Co-Immobilizing Two Glycosidases Based on Cross-Linked Enzyme Aggregates to Enhance Enzymatic Properties for Achieving High Titer Icaritin Biosynthesis

Icaritin is a rare and high-value isopentane flavonoid compound with remarkable activities. Increasing yields while reducing cost has been a great challenge in icaritin production. Herein, we first reported a high titer icaritin biosynthesis strategy from epimedin C through co-immobilizing α-l-rhamnosidase (Rha1) and β-glucosidase (Glu4) using cross-linked enzyme aggregates (CLEAs). The created CLEAs exhibited excellent performances in terms of catalytic activity, thermal stability, pH stability, and reusability. Notably, Rha1-CLEAs (Ki: 1 M) and Glu4-CLEAs (Ki: 0.1 M) were more tolerant to sugars (glucose or rhamnose) than free enzymes (0.1 M for Rha1 and 0.007 M for Glu4) by immobilization, achieving the highest icaritin productivity under the highest substrate concentration ever reported. Finally, about 34.24 g/L icaritin could be obtained from 100 g/L epimedin C within 8 h, indicating the great potential for industrialization. This study also provides a promising strategy for the low-cost production of other high-value aglycone compounds by solving poor stability and sugar inhibition of glycosidase.