A NOVEL THERMOSTABLE L-ASPARAGINASE WITH A PROMISING ANTI-CANCER ACTIVITY
Bishwajit Kundu
Indian Institute of Technology, School of Biological Sciences, Block-1, IIT Delhi Hauz Khas, New Delhi-110016, India
Abstract:
As far as chemotherapy of leukemia is concerned, the enzyme L-asparaginase holds the center stage. In spite of availability from various sources, research inclined towards making more stable, glutaminase-free, L-asparaginase has drawn attention of researchers. To contribute to the field we cloned L-asparaginase from Pyrococcus furiosus and expressed in E. coli. In order to combine properties of stability, specificity, high substrate affinity and overall antineoplastic activity, three active site (two single mutants K274E and T53Q, and one double mutant K274E/T53Q) mutants of the enzyme were developed. A detailed study of the stability, activity, and cytotoxicity properties of the enzymes were carried out after purification from cloned E.coli host. All the four variants were found to be lacking glutaminase activity, displayed wide range of temperature, pH stability and considerable substrate affinity. Additionally, all the enzymes were found to be cytotoxic against leukemic cell lines (HL60 and K562) as well as breast cancer cell line (MCF-7). Among the enzymes, K274E and T53Q were found to be more promising with higher cytotoxic effect and low LC50. These singly mutated enzymes may serve as additional and alternative chemotherapeutic agents.
