Institute of Biotechnology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia.
Molecules. 2020 Sep 18;25(18):4278. doi: 10.3390/molecules25184278.
The co-immobilization of ketoreductase (KRED) and glucose dehydrogenase (GDH) on highly cross-linked agarose (sepharose) was studied. Immobilization of these two enzymes was performed via affinity interaction between His-tagged enzymes (six histidine residues on the N-terminus of the protein) and agarose matrix charged with nickel (Ni ions). Immobilized enzymes were applied in a semicontinuous flow reactor to convert the model substrate; α-hydroxy ketone. A series of biotransformation reactions with a substrate conversion of >95% were performed. Immobilization reduced the requirement for cofactor (NADP+) and allowed the use of higher substrate concentration in comparison with free enzymes. The immobilized system was also tested on bulky ketones and a significant enhancement in comparison with free enzymes was achieved.
研究了酮还原酶（KRED）和葡萄糖脱氢酶（GDH）在高度交联琼脂糖（琼脂）上的共固定化。通过固定在 N 端的 His 标签酶（蛋白质上的六个组氨酸残基）和带负电荷的琼脂糖基质与镍（Ni 离子）之间的亲和相互作用来固定这两种酶。固定化酶应用于半连续流反应器中，以转化模型底物；α-羟基酮。进行了一系列转化率超过 95%的生物转化反应。与游离酶相比，固定化降低了对辅因子（NADP+）的需求，并允许使用更高的底物浓度。固定化系统还在大体积酮上进行了测试，与游离酶相比，取得了显著的增强效果。
