Efficient production and purification of extracellular 1,2-alpha-L-fucosidase of Bacillus sp. K40T.
When Bacillus sp. K40T was cultured in the presence of L-fucose, 1,2-alpha-L-fucosidase was found to be produced specifically in the culture fluid. The enzyme was purified to homogeneity from a culture containing only L-fucose by chromatography on hydroxylapatite and chromatofocusing. The molecular weight of the enzyme was estimated to be 200,000 by gel filtration on Sephadex G-200. The enzyme was optimal at pH 5.5-7.0 and was stable at pH 6.0-9.0. The enzyme hydrolyzed the alpha-(1-->2)- L-fucosidic linkages in various oligosaccharides and glycoproteins such as lacto-N-fucopentaose (LNF)-I <O-alpha-L-fucose-(1-->2)-O-beta-D-galactose-(1-->3)-N-acetyl-O-beta-D- glucosamine-(1-->3)-O-beta-D-galactose-(1-->4)-D-glucose>, porcine gastric mucin, and porcine submaxillary mucin. The enzyme also acted on human erythrocytes, which was confirmed by the hemagglutination test using Ulex anti-H lectin. The enzyme did not hydrolyze alpha-(1-->3)-, alpha-(1-->4)- and alpha-(1-->6)-L-fucosidic linkages in LNF-III <O-beta-D-galactose-(1-->4)[O- alpha-L-fucose-(1-->3)-]-N-acetyl-O-beta-D-glucosamine-(1-->3)-O-beta-D- galactose-(1-->4)-D-glucose>, LNF-II <O-beta-D-galactose-(1-->3)[O-alpha-L- fucose-(1-->4)-]-N-acetyl-O-beta-D-glucosamine-(1-->3)-O-beta-D- galactose-(1-->4)-D-glucose> or 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine.
Kurimura
Y
Y
Research Laboratory of Higashimaru Shoyu Co., Ltd., Hyogo, Japan.
Tsuji
Y
Y
Yamamoto
K
K
Kumagai
H
H
Tochikura
T
T
D001407:Bacillus / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D005644:alpha-L-Fucosidase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
D005644:alpha-L-Fucosidase