凡纳滨对虾组织蛋白酶L性质分析及其对肌肉蛋白的降解

摘要

本研究从凡纳滨对虾肝胰腺中纯化获得一种组织蛋白酶L,其分子质量约为31kD,肽质量指纹图谱分析得到8个片段共112个氨基酸残基,与报道的凡纳滨对虾组织蛋白酶L序列完全一致.该酶的最适温度和最适pH值分别为35℃和5.5,且在0～40℃以及pH 5.5～6.5之间酶活力稳定.该酶仅对底物Z-Phe-Arg-MCA特异分解.半胱氨酸蛋白酶抑制剂E-64和Leupeptin对其有明显的抑制作用,而金属蛋白酶抑制剂乙二胺四乙酸和乙二醇二乙醚二胺四乙酸对其有少量的激活作用.扫描电子显微镜观察结果显示,随着低温贮藏时间的延长,对虾肌肉纤维的断裂程度不断增加.十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示,组织蛋白酶L可使肌肉蛋白发生分解,推测其可能参与对虾低温贮藏过程中肌肉的降解.%This study aimed to investigate the enzyme that might be involved in this process.Cathepsin L was purified to homogeneity from the hepatopancreas of Litopenaeus vannamei,and its molecular mass was approximately 31 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Peptide mass fingerprinting revealed that 8 peptide fragments with a total of 112 amino acid residues were completely identical to the cathepsin L gene of L.vannamei.Using Z-Phe-Arg-MCA as substrate,the proteinase revealed optimal activity at 35 ℃ and pH 5.5,respectively.Purified cathepsin L was stable at temperature up to 40 ℃ and in the pH range from 5.5 to 6.5.It exhibited high specificity towards the substrate Z-Phe-Arg-MCA.Its enzymatic activity was significantly suppressed by the cysteine proteinase inhibitors E-64 and leupeptin,while it could be slightly activated by the metalloproteinase inhibitors ethylene diamine tetraacetic acid (EDTA) and ethylene glycol tetraacetic acid (EGTA).In addition,the fibrous structure of shrimp meat was increasingly destroyed with the prolongation of cold storage time as observed by scanning electron microscope (SEM).Purified cathepsin L effectively hydrolyzed muscular proteins as detected by SDS-PAGE,suggesting its potential involvement in the degradation of shrimp muscular proteins during cold storage.