Biochemical characterization of chymotrypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus).
Two chymotrypsins (chymotrypsins A and B) have been purified to homogeneity from the hepatopancreas of Japanese sea bass ( Lateolabrax japonicus ) by ammonium sulfate fractionation and chromatographies on DEAE-Sepharose and Phenyl-Sepharose. Two-dimensional electrophoresis (2-DE) analysis revealed that the molecular masses of chymotrypsins A and B were approximately 27.0 and 27.5 kDa, respectively. Their respective isoelectric points were 8.0 and 7.0. Purified chymotrypsins also revealed a single band on native-PAGE, whereas their mobilities were quite different. Optimum temperature and pH of chymotrypsins A and B were 45 degrees C and 8.0, respectively. Both enzymes were strongly inhibited by chymostatin, phenylmethanesulfonyl fluoride (PMSF), and Pefabloc SC, but slightly inhibited by metalloproteinase inhibitor of 1,10-phenanthroline and EDTA. Using Suc-Leu-Leu-Val-Tyr-MCA as substrate, apparent K(m) values of chymotrypsins A and B were 0.8 and 1.1 microM and k(cat) values were 2.7 and 2.0 s(-1), respectively. The N-terminal amino acid sequences of chymotrypsins A and B were determined to the 21st and 18th residues, respectively, and were identical. These sequences exhibited high identities to chymotrypsins from other animals. The digestive effect of the two chymotrypsins on myofibrillar proteins indicated their effectiveness in the degradation of food proteins.
Jiang
Yu-Kun
YK
College of Biological Engineering, The Key Laboratory of Science and Technology for Aquaculture and Food Safety, Jimei University, Jimei, Xiamen, China 361021.
Sun
Le-Chang
LC
Cai
Qiu-Feng
QF
Liu
Guang-Ming
GM
Yoshida
Asami
A
Osatomi
Kiyoshi
K
Cao
Min-Jie
MJ
D000818:Animals; D001492:Bass; D002918:Chymotrypsin / Q000737:chemistry* / Q000302:isolation & purification; D004795:Enzyme Stability; D029941:Fish Proteins / Q000737:chemistry* / Q000302:isolation & purification; D043143:Hepatopancreas / Q000737:chemistry / Q000201:enzymology*; D007526:Isoelectric Point; D008970:Molecular Weight
D029941:Fish Proteins; D002918:Chymotrypsin