Purification and Characterization of Protease Q: A Detergent- and Urea-Stable Serine Endopeptidase from Bacillus pumilus

An extracellular endopeptidase from a strain of Bacillus pumilus displaying high stability in 10% (w/v) sodium dodecyl sulfate (SDS) and 8 M urea has been purified and characterized. The enzyme, named protease Q, is a basic protein (pI = 9.35) composed of 309 amino acids with a molecular weight of 31 100. It is stable at 25 °C in the pH range 5−12 and most active at pH 10.5. Like other bacterial subtilisins, protease Q contains no cysteine and cystine residues. Protease Q possessed neither esterase nor amidase activity on synthetic substrates with arginine at position 1 and substrates of trypsin-like proteases. However, the enzyme showed strong activity on substrates with phenylalanine at position 1, substrates for chymotrypsin-like proteases. It also exhibited keratinase-like activity on keratin in 8 M urea. Inhibition studies indicated that protease Q was not sensitive to either trypsin-specific inhibitors, such as tosyllysine chloromethyl ketone (TLCK) and soybean trypsin inhibitor (SBTI), or chymotrypsin-specific inhibitors, such as tosylamido-2-phenylethyl chloromethyl ketone (TPCK). However, it was irreversibly inhibited by serine protease inhibitors such as PMSF. Although the N-terminal sequence of protease Q exhibited a high degree of homology with those of subtilisin Carlsberg and subtilisin BPN‘, it did not exactly match with any of the known enzymes in the protein data bank. The specificity of protease Q on the oxidized insulin B-chain was distinctly different from those of subtilisin Carlsberg and proteinase K. One of the distinguishing properties of protease Q is that it is remarkably more stable than subtilisin Carlsberg in 5% SDS at 50 °C. These data suggest that protease Q is a new member of the subtilisin family of endopeptidases.