IADR Abstract Archives
Glucanohydrolases to Degrade Exopolysaccharide Matrix and Remove Cariogenic Biofilm
Objectives: Extracellular polysaccharides (EPS) contribute to bacterial adherence and enhance biofilm formation and maintenance, favoring the retention of acids and caries development. Therefore, we evaluated the effect of α-(1→3) and α-(1→6)-glucanases from, respectively, Prevotella melaninogenica and Capnocytophaga ochraceae on EPS degradation and cariogenic biofilm removal.
Methods: First, enzymes were cloned, expressed heterologously and purified, having their optimal pH and temperature determined. Early S. mutans UA159 biofilms were formed for 24 h in 96-well plates using UTEYB medium supplemented with 1% sucrose. For time-response-assay, biofilms were treated with each enzyme separately at 1 mg/ml in PBS for 0.5; 1; 2; 4 or 24 h. For dose-response-assay, biofilms were treated for 1 h with enzymes separately or combined at 0.125; 0.25; 0.5 and 1 mg/mL in PBS. For both assays, PBS only was used as control. After enzymatic treatment, the solution containing the enzymes was collected for determination of hydrolytic products by HPAEC system. Biomass was estimated by crystal violet assay. EPS was extracted with 1 M NaOH and quantified colorimetrically by the phenol-sulfuric-method. Biofilm architecture was visualized by CLSM microscopy. The % of biomass (n=3) and EPS (n=2) reduction was calculated for each treatment group in relation to the control, as well as the degree of synergism (DS). The % of biomass and EPS reduction were analyzed by two-way ANOVA, Tukey test (α=5%), considering enzymes and treatments as factors.
Results: The % of biomass and EPS reduction ranged, respectively, for α-(1→3)-glucanase: from 6.9±3.0 to 92.0±0.7 and from 52.2±4.4 to 99.3±0.4; and for α-(1→6)-glucanase: from 9.2±4.7 to 85.6±0.9 and from 75.9±0.0 to 99.4±0.0. Synergism was found for biomass reduction (DS from 1.4 to 3.2) at all concentrations tested, but not for EPS (DS from 0.5 to 0.7).
Conclusions: The data suggest that α-(1→3) and α-(1→6)-glucanases tested were effective on EPS degradation and cariogenic biofilm removal.
Methods: First, enzymes were cloned, expressed heterologously and purified, having their optimal pH and temperature determined. Early S. mutans UA159 biofilms were formed for 24 h in 96-well plates using UTEYB medium supplemented with 1% sucrose. For time-response-assay, biofilms were treated with each enzyme separately at 1 mg/ml in PBS for 0.5; 1; 2; 4 or 24 h. For dose-response-assay, biofilms were treated for 1 h with enzymes separately or combined at 0.125; 0.25; 0.5 and 1 mg/mL in PBS. For both assays, PBS only was used as control. After enzymatic treatment, the solution containing the enzymes was collected for determination of hydrolytic products by HPAEC system. Biomass was estimated by crystal violet assay. EPS was extracted with 1 M NaOH and quantified colorimetrically by the phenol-sulfuric-method. Biofilm architecture was visualized by CLSM microscopy. The % of biomass (n=3) and EPS (n=2) reduction was calculated for each treatment group in relation to the control, as well as the degree of synergism (DS). The % of biomass and EPS reduction were analyzed by two-way ANOVA, Tukey test (α=5%), considering enzymes and treatments as factors.
Results: The % of biomass and EPS reduction ranged, respectively, for α-(1→3)-glucanase: from 6.9±3.0 to 92.0±0.7 and from 52.2±4.4 to 99.3±0.4; and for α-(1→6)-glucanase: from 9.2±4.7 to 85.6±0.9 and from 75.9±0.0 to 99.4±0.0. Synergism was found for biomass reduction (DS from 1.4 to 3.2) at all concentrations tested, but not for EPS (DS from 0.5 to 0.7).
Conclusions: The data suggest that α-(1→3) and α-(1→6)-glucanases tested were effective on EPS degradation and cariogenic biofilm removal.