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 degrees 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 nl: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 degrees C. These data suggest that protease Q is a new member of the subtilisin family of endopeptidases.