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beta-Mannosidase from the mushroom Polyporus sulfureus
Abstract:
beta-D-Mannosidase (EC 3.2.1.25), a useful tool for the structural studies of heterosaccharide chains, has been isolated in a highly purified form from the fruiting bodies of the mushroom Polyporus sulfureus. This mushroom is unique among reported sources of this enzyme in that it has the advantage of being almost free of alpha-mannosidase activity. The purification procedure involves ammonium sulfate fractionation followed by Sephadex G-100 filtration and chromatography on columns of DEAE-cellulose and hydroxylapatite. The final enzyme preparation gives essentially a single band on disc gel electrophoresis. The purified enzyme liberates the beta-D-mannopyranosyl unit from various natural substrates such as the core glycopeptide, Man(GlcNAc)2-Asn isolated from ovalbumin, from Taka-amylase A, and from human alpha1-acid glycoprotein. It also hydrolyzes (Man)2-GlcNAc from the urine of an alpha-mannosidosis patient, 1,4-D-mannobiose and mannotriose isolated from ivory nut mannan, 4-O-beta-D-mannopyranosyl-L-rhamnose, 6-O-beta-D-mannopyranosyl-D-galactose and 4-O-beta-D-mannopyranosyl-N-acetylglucosamine. The molecular weight of this enzyme is estimated to be about 64,000 by gel filtration. For p-nitrophenyl-beta-D-mannopyranoside, the pH optimum is between 2.4 and 3.4 and the Km is 1.6 mM.
Insights
Highly purified beta-D-mannosidase was isolated from Polyporus sulfureus mushrooms, offering a unique, alpha-mannosidase-free enzyme for glycosaccharide structural studies.
Area of Science:
- Biochemistry
- Enzymology
- Glycobiology
Background:
- Beta-D-mannosidase (EC 3.2.1.25) is crucial for analyzing complex carbohydrate structures.
- Enzymes with high specificity are valuable tools in biochemical research.
- Polyporus sulfureus is a novel source for enzyme isolation.
Purpose of the Study:
- To isolate and purify beta-D-mannosidase from Polyporus sulfureus.
- To characterize the enzyme's properties and substrate specificity.
- To evaluate its potential for structural studies of heterosaccharides.
Main Methods:
- Enzyme purification using ammonium sulfate fractionation, Sephadex G-100 filtration, DEAE-cellulose, and hydroxylapatite chromatography.
- Analysis of enzyme purity via disc gel electrophoresis.
- Determination of molecular weight using gel filtration.
- Enzyme activity assays on various natural and synthetic substrates.
Main Results:
- A highly purified beta-D-mannosidase preparation was obtained from Polyporus sulfureus, exhibiting minimal alpha-mannosidase activity.
- The enzyme preparation showed a single band on disc gel electrophoresis, indicating high purity.
- The purified enzyme successfully hydrolyzed beta-D-mannopyranosyl units from diverse substrates, including glycopeptides and oligosaccharides.
- Molecular weight was estimated at approximately 64,000 Da, with optimal activity between pH 2.4-3.4 and a Km of 1.6 mM for p-nitrophenyl-beta-D-mannopyranoside.
Conclusions:
- Polyporus sulfureus is a valuable and unique source of highly purified beta-D-mannosidase.
- This enzyme is suitable for structural analysis of complex carbohydrates due to its specificity.
- The characterized properties provide a foundation for its application in glycobiology research.