Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

Abstract

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS-PAGE. Optimum pH and temperature were 9.0 and 65°C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0-11.5) and at 55°C. The kinetic parameters K m , k cat and k cat /K m were 0.47 ± 0.042 mM, 1.33 s À1 and 2.82 s À1 mM À1 , respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe 2+ , Hg 2+ , Zn 2+ , Al 3+ , Pb 2+ , and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).

Study on enzymes of industrial interest in digestive viscera: Greater amberjack (Seriola dumerili)

“…The activity of the trypsin from crude extract tested in the present study was higher than that from crude extract of Japanese sea bass Lateolabrax japonicus (0.30 IU/mg) [20], zebra blenny Salaria basilisca (0.12 IU/mg) [11], pirarucu A. gigas (0.37 IU/mg [8], tropical gar A. tropicus (0.000 006 IU/mg) [13] and hybrid catfish (Clarias microcephalus x Clarias gariepinus) (0.18 IU/mg) [21].…”

“…The release of p-nitroaniline was monitored by a microplate reader at 405 nm and the enzymatic activity was calculated as described in section 2.3. The activity values obtained for each substrate concentration were plotted on a Michaelis-Menten graph using the MicroCal™ Origin 襆 Version 8.0 (MicroCal, Northampton, MA, USA) [8].…”

“…Then, 30 μL aliquots were withdrawn and used to assess the activity of the enzyme at the optimum pH presented using 8 mmol/L substrate. The activity was calculated as the ratio between the enzymatic activity observed at the end of each incubation run and that at the beginning, and expressed as percentage (%) [8]. …”

“…Moreover, the use of neglected fish viscera contributes to reducing environmental damage. In this context, fish enzymes can minimize production costs and can be a cost-effective alternative to biomolecules for the fishery and aquaculture production chain [7,8].…”

“…Digestive serine proteases have already been extracted from the guts of different types of fishes, such as trypsin from the Silver mojarra Diapterus rhombeus [7], sardinelle Sardinella aurita [10], zebra blenny Salaria basilisca [11], barbel Barbus callensis [12], pirarucu Arapaima gigas [8], crevalle jack Caranx hippos [1], tropical gar Atractosteus tropicus [13] and Nile tilapia Oreochromis niloticus [14]; and chymotrypsin from the cuttlefish Sepia officinalis [15], monterey sardine Sardinops sagax caerulea [16], tropical gar A. tropicus [13] and Nile tilapia O. niloticus [9].…”