Isolation and characterisation of sialidase from a strain of Streptococcusoralis

Streptococcus oralis, the most virulent of the viridans streptococci, produ ces a sialidase and this exo-glycosidase has been implicated in the disease process of a number of pathogens. The sialidase of S. oralis strain AR3 wa s purified in order to understand the characteristics of this putative viru lence determinant. The enzyme isolated as a high mol. wt aggregate (c. 325 kDa) was purified 4520-fold from late exponential phase cultures by a combi nation of ultrafiltration, ammonium sulphate precipitation, ion-exchange an d gel filtration chromatography. The sialidase component had a mol. wt of 1 44 kDa as determined by SDS-PAGE analysis. The purified sialidase released N-acetylneuraminic acid from a range of sialoglycoconjugate including human al-acid glycoprotein, bovine submaxillary mucin, colominic acid and sialyl -alpha 2,3- and sialyl-alpha 2,6-lactose. Also, N-glycolylneuraminic acid w as cleaved from bovine submaxillary mucin. The sialidase had a K-m of 11.8 mu M for alpha(1)-acid glycoprotein, was active over a broad pH range with a pH optimum of 6.0 and cleaved alpha 2,3-, alpha 2,6- and alpha 2-8-sialyl glycosidic linkages with a marked preference for alpha 2,3-linkages. The e nzyme was competitively inhibited by the sialic acid derivative, 2,3-dehydr o-N-acetylneuraminic acid, with a K-IC of 1.2 mu M. The characteristics of the purified sialidase would support a nutritional role for this enzyme tha t may be significant in the proliferation of this organism in the oral cavi ty and at extra-oral sites in association with life-threatening infections.