Purification and characterization of thermostable organic solvent-stable protease from Aeromonas veronii PG01

Abstract

A mesophilic bacterium, Aeromonas veronii PG01, isolated from industrial wastes produced an extracellular thermostable organic solvent tolerant protease. The optimum condition for cell growth and protease production was pH 7.0 and 30 °C. The protease produced was purified 53-fold to homogeneity with overall yield of 32%, through ammonium sulphate precipitation, ion-exchange and gel permeation chromatography (GPC). The molecular weight, as determined by GPC–HPLC, was found to be about 67 kDa. SDS-PAGE revealed that the enzyme consisted of two subunits, with molecular weight of 33 kDa. The protease was active in broad range of pH from 6.0 to 10.0 with optimum activity at pH 7.5. The optimum temperature for this protease was 60 °C. The enzyme remained active after incubation at 50–60 °C for 1 h. This enzyme was stable and active after incubation with benzene and it was activated 1.3- and 1.5-fold by n-hexane and n-dodecane, respectively. This protease was inhibited completely by the classic metalloprotease inhibitor 1,10-phenanthroline and partially by the metal chelator EDTA but not by the serine protease inhibitor PMSF. The PG01 protease was found to contain 1.901 mol of zinc per mole of enzyme upon analysis by Inductively coupled plasma-optical emission spectroscopy. The thermostable and solvent tolerance property make it an attractive and promising biocatalyst for enzyme mediated synthesis.