ABSTRACT
Proteolytic enzymes have an important role in variety of physiological and pathological functions. They have been used in therapeutic and pharmaceutical applications. Characterizations of extracellular proteases from various strains of S. marcescens indicate that most strains produce a very similar major metalloprotease. This metalloprotease [serrapeptidase, serrapeptase] is an important pharmaceutical agent. Serrapeptase has been used in Asian and European countries for the treatment of inflammatory diseases, cardiovascular disorders, and bacterial infections. In the present study, purification and characterization of extracellular metalloprotease from Serratia sp. ZF03 for therapeutic purposes were reported. In this study the protease gene encoding a zinc-metalloprotease was isolated from the previously isolated red-pigmented Serratia sp. ZF03. The gene was sequenced and submitted to the GenBank. Proteolytic activity was detected by skim milk agar plate method and zymography. This fragment was found to encode an extracellular zincmetalloendopeptidase with a molecular weight of approximately 50 kDa. The metalloprotease was purified by ammonium sulfate precipitation and dialysis, and then characterized. The effects of various inhibitors and reagents on protease activity and its kinetic parameters were also determined. The nucleotide sequence demonstrated that deduced amino acid sequence has a higher identity with those of metalloprotease from serralysin family. Production of metalloprotease was highest at 48[th] h of cultivation. Optimum protease activity occurred at a temperature range of 50-55[degree sign]C and a pH range of 8.0-10. EDTA as a metal chelator, significantly inhibited protease activity. Zymography and inhibition assays showed that metalloprotease is the major secreted protease of Serratia sp. ZF03. The kinetic parameters, K[m] and V[m], were 0.00105 mg/ml and 0.0531 mM/min, respectively. Since the metalloprotease of this strain has strong proteolytic properties and good stability, it would be a suitable candidate to be used as an effective drug in the medicine and pharmaceutical industries