Abstrait

Isolation and characterisation of visceral trypsin of Japanese threadfin bream (Nemipterus japonicus)

Krishna Prasad Nooralabettu, Adel DÃ?Â?Ã?Â?Ã?Â?Ã?Â?cunha, Priyanka Miranda, Swetha Nayak, Anantharamakrishna

Trypsin was purified to homogeneity and characterised from the pyloric caeca of Japanese threadfin bream (Nemipterus japonicus). Trypsin was optimally recovered fromthe tissues by homogenization at 3000 rpmfor 10 minutes, followed by centrifugation at 10000 rpmfor 30minutes, precipitation at 45%ammoniumsulphate saturation level, and subsequent chromatography using Sephacryl S-200, DEAE-cellulose and Sephadex G-50. Molecularmass of the enzyme was 25kDa and showed esterase activity on Ná-p-tosyl-L-arginine methyl ester hydrochloride (TAME) as a substrate. Trypsin showed temperature optimum of 60 °C and pH optimum of 8 for catalytic activity, and it exhibited sharp fall in its activity above 60 oC and below pH 6. The remaining activity of the trypsin pre-incubated in the presence of 10mM concentration of CaCl2 was stable up to two hours when at 30 oC for pH 8, but in the presence of 10mM concentration of EDTAat 30 oC for pH 8 enzyme activity reduced sharply to negligible level within 5 hours.