Purification and some properties of free and cell-associated dextransucrase from Streptococcus sanguis.
Dextransucrase of Streptococcus sanguis occurred in cell-free and cell-associated forms. Cell-free dextransucrase was purified by four successive chromatographies on Bio-Gel P 60, DEAE-cellulose, and Bio-Gel P 200 from the culture supernatant. The purification of cell-associated dextransucrase was made from the pellet of Streptococcus sanguis culture. Bacterial pellet was extracted with 1 M phosphate buffer (pH 6.0) and chromatographied by using an immunosorbent column. The two enzymes gave single bands in polyacrylamide gel electrophoresis. The molecular weight determined by sodium dodecyl sulfate polyacrylamide gel was about 100 000 daltons for the two forms of dextransucrases. The optimum pH of the cell-free and cell-associated enzymes was around 6 and the temperature optimum was broad for the two enzymes. The KM values for sucrose were respectively 2 mM and 3 mM for cell-free and cell-associated enzymes. When primer dextran was added, the reaction velocity increased but the KM for sucrose remained the same, and the KA for dextran was 200 muM for the two dextransucrases. Trehalose and maltose acted also as glucosyl residue acceptors. Purified enzymes had dextran synthesising activity and invertase-like activity. The same properties of the two forms of enzymes and the positive cross reaction against anti free and anti cell-associated globulins stongly suggest the identity of the two enzymes.
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D003600:Cytosol / Q000201:enzymology; D003911:Dextrans / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007700:Kinetics; D008970:Molecular Weight; D013295:Streptococcus mutans / Q000201:enzymology; D013298:Streptococcus sanguis / Q000201:enzymology*; D013393:Sucrase
D003911:Dextrans; D005964:Glucosyltransferases; C020025:dextransucrase; D013393:Sucrase