ImobilizaÃÃo de tripsina de TilÃpia NilÃtica (Oreochromis niloticus) em Dacron ferromagnÃtico: caracterizaÃÃo do derivado e sua aplicaÃÃo na detecÃÃo de inibidores de protease

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

Nile tilapia (Oreochromis niloticus) is the most produced fish in Brazilian aquaculture, originating byproducts (viscera) that can be used as a source of biomolecules with potential use in biotechnology. In this work trypsin from tilapia intestine was covalently immobilized onto ferromagnetic Dacron (polyethyleneterephthalate or PET) via glutaraldehyde, characterized and applied in the detection of antinutritional factors (protease inhibitors). The following derivative properties were investigated: optima pH and temperature, thermal stability, effect of ions and inhibitors, kinetics, usage and storage stabilities, and compared to the soluble fish trypsin. The derivative was employed to detect inhibitor in Cratylia mollis seeds crude extract, a potential feedstuff for animal feeds popularly known as camaratu bean. Previously, the C. mollis extract had its nutritional composition determined, including the level of minerals and amino acid composition. The immobilized protein and trypsin activity acting on benzoil-arginine-p-nitroanilide were 26 μg/mg of ferromagnetic Dacron and 27%, respectively. The immobilized enzyme showed an apparent Km (0,132 Â 0,044 mM) about five times lower than that estimated for the soluble trypsin (0,735 Â 0,141). The derivative presented an optimum pH (7,0) lower than the soluble enzyme (8,5 â 9,5). It was noticed a slight increase in the thermal stability, while the optimum temperature was lowered to 50ÂC after immobilization. Benzamidine and tosyl-lysine-chloromethyl-ketone inhibited the immobilized trypsin 82% and 89%, respectively. The derivative was also inhibited by ions, noticeably by aluminum and copper. It was stable for about two months stored in buffer solution at 10ÂC, and after eight utilizations it presented a slight loss of activity (18%). C. mollis presented a low level of protein (13,74 Â 1,22%) and lipid (2,2 Â 0,25%), and a high level of carbohydrate (64,47 Â 0,19%). The minerals content of the C. mollis seeds was similar to other beans. The amino acid composition showed a high level of essential amino acids, except that methionine was found to be 50,8% lower that recommended. To end with, the C. mollis seeds ketonic extract inhibited the porcine (26%) (used as a control) and O. niloticus (49%) immobilized trypsins. The electrophoretic pattern of the polypeptides recovered from theses derivatives was very distinct from one another. This result shows that a specie-specific interaction occurred between trypsin from different sources and inhibitors present in the C. mollis extract. Therefore, fish trypsin immobilized onto ferromagnetic Dacron proved to be an efficient tool for the detection of trypsin inhibitors in alternative nutritional sources

ASSUNTO(S)

immobilized trypsin bioquimica oreochromis niloticus oreochromis niloticus ferromagnetic dacron cratylia mollis dacron ferromagnÃtico tripsina imobilizada cratylia mollis

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