a lower eukaryote is disclosed. Hydrolysis of these linkages on oligosaccharides produces substrates for further N-glycan processing in the secretory pathway.

The invention features a method of producing a high mannose glucocerebrosidase (hmGCB) which includes: providing a cell which is capable of expressing glucocerebrosidase (GCB), and allowing production of GCB having a precursor oligosaccharide under conditions which prevent the removal of at least one mannose residue distal to the pentasaccharide core of the precursor oligosaccharide of GCB, to thereby produce an hmGCB preparation. Preferably, the condition which prevents the removal of at least one mannose residue distal to the.pentasaccharide core is inhibition of a class 1 processing mannosidase and / or a class 2 processing mannosidase. The invention also features an hmGCB preparation and methods of using an hmGCB preparation.

Improved host cells and culture methods involving overexpression of MAN1C1 activity to improve protein production are provided.

The invention discloses an extremely-heat-resistant beta-mannosidase gene as well as an expression protein and an application thereof, wherein the DNA (deoxyribonucleic acid) sequence of the extremely-heat-resistant beta-mannosidase gene is as shown in SEQ NO: 1, and the amino acid sequence of the extremely-heat-resistant beta-mannosidase expressed by the extremely-heat-resistant beta-mannosidase gene is as shown in SEQ NO: 2. The extremely-heat-resistant beta-mannosidase has extremely strong heat-resistant performance and the characteristic of high activity in a neutral pH condition, and the enzymatic activity is the highest and the specific enzyme activity achieves 144.6 U / mg in the conditions of 85 DEG C and a pH of 5.5; and the protease has a high enzyme activity at a temperature ranging from 70 to 95 DEG C and a pH ranging from 5.0 to 7.5. The mannosidase is extremely high in heat-resistant performance, and the residual enzyme activity is about 70% in the case that the mannosidase is heat-insulated for 2h at 80 DEG C. Via the characteristics aforementioned, the mannosidase expressed by the extremely-heat-resistant beta-mannosidase gene disclosed by the invention has more advantages than the existing mannosidase, is suitable for degradation for hemicellulose in the conditions of a high temperature of greater than 80 DEG C and a slightly acidic pH, and has a potential industrial application value.

The invention discloses an enzyme solution with a better enzyme activity ratio of beta-mannase to alpha-galactosidase as well as a preparation method and application of the enzyme solution, and belongs to the technical field of microbial culture. According to the preparation method, trichoderma reesei is taken as an enzyme-producing strain, microcrystalline cellulose and / or melibiose is taken as a carbon source, the enzyme solution is produced by adopting a fermentation method, the enzyme activity of the beta-mannase in the enzyme solution is not higher than 0.05 U / mL, and the enzyme solution has a better enzyme activity ratio of beta-mannase to the alpha-galactosidase. The enzyme solution can directly hydrolyze the galactomannan to prepare micromolecular galactomannan and galactomannan-oligosaccharide without purification, the yield of the micromolecular galactomannan and the galactomannan-oligosaccharide can be effectively increased, the production cost is reduced, and the enzyme solution has a very good application prospect.

A method is described for producing protein compositions having reduced amounts of O-linked glycosylation. The method includes producing the protein in cells cultured in the presence of an inhibitor of Pmt-mediated O-linked glycosylation and / or in the presence of one or more α-1,2-mannosidases.

The present invention relates to a process for purification of recombinant alpha-mannosidase, a process for production of alpha-mannosidase, a composition comprising alpha-mannosidase, use of the composition as a medicament, use as a medicament for the treatment of alpha-mannosidosis and a method of treating alpha-mannosidosis and / or alleviating the symptoms of alpha-mannosidosis.

Provided are methods and compositions for treating an individual with cancer by administering to the individual a composition that includes a Dectin-2 stimulating agent that stimulates Dectin-2 signaling in myeloid cells (e.g., induces Dectin-2 clustering on the cell surface), thereby stimulating an anti-cancer immune response in the individual. In some cases, the myeloid cells are tumor-associated myeloid (TAM) cells. Methods and compositions are also provided for: treating an individual with cancer via contacting a cancer cell from the individual with an alpha-mannosidase class 1 inhibitor (e.g., to increase the display and / or density of terminal mannose / mannobiose residues on the surface of target cells) in vitro or ex vivo and introducing the contacted cancer cell into the individual; stimulating an antigen presenting cell (APC) via contacting a cancer cell with an alpha-mannosidase class 1 inhibitor and contacting the APC with the inhibitor-contacted cancer cell; and stimulating an APC via contacting it with a subject Dectin-2 stimulating agent.

The present invention provides genetically engineered strains of Pichia capable of producing proteins with reduced glycosylation. In particular, the genetically engineered strains of the present invention are capable of expressing either or both of an α-1,2-mannosidase and glucosidase II. The genetically engineered strains of the present invention can be further modified such that the OCH1 gene is disrupted. Methods of producing glycoproteins with reduced glycosylation using such genetically engineered stains of Pichia are also provided.

A method, and associated kit, for assaying the activity of lysosomal enzymes present in dried bodily fluids and cell tissue samples, such as α-L-iduronidase, β-D-galactosidase, β-D-glucosidase, chitotriosidase, total α-D-galactosidase and α-D-galactosidase A, hexosaminidase A and B, α-D-mannosidase, β-D-mannosidase, α-L-fucosidase, N-acetyl-α-galactosaminidase, arylsulfatases, sphingomyelinase, β-galactocerebrosidase, iduronate-2-sulfatase and β-D-glucuronidase. The method includes: (a) combining with a dried bodily fluid or cell tissue sample containing at least one type of lysosomal enzyme: (1) an eluent, (2) an incubation buffer and (3) a substrate or substrates capable of reacting with the assayed lysosomal enzymes and producing their corresponding enzyme product or products, (b) allowing the dried bodily fluid or cell tissue sample to react with the eluent, incubation buffer and substrate or substrates for an adequate time and temperature, and (c) applying measuring means to the enzyme product to determine the activities of the lysosomal enzymes present.

The invention relates to an animal cell strain and method for glycoprotein production and glycoprotein and application thereof. The animal cell strain which is used for producing glycoprotein with high-mannose carbohydrate chains as main N-carbohydrate chain structures is characterized in that at least two genes of golgi apparatus mannosidase genes and endoplasmic reticulum mannosidase genes of the cell strain are damaged or knocked out. By adoption of the method, glycoprotein which is sharply reduced in compound carbohydrate chain content and takes high-mannose type N-carbohydrate chains suchas Man9-GlcNAc2 and Man8-GlcNAc2 as the main structures can be obtained. The glycoprotein with the high-mannose carbohydrate chains as the main N-carbohydrate chain structures can be used for production of biological pharmaceutical molecules including lysosomal enzymes, antibodies and the like, and accordingly the glycoprotein can be used for treatment of various lysosomal storage diseases.

A method for producing human-like glycoproteins by expressing a Class 2 α-mannosidase having a substrate specificity for Manα1,3 and Manα1,6 glycosidic linkages in a lower eukaryote is disclosed. Hydrolysis of these linkages on oligosaccharides produces substrates for further N-glycan processing in the secretory pathway.

A method for producing human-like glycoproteins by expressing a Class 2 α-mannosidase having a substrate specificity for Manα1,3 and Manα1,6 glycosidic linkages in a lower eukaryote is disclosed. Hydrolysis of these linkages on oligosaccharides produces substrates for further N-glycan processing in the secretory pathway.

The invention relates to a processing method for a peanut cake meal feed for prevention and control of poultry diseases, and belongs to the field of peanut cake meal high-valued utilization; the cake meal obtained after oil manufacture of peanut is used as a raw material in the method, and the processing method includes the following steps: Chinese herbal medicine soaking with ethanol, enzymatic hydrolysis, fermentation, grinding into thick liquid, homogenizing, spray drying and the like. With use of the method, garlic, tartary buckwheat, folium artemisiae argyi, fructus forsythiae, fruit of cubeb litsea tree, malt, dried tangerine or orange peel, spina date seed, cassia bark, anise and the like are added during Chinese herbal medicine soaking; the enzymatic hydrolysis adopts pancreas, cellulase and beta-mannosidase; the microbial fermentation adopts candida utilis, bifidobacterium animalis, butyric acid bacteria and aspergillus candidus; the cake meal feed prepared by the method has the advantages of high sale price, good protein solubility and high absorption utilization degree, has no aflatoxin contamination, can effectively prevent and control various poultry diseases and insect pests when used as a feed, allows poultry to grow fast, has remarkable economic benefits and social benefits, and realizes the high-valued utilization of the peanut cake meal.

The invention discloses a heat-resistant mannosidase gene as well as an expression protein and application thereof, and mannosidase better meeting requirements is obtained by carrying out directional modification on enzyme through related technical means of protein engineering. The mannosidase has the characteristics of better high temperature resistance and high activity under a slightly acidic pH (Potential of Hydrogen) condition. The enzyme activity is the highest under the conditions that the temperature is 75 DEG C and the pH value is 6.0, and the specific enzyme activity reaches 8.2 mu mol / mg min; the mannosidase has relatively high enzyme activity at the temperature of 70 to 75 DEG C and the pH value of 6 to 6.5. The heat resistance of the mannosidase is extremely high, and the enzyme activity can be kept above 80% after the mannosidase is incubated for 1 hour in an environment with the temperature of 70-75 DEG C and the pH value of 4.5-7. Compared with the mannosidase before modification, the mannosidase has the transglycosylation capability, mannodisaccharide and mannotriose with the total sugar content of about 10% can be converted after 2 days under the conditions that the mannose concentration is 60% (w / w), the pH value is 6, and the temperature is 55 DEG C. Compared with the mannodisaccharide and mannotriose produced by an existing enzymolysis method, the mannosidase has the advantages that the product is relatively single and pure, the implementation is simple and convenient, and the mannosidase has greater superiority.

The invention discloses a preparation method of alpha-galactosidase with the low beta-mannosidase content. Alpha-galactosidase liquid containing beta-mannosidase with the enzyme activity not higher than 0.05 U / mL is produced by taking trichoderma reesei as an enzyme producing strain and taking galacto-mannan-oligosaccharides as a carbon source through a fermentation method. According to the method, the alpha-galactosidase is produced by adopting the trichoderma reesei and taking the galacto-mannan-oligosaccharides as the carbon source and an inducer through fermentation, and the content (activity) of the beta-mannosidase in the enzyme liquid is very low while the high activity of the alpha-galactosidase with is achieved; the enzyme liquid can directly cooperate with the beta-mannosidase to hydrolyze galactomannan to prepare micromolecular galactomannan and the galacto-mannan-oligosaccharides without being purified to remove the beta-mannosidase, and therefore the production cost that the micromolecular galactomannan and the galacto-mannan-oligosaccharides are prepared by directionally degrading the galactomannan can be effectively reduced.

Provided are an animal cell strain for use in producing a glycoprotein which uses a high-mannose sugar chain as a main N-glycan structure, a method for use in producing a glycoprotein by using the cell strain, a glycoprotein produced by using the method, and a use thereof. At least two genes from among a Golgi mannosidase and an endoplasmic reticulum mannosidase gene of the cell strain are damaged or knocked out.

The present invention relates to a process for purification of recombinant alpha-mannosidase, a process for production of alpha-mannosidase, a composition comprising alpha-mannosidase, use of the composition as a medicament, use as a medicament for the treatment of alpha-mannosidosis and a method of treating alpha-mannosidosis and / or alleviating the symptoms of alpha-mannosidosis.

The invention discloses a phosphorus and potassium compounded coloring sugar-increasing fertilizer and a preparation method thereof. The fertilizer comprises the following components: serine, cystine, glutamic acid, lysine, arginine, glycine, threonine, histidine, cysteine, nucleotide, rare earth, titanium citrate, mannosidase, xylosidase, glucuronidase, glucose, glycerol, griseofulvic acid, dipotassium phosphate and water. The compound phosphorus-potassium coloring sugar-increasing fertilizer is simple in preparation method, good in use effect, high in nutrition, good in absorption, good in coloring, high in sugar content and good in quality and is an innovation in crop fertilizer, trace mineral substances necessary for fruit trees in the fertilizer are increased through rare earth and citric acid titanium, the trace mineral substances are slowly and uniformly released in soil, absorption of the fruit trees is fully guaranteed, and the yield of the fruit trees is increased. The griseofulvic acid can inhibit fungal mitosis, break a spindle structure of the mitosis, terminate metaphase cell division and prevent fungal infection of fruit trees.

Novel phosphotetrahydropyran compounds that mimic mannose-6-phosphate but that are more resistant to phosphatases and mannosidases, and pharmaceutical compositions thereof, are disclosed. These compounds and compositions inhibit T lymphocyte migration from blood to tissues or to other extravascular sites. By inhibiting such migration, these compounds are useful for treating diseases or disorders that are mediated at least in part by such T lymphocyte migration. Such diseases and disorders include rheumatoid arthritis, multiple sclerosis, acute disseminated encephalomyelitis, psoriasis, inflammatory bowel disease, T cell-mediated dermatitis, stromal keratitis, uveitis, thyroiditis, sialitis and type I diabetes.