Vol. 5, Issue 12 (2016)

Author(s):
Khaled Aziz Hashem, Sawsan Hassan Authman and Luqaa Hameed Mahdi

Abstract:
A hundred and fifty samples of stools were collected from patients suffering diarrhea. Hundred and thirty bacterial isolated (86.7%) were identified 84 (64.6%) belonged to Escherichia coli, 23(17.7%) to Klebsiella pneumoniae, 10 (7.7%) Enterobacter cloacae, 7 (5.4%) to Shigella dysenteriae, 4(3.1%) to Salmonella typhi and 2(1.5%) to Pseudomonas aeruginosa. The ability of E.coli isolates to produce alkaline phosphatase enzyme was tested on MM agar medium along with pNPP. ALP was extracted, the crude extract activity and specific activity was 1.86 unit/ml and 20.44 unit/mg protein respectively. The enzyme purified by precipitating with ammonium sulfate (50-75%) saturation then using ion-exchange chromatography in DEAE-Cellulose ionic exchange column and gel filtration chromatography by using sepharose-6B gel filtration column, to obtained pure ALP enzyme with specific activity 272.73unit/mg, with 13.34 fold purification and yield of enzyme 56.45%. The in vivo investigation for the effect of ALP enzyme on the infection with P. aeruginosa was done using BLBC mice. The results shows that ALP degreased the number of bacteria in liver, spleen and lung with significant differences (P<0.05). Also the results shows a significant differences (P<0.05) in elevated levels of cytokines (IL-6, IL-10 and IL-12) compering with control group.