از باكتري باسيلوس آميلوليكوفاسينس BAM H I جداسازي تخليص آنزيم اندونوكلياز

Isolation, Purification of Endoneuclease BamH1 from Bacillus Amyloliquefaciens

Objective: The aim of study was to introduce a new method for purification of the enzyme BamHI endonuelease from bacillus amyloliduetaciens which can be used in molecular biology and genetic engineering laboratories. Materials and Methods: The BamH1 specific endonuclease was purified from Bacillus amyloficluefaciens (BS2) culture. The productive bacteria grown in specific medium (SLBH) were collected at the end of log phase. The cells were lysed by sonication and the extract was collected. The enzyme was precipitated from cell extract by ammonium sulfide at 45-50% and subsequently dialyzed against 25mM phosphate buffer. It was then concentrated with PEG8000 and was further purified using gel filtration and ion-exchange chromatography. The product obtained there by was analyzed by SDS-PAGE. The specific activity of the enzyme was determined by digestion of A phase DNA. Results: The results indicate that the maximum production of the enzyme was at the end of log phase. Also the cell mass obtained from bacterial culture was more than those reported earlier. The high enzyme activity in this research was due to application of shortened protocol. Conclusion: The results obtained indicate that the enzyme production in the above medium by the bacteria was satisfactory. The purity of the enzyme was 90%. The enzyme exhibited about 80% specific activity.