Purification and characterization of cathepsin L from the muscle of silver carp (Hypophthalmichthys molitrix).
Cathepsin L in silver carp musle was purified to 48.4-fold by acid-heat treatment and ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 30 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64 and insensitive to PMSF and pepstatin A, suggesting that the purified enzyme belongs to a family of cysteine proteinase. Consistent with this conclusion, Zn2+, Cu2+, Co2+, Ni2+, and Fe2+ could strongly inhibit the activity of this enzyme. The optimal pH and temperature were 5.0 and 55 degrees C, respectively. The enzyme catalyzed the hydrolysis of Z-Phe-Arg-MCA with a parameter of K(m) (8.27 microM) and K(cat) (28.7 s(-1)) but hardly hydrolyzed Z-Arg-Arg-MCA, Arg-MCA, and Boc-Val-Leu-Lys-MCA. The microstructure analysis by scanning electron microscopy showed that this proteinase is capable of destroying the network structure of silver carp surimi gels. The enzyme exhibited a higher hydrolytic activity on surimi protein at 65 degrees C than at 40 degrees C.
Liu
Huan
H
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
Yin
Lijun
L
Zhang
Nan
N
Li
Shuhong
S
Ma
Changwei
C
D000818:Animals; D002347:Carps; D056668:Cathepsin L; D002403:Cathepsins / Q000302:isolation & purification* / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009132:Muscles / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L