Purification and properties of a novel fungal alkaline keratinase from Cunninghamella echinulata

Abstract

Aim: The purification and characterization of an alkaline keratinase from Cunninghamella echinulata. Materials and Methods: Feather-meal medium was used to isolate and screen the fungi. Acetone precipitation and lectin agarose affinity column were used in the purification. The effect of pH, temperature, metal ions and group modifying chemicals was tested. Results: The purified keratinase is a serine protease with a molecular mass of 33kDa with an optimal pH of 4.5 and 10.0, and an optimum temperature of 30°C and 60oC. A 13.2-fold purification was obtained with affinity methods. Discussion: C.echinulata keratinase was inhibited by PMSF, thus it could be a serine pro- tease. Enzyme was inhibited by group specific reagents like TLCK, IAA, NEM and NAI indicating that serine, cysteine, tyrosine and lysine play an important role in the catalytic activity. There was no effect of metal-chelating agent on enzyme activity indicating that the enzyme is not a metalloenzyme; however, it is a metal-activated enzyme as the activity was enhanced by Mn²+. Inhibitory effects of group specific reagents indicated that the enzyme is a serine protease which does not have any divalent ion requirement. The enzyme isolated also has appreciable activity at two different temperatures and pH values making it a versatile organism for industrial applications.