关键词: 鱿鱼肝脏, 酶解, 抑制剂, 荧光底物, 组织蛋白酶L

Abstract: In order to understand the applicability of endogenous proteases from squid liver as a rich source of proteases, the endogenous proteases of squid liver were identified and characterized with common carp myofibrillar protein as substrate by adding a variety of protease inhibitors. It was found that the degradation of myosin heavy chain (MHC) was significantly inhibited by addition of E-64, 1,10-phenanthroline and PMSF to the reaction system indicating that squid liver contained three kinds of proteases, metalloprotease, cysteine protease and serine protease. The cysteine protease had the best thermal stability and highest activity, which still maintained a high activity at temperatures higher than 50 ℃, and it could hydrolyze myofibrillar proteins into small molecules. Substrate specificity analysis indicated that the cysteine protease could only hydrolyze Z-phe-Arg-MCA, and was inhibited 100% by leupeptin and 99.4% by E-64, indicating that the enzyme consisted mainly of cathepsin L. At last, cathepsin L was purified to homogeneity by ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The purified enzyme showed a molecular weight of about 25 kD.

Key words: squid liver, enzymatic hydrolysis, inhibitor, fluorogenic?substrate, cathepsin L