Objective: The enzyme elastase is an important virulent factor of the opportunistic pathogen Aeromonas hydrophila. To know better about the molecular characterization of this enzyme, we purified the elatase and investigated its property and activity.
Methods: We cloned a structural gene ahyB encoding for the extracellular elastase of A. hydrophila J-1 and expressed it in E. coli BL21 by using pET-32a as vector. We purified the recombinant enzyme using His Bind Resin Purification Kit and recovered the elastolytic activity of the purified protein by incubation in a buffer containing 6 mol/L guanidine HCl and subsequent removal of denaturant by dilution. In addition, we also purified the native enzyme from the culture supernatant of A. hydrophila J-1 by 30 to 60% ammonium sulfate fractionation, anion exchange chromatography and sephaceryl chromatography. We compared the properties of the two elastase preparations.
Results: Native enzyme showed a pH optimum at 8.5, but recombinant enzyme at 10.0. Compared with the native enzyme, the recombinant enzyme was more stable for heat. Both the elastase preparations showed some identical properties concerning inhibitors,which were both mainly inhibited by EDTA, the cation chelator, and OPA, a Zn2+ /Fe2+ protease inhibitor. However,the recombinant elastase had higher tolerance than native enzyme against the inhibitors. And the metal cation Zn2+ and Fe2+ strongly inhibited the enzyme activity.
Conclusion: The recombinant product showed the similar enzymatic characteristics as the native enzyme from A. hydrophila J-1 and the two elastases belonged to the zinc-and-iron-dependant metalloendopeptidase. This is the first report on the recombinant expression and subsequent molecular chracterization analysis of A. hydrophila elastase.