Abstract
Trypsin was purified to homogeneity from hepatopancreas of freshwater prawn (HFWP) (Macrobrachium rosenbergii) using a series of chromatographies including Q-Sepharose, Superdex 75 and MonoQ columns. HFWP trypsin was purified 525-fold with a yield of 10.6%. Based on native-PAGE, the purified trypsin showed a single band. Trypsin had a molecular weight of 17 kDa as estimated by SDS-PAGE. The optimal pH and temperature for Boc-Val-Pro-Arg-MCA hydrolysis were 8.0 and 55 C, respectively. Trypsin was sta- ble to heat treatment up to 40 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 par- tially inhibited by ethylenediaminetetraacetic acid (EDTA). Apparent Km value of trypsin was 0.24 lM and Kcat value was 607.56 s 1 for Boc-Val-Pro-Arg-MCA. The N-terminal amino acid sequence of 20 residues of HFWP trypsin was IVGGDEAAPGEFPHQISMQV, which was highly homologous with those from other spe- cies of prawn. HFWP trypsin also showed high collagenolytic activity toward prawn, shrimp and fish col- lagens, suggesting its possible role in muscle softening of freshwater prawn during extended storage. 2012 Elsevier Ltd. All rights reserved.
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