تاثير الانزيمين الكلوكواميليز والكلوكوز اوكسيديز في تثبيط بكتريا Streptococcus mutans المسببة لتسوس الاسنان == The Effect of Glucoamylase And Glucose Oxidase On Inhibition of Streptococcus Mutans Causes The Dental Caries
Author name:
بيداء عبود حسن الجنابي
Supervisor name:
زهرة محسن علي | محمد عبد الله جبر
General topic:
Biology
Specific topic:
Microbiology
Degree:
Doctorate
University:
University of Kufa - College Of Science - Department Of Biology
Language:
English
University location:
Najaf
First pages:
24T2760 - p.pdf
Abstract:
تضمنت الدراسة استخلاص الكلوكواميليز والكلوكوز اوكسيديز من الفطرين Aspergillus niger وPenicillum notatum على التوالي وتنقية وتوصيف الانزيمين لغرض زيادة تركيز بيروكسيد الهيدروجين المحفز لانزيم اللاكتوبيروكسيد الموجود باللعاب على تكوين ايونات الهايبوثايوسيان | This study aims at producing glucoamylase and glucose oxidase from the Aspergillus niger and Penicillium notatum respectively , purifying and characterizing the enzymes to increase H2O2 concentration which induce lactoperoxidase in saliva to the formation of hypothiocyanite ion to inhibite Streptococcus mutans which causes the dental caries. For glucoamylase and glucose oxidase production, the results shown the the highest enzymes production were occurred using the synthetic medium, it gave high titer of glucoamylase and glucose oxidase activity (0.212, 0.605 U/ml) respectively, the optimum incubation period for glucoamylase was occurred after 96 h (0.125 U/ml), while the maximum activity of glucose oxidase was occurred after 72 h (0.662 U/ml), the optimum temperature for enzymes production was occurred in 30°C, (0.257, 0.690 U/ml) respectively, the maximum production of glucoamylase and glucose oxidase were occurred in the pH = 6 (0.174, 0.391 U/ml) respectively, the optimum nitrogen source for enzymes production was yeast extract and potassium nitrate (0.157, 0.571 U/ml) respectively, the optimum carbon source for glucoamylase and glucose oxidase production was starch and glucose (0.167, 0.459 U/ml) respectively. For glucoamylase and glucose oxidase purification, the results found the ammonium sulfate (80 %) was selected as the best ratio for precipitate of glucoamylase , the specific activity reached to (3.626 U/mg) , with a purification fold (1.241) and yield (0.38) % , while the (60 %) was selected as the best ratio for precipitate of glucose oxidase, the specific activity reached to (10.304 U/mg) , with a purification fold (1.219) and yield (0.34) % , two peaks of glucoamylase were appeared in the gel filtration the specific activity of the first form (4.195 U/mg) with purification fold (1.436), while the second (31.214 U/mg) with purification fold (10.689), while one peak of glucose oxidase was appeared in the gel filtration, the specific activity was reached (62.382 U/mg) with purification fold (7.385). For characterization of enzymes, the results reported the highest activity of glucoamylase (Aand B) occurred in pH = 8, 6.5 (0.253U/ml, 0.511 U/ml) respectively , while the maximum activity of glucose oxidase (1.289 U/ml) in pH = 5.5, the optimum temperature of glucoamylase (Aand B) activity (0.243U/ml, 0.703 U/ml) in 40, 30 °C respectively , while the optimum temperature of glucose oxidase activity (1.424 U/ml) in 45 °C, the optimum pH for glucoamylase (B) stability ranging between (5.5 - 6.5), while the optimum pH for glucose oxidase stability ranging between (5 - 6) , the optimum temperature for glucoamylase (B) stability ranging between (10 - 35) °C for 30 min, while the optimum temperature for glucose oxidase stability ranging between (25 - 35) °C for 15min, the molecular weights of glucoamylase (Aand B) approximately 52 & 66 kDa respectively using the electrophoresis technique, while approximately 68 kDa for glucoamylase (B) using the gel filtration technique, the molecular weight of glucose oxidase approximately 78 and 87 kDa electrophoresis electrophoresis and gel filtration respectively, the values of Km and Vmax of glucoamylase and glucose oxidase were (2.4 mM , 9.6 mM/min and 19.6 mM, 7.5 mM/min) respectively. The result of this study showed the Streptococcus mutans growth was killed in the using the first and the second concentration of glucoamylase and glucose oxidase (0.09, 0.009 and 0.3, 0.03 µl) respectively , while the growth was appered in third concentration of enzymes (0.0009, 0.003 µl) respectively. The NaF, ZnF2 , C 12H 7 Cl 3 FNaO2 , NaCl , NaHCO3 and KSCN with (0.5 and 1M ) leads to increase the enzymes activity , while Na3PO4 , SrCl2 , Fe2O3 and Ca(HCO3) 2 caused decreased the activity of enzymes when treated with (0.5 and1 M) from these solutions, the glucoamylase activity also decreased when it treated with (0.5 and 1M) from KNO3 solution but this solution increased the activity of glucose oxidase when it treated with (0.5 and 1 M) from this solution
