Purification and characterisation of cathepsin L2 from dorsal muscle of silver carp (Hypophthalmichthys molitrix)

Cathepsin L2 was purified to homogeneity from silver carp muscle using an array of chromatography methods. The enzyme showed affinity to con A-sepharose. Although it appeared to be 78 kDa on non-reducing SDS–PAGE and gel–substrate-activity SDS–PAGE, it completely degraded into 31 kDa and 26 kDa sub-units, as well as some small polypeptides on reducing SDS–PAGE. The optimum pH and temperature of cathepsin L2 for hydrolysis of Z-Phe-Arg-MCA were pH 4.5–5.5 and 45 °C, respectively. It was stable at pH 5.5 and below 40 °C, but almost inactivated at pH 7.0 and 60 °C. Substrate specificity analysis indicated that it could hydrolyse Z-Phe-Arg-MCA but not Z-Arg-Arg-MCA or l-Arg-MCA. Cathepsin L2 was efficiently activated by Cys, DTT and β-ME, but was completely inhibited by E-64. P 2 O 7 4 - and Cl− have inhibitory effects on its activity. Cathepsin L2 showed a high Km value of 9.5 μmol/l, but extremely low Kcat and Kcat/Km values of 0.8 s−1 and 84.2 s−1 mM−1, respectively. Except for under optimum conditions (pH 5.0, 35 °C), silver carp cathepsin L2 could also hydrolyse myosin heavy chain at softening temperatures ranging from 50 to 60 °C and at surimi pH of 6.5.