Two sialidases which preferentially hydrolyze sialyl alpha 2-8 linkage from Bacteroides fragilis SBT3182.
Bacteroides fragilis SBT3182 produced two sialidases which differ in molecular weight on SDS-PAGE. These sialidases, a 50 kDa and a 55 kDa enzymes, were purified separately and their properties were compared. Both enzymes preferentially hydrolyze sialyl alpha 2-8 linkage rather than alpha 2-3 and alpha 2-6 bonds. The Km values for Neu5Ac alpha 2-3lactose, Neu5Ac alpha 2-6lactose, and colominic acid, which is a homopolymer of N-acetylneuraminic acid linked by alpha 2-8 bonds, were identical between the two enzymes. These enzymes had Km value of 1.0-1.2 mM for Neu5Ac alpha 2-3lactose and 1.3-1.5 mM for Neu5Ac alpha 2-6lactose, which are in the ranges reported for other sialidases. However, the Km values for colominic acid (0.03-0.04 mM) were lower than those of other sialidases, indicating that sialidases from B. fragilis SBT3182 show high affinity for the sialyl alpha 2-8 linkage. The two sialidases also had identical N-terminal amino acid sequences and did not reveal any homology to known sialidases. PAS-staining suggested that these two sialidases were glycoproteins. In the lectin analysis, the 50 kDa enzyme was stained with Con A, DBA, and UEA-I while the 55 kDa sialidase was stained only with Con A. This suggested that the difference in molecular weight may be due to the carbohydrate composition. When the 50 kDa enzyme was incubated with UEA-I, which is a lectin specific for alpha-fucose residue, the activity decreased by 20%.
Tanaka
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Technical Research Institute, Snow Brand Milk Products, Co., Ltd., Saitama.
Ito
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Iwasaki
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T
D000595:Amino Acid Sequence; D001441:Bacteroides fragilis / Q000201:enzymology*; D015153:Blotting, Western; D002236:Carbohydrate Conformation; D004591:Electrophoresis, Polyacrylamide Gel; D006001:Glycoconjugates / Q000378:metabolism; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D037102:Lectins / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009439:Neuraminidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D013194:Staining and Labeling; D013379:Substrate Specificity
D006001:Glycoconjugates; D007527:Isoenzymes; D037102:Lectins; D011134:Polysaccharides; C014420:colominic acid; D009439:Neuraminidase