Charactrization of glucosyl transferase (GTF) of mutans streptococci streptococcus sobrinus (serotype G)(functional and immunological assessment). +CD

Fifty plaque samples were collected from teeth. Forty five samples were considered to be positive bacterial isolates about (104 bacteria/ml) using selective Ms-agar (Mitis-Salivarius agar) medium. Thirty isolates were considered to be related to the genus Streptococcus and specially to the mutans streptococci of various group; S. sobrinus (serotype D, G), S. mutans (serotype C, F), S. cricetus (serotype A) and S. rattus (serotype B) with percentages of (39.29%), (30.30%), (18.18%) and (3.03%) respectively, depending on biochemical and Lancefield grouping identification systems. Seventeen isolates; related to different serotype groups, were tested for production of an extracellular Glucosyltransferase (GTF) through determination of their enzyme specific activity. All isolates were able to produce the enzyme. Mutans streptococci isolate N10 which identified as (S. sobrinus serotype G) was selected as the highest producible bacterial isolate for GTF with a specific activity of (0.752U/mg). Productivity phase of growth and GTF activity for the isolate N10 (the chosen isolate) was specified prior to determine maximal productivity. It was found that GTF was produced during the stationary phase of growth (20-40 hr.) and its maximal productivity was recorded at 24 hr. The effect of ice/freezing preservation at (-20oC) on the activity of crude GTF with and without the presence of glycerol was done ,and the effect of concentration by polyethylene glycol (PEG) and sucrose powders, freezing/drying (lyophilization) system and an Amicone P50 in (ultrafiltation-cell system) on GTF activity and protein concentration was recorded . For long term storage of GTF, the addition of 15% glycerol then ice/freezing preservation at (-20°C) gives good results and the best method to concentrate GTF was done by using an Amicon-filter P50 in (ultrafiltration-cell) which gave a specific activity of (18.42 U/mg protein). Large scale production, concentration and purification of mutans streptococci (S.sobrinus) (serotype G) N10 GTF were done by ultrafiltration-method using an Amicone-filter P50, adsorption chromatography (hydroxyapatite beads), ion-exchange chromatography (DEAE-cellulose column) and gel-filtration chromatography using (Sephacryl S-200) column. Three purified GTF enzymes (GTF-Ia, GTF-Ib, GTF-II) were detected with a specific activity of (31.60U/mg protein), (31.50U/mg protein) and (66.270U/mg protein) after (27.59), (27.92) and (58.75) folds of purification with yield of (42.05%). Detrmination of purified GTF enzymes (GTF-Ia, GTF-Ib, GTF-II) molecular weights was done by using gel-filtration chromatography (Sephacryl S-200) column with the presence of high-molecular weights standards proteins. Two GTF enzymes (GTF-Ia and GTF-Ib) were considered as isozymes with a molecular weight of (128882 dalton) and the third (GTF-II) had a molecular weight of (186208 dalton). The ability of GTF to stimulate the immune system was tested in this study. The subcutaneous route injections of purified antigen (GTF-Ib) in the back of experimental rabbits were done. A double immunodiffusion test for detection of the immune response between anti-GTF-Ib enzyme antibody and purified (GTF-Ia, GTF-Ib and GTF-II) antigens were recorded. The same immunological response was indicated (by the appearance of the precipitation lines) on the surface of agarose-gel between the two GTF isozymes and the anti-GTF-Ib enzyme antibody. The effect of different concentrations of inhibitors (EDTA, sodium fluoride, chlorohexidine) Zak (mouthrinse) and anti-GTF-Ib enzyme antibody on the growth of bacteria was tested using broth dilution method and diffusion method on solid medium. Anti-GTF-Ib enzyme antibody and EDTA had no effect on the growth of mutans streptococci N10 (S. sobrinus serotype G), while sodium fluoride, chlorohexidine at concentrations (18mM) and (20mM) respectively were capable to produce a complete bacterial growth inhibition, ZAK (mouthrinse) at concentration (12mM) was capable to inhibit the growth of bacteria when broth-dilution method was used. The effects of different concentrations of these inhibitors and anti-GTF-Ib enzyme antibody on the activity of purified GTF-Ib enzyme were also tested. Anti-GTF-Ib enzyme antibody at concentration 0f (1.5x10-3mM) was capable to inhibit (87.33%) of the purified GTF-Ib enzyme activity followed by sodium fluoride (18mM), chlorohexidine (20mM) and ZAK (mouthrinse) (12mM) which were capable to inhibit (81.64%), (75.55%) and (60.40%) of the purified GTF-Ib enzyme activity respectively. However EDTA had no effect on the purified GTF-Ib enzyme activity. Deterermination of the kinetic constants, Mechalis Menton constant (Km) value and the maximum velocity (Vmax) value for the purified GTF-Ib enzyme was specifies as (11mM) and (0.05mM/min-1) respectively using (Lineweaver Burk plot).