The present invention relates to a novel mutant of L-asparaginase enzyme characterized in having high thermostability, pH stability and no glutaminase activity useful for therapeutics and the process of preparing the same. The present invention specifically relates to mutant's MTCC 5580, MTCC 5581 and MTCC 5582 characterized in having higher stability, no glutaminase activity etc., to allow their usage in the form of improved protein therapeutics. A thermostable L-asparaginase from P. furiosus was cloned and expressed in E. coli host. The enzyme was engineered at its active site to create three different mutants based on structural and sequence analysis with a E. coli—derived enzyme homologue. The mutants MTCC 5580, MTCC 5581 and MTCC 5582 were tested for their stability, substrate affinity, optimum pH and temperature of activity and cytotoxicity. Based on the studies, all the three enzymes were found thermostable and with no glutaminase activity as compared to other available enzyme EcA II. MTCC 5579 and the above said three mutants showed the cytotoxicity on the leukemic cell lines. The present study showed that these enzymes are promising candidates for the treatment of leukemia.
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