L-asparaginase production from bacteria and using it in cancer cells inhibition. 3.Purification and characteraization for L-asparaginase  from Erwinia carotovora MM3

O. Mohhideen, Mohammed

	L-asparaginase production from bacteria and using it in cancer cells inhibition. 3.Purification and characteraization for L-asparaginase from Erwinia carotovora MM3
Tikrit Journal of Pure Science
Article 1, Volume 16, Issue 1, 2011, Pages 147-159
Author
Mohammed O. Mohhideen
Abstract
The aim of this study is to Purification and characteraization for L-asparaginase from Erwinia carotovora MM3 and study its kinetics and effective factors in enzyme production .The purification of L-asparaginase by ammonium sulfate precipitation was done , the yield and purification number was 44% and 1.46 times respectively. Ion exchange by DEAE-sephacyl was done , the yield and purification number was 42.57% and 3.05 time respectively. The gel filtration by sephacryl S-300 gel showed two peaks for enzyme,s activity, ERI and ERII , the yield and purification number for ERI was 21.6% and 3.36 times respectively, the yield and purification number for ERII 32.17% and 5.03 times respectively . each peak was separated by gel filtration in the same conditions ERI fraction has a yield and purification number 18.30% and 7.92 times respectively, while ERII has a yield and purification number 29.19% and 12.50 times respectively. Both ERI and ERII are running on Polyacrylamide Gel Electrophoresis PAGE (7%) to explain the purity of enzymes , ERI has one band while ERII has three bands. The molecular weight for L-asparaginase was determined by gel filtration and PAGE-SDS showed the different values according to the useful method . The molecular weight for ERI and ERII was 39800 and 10200 daltons respectively by gel filtration method and 45700 and 16600 daltons respectively in PAGE-SDS method. The optimum temperature for ERI and ERII was 30°C for both enzymes, the activation energy for ERI was 13.5 X 103 cal/mole and ERII was 10.9 X 103 cal/mole , while the activation energy for denaturation for ERI was 4.6 X 103 cal/mole and ERII was 9.4 X 103 cal/mole . The optimum pH for both enzymes was neutral equal 7.00 , ERI keeps all its activity when treated by pH = 7.00 for 60 min. while ERII keeps all activity when treated by pH= 7.00 for 30 min. Km for ERI was 6 X 10 –3 molar and ERII was 3 X 10 – 3 molar , V max foe ERI was 3.2 molar/ min. and ERII was 6.1 molar/ min when they were estimated by Michaelis – Menten plot method. Both ERI and ERII resistance NaCl and KCl in 0.2% concentration where keeps all activity in this concentration, the activity of ERI and ERII decreased when increased concentration of EDTA and 2- mercaptoethanol.

Statistics Article View: 106 PDF Download: 91