Purification and partial characterisation of a trypsin from the processing waste of the silver mojarra (Diapterus rhombeus)

Abstract

An alkaline peptidase was purified from the viscera of the silver mojarra (Diapterus rhombeus) in a three-step process: heat treatment, ammonium sulphate fractionation and molecular exclusion chromatography (Sephadex® G-75), with final specific activity 86-fold higher than the enzyme extract and yield of 22.1%. The purified enzyme had an estimated molecular mass of 26.5kDa and NH2-terminal amino acid sequence IVGGYECTMHSEAHE. Higher enzyme activity was observed at pH 8.5 and between 50 and 55°C. The enzyme was completely inactivated after 30min at 55°C and it was significantly more stable at alkaline pH. Km, Kcat and Kcat·Km(-1) values, using BApNA as substrate, were 0.266mM, 0.93s(-1) and 3.48mM(-1)s(-1), respectively. Enzyme activity increased in the presence of the ions (1mM) K(+), Li(+) and Ca(2+), but was inhibited by Fe(2+), Cd(2+), Cu(2+), Al(3+), Hg(2+), Zn(2+) and Pb(2+) as well as by the trypsin inhibitors TLCK and benzamidine.

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

“…The K m is used to assess the affinity of the tested enzyme to the substrate and constitutes one of the characteristics observed by the industry during the prospection process for novel commercial enzymes. The results for trypsin showed similar values to that of alkaline trypsin from silver mojarra D. rhombeus [7], pirarucu A. gigas [8] and crevalle jack C. hippos [1]; while chymotrypsin values diverged from that reported by Castillo-Yañez et al [25]. The effect of temperature on the activity of trypsin in this work was similar to that of Brownstripe red snapper Lutjanus vitta [26] and zebra blenny Salaria basilisca [11].…”

“…To test thermal stability, the enzyme was incubated in a water bath for 30 min and the remaining activity was then measured at 25 °C . The activity was calculated as the ratio between the enzymatic activity at the end of each incubation run and that at the beginning, and expressed as percentage (%) [7].…”

“…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].…”

Apparent digestibility of nutrients and energy of conventional ingredients for the silver mojarra, Diapterus rhombeus

“…Studies with silver mojarra have been conducted on its biology (ETCHEVERS, 1978;CHAVES;OTTO, 1998;SILVA et al, 2011) and distribution aspects (ARAÚJO; SANTOS, 1999;AYALA-PEREZ et al, 2001;COSTA et al, 2012). However, the digestibility of ingredients has not been the subject of research and this is a major step for the development of an efficient diet to promote a better nutritional status for the animals.…”