2 SIALIDASES WHICH PREFERENTIALLY HYDROLYZE SIALYL ALPHA-2-8 LINKAGE FROM BACTEROIDES-FRAGILIS SBT3182

Bacteroides fragilis SBT3182 produced two sialidases which differ in m olecular weight on SDS-PAGE. These sialidases, a 50 kDa and a 55 kDa e nzymes, were purified separately and their properties were compared. B oth enzymes preferentially hydrolyze sialyl alpha 2-8 linkage rather t han alpha 2-3 and alpha 2-6 bonds. The K-m values for Neu5Ac alpha 2-3 lactose, Neu5Ac alpha 2-6lactose, and colominic acid, which is a homop olymer of N-acetylneuraminic acid linked by alpha 2-8 bonds, were iden tical between the two enzymes. These enzymes had K-m value of 1.0-1.2 mM for Neu5Ac alpha 2-3lactose and 1.3-1.5 mM for Neu5Ac alpha 2-6lact ose, which are in the ranges reported for other sialidases. However, t he K, values for colominic acid (0.03-0.04 mM) were lower than those o f other sialidases, indicating that sialidases from B. fragilis SBT318 2 show high affinity for the sialyl alpha 2-8 linkage. The two sialida ses also had identical N-terminal amino acid sequences and did not rev eal any homology to known sialidases. PAS-staining suggested that thes e two sialidases were glycoproteins. In the lectin analysis, the 50 kD a enzyme was stained with Con A, DBA, and UEA-I while the 55 kDa siali dase was stained only with Con A. This suggested that the difference i n molecular weight may be due to the carbohydrate composition. When th e 50 kDa enzyme was incubated with UEA-I, which is a lectin specific f or alpha-fucose residue, the activity decreased by 20%.