Marín-Zamora María Elisa, Rojas-Melgarejo Francisco, García-Cánovas Francisco, García-Ruiz Pedro Antonio
Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100, Espinardo, Murcia, Spain.
J Biotechnol. 2007 Sep 30;131(4):388-96. doi: 10.1016/j.jbiotec.2007.05.004. Epub 2007 May 17.
Mushroom tyrosinase was immobilized from an extract onto glass beads covered with one of the following compounds: the crosslinked totally cinnamoylated derivatives of glycerine, D-sorbitol, D-manitol, 1,2-O-isopropylidene-alpha-D-glucofuranose, D-glucuronic acid, D-gulonic acid, sucrose, D-glucosone, D-arabinose, D-fructose, D-glucose, ethyl-D-glucopyranoside, inuline, dextrine, dextrane or starch, or the partially cinnamoylated derivative 3,5,6-tricinnamoyl-D-glucofuranose which was obtained by the acid hydrolysis of 1,2-O-isopropylidene-alpha-d-glucofuranose. The enzyme was immobilized by direct adsorption onto the support and the quantity of tyrosinase immobilized was found to increase with the hydrophobicity of the supports. The kinetic constants of immobilized tyrosinase acting on the substrates, 4-tert-butylcatechol, dopamine and DL-dopa, were studied. When immobilized tyrosinase acted on 4-tert-butylcatechol, the values of K(m)(app) were lower than these obtained for tyrosinase in solution while, when dopamine and DL-dopa were used, the K(m)(app) were higher. The order of the substrates as regards their ionizable groups, DL-dopa (two ionizable groups)>dopamine (one ionizable group)>4-tert-butylcatechol (no ionizable group) coincided with the order of the K(m)(app) values shown by tyrosinase immobilized on the hydrophobic supports, and was the inverse of that observed for tyrosinase in solution. The K(m)(app) values of immobilized tyrosinase were in all cases higher than those of soluble tyrosinase and depended on the nature of the support and the hydrophobicity of the substrate, meaning that it is possible to design supports with different degrees of selectivity towards a mixture of enzyme substrates in the reaction medium.
甘油、D-山梨醇、D-甘露糖醇、1,2-O-异亚丙基-α-D-呋喃葡萄糖、D-葡萄糖醛酸、D-古洛糖酸、蔗糖、D-葡糖酮、D-阿拉伯糖、D-果糖、D-葡萄糖、乙基-D-吡喃葡萄糖苷、菊粉、糊精、葡聚糖或淀粉的交联全肉桂酰化衍生物，或通过1,2-O-异亚丙基-α-D-呋喃葡萄糖酸水解得到的部分肉桂酰化衍生物3,5,6-三肉桂酰-D-呋喃葡萄糖。通过直接吸附将酶固定在载体上，发现固定的酪氨酸酶量随载体的疏水性增加。研究了固定化酪氨酸酶作用于底物4-叔丁基邻苯二酚、多巴胺和DL-多巴的动力学常数。当固定化酪氨酸酶作用于4-叔丁基邻苯二酚时，K(m)(app)值低于溶液中酪氨酸酶的值，而当使用多巴胺和DL-多巴时，K(m)(app)值较高。底物关于其可电离基团的顺序，DL-多巴（两个可电离基团）>多巴胺（一个可电离基团）>4-叔丁基邻苯二酚（无可电离基团）与固定在疏水载体上的酪氨酸酶显示的K(m)(app)值顺序一致，并且与溶液中酪氨酸酶观察到的顺序相反。固定化酪氨酸酶的K(m)(app)值在所有情况下都高于可溶性酪氨酸酶的值，并且取决于载体的性质和底物的疏水性，这意味着可以设计对反应介质中酶底物混合物具有不同选择性程度的载体。
