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انتاج انزيم الفوسفاتيز القاعدي من العزلة المحلية لبكتريا Bacillus sp. وتنقيته جزئيا
Author name:
جعفر انور قدوري الخاجي
Supervisor name:
رباب عمران راضي الجيلاوي
General topic:
Biology
Specific topic:
Biotechnologies
Degree:
Master
University:
University of Babylon - College Of Science - Department Of Biology
Language:
Arabic
University location:
Babylon
First pages:
24T2758 - p.pdf
Abstract:
Fifteen local isolates of Bacillus bacteria and previously isolated were screening a from laboratory of advanced biotechnology and Genetic Engineering in Babylon University, College of Science to know their ability on production of extracellular alkaline phosphatase after its growth in liquid production in PH8 medium for 4 days and temperature 37°C. and destinguished 11 isolates by their different abilities on produc of enzyme and Bacillus sp.I bacteria was their best and perfected it selection to complete the study and described by depending on microscopic and cultured and biochemical characteristics studying of effect some of environmental condition for production of alkaline phosphatase enzyme by submerged cultural method were appeared that the optimum PH for production was 8, and optimum temperature 40°C, after incubation time 4days and explain that the production by static incubator was better from shaker incubator.Alkaline phosphatase of the isolate Bacillus sp.I was purified partial by fractionating with ammonium sulphate and the best ratio for saturation 95%. And on purification of enzyme by ion exchange chromatography with batch wise by using DEAE - cellulose and enzymatic recovery was 37.1%, and fold 3.64 once, and enzyme passed after concentrated on gel filtration column (Sepharcyl S - 200) one peak was appeared for enzyme and enzymatic recovery was 23 % and fold 36.80 once.Results of electrophoresis by presense of denaturation condition appeared that the molecular weight of alkaline phosphatase produced from gel filteration by using Sepharcyl S - 200 gain approximately 29.5 KDa and 28.8 KDa and this enzyme composed of homodimer.The characterization results for the enzyme indicated that the optimal pH for activity of enzyme was (9), while the optimal pH for the enzyme stability was (8 - 9), and found that the optimal temperature for activity of anzyme was 30 ? C, and on studying optimal temperature for stability of enzyme indicated that better stability for this enzyme was on temperature 0 ?C while the activity lost completely on temperature 60°C. and on studying effect some chemical compounds in activity of enzyme found that the enzyme needs to cofactor and it is zinc andmagnessium ion to increase catalytic activity and needs to calessium ion to increase stability of enzyme. At that time potassium and sodium ions appeared a negatively effect on activity, and heavy metals ions Fe and Hg appeared an inhibited effect to activity and so that the enzyme was inhibited by presence EDTA and and potassium ferro cyanide indicating that the enzyme from metal enzymes.Determination of constant kinitics of the enzyme was completed of Michaelis - Menten constant (Km) of para - nitrophenyl phosphate and this gained 500 micromolar and maximum velocity of this enzyme (Vmax), and this gained 2500 unit/ml.
