Co-immobilization of enoate reductase with a cofactor-recycling partner enzyme

Herein we established co-immobilized methods for enoate reductases (ERs) and glucose dehydrogenase (GDH), forming a cofactor regeneration system. In cross-linked enzyme aggregates (CLEAs), ammonium sulfate and oxidized dextran were selected as a precipitant and a cross-linker, respectively. In biomimetic immobilization (BI), ER-GDH-silica particles (ER-GDH-SPs) were rapidly formed through a one-step approach by using a silicic acid precursor. Under the optimal conditions, the ER activity recovery in ER-GDH-CLEAs and ER-GDH-SPs were 44.9 +/- 1.8% and 44.5 +/- 2.1%, and the immobilization efficiency was 93.5 +/- 1.2% and 92.4 +/- 1.2%, respectively. ER-GDH-CLEAs and ER-GDH-SPs exhibit excellent thermal and pH stability, and superior reusability. The activity of ER-GDH-SPs toward the substrate is also better than that of free ER and GDH in reduction of 4-(4-Methoxyphenyl)-3-buten-2-one. This study introduces simple and inexpensive co-immobilization strategies to construct novel and efficient ER-GDH-CLEAs and ER-GDH-SPs with high activity and stability.