Heterologous expression, refolding and characterization of a salt activated subtilase from Pleurotus ostreatus

The submerged cultivation of Pleurotus ostreatus var. florida on wheat gluten led to an increased solubility of the substrate. The coding sequence of one of the extracellular peptidases was obtained by PCR with degenerated primers and RACE. The sequence amounted to 1161 bp, translating into 386 aa with a molecular mass of 38.7 kDa. By comparison with homologous proteins the new enzyme was classified as a subtilisin-like peptidase of the proteinase K subfamily. P. ostreatus peptidase 1 (POP1) was expressed as a preproenzyme, whose 19 aa signal sequence was cleaved off during export into the extracellular space. The prosequence acted as an intramolecular chaperone and temporary inhibitor and was degraded in an autocatalytic process. The heterologous expression in Escherichia coli resulted in the formation of inclusion bodies, from which the peptidase was refolded by using β-cyclodextrin and CTAB. POP1 showed optima at pH 7.5 and 37 °C. It was most stable at pH 8 and 30 °C. The peptidase was completely inhibited by PMSF, antipain and Cu2+, while Ca2+ significantly enhanced its performance. The hydrolysis of N-Suc-AAPF-pNA was almost tripled in the presence of 5 M NaCl.