Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft

Ak,1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB551 7. It is active against substrates containing neutral or hydrophobic branch ed-chain amino acids at the P-1 site, such as valine, alanine or phenylalan ine, The K-m and k(cat) of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specifi city changes with temperature. The protease is markedly stabilized by Ca2ions. At 70 degrees C, a in-fold increase in Ca2+ concentration increases t he half-life by three orders of magnitude. Ak.1 protease is stabilized by C a2+ to a greater extent than is thermitase. This may be due, in part, to th e presence of an extra Ca2+-binding site in Ak.1 protease. Other metal ions , such as Sr2+, increase the thermostability of the enzyme, but to a signif icantly lower degree than does Ca2+. The structure of the protease showed t he presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide b ond appears to have a dual role: maintaining the integrity of the substrate binding cleft and increasing the thermostability of the protease. The prote ase was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored furth er.