Abstract
Trypsin was purified to homogeneity from hepatopancreas of royal red prawn by ammonium sulphate (400 g L−1–600 g L−1 saturation) precipitation, Benzamidine affinity column, and MonoQ column chromatography. Trypsin was purified to 91.0-fold with a yield of 12.1% and showed a single band on native-PAGE. Trypsin had a molecular weight of 27 kDa as estimated by SDS-PAGE. The optimal pH and temperature for Boc-Val-Pro-Arg-MCA hydrolysis were 9.0 and 50°C, respectively, while purified enzyme was stable to heat treatment up to 50°C and over a pH range of 7.0-11.0. Trypsin activity was strongly inhibited by soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone (TLCK), and Pefabloc SC and was partially inhibited by ethylenediaminetetraacetic acid (EDTA). Apparent Km value of trypsin was 0.28 μM and kcat value was 656.44s−1 for Boc-Val-Pro-Arg-MCA. The N-terminal amino acid sequence of 20 residues of trypsin was IVGGTVATPYEFPYQISFQD, which was highly homologous with those from other species of prawn. Purified trypsin also showed high collagenolytic activity toward prawn and shrimp collagens, suggested that it is possible to use for collagen’s hydrolysis.
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