A thermostable trypsin from freshwater fish Japanese dace (Tribolodon hakonensis): a comparison of the primary structures among fish trypsins.

Trypsin from Japanese dace (Tribolodon hakonensis) (JD-T) living in freshwater (2-18 °C) was purified. JD-T represented typical fish trypsin characteristics regarding the effects of protease inhibitor, calcium-ion, and pH. For the effect of temperature, JD-T quite resembled to the trypsins from tropical-zone marine fish and freshwater fish (the catfish cultured in Thailand), i.e., the optimum temperature was 60 °C, and it was stable below 60 °C at pH 8.0 for 15 min incubation. From the data, it seemed that the trypsin from freshwater fish is thermostable in spite of the fact that their habitat temperatures are low. So, we determined the primary structure of JD-T to discuss its thermostability-structure relationship. JD-T possessed basic structural features of fish trypsin such as the catalytic triad, the Asp189 residue for substrate specificity, 12 Cys residues forming six disulfide-bridges, and the calcium-ion-binding loop. On the other hand, the contents of charged amino acid residues in whole JD-T molecule (16.2%) and N-terminal region (13.8%) were similar to those of tropical-zone marine fish and other freshwater fish trypsins. Then, JD-T conserved the five amino acid residues (Glu70, Asn72, Val75, Glu77, and Glu80) coordinate with calcium-ion, and the proportion of negatively charged amino acids to charged amino acids in the calcium-ion-binding region of JD-T (75.0%) was equivalent to those of tropical-zone marine fish and freshwater fish trypsins. Therefore, it was suggested that the high thermostability of JD-T are stemmed from these structural specificities.