Minor thermodtable alkaline protease produced by Thermoactinomyces sp. E79

Abstract

Thermoactinomyces sp. E79 produced two types of thermostable alkaline proteases extracellularly. A minor protease was separated from a major protease by using DEAE-column chromatography. This enzyme was purified to homogeneity by ammonium sulfate and DEAE-Sepharose ion-exchange chromatography. The purified minor protease showed different biochemical properties compared to the major protease. The molecular mass of the purified enzyme was estimated by SDS-PAGE to be 36 kDa. Its optimum temperature and pH for proteolytic activity against Hammarsten casein were 70°C and 9.0, respectively. The enzyme was stable up to 75°C and in an alkaline pH range of 9.0-11.0. The enzyme was inhibited by phenylmethylsulfonyl fluoride (PMSF) and Hg2+, indicating that the enzyme may be a cysteine-dependent serine protease. In addition, the enzyme cleaved the endoproteinase substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and the K(m) value for the substrate was 1.2 mM.