Colagenase de pescada branca: extração, purificação parcial, caracterização e teste de especificidade ao colágeno para aplicação industrial

Abstract

Fish processing residues are rich sources of biomolecules with industrial potential, such as enzymes with collagenolytic properties applied in the pharmaceutical, textile and leather sectors. Here, collagenolytic serine proteases were partially purified from the waste (viscera) of smooth weakfish Cynoscion leiarchus and characterized for the purpose of obtaining a value-added product from fisheries resources. The higher activity of the enzyme (72.5 U mL -1 ) was verified in optimal temperature and pH of 55°C and 8.0, respectively. The enzyme was stable in wide ranges of temperature (25-60°C) and pH (6.5 to 11.5). The ions Ca 2+ and Mg 2+ increased the protease activity, whilst Pb 2+, Al 3+ and Cu 2+ had an inhibitory effect, as observed in the presence of Benzamidine and TLCK (inhibitors of serine proteases). Hydrolysis was detected after 48 hours, when the enzyme and bovine collagen type I were incubated together. Thus, digestive viscera of C. leiarchus is suggested as an alternative source of enzymes capable of cleaving type I collagen, with similar biochemical properties to bacterial collagenases commonly employed in industrial processes, reducing costs, adding value to the fisheries product and minimizing the environmental impact caused by its waste. , com temperatura e pH ótimos de 55°C e 8.0, respectivamente. A enzima manteve-se estável em faixas amplas de temperatura (25-60 °C) e pH (6,(5)(6)(7)(8)(9)(10)(11)5 , Al 3+ e Cu 2+ inibiram essa atividade assim como os inibidores de serino-proteases (Benzamidina e TLCK). A hidrólise foi detectada após 48h de incubação com colágeno bovino tipo I. Assim, sugere-se vísceras digestivas de C. leiarchus como fonte alternativa para o fornecimento de enzimas com capacidade de clivar o colágeno do tipo I e com propriedades bioquímicas semelhantes às colagenases bacterianas já empregadas nas etapas de processamento industrial, como forma de redução de custo, agregação de valor ao produto pesqueiro e contribuindo para minimizar o impacto ambiental deste tipo de resíduo.

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

“…High collagenolytic activity was reported for intestinal crude extract of smooth weakfish Cynoscion leiarchus (72.5 IU/mg) [4], and Daboor et al [17] reported the collagenolytic activity (11.63 IU/mg) using a mixture of haddock, herring, flounder and ground fish crude extracts. Souchet and Laplante [28] also detected the collagenolytic activity (13.3 IU/mg) in byproducts of snow crab Chionoecetes opilio.…”

“…Samples of 300 g intestine were collected, packaged in plastic containers, kept on ice and transported to the Laboratory of Enzymology, Center of Biological Sciences, Federal University of Pernambuco, Recife, Pernambuco, Brazil. The enzymatic extraction was performed according to the method described by Oliveira et al [4]. The ratio of viscera to extraction buffer (0.05 mol/L Tris-HCl, pH 7.5, containing 5 mmol/L CaC1 2 ) was 1:5 (w/v).…”

“…The test for collagenolytic activity of the crude extract was performed according to method of Oliveira et al [4], using Azo dye-impregnated collagen (azocoll) as substrate. A reaction mixture containing azocoll (5 mg), 50 mmol/L Tris-HCl buffer (500 μL, pH 7.5, contained 5 mmol/ L CaCl 2 ) and crude extract (500 μL) was typically incubated at 55 °C for 30 min with stirring.…”

“…Solid wastes (carcasses, skin, bones, fins and heads) discarded during fish processing are potential sources of biologically active molecules of industrial interest [1][2][3][4][5]. The proteases recovered from these sources have physicochemical and kinetic properties similar to the mammalian enzymes commonly employed in the production processes of the textile, pharmaceuticals and foods [6].…”

“…Serinocollagenases from aquatic sources have been reported in a mixture of haddock, herring, ground fish and flounder crude extracts [17] and intestinal viscera of smooth weakfish Cynoscion leiarchus [4]. To date, no fibrinolytic proteases have been identified in fish species, and these species constitute alternative sources because of the industrial/biomedical importance of this enzyme as a thrombolytic agent.…”