Purification and characterization of extracellular 1,2-alpha-L-fucosidase from Bacillus cereus.
Bacillus cereus isolated from a soil sample, inductively produced alpha-L-fucosidase in culture medium containing porcine gastric mucin (PGM). The production of the enzyme was also weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose. The enzyme was purified 61-fold with an overall recovery of 1.8% from the culture fluid supplemented with PGM by ammonium sulfate precipitation, acetone fractionation, and subsequent column chromatography. The purified enzyme was found homogeneous by SDS-PAGE and its molecular mass was estimated to be approximately 196,000 kDa. Its optimum pH was 7.0 and it was stable in the pH range of 5.0 to 9.0. The enzyme hydrolyzed the alpha-(1-->2)-L-fucosidic linkage in oligosaccharides such as Fucalpha1-2Galbeta1-4Glc (2'-fucosyllactose), Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose I), and the glycoprotein PGM. The enzyme was inactive on p-nitrophenyl alpha-L-fucoside, the alpha-(1-->3)-L-fucosidic linkages in Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose III) and orosomucoid, the alpha-(1-->4)-L-fucosidic linkage in Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose II), and the alpha-(1-->6)-L-fucosidic linkage in thyroglobulin.
Miura
Toyokazu
T
Department of Biotechnology, Faculty of Engineering, Tottori University, 4-101 Koyamacho-Minami, Tottori, Tottori 680-8552, Japan.
Okamoto
Kenji
K
Yanase
Hideshi
H
D000818:Animals; D001409:Bacillus cereus / Q000145:classification / Q000201:enzymology*; D004789:Enzyme Activation; D004795:Enzyme Stability; D045604:Extracellular Fluid / Q000737:chemistry*; D005752:Gastric Mucins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D012988:Soil Microbiology; D013379:Substrate Specificity; D013552:Swine; D005644:alpha-L-Fucosidase / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification
D005752:Gastric Mucins; D005644:alpha-L-Fucosidase