Partial Purification and Characterization of Protease from Local Isolate of Beuveria bassiana

Abstract

Objectives: Entomopathogenic fungi were identified as effective alternative to chemicals in insect pest control. Extra cellular proteases of these fungi are implicated as components of the insect cuticle degradation process. This study aimed to the purification and characterization of a protease from a local isolate of the entomopathogenic fungus Beuveria bassiana (B. bassiana). Methods: The purification process of the protease from the culture filtrate of B. bassiana was carried out by dialysis, ethanol precipitation and DEAE-cellulose ion exchange chromatography. After that, the enzyme was characterized to establish the identity of the enzyme. Results: The protease was partially purified by 2.3 folds with 16.3 yield. The results of enzyme characterization showed that the Km values were (4.43, 6.26 and 6.66) mg/ml using casein, gelatin and bovine serum albumin as substrates respectively. Furthermore, the Vmax values were (16.3, 16.66 and 16.66) mg/ml.min for the same substrates respectively. The optimum pH for the protease activity was 9 with heat stability range of 20-40 C°. The effects of protease inhibitors and activators showed that the enzyme was totally inhibited by PMSF at 1 and 5 mM. This indicated that the protease belonging to serine proteases. Conversely, calcium chloride and magnesium chloride showed stimulating effect on the enzyme activity. Conclusion: : A protease was partially purified from an Iraqi isolate of B. bassiana. The enzyme can be used to degrade different types of insect cuticles as it showed a range of specificity toward different substrates.