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

Abstract

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 μmol min−1 mg−1. The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 °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 °C and 40 min at 50 °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 Km and Vmax values were 2.4 mM and 265 μmol 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.