Purification and Characterization of a Membrane-Unbound Highly Thermostable Metalloprotease from Aeromonas Caviae

Abstract

The main objective of the current study was to purify and characterize an alkaline thermostable metalloprotease from Aeromonas caviae. The enzyme was subjected to a two-step purification scheme using ammonium sulfate [(NH4)2SO4] fractionation and ion-exchange chromatography on a DEAE-sepharose fast flow column. The fraction which precipitated with 40–60 % (w/v) of [(NH4)2SO4] exhibited the highest enzyme activity. Anion-exchange chromatography resulted in approximately 51-fold purification, with a yield of 64.7 %. The enzyme was successfully purified to homogeneity, and this was confirmed on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). A single band with an approximate molecular mass of ∼42 kDa was observed. The maximum enzymatic activity was observed at pH 8 with azocasein as the substrate, and the enzyme was stable in the pH range of 6–10 after 5 h of incubation. The purified enzyme was active in the temperature range between 50 and 60 °C and showed 80 % loss in enzymatic activity upon heating at 70 °C. The K m and V max values for the purified protease, with azocasein as substrate under optimal reaction conditions (pH 8 and 50 °C) were 0.74 and 122.4 mg/ml, respectively. The protease was investigated for its application for the degradation of chicken feathers. The high enzymatic activity, pH, and thermal stability of AU04 protease make it an industrially important enzyme.