Purification and preliminary characterization of exo-beta-D-fructosidase in Streptococcus salivarius KTA-19.
Streptococcus salivarius fructosidase (beta-D-fructan fructohydrolase, EC 3.2.1.80) was purified to homogeneity. The molecular weight of the fructosidase was estimated to be 83,000 to 85,000 by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pH optimum of the enzyme was 7.0, and the isoelectric point was pH 4.7. The purified enzyme preparation hydrolyzed levan, inulin, and several 2-beta-linkage-containing oligosaccharides such as sucrose and raffinose, but not melezitose, dextran, and pseudonigeran. The fructosidase was inhibited by Fe3+, Cu2+, Hg2+, and Ag+, but not by Ca2+, Co2+, Mg2+, and Zn2+, at a concentration of 10(-3) M. Mn2+ was particularly effective in stimulating activity at the same concentration. The presence of either EDTA or KCN also increased fructosidase activity by 20 to 30%. The enzyme was susceptible to sulfhydryl reagents since p-chloromercuribenzoate (10(-7) M) produced 63% inhibition of the activity. However, this inhibition was overcome in the presence of cysteine. This enzyme acts as an exofructosidase since thin-layer chromatographic analysis revealed that D-fructose was formed from levan or inulin by the action of the enzyme.
D002412:Cations; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003773:Dental Plaque / Q000382:microbiology; D004589:Electrophoresis, Disc; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013291:Streptococcus / Q000201:enzymology* / Q000302:isolation & purification; D013379:Substrate Specificity
D002412:Cations; D006026:Glycoside Hydrolases; C044118:fructan beta-fructosidase