رسائل دكتوراة
English
2006
Characterization of Glucosyltransferase (GTF) of Mutans Streptococci Streptococcus sobrinus (Serotype G): Functional and Immunological Assessment
عبد الواحد الشيباني, ندى هشام عبد اللطيف المدلل
كلية العلوم-جامعة النهرين · العراق
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 (ultrafiltationcell 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 ultrafiltrationmethod 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%).
Determination 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 antiGTF-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-GTFIb 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.
Determination 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).