Purification and characterization of alpha-L-fucosidase from Bacillus circulans grown on porcine gastric mucin.
Bacillus circulans isolated from soil was found to produce two types of alpha-L-fucosidase differing in substrate specificity. One was able to liberate L-fucose from porcine gastric mucin (PGM), but not from artificial substrates, including p-nitrophenyl and methyl alpha-L-fucosides, while the other acted not on PGM but on p-nitrophenyl alpha-L-fucoside. The production of the former enzyme was enhanced about 150 times as much by PGM added to the medium as by glucose. The alpha-L-fucosidase acting on PGM was purified from the culture fluid obtained with PGM medium by ammonium sulfate fractionation and subsequent column chromatography. The purified enzyme was found to be homogeneous by PAGE and its molecular weight was estimated to be approximately 285,000. The optimum pH was found to be 5.5 to 6.5 and the stable pH range was 4.5 to 9.0. The enzyme decomposed various blood group O(H) active substances such as PGM, human milk and human saliva, and moreover acted on A-, B-, and O-erythrocytes. The enzyme was shown to cleave alpha-(1----2)-, (1----3)-, and (1----4)-L-fucosidic linkages in various glycoproteins and oligosaccharides, but failed to hydrolyze alpha-(1----6)-L-fucosic linkages in 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine and intact bovine thyroglobulin.
Tsuji
Y
Y
Research Laboratory of Higashimaru Shoyu Co., Ltd., Hyogo.
Yamamoto
K
K
Tochikura
T
T
Seno
T
T
Ohkubo
Y
Y
Yamaguchi
H
H
D000017:ABO Blood-Group System; D000818:Animals; D001407:Bacillus / Q000201:enzymology*; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D003470:Culture Media; D004912:Erythrocytes / Q000187:drug effects; D005752:Gastric Mucins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007527:Isoenzymes / Q000096:biosynthesis; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine; D013696:Temperature; D005644:alpha-L-Fucosidase / Q000096:biosynthesis / Q000302:isolation & purification*
D000017:ABO Blood-Group System; D003470:Culture Media; D005752:Gastric Mucins; D007527:Isoenzymes; D008670:Metals; D013439:Sulfhydryl Reagents; D005644:alpha-L-Fucosidase