Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence

The gene encoding subtilisin-like protease T, kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T . kodakaraensis subtilisin is a member of the subtilisin family and compose d of 422 amino acid residues with a molecular weight of 43,783, It consists of a putative presequence, prosequence, and catalytic domain. Like bacteri al subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilize d in the presence of 8 M urea, and refolded and converted to an active mole cule. However, unlike bacterial subtilisins, in which the prosequence was r emoved from the catalytic domain by autoprocessing upon refolding, T, kodak araensis subtilisin was refolded in a form with a putative prosequence. Thi s refolded protein of recombinant T. kodakaraensis subtilisin which is comp osed of 398 amino acid residues (Gly(-82) to Gly(316)), was purified to giv e a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and ch aracterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtili sin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the p resence of the Ca2+ ion with an optimal pH and temperature of pH 9.5 and 80 degreesC, Like bacterial subtilisins, it showed a broad substrate specific ity, with a preference for aromatic or large nonpolar P1 substrate residues . However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of > 60 min at 80 degreesC, 20 min at 90 degreesC, and 7 min at 100 degreesC.