Comparative evaluation of different cell disruption methods for the release of L-asparaginase from Erwinia carotovora MTCC 1428 Sarita, Azmi Wamik* Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, Himachal Pradesh, India *E-mail of corresponding author: wamikazmi@rediffmail.com
Online published on 9 November, 2012. Abstract L-asparaginase (E.C. 3.5.1.1.) is a therapeutic enzyme extensively used in the treatment of acute lymphoblastic leukemia and its clinical preparations are of microbial origin. However, many Gram's negative bacteria do not typically excrete proteins into the culture medium since the cell wall is covered by an outer membrane. L-asparaginase obtained from bacterial culture Erwinia carotovora MTCC1428 is an intracellular enzyme, hence cell disruption is mandatory to release the enzyme for further purification. Cells of E. carotovora were lysed by different physical (vortex, pestle and mortar, sonicator, Bead Beater), chemical (alkali lysis, acetone powder, guanidine-HCl and triton X-100) and enzymatic (lysozyme) methods. Among various methods evaluated, L-asparaginase release was found to be the best when cells were disrupted by sonication. Different parameters considered important include cell concentration, cell volume, number of cycles and amplitude of sonication that were optimized for the release of enzyme. The recovery of enzyme was found 70% and the specific activity of the released L-asparagianse was found to be 0.16 U/mg of protein with sonication. Moreover, the loss in enzyme activity was also found to be minimum (25%) when E. carotovora MTCC1428 cells were disrupted by sonication. Top Keywords L-asparaginase, Erwinia carotovora MTCC1428, Intracellular enzyme, Cell disruption, Sonication. Top |