Department for Innovation in Biological, Agro-food and Forest Systems-DIBAF, University of Tuscia, via S. Camillo de Lellis, 01100 Viterbo, Italy.
Appl Biochem Biotechnol. 2012 Apr;166(7):1736-46. doi: 10.1007/s12010-012-9577-z. Epub 2012 Feb 11.
In order to use lysozyme as an anti-microbial agent during the winemaking process, hen egg-white lysozyme (LYZ) was covalently immobilized on two different micro-size magnetic particles (tosyl-activated and carboxylated, TSA and CA, respectively). A cell suspension of Oenococcus oeni, an oenological strain involved in the winemaking process, was utilized as LYZ substrate. Both a kinetic study and a study of the stability of free and immobilized LYZ were performed in McIlvane buffer at pH 3.2, that is the average minimum pH value in wine. The activity and kinetic parameters measured for the free LYZ at pH 3.2 are lower than those reported at the optimum pH (4.5); however the residual activity at pH 3.2 is sufficient to be of interest for further immobilization and applications in winemaking. All kinetic parameters of both biocatalysts (LYZ-CA and LYZ-TSA) are altered after immobilization, probably due to the structural modifications in the active site caused by covalent attachment to the supports. The half-life calculated at 25 °C was 39 h for free LYZ, while it increased to 280 and 134 h for LYZ-TSA and LYZ-CA, respectively. This result indicates that immobilization improves the enzyme stability and that LYZ can be utilized in wine applications in its immobilized forms. In addition, LYZ-TSA seems to be the best biocatalyst for further applications in winemaking.
为了在酿酒过程中使用溶菌酶作为一种抗菌剂，将蛋清溶菌酶（LYZ）共价固定在两种不同的微尺寸磁性颗粒（对甲苯磺酰基活化和羧基化，分别为 TSA 和 CA）上。利用酿酒酵母（Oenococcus oeni）的细胞悬浮液作为 LYZ 底物，该酵母是参与酿酒过程的一种酿酒酵母。在 McIlvane 缓冲液（pH 3.2）中进行了游离和固定化 LYZ 的动力学研究和稳定性研究，这是葡萄酒中平均最小 pH 值。在 pH 3.2 下测量的游离 LYZ 的活性和动力学参数低于在最佳 pH（4.5）下报告的值；然而，在 pH 3.2 下的剩余活性足以引起进一步固定化和在酿酒中的应用的兴趣。两种生物催化剂（LYZ-CA 和 LYZ-TSA）的所有动力学参数在固定化后都发生了变化，这可能是由于活性位点的结构修饰所致，这些结构修饰是由于与载体的共价结合引起的。在 25°C 下计算的半衰期为游离 LYZ 的 39 小时，而 LYZ-TSA 和 LYZ-CA 的半衰期分别增加到 280 和 134 小时。这一结果表明，固定化提高了酶的稳定性，并且可以将固定化的 LYZ 用于葡萄酒应用。此外，LYZ-TSA 似乎是进一步在酿酒中应用的最佳生物催化剂。
