A PEPSTATIN-INSENSITIVE ASPARTIC PROTEINASE FROM A THERMOPHILIC BACILLUS SP

Bacillus sp. strain Wp22.A1 produced a cell-associated aspartic protei nase which was purified to homogeneity using phenyl-Sepharose (hydroph obic and affinity chromatography) and Mono Q. The proteinase has a mol ecular mass of 45 kDa by SDS/PAGE and a pI of 3.8. It is insensitive t o pepstatin, but is sensitive to the other aspartic proteinase-specifi c inhibitors diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epox y-3-(p-nitrophenoxy)propane. Inactivation by DAN was only partial, sug gesting that it had non-specifically modified an aspartate residue at a site other than the active site. The enzyme was not inhibited by any of the serine or cysteine proteinase inhibitors tested. Maximum prote olytic activity was observed at pH 3.5. The proteinase had a higher ac tivity with haemoglobin, but was more specific (V-max/K-m) for cytochr ome c. Substrate inhibition was observed with both these substrates. T he cleavage of oxidized insulin B chain tended to occur at sites where the P1 amino acid was bulky and non-polar, and the P1' amino acid was bulky and polar, such as its primary cleavage site of Val(2)-Asn(3). The proteinase was stable in the pH range 2.5-5.5. Thermostability was increased in the presence of Ca2+, although to a lesser extent at hig her temperatures. The thermostabilities at 60, 70, 80 and 90 degrees C were 45 h, 102, 21 and 3 min respectively in the presence of Ca2+.