Purification and partial characterization of chymotrypsin-like proteases from the digestive gland of the scallop Pecten maximus

Abstract

Three variants of a chymotrypsin-like protease were purified from scallop digestive glands successively by ion-exchange, gel filtration and high-performance liquid chromatographies. Enzyme activity was detected using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a specific synthetic substrate for chymotrypsin. This proteinase was inhibited by chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. Estimated molecular mass of the purified enzyme is around 32 kDa. These isoenzymes exhibit very low activities in hydrolyzing small synthetic specific substrates used for trypsic, elastolytic and collagenolytic measurements and referred mainly to a chymotrypsin-like proteinase. Very few differences were measured concerning pH profiles among the three isoenzymes. Stability is higher at low temperature for two variants. An N-terminal analysis was performed on one variant (B) among the three isoenzymes. The alignment of the N-terminal amino acid sequence indicates some homologies with abalone chymotrypsin-like protein and arthropod chymotrypsin proteases as well as with vertebrate serine protease counterparts (trypsin, chymotrypsin and elastase).