Fernanda Braz Candiotto (2013) Characterization of sole Paralichthys orbignyanus digestive enzymes

Characterization of sole Paralichthys orbignyanus digestive enzymes

Author: Fernanda Braz Candiotto (Currículo Lattes)
Supervisor: Dr Marcelo Borges Tesser


In Europe and Asia, three species known as sole are produced (Psetta maximum, Paralichthys olivaceus and Cynoglossus semilaevis). In Brazil there is no commercial production of sole, however the main target species of the studies is Paralichthys orbignyanus. This species was once an important fishing resource in the south of the country, however, fishing is stagnant. Thus, the production of this sole may be an alternative to increase the supply of fish in the region, due to the characteristics that indicate the viability of breeding as tolerance to high concentrations of nitrogen compounds, temperature variations, good growth in salt and brackish water, reproduction and dominated captive larviculture. However, there is still a great lack of research related to the species' dysgestory physiology.For this purpose, the activity of digestive enzymes present in the liver, intestine and stomach of sole (Paralichthys orbignyanus) was determined and characterized, through nonspecific and specific substrates, in addition to specific inhibitors. The physical-chemical parameters were also determined using specific substrates. For the analysis of digestive enzymes present in the crude extract of the intestine and liver, in vitro tests were carried out in the presence of long chain substrates (1% azocasein and 2% starch), p-nitroanilide (BApNA, SApNA and Leu-p-Nan ). For the analysis of digestive enzymes present in the crude extract of the stomach, hemoglobin substrate was used. For enzymatic analysis of the aminopeptidases present in the crude extract of the intestine, the substrate β-naphthylamide (alanine, arginine, leucine, proline, tyrosine, serine,glycine, isoleucine and histidine). Enzymatic activity present in the crude extract of the intestine was: Azocasein (0.3645 ± 0.25mU mg-1), BApNA (0.5119 ± 0.2 mU mg-1), SApNA (2.6281 ± 1.8 mU mg - 73 1), Leu-p-Nan (0.9709 ± 0.83 mU mg-174), starch (without activity). The enzymatic activity present in the crude liver extract: Azocasein (0.27 ± 0.01mU mg-175), BApNA (0), 4 SApNA (0.7 ± 0.08 mU mg-1), Leu-p-Nan (0), starch (33.25 ± 76 0.8 mU mg-1). The effect of specific inhibitors on the residual activity of peptidases present in the crude extract of the intestine presented the following profile: PMSF (BapNA = 52 ± 2, 3; SapNa = 42 ± 1.2; Leu-p-Nan = 43 ± 1.4%), TLCK (BapNA = 33 ± 1.8; SapNa = 45 ± 0.3; Leu-p-Nan = 44 ± 4.1%), TPCK (BapNA = 100 ± 1.2; SapNa = 79 ± 4.5; Leu-p-Nan = 60 ± 1.8%), Benzamidine (BapNA = 41 ± 2.3; SapNa = 58 ± 0.5; Leu-p-Nan = 40 ± 1.2%).Patterns of peculiar inhibition can be explained due to the use of the crude extract that has other enzymes, in addition to these inhibitors being based on mammalian enzymes. The optimum pH observed for trypsin, chymotrypsin, leucine aminopeptidase, amylase and pepsin activity was pH 9.5, 9, 8, 7.5 3.5, respectively. The optimum temperature observed for the activity of these enzymes was 50, 50, 50, 40 and 45 ° C, respectively. Alanine and Leucine aminopeptidases showed a high activity compared to other evaluated aminopeptidases. Trypsin and leucine aminopeptidase have been shown to be thermostable up to 70 and 50 ° C, respectively. Features that indicate the possibility of biotechnological application.