Comparative study on thermal stability of trypsin from the pyloric ceca of threadfin hakeling (Laemonema longipes)

Trypsin was purified from the pyloric ceca of threadfin hakeling (Laemonema longipes. ). Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Activity of the enzyme was inhibited by serine protease inhibitors, such as soybean trypsin inhibitor, N. ^α-tosyl-L-lysine chloromethyl ketone and squid trypsin inhibitor. The molecular weight of the trypsin was estimated to be 24 kDa by SDS-PAGE. The N. -terminal amino acid sequence of the trypsin, IVGGKECAKHSQRHQVSLNS, was found, and the sequential identity between the threadfin hakeling trypsin with Frigid Zone fish trypsin was relatively higher (ca. 80%) than Temperate Zone fish trypsin (ca. 74%), Tropical Zone fish trypsin (ca. 75%) and mammalian trypsin (ca. 58%). The trypsin had maximal activities at around pH 8.0 and 50C for hydrolysis of N^α-p-tosyl-L-arginine methyl ester hydrochloride. The trypsin was unstable at below pH 5.0 and at above 30C. It was stabilized by calcium ion. Similar to other trypsin from the Frigid Zone fish, threadfin hakeling trypsin was thermo unstable than the trypsin from the Temperate Zone fish and Tropical Zone fish. The relationship between habitat temperature of fish and thermo stability of the fish trypsin indicated strong positive correlation.