Liu Huan, Yin Lijun, Zhang Nan, Li Shuhong, Ma Changwei
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
J Agric Food Chem. 2006 Dec 13;54(25):9584-91. doi: 10.1021/jf062038m.
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.
通过酸热处理和硫酸铵分级分离，随后进行一系列色谱分离，鲢鱼肌肉中的组织蛋白酶L被纯化至48.4倍。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，纯化酶的分子量为30 kDa。纯化酶被二硫苏糖醇和半胱氨酸激活，而被E-64强烈抑制，对苯甲基磺酰氟和胃蛋白酶抑制剂A不敏感，表明纯化酶属于半胱氨酸蛋白酶家族。与此结论一致，Zn2+、Cu2+、Co2+、Ni2+和Fe2+可强烈抑制该酶的活性。最佳pH和温度分别为5.0和55℃。该酶催化Z-苯丙氨酸-精氨酸-甲基香豆素酰胺水解，参数K(m)为8.27 microM，K(cat)为28.7 s(-1)，但几乎不水解Z-精氨酸-精氨酸-甲基香豆素酰胺、精氨酸-甲基香豆素酰胺和Boc-缬氨酸-亮氨酸-赖氨酸-甲基香豆素酰胺。扫描电子显微镜的微观结构分析表明，这种蛋白酶能够破坏鲢鱼鱼糜凝胶的网络结构。该酶在65℃时对鱼糜蛋白的水解活性高于40℃。
