Purification and characterization of cell-associated glucosyltransferase synthesizing water-insoluble glucan from serotype c Streptococcus mutans.
Strains of Streptococcus mutans (serotypes c, e and f) were found to possess high levels of glucosyltransferase (GTase) activity, both cell-associated and in the culture medium, when grown in either sucrose-free or sucrose-containing broth media. The cell-associated GTase of S. mutans MT8148 (serotype c) was effectively extracted by treatment with 8 M-urea at 25 degrees C for 1 h. Approximately 95% of the GTase activity was solubilized by this treatment. The crude extract was purified by DEAE-Sephacel and hydroxylapatite column chromatography. For comparison, extracellular GTase was also purified from the culture supernatant of the same strain by ammonium sulphate precipitation, chromatofocusing and hydroxylapatite chromatography. The molecular masses of the cell-associated and extracellular GTase proteins were similar (156 kDa) as determined by SDS-PAGE. However, the pH optima for maximum GTase activity were different: pH 6.7 to 7.0 for the cell-associated enzyme and pH 5.5 to 6.5 for the extracellular enzyme. The product of cell-associated GTase from sucrose was almost exclusively water-insoluble glucan. On the other hand, extracellular GTase produced mainly water-soluble glucan from sucrose. This indicates that GTase synthesizing water-insoluble glucan is present primarily in a cell-associated form in serotype c S. mutans. Insoluble glucan synthesis by the cell-associated GTase from sucrose was not enhanced by addition of primer dextran T10 to the reaction mixture. The extracellular and cell-associated GTases were immunologically unrelated as determined by ELISA using monoclonal antibodies.
Hamada
S
S
Department of Oral Microbiology, Osaka University Faculty of Dentistry, Japan.
Horikoshi
T
T
Minami
T
T
Okahashi
N
N
Koga
T
T
D002845:Chromatography; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005936:Glucans / Q000096:biosynthesis*; D005964:Glucosyltransferases / Q000302:isolation & purification*; D012703:Serotyping; D013045:Species Specificity; D013295:Streptococcus mutans / Q000201:enzymology*
D003470:Culture Media; D005936:Glucans; D005964:Glucosyltransferases