Gene cloning and characterization of recombinant L-Asparaginase from Bacillus subtilis strain R5

L-asparaginase gene from Bacillus subtilis strain R5 (Asn-R5), comprising 990 nucleotides corresponding to a polypeptide of 329 amino acids, was cloned and expressed in Escherichia coli. Recombinant Asn-R5 was produced in soluble and active form exhibiting a specific activity of 223 mu mol min(-1) mg(-1). The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 A degrees C and 9.0, respectively. Asn-R5 displayed a 50% activity with D-asparagine and 2% with L-glutamine compared to 100% with L-asparagine. No activity could be detected when D-glutamine was used as substrate. Half-life of the enzyme was 180 min at 35 A degrees C and 40 min at 50 A degrees C. There was no effect of metal ions and EDTA on the activity indicating that Asn-R5 enzyme activity is not metal ion dependent. The K (m) and V (max) values were 2.4 mM and 265 mu mol min(-1) mg(-1), respectively. Activation energy for reaction catalyzed by Asn-R5 was 28 kJ mol(-1). High L-asparaginase activity and thermostability of recombinant Asn-R5 may be beneficial for industrial production and application.