Purificação parcial e caracterização da(s) tripsina(s) da traíra (Hoplias malabaricus)

Abstract

Fish viscera are part of the solid waste discarded after consumption and processing of these animals. However, they can be utilized as a rich resource of enzymes. The traíra, Hoplias malabaricus (Bloch, 1794), little studied fish and having scarce of information about the enzymes produced by it, is a fish with great potential to be explored. In addition, the enzyme trypsin can be isolated from the traíra and present application in the scientific and technological field. However, it is necessary to know its structural and biological characteristics. With this information, this work pursued the purification and characterization of trypsin from traíra. With the traíra’s pyloric cecum was made a crude extract, which was fractionated by the precipitation with organic solvent and the precipitation with ammonium sulphate. Enzymatic hydrolysis tests were performed with the substrate N-α-benzoyl-L-arginine-4-nitroanilide (BApNA) in all fractions of the precipitations and it was observed that the fraction 20-40% of the saline precipitation had higher activity. Gel filtration chromatography (Sephacryl S-100 – 60 x 1 cm) of the 20-40% fraction showed a peak with high trypsin hydrolytic activity and low protein concentration, in others words, high specific activity. SDS-PAGE demonstrated the isolation of trypsin after saline precipitation and gel filtration on the Sephacryl S-100 column. . The enzyme isolated was characterized and showed high activity at pH 9.0 and temperature of 50 ºC, besides having inhibition of 98,95% by phenylmethylsulfonyl fluoride (PMSF), 94.04% by the soybean trypsin inhibitor (SBTI) and 94,74% by benzamidine, showing to be a serine protease The kinetic constant for this enzyme had Km with 1.62 mM and Vmax with 0.69 mM s-1. It is concluded in this work that trypsin from traira was partially isolated by two purification methods and that the enzyme showed catalytic efficiency in temperature and pH conditions distinct from others trypsins isolated from fish, thus demonstrating great potential for possible biotechnological applications.