ELUCIDATION OF THE THERMAL-STABILITY OF THE NEUTRAL PROTEINASE-II FROM ASPERGILLUS-ORYZAE

The neutral proteinase II from Aspergillus oryzae (NpII) is a zinc pro teinase with three intramolecular disulfide bonds. NpII is most unstab le after 10 min at about 75 degrees C, but regains stability beyond th is temperature and is relatively stable at 100 degrees C. We analyzed the thermal stability of wild-type NpII and apo NpII. The results sugg ested that NpII unfolds reversibly upon incubation up to 100 degrees C , and that the irreversible inactivation observed is mainly due to aut oproteolysis. To further understand the stability, a mutant NpII (Cys( 78) --> Ala) lacking one of the disulfide bonds, was produced in a het erologous yeast expression system. The mutant NpII showed a similar st ability profile, but the most unstable temperature and the most cataly tically active temperature decreased to the same extent (around 10 deg rees C), confirming that autoproteolysis is the main cause of the irre versible inactivation. Several lines of evidence presented in this stu dy demonstrated that the thermal stability of NpII is attributed to re versible thermal unfolding and autoproteolysis.