L-asparaginase is an important enzyme with diverse applications in food industry and therapeutics. However, the enzyme currently employed in the treatment of leukaemias, comes with undesired L-glutaminase activity. A gene encoding 38 kDa L-asparaginase form Anoxybacillus flavithermus was cloned and expressed in Escherichia coli as a soluble and active enzyme. Heat treatment and Ni-affinity column chromatography provided highly purified enzyme possessing a specific activity of 165 units mg. The enzyme exhibited allosteric behaviour with a Hill coefficient of 1.60 and K of 25 mM with L-asparagine as specific substrate. No detectable activity was observed in the presence of D-asparagine, l-glutamine and d-glutamine. Purified AfASNase showed optimum activity at 60 °C and pH 7.0. The enzyme had ability to withstand up to 6 M urea and showed complete inactivation when treated with 1 M guanidine hydrochloride. Protein-Ligand docking and molecular dynamic simulations indicated that the regulatory site is formed by T262-T263-C265-G269-Thr294 and is located on a domain different from the one carrying the well-established active site. AfASNase is reported as first thermostable L-asparaginase with allosteric regulation. Hitherto, AfASNase presents the first characterization of recombinant L-asparaginase from the genus Anoxybacillus.
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