Premium
Purification and Characterization of Trypsin from Hepatopancreas of P acific White Shrimp
Author(s) -
Senphan Theeraphol,
Benjakul Soottawat,
Kishimura Hideki
Publication year - 2015
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/jfbc.12147
Subject(s) - hepatopancreas , trypsin , kunitz sti protease inhibitor , litopenaeus , ammonium sulfate precipitation , protease , chemistry , proteases , chromatography , biochemistry , hydrolysis , shrimp , chymotrypsin , enzyme , biology , fishery , size exclusion chromatography
Trypsin from hepatopancreas of P acific white shrimp ( L itopenaeus vannamei ) was purified to homogeneity using ammonium sulfate precipitation and a series of chromatographies including diethylaminoethyl sepharose and soybean trypsin inhibitor s epharose 4 B 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 60 C 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 M ichaelis– M enten constant ( K m ) and catalytic constant ( k cat ) of 1.60 mM and 3.33 s −1 , respectively, when BAPNA was used as the substrate. Trypsin with high k cat indicated its high capacity of hydrolysis and it could serve as a promising protease. Practical Applications P acific white shrimp hepatopancreas generally serves as a major source of proteases, especially trypsin and chymotrypsin, which can be used as an alternative food processing aid. Proteases in the hepatopancreas can be recovered and further used, in which the cost of commercially available proteases can be reduced. Furthermore, the by‐product can be better exploited and the extracted proteases can increase the revenues for the shrimp processor.