Purification and Characterization of Trypsin from the Intestine of Hybrid Tilapia (Oreochromis niloticus x O.aureus)

摘要

Trypsin from the intestine of hybrid tilapia (Oreochromis niloticus x O.aureus) was purified by the following techniques: acetone precipitation, ammonium sulfate fractionation, Sephacryl S-200 gel filtration, and DEAE-sephacel ion exchange chromatography. The purified enzyme was determined to be homogeneous by polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight was estimated as 22,000 Da. The optimum pH and temperature of the enzyme for the hydrolysis of casein were determined to be 9.0 and 60°C, respectively. The enzyme was stable over a broad pH range from 7.0 to 12.0 at 30 °C, and the enzyme was inactive at temperatures above 50 °C. The behavior of the enzyme for the hydrolysis of casein followed Michaelis-Menten kinetics with K_m of 0.46 mg/mL The purified enzyme was inhibited by the general serine protease inhibitor phenyl methyl sulphonyl fluoride (PMSF) and also by the specific trypsin inhibitor N-p-tosyl-L-lysine chloromethyl ketone (TLCK) using N_α-CBZ-L-lysine p-nitrophenyl ester hydrochloride (CBZ-LyS·pNP) as a substrate. The protease was inhibited by the following ions in decreasing order: Zn~(2+) > Fe~(3+) > Cu~(2+) > Al~(3+) > Co~(2+) = Pb~(2+) > Cd~(2+) > Mn2+. The ions Li~+, Na~+, K~+, Mg~(2+)., and Ba~(2+) had little effect on the enzyme, and Ca~(2+) can partially promote its activity at low concentration.