EXTRACTION, PURIFICATION AND CHARACTERIZATION OF EXTRACELLULAR PULLULANASE BY KLEBSIELLA PNEUMONIAE ISOLATED FROM SOIL

Abstract

Many soil samples collected from different locations in Mosul city in Iraq showed existence of bacterial isolates
produce pullulanases, seven isolates were obtained and the most prominent one was denoted No5 in terms of the
decomposition halo diameter, which reached 8.46 mm, and when the isolates subjected to primary screening by
cultured on pullulan-supported medium it was identified as Klebsiella pneumonia after a number of diagnostic and
biochemical tests. A number of sequenced steps for enzyme purification included precipitation with 75% saturation
of ammonium sulphate which record enzyme activity 93.05 U/ml, followed by dialysis step for 24 hours. The
purification step by using Sephadex-G100 gave 19.013% as enzymatic yield with 74.581 folds. It is cleared by using
electrophoresis, that the molecular weight of the enzyme was 94 KD. By studying some enzyme characteristics, the
results showed that optimum pH for activity was 6 while the optimum pH for stability was 6-7, and the optimum
temperature was 60⁰C, while 95% of enzyme activity was retained at 50-60⁰C.