Abstract
Trypsin from hepatopancreas of Pacific white shrimp (Litopenaeus vannamei) was purified to homogeneity using ammonium sulfate precipitation and a series of chromatographies including diethylaminoethyl sepharose and soybean trypsin inhibitor sepharose 4B columns. Trypsin was purified to 50.4-fold with a yield of 13.7%. Based on native-polyacrylamide gel electrophoresis (PAGE), the purified trypsin showed a single band. Trypsin had a molecular weight of 24 kDa as estimated by sodium dodecyl sulphate-PAGE. The optimal pH and temperature for α-N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) hydrolysis were 8.0 and 60C, respectively. Trypsin was stable to heat treatment up to 60C and over a pH range of 7.0‐11.0. The activity was strongly inhibited by soybean N-ρ-tosyl-L-lysine chloromethyl ketone. Purified trypsin had Michaelis‐Menten constant (Km) and catalytic constant (kcat) of 1.60 mM and 3.33 s −1 , respectively, when BAPNA was used as the substrate. Trypsin with high kcat indicated its high capacity of hydrolysis and it could serve as a promising protease.
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