34 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.

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 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.

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.

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.

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.