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PURIFICATION AND CHARACTERIZATION OF POLYMORPHONUCLEAR LIPASES FROM MASTITIS MILK OF COWS | ||
| IRAQ JOURNAL OF AGRICULTURE | ||
| Article 1, Volume 14, Issue 4, 2009, Pages 0-0 | ||
| Authors | ||
| A. J. Al- Mosowy; K. M. Azez; A. M. A. Al. Salih | ||
| Abstract | ||
| The obtained lipases extract of PMN were purified by ion exchange chromatography through DEAE–cellulose (1.5×28) cm and gel– filtration chromatography using sephadex (G-50) column (1.5 ×32) cm.Three enzymes (A, B, C) were found with a yield of 48.97, 23.87 and 37.16%, purification fold were 24.19, 13.30 and 19.01 times and with specific activities of 63.63, 35 and 50 unit/mg protein respectively. The purity of enzymes were examined by polyacrylamide gel electrophoresis. The purified enzymes were characterized by determining molecular weight of the enzymes A, B, C which were 6000, 13600, 15800 Dalton respectively when estimated by gel filtration using sephadex G-50 column. The optimum pH for enzyme activity was 7.5 for A and 6.5 for both B and C enzymes, while the Optimum pH for enzymes stability was 6-8, 5.5-7.5 and 6-7 for enzyme A, B and C respectively. The optimum temperature for enzyme activity was 40, 30 and 45oC for A, B and C respectively. Enzyme A retained 100% of its original activity over 30 min incubation at 20-40 oC and lost 62% of activity at 70oC, while enzyme B was stable for 30 min at 20-37 oC and lost 80% of activity at 60oC, and enzyme C was stable for 30 min at 20-50 oC and lost 25% of its activity at 70oC. The Activition energy of conversion were 9.57, 10.68 and 6.58 Kcal/mol for A, B and C enzymes respectively.Calcium and Magnesium chlorides had slight effect at concentration of 1, 5 mM, while copper, iron and mercury chlorides had an inhibitory effect at the same concentration. PMSF had no effect on enzymes activity when incubated at these two concentration, while the activity was slighty inhibited by the chelating agents such as EDTA, the activity was also inhibited significantly by the presence of 2- Mercaptoethanol. The purified A, B and C enzymes were found to contain glycoprotein which rated 19.25, 15.47 and 28.65% carbohydrate respectively. | ||
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