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Purification and Characterization of Trypsin from Hepatopancreas of Pacific White Shrimp

Author:
Senphan, Theeraphol, Benjakul, Soottawat, Kishimura, Hideki
Source:
Journal of food biochemistry 2015 v.39 no.4 pp. 388-397
ISSN:
0145-8884
Subject:
Litopenaeus vannamei, agarose, ammonium sulfate, chymotrypsin, enzyme kinetics, food processing, heat stability, heat treatment, hepatopancreas, hydrolysis, income, molecular weight, pH, polyacrylamide gel electrophoresis, shrimp, sodium, soybeans, temperature, trypsin, trypsin inhibitors
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. PRACTICAL APPLICATIONS: Pacific 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.
Agid:
3408303