A novel extracellular subtilisin-like protease from the hyperthermophile Aeropyrum pernix K1:: biochemical properties, cloning, and expression

A novel extracellular serine protease designated Pernisine was purified to homogeneity and characterized from the archaeon Aeropyrum pernix K1. The molecular mass, estimated by SDS-PAGE analysis and by gel filtration chromatography, was about 34 kDa suggesting that the enzyme is monomeric. Pernisine was active in a broad range of pH (5.0-12.0) and temperature (60-120 degreesC) with maximal activity at 90 degreesC and between pH 8.0 and 9.0. In the presence of 1 mM CaCl2 the activity, as a function of the temperature, reached a maximum at 90 degreesC but at 120 degreesC the enzyme retained almost 80% of its maximal activity. Activity inhibition studies suggest that the enzyme is a serine metalloprotease and biochemical data indicate that Pernisine is a subtilisin-like enzyme. The protease gene, identified from the sequenced genome of A. pernix, was amplified from total genomic DNA by PCR technique to construct the expression plasmid pGEX-Pernisine. The Pernisine, lacking the leader sequence, was expressed in Escherichia coli BL21 strain as a fusion protein with glutathione-S-transferase. The biochemical properties of the recombinant enzyme were found to be similar to those of the native enzyme.