Protein and Peptide Sciences (Track)
Active site engineered thermostable L-asparaginase with anti-cancer activity
Saurabh Bansal
Department of Biochemical Engineering & Biotechnology
Indian Institute of Technology, Delhi
Abstract:
Thermophilic enzymes are characterized by their capacity to withstand extremes of physicochemical conditions and displaying optimum activity at high-temperatures. Although stable enzyme is always a desirable characteristic but use of thermophile-derived enzymes for therapeutic purposes invites serious criticism. This is because they fail to act optimally at physiological conditions (temperature and pH) as opposed to mesophilic enzymes. To overcome the problem, we attempted to combine thermophilic scaffold stability with mesophilic enzymatic activity. For this purpose an L-asparaginase derived from hyperthermophile Pyrococcus furiosus (PfA) was cloned, expressed and purified from E.coli host. We made three active sites mutants (two single and one double) of PfA through comparative structural and sequence analysis of mesophilic and thermophilic counterparts. In addition to high substrate affinity and activity at physiological conditions, the mutants were found to be stable and free of any glutaminase activity (a desirable characteristic to reduce side effects). Most significantly, the strikingly high anti-proliferative activity of the mutants (in particular K274E and T53Q) on leukemic cell lines as compared to E.coli asparaginase led us to propose these mutants as potential anti-leukemic drug.
