Highly active metallocarboxypeptidase from newly isolated Geobacillus strain SBS-4S: cloning and characterization

The carboxypeptidase gene from Geobacillus SBS-4S was cloned and sequenced. The sequence analysis displayed the gene consists of an open reading frame of 1503 nucleotides encoding a protein of 500 amino acids (CBP(SBS)). The amino acid sequence comparison revealed that CBP(SBS) exhibited a highest homology of 41.6% (identity) with carboxypeptidase Taq from Thermus aquaticus among the characterized proteases. CBP(SBS) contained an active site motif (265)HEXXH(269) which is conserved in family-M32 of carboxypeptidases. The gene was expressed with His-Tag utilizing Escherichia coli expression system and purified to apparent homogeneity. The purified CBP(SBS) showed highest activity at pH 7.5 and 70°C. The enzyme activity was metal ion dependent. Among metal ions highest activity was found in the presence of Co(2+). Thermostability studies of CBP(SBS) by circular dichroism spectroscopy demonstrated the melting temperature of the protein around 77°C. The enzyme exhibited K(m) and V(max) values of 14 mM and 10526 μmol min(-1) mg(-1) when carbobenzoxy-alanine-arginine was used as substrate. k(cat) and k(cat)/K(m) valves were 10175 s(-1) and 726 mM(-1) s(-1). To our knowledge this is the highest ever reported enzyme activity of a metallocarboxypeptidase and the first characterization of a metallocarboxypeptidase from genus Geobacillus.