酪氨酸酶、漆酶和谷氨酰胺转氨酶催化交联蛋白质的比较分析

Comparison of protein cross-linking by tyrosinase,laccase and transglutaminase

蛋白质交联在食品加工、组织工程、酶工程和药物传递等领域具有广泛用途。以酪蛋白和牛血清白蛋白(BSA)为模式蛋白,考察酪氨酸酶、漆酶和谷氨酰胺转氨酶催化蛋白质交联的底物特异性及交联规律,揭示酶对底物蛋白质结构及反应条件的要求。采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析酶催化蛋白质交联规律,激光粒度分布仪测量交联产物粒径。结果表明:酪氨酸酶、漆酶和谷氨酰胺转氨酶对底物的特异性有共同特征,即均可以催化结构松散的蛋白质分子(酪蛋白)交联,但不能催化结构紧密的蛋白质分子(BSA)交联;还原剂二硫苏糖醇(DTT)的加入能促进酶催化BSA交联反应;DTT对酪氨酸酶和谷氨酰胺转氨酶催化酪蛋白交联无影响,但抑制漆酶对酪蛋白的交联。

The covalent cross-linking of protein molecules is widely used in food processing,tissue engineering,enzyme engineering and pharmaceutical delivery.To investigate the substrate specificity of tyrosinase,laccase and microbial transglutaminase(mTG),bovine casein and bovine serum albumin(BSA)were selected as substrate models.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)was used to determine the cross-linking effects.Laser particle size analyzer was used to determine the size of the cross-linking proteins.The results demonstrated that tyrosinase,laccase and mTG could readily cross-link flexible proteins(bovine casein),but could not cross-link compact proteins(BSA).The presence of reducing agent,dithiothreitol(DTT),improved the cross-linking of BSA,whereas it prevented the cross-linking of casein catalyzed by laccase.