One-pot cascade reaction systems offer several process and technological benefits, completed by the opportunity of the second enzyme to act on a convenient concentration of its substrate. Most of the reported cascade reactions involve immobilization of both biocatalysts on a single support, but in this case the catalytic performance of the whole system is affected if the activity of either enzyme drops. In this work, tailor-made immobilization of two β-galactosidases (from Kluyveromyces lactis and Aspergillus oryzae) and a glucose oxidase (from Aspergillus niger) was investigated, to subsequent utilization of the best performing immobilized biocatalyst of each type for the one-pot conversion of lactose to gluconic acid and galacto-oligosaccharides. The immobilization was performed by covalent binding onto amino-terminated Ni_0.4Co_0.2Zn_0.4Fe₂O₄ and Ni_0,5Zn_0,5Fe₂O₄ magnetic particles and commercial amino- and epoxy-functionalized methacrylate supports. The highest activity for β-galactosidases was achieved on Ni_0.5Zn_0.5-Fe₂O₄ magnetic particles, meanwhile the glucose oxidase showed higher affinity for the supports with epoxy active groups. The immobilized biocatalysts have been characterized in detail, demonstrating enhanced storage, pH, and thermal stability as well as better operational stability compared to the soluble enzymes.

一锅级联反应系统具有多种工艺和技术优势，通过第二种酶以便利的浓度作用于其底物的机会得以完成。大多数报道的级联反应都涉及将两种生物催化剂固定在单一载体上，但是在这种情况下，如果任何一种酶的活性下降，则会影响整个系统的催化性能。在这项工作中，两个β-半乳糖苷酶（来自乳酸克鲁维酵母和米曲霉）和一个葡萄糖氧化酶（来自黑曲霉）的量身定制的固定化）进行了研究，然后将每种类型的性能最佳的固定化生物催化剂用于从一锅法将乳糖转化为葡萄糖酸和低聚半乳糖。固定化是通过共价结合到氨基末端的Ni _0.4 Co _0.2 Zn _0.4 Fe ₂ O ₄和Ni _0.5 Zn _0.5 Fe ₂ O ₄磁性颗粒以及氨基和环氧官能化的甲基丙烯酸酯载体上进行的。在Ni _0.5 Zn _0.5 -Fe ₂ O ₄上达到了β-半乳糖苷酶的最高活性磁性颗粒，同时葡萄糖氧化酶对具有环氧活性基团的载体表现出更高的亲和力。已对固定化的生物催化剂进行了详细表征，与可溶性酶相比，具有更高的储存性，pH和热稳定性以及更好的操作稳定性。