title: Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity.
abstract: BACKGROUND
  Fish proteases, especially trypsin, could be used to prepare fish protein hydrolysates with antioxidative activities. In this study, trypsin from the pyloric caeca of unicorn leatherjacket was purified by ammonium sulfate precipitation and soybean trypsin inhibitor (SBTI)-Sepharose 4B affinity chromatography. Hydrolysate from Indian mackerel protein isolate with different degrees of hydrolysis (20, 30 and 40% DH) was prepared using the purified trypsin, and antioxidative activities (1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activities, ferric-reducing antioxidant power and ferrous-chelating activity) of the hydrolysate were determined.
  
  
  RESULTS
  Trypsin was purified 26.43-fold with a yield of 13.43%. The purified trypsin had a molecular weight (MW) of 23.5 kDa and optimal activity at pH 8.0 and 55 °C. It displayed high stability in the pH range of 6.0-11.0 and was thermally stable up to 50 °C. Both SBTI (0.05 mmol L(-1)) and N-p-tosyl-L-lysine-chloromethylketone (5 mmol L(-1)) completely inhibited trypsin activity. Antioxidative activities of the hydrolysate from Indian mackerel protein isolate increased with increasing DH up to 40% (P < 0.05). Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the hydrolysate with 40% DH had a MW lower than 6.5 kDa.
  
  
  CONCLUSIONS
  The purified protease from unicorn leatherjacket pyloric caeca was identified as trypsin based on its ability to hydrolyze a specific synthetic substrate and the response to specific trypsin inhibitors. The purified trypsin could hydrolyze Indian mackerel protein isolate, and the resulting hydrolysate exhibited antioxidative activity depending on its DH.
authors.affiliation: Fisheries Department, Faculty of Natural Resources and Environmental, Malayer University, Malayer, Hamedan, Iran.
authors.forename: Abbas
authors.initials: A
authors.lastname: Zamani
authors.affiliation: Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, 90112, Thailand.
authors.forename: Soottawat
authors.initials: S
authors.lastname: Benjakul
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000378:metabolism / Q000494:pharmacology*; D002432:Cecum / Q000378:metabolism; D029941:Fish Proteins / Q000378:metabolism*; D019649:Food Industry; D063427:Food Quality; D006801:Humans; D010473:Perciformes; D011492:Protein Hydrolysates / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000378:metabolism
chemical_list: D000975:Antioxidants; D029941:Fish Proteins; D011492:Protein Hydrolysates; D014361:Trypsin Inhibitors; D014357:Trypsin
keywords: Indian mackerel; antioxidative activity; protein hydrolysate; purification; trypsin; unicorn leatherjacket