Purification and Characterization of α – Amylase Produced by The Local Isolate Xanthomonas campestris H6

number: 
1145
إنجليزية
Degree: 
Author: 
Yaarub Raji Al-Hussuna
Supervisor: 
Dr.Majed H.. Al-Gelawi
Dr.Hamid M.Al-Dulaimi
year: 
2005

    The ability of Xanthomonas campestris strain H6 for the production of α – amylase  was studied and it was found that Xc H6 is capable of producing the e xtracelluar α – amylase enzyme.  Optimal conditions for the α – amylase production in PM1 medium were studied and the higher activity of this enzyme was obtained in PM1 medium  supplemented with  glucose 3.5 %, peptone 2 %, K2HPO4 2.5 %    at 30 ºC and pH 7.0.   Results obtained demonstrate that α – amylase  produced by Xc H6 is a constitutive enzyme that is not subjected to catabolite repression by glucose like many other amylases.This enzyme was initially purified by ammonium sulphate precipitation with saturation ratio or 60 – 80 % which gave a specific activity of 12.97 U/mg with 1.4 purification fold, then it was purified by gel filtration using Sepharose CL – 6B which resulted in specific activity of 42.63, 4.7  purification fold and an overall yield of 21.69%.   Purity of the  purified α – amylase was confirmed using poly acrylamide gel electrophoresis under non denatured condition which demonstrated only single protein band.  Characterization experiments showed that the molecular weight of the α – amylase produced by Xc H6 is about 14000 Dalton, on the other hand it was found that  optimum pH for the activity of the   purified enzyme  was 7.5 and the enzyme retained its complete activity when it was incubated at pH 6.5 and 7.0 and it retained more that 90% of its activity at pH 7.5.    Thermostability of the  purified α – amylase was tested and the enzyme showed 100% of its activity at 30, 35 and 40 ˚C and about 95% at 45 ˚C. Activity of the enzyme was 50% when it was incubated at 60 ˚C.    The effect of chelating agents on α – amylase activity was investigated and results showed that there was no significant decrease in the activity of the  α – amylase when it was incubated with different concentrations of EDTA, indicating that chelating agent have no effect on α – amylase enzyme and its is a non – metalloenzyme.