[50] Thermolysin and other neutral metalloendopeptidases

Thermolysin from Bacillus thermoproteolyticus and the neutral proteases of Bacillus subtilis are neutral metalloendopeptidases having similar size, metal content, amino acid composition, and substrate specificity. Recent evidence indicates that their amino acid sequences have regions of identity, suggesting that these enzymes have diverged from a common ancestor. Problems of self-digestion, which have been encountered in the purification of these and other proteolytic enzymes, can be minimized by rapid procedures of affinity chromatography. Because Cbz-Gly-D-Phe is a competitive inhibitor of both enzymes, affinity adsorbents were developed using Gly-D-Phe covalently attached by appropriate spacers to a Sepharose matrix. The enzymes were selectively adsorbed at low pH and eluted by raising the pH. Three effective adsorbents have been described that can be used interchangeably for thermolysin and for neutral protease and differ only in the length of their spacers. Preparation of the simplest adsorbent is described in this chapter.