Cloning and expression of an intracellular alkaline protease gene from alkalopihilic thermoactinomyces sp. Η8682

Abstract

An intracellular alkaline serine protease gene of alkalophilic Thermoactinomyces sp. HS682 was cloned and expressed in Escherichia coli. Sequence analysis showed a putative promoter region, a putative transciptional termination signal, and an open reading frame of 963 bases, coding for a polypeptide of 321 amino acids. The protease expressed in E. coli was purified by DEAE-Toyopearl 650M and Sephadex G-75 chromatography. The N-terminal sequence (30 amino acids) of the purified protein was coincident with Aspl6–Val45 of the deduced amino acid sequence of the ORF. Fifteen amino acids in the N-terminal region were removed during the purification procedures. The deduced amino acid sequence showed high similarity with microbial intracellular serine proteases. The molecular mass of this enzyme was estimated to be 38 kDa by SDS–PAGE. The enzyme was stable at pH 6.0–12.0 and below 60°C in the presence of Ca2 +. The temperature and pH optima of the enzyme were 65°C and pH 11.0, respectively. The enzyme was inhibited by DFP and PMSF, but not by MIA and EDTA. © 1997, Taylor & Francis Group, LLC. All rights reserved.