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 (CBPSBS). The amino acid sequence comparison revealed that CBPSBS exhibited a highest homology of 41.6% (identity) with carboxypeptidase Taq from Thermus aquaticus among the characterized proteases. CBPSBS contained an active site motif 265HEXXH269 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 CBPSBS 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 Co2+. Thermostability studies of CBPSBS by circular dichroism spectroscopy demonstrated the melting temperature of the protein around 77°C. The enzyme exhibited Km and Vmax values of 14 mM and 10526 μmol min− 1 mg− 1 when carbobenzoxy–alanine–arginine was used as substrate. kcat and kcat/Km 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.