Purification and characterization of trypsin from the intestine of hybrid tilapia (Oreochromis niloticusxO.aureus).
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 degrees C, respectively. The enzyme was stable over a broad pH range from 7.0 to 12.0 at 30 degrees C, and the enzyme was inactive at temperatures above 50 degrees C. The behavior of the enzyme for the hydrolysis of casein followed Michaelis-Menten kinetics with Km 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 Nalpha-CBZ-L-lysine p-nitrophenyl ester hydrochloride (CBZ-Lys.pNP) as a substrate. The protease was inhibited by the following ions in decreasing order: Zn2+>Fe3+>Cu2+>Al3+>Co2+=Pb2+>Cd2+>Mn2+. The ions Li+, Na+, K+, Mg2+, and Ba2+ had little effect on the enzyme, and Ca2+ can partially promote its activity at low concentration.
Wang
Qin
Q
Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Science, Xiamen University, Xiamen 361005, China. qwang@xmu.edu.cn
Gao
Zhi-Xing
ZX
Zhang
Nong
N
Shi
Yan
Y
Xie
Xiao-Lan
XL
Chen
Qing-Xi
QX
D000818:Animals; D004795:Enzyme Stability; D029941:Fish Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D007422:Intestines / Q000737:chemistry / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D017210:Tilapia / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry* / Q000302:isolation & purification*
D029941:Fish Proteins; D014357:Trypsin