Purification and characterization of three trypsin isoforms from viscera of sardinelle (Sardinella aurita)

Abstract

Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20-70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-DL: -arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0-11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K (m) and k (cat) were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s(-1), respectively. The N-terminal sequences of the first 10 amino acids were "I V G G Y E C Q K Y" for trypsin A and "I V G G Y E A Q S Y" for trypsins B and C. These sequences showed highly homology to other fish trypsins.

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

“…Optimum pH of trypsin is consistent with that reported by Ben Khaled et al [10], Freitas-Junior et al [8], Ktari et al [11] and Sila et al [12] for the fish species sardinelle S. aurita, pirarucu A. gigas, zebra blenny S. basilisca and barbel B. callensis, respectively, indicating an optimum pH range between 8.0 and 10. In this study, peak activity of trypsin was found in the pH 9.0.…”

“…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].…”

Purification and characterization of trypsin from Luphiosilurus alexandri pyloric cecum

“…disjunctivus0.131.4611.2425Villalba et al (2013)S. aurita A0.132.2417.9225Khaled et al [10]E. encrasicholus0.663.24.8425Martínez et al [23]G.…”

“…One of the major obstacles faced by breeders is related to production cost, particularly food costs that, depending on the species, can reach 70% of total expenses. To counterbalance the excessive costs of production, breeders should dispose all of the animal mass potential and not just the commercialization of muscle fiber, because approximately 30% of the assessed weight corresponds to innards, primarily categorized as subproducts [10]. These residues have significant biotechnological potential by providing, for example, a source of enzymes that can be applied in several industries.…”

“…Normally, trypsin isolated from either fresh or salt water fish shows satisfactory levels of reactivity in extreme conditions, such as high alkalinity and temperature as well as in the presence of surfactant agents. Consequently, studies have been conducted on the purification and characterization of these molecules from biological sources in order to identify low-cost methodologies with high level of recuperation: spotted goatfish ( Pseudupeneus maculatus ) [19], brown stripe red snapper ( Lutjanus vitta ) [13], hybrid catfish ( Clarias microcephalus and Clarias gariepinus ) [16], sardines ( Sardinella aurita ) [10], silver mojarra ( Diapterus rhombeus ) [29], and zebra blenny ( Salaria basilisca ) [20].…”