A NOVEL THERMOSTABLE NEUTRAL PROTEINASE FROM SACCHAROMONOSPORA-CANESCENS

A novel thermostable neutral proteinase, called NPS, was purified to e lectrophoretic homogeneity from the culture broth of Saccharomonospora canescens sp. novus, strain 5, The molecular mass was determined by S DS-polyacrylamide gel electrophoresis to be 35 000Da, The enzyme exhib its a sharp pH optimum of proteolytic activity at pH 6.7, NPS was comp letely inactivated with inhibitors, typical for metalloendopeptidases, EDTA and 1,10-phenantroline, whereas the serine proteinase inhibitor PMSF had no effect, Atomic absorption measurements showed that the pro teinase binds a single zinc and four calcium ions, The enzyme thermost ability was characterized in the absence and presence of added calcium , Melting temperature, T-m = 77 degrees C and an activation energy, E- a, for the thermal deactivation of the excited protein fluorophores of 72.13 kJ mol(-1) were calculated in the presence of 100 mM CaCl2, The E-a-value is considerably higher than those obtained for a number of proteinases from microorganisms and was explained by the thermostable structure of the enzyme. Effective radiationless energy transfer from phenol groups to indole rings was observed, 68% of the light absorbed by tyrosyl residues is transfered to tryptophyl side chains. No homolo gy was found after comparison of the NPS N-terminal sequence, includin g the first 26 residues, with those of other neutral proteinases from microorganisms. In contrast to the well-known bacterial neutral protei nase thermolysin and related enzymes from microorganisms, NPS possesse s arylamidase and esterase activities, Further crystallographic studie s will reveal the structural reasons for this specificity. Epoxy and e pithio pyranosides are inhibitors of the proteinase arylamidase activi ty. (C) 1998 Elsevier Science B.V.