Pcal_0970: an extremely thermostable l-asparaginase from Pyrobaculum calidifontis with no detectable glutaminase activity

The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with l-asparaginases. In search of a thermostable l-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100 degrees C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing d-asparagine with a 20% activity as compared to 100% with l-asparagine. Pcal_0970 did not show any detectable activity when l-glutamine or d-glutamine was used as substrate. Pcal_0970 exhibited a K-m value of 4.5 +/- 0.4mmol/L and V-max of 355 +/- 13molmin(-1)mg(-1) towards l-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 +/- 0.6kJmol(-1). To the best of our knowledge, Pcal_0970 is the most thermostable l-asparaginase with a half-life of more than 150min at 100 degrees C and this is the first report on characterization of an l-asparaginase from phylum Crenarchaeota.