Low molecular weight trypsin from hepatopancreas of freshwater prawn (Macrobrachium rosenbergii): Characteristics and biochemical properties

• Published in: Food chemistry Vol. 134; no. 1; pp. 351 - 358
• Main Authors: Sriket, Chodsana, Benjakul, Soottawat, Visessanguan, Wonnop, Hara, Kenji, Yoshida, Asami, Liang, Xiao
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier Ltd 01.09.2012; Elsevier
• Subjects: amino acid sequences; Biological and medical sciences; chromatography; collagen; Collagenolytic activity; EDTA (chelating agent); fish; Fish and seafood industries; Food industries; Freshwater; Freshwater prawn; Fundamental and applied biological sciences. Psychology; heat stability; heat treatment; Hepatopancreas; hydrolysis; ketones; Macrobrachium rosenbergii; molecular weight; muscles; Purification; shelf life; shrimp; soybeans; temperature; Trypsin; trypsin inhibitors; Trypsin; Purification; Freshwater prawn; Collagenolytic activity; Hepatopancreas; Serine endopeptidases; Enzyme; Properties; Macrura; Crustacea; Peptidases; Edible crustacean; Arthropoda; Hydrolases; Decapoda; Invertebrata; Molecular mass; Shrimp
• ISSN: 0308-8146; 1873-7072
• DOI: 10.1016/j.foodchem.2012.02.173

► Trypsin from hepatopancreas of freshwater prawn (Macrobrachium rosenbergii) was purified and characterised. ► Low molecular weight (17kDa) trypsin was firstly reported in crustacean and fish. ► Purified trypsin hydrolysed prawn and fish collagens effectively. 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 17kDa 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 stable 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 partially inhibited by ethylenediaminetetraacetic acid (EDTA). Apparent Km value of trypsin was 0.24μM and Kcat value was 607.56s−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 species of prawn. HFWP trypsin also showed high collagenolytic activity toward prawn, shrimp and fish collagens, suggesting its possible role in muscle softening of freshwater prawn during extended storage.