Trypsin from digestive tract of harpiosquillid mantis shrimp: Molecular characteristics and the inhibition by chitooligosaccharide and its catechin conjugate.

Abstract

Trypsin from the digestive tract of harpiosquillid mantis shrimp (HMS) was purified using ammonium sulfate precipitation and a soybean trypsin inhibitor-CNBr-activated Sepharose 4B affinity column. The purified trypsin (PTRP-HMS) had a purity of 30.4-fold, and a yield of 14.5% was obtained. PTRP-HMS had the molecular weight of 23.0 kDa as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and only one isoform was detected by native-PAGE. Its optimal temperature and pH were 55°C and 8.5, respectively. TLCK almost completely inhibited the activity of trypsin. The PTRP-HMS had a Michaelis-Menten constant (K_m) and catalytic constant (K_cat) of 0.87 mM and 13.04 s^-1, respectively, toward Nα-benzoyl-l-arginine 4-nitroanilide hydrochloride. When chitooligosaccharide (COS) and COS-catechin (COS-CAT) conjugates were examined for inhibition toward the PTRP, the latter exhibited higher efficacy in inhibiting the trypsin. Both COS and COS-CAT conjugate showed mixed-type inhibition kinetics. As a consequence, COS and COS-CAT conjugate could be used as natural additives for inhibiting trypsin in whole HMS, thus retarding the softening and lengthening the shelf-life of HMS during the iced storage. PRACTICAL APPLICATION: Harpiosquillid mantis shrimp (HMS) is of high demand due to its delicacy. However, its meat undergoes rapid softening within 2-3 days when stored in ice. Understanding causative proteolytic enzymes, especially trypsin from digestive tract, paves a way for preventing their negative impact on HMS eating quality. Employment of safe inhibitors, for example, chitooligosaccharide (COS) or COS conjugated with catechin, could inhibit HMS trypsin. Overall, softening of whole HMS containing trypsin in its digestive tract can be impeded, especially when treated with COS-CAT. This finding is beneficial for the HMS local vendor or exporter, in which HMS quality can be maintained.