Engineering artificial fusion naringinase for enhancing naringenin biosynthesis

Naringinase (NGase) is a multi -compound enzyme with the subunits of alpha-L-rhamnosidase (Rha) and beta-Dglucosidase (BGL). The biotransformation of naringin into the more valuable naringenin was impeded by the uncoordinated activity and instability of natural naringinases. In this study, the efficient artificial fusion naringinases of Rha from Spirochaeta thermophila (StRha) and BGL from Pyrococcus furiosus (PfBGL) were constructed and utilized in biosynthesis of naringenin. Fusion naringinases StRha-PfBGL (DL-NGase), StRha-(GGGGS)-PfBGL (FL-NGase) and StRha-(EAAAK)-PfBGL (RL-NGase) were obtained by fusing StRha and PfBGL through direct fusion or mediated by peptide linkers. DL-NGase showed more coordinated activity and better thermostability than FL-NGase and RL-NGase. The optimal temperature of StRha and PfBGL of DL-NGase was above 65 C-degrees, and both retained above 75% of the initial activities at 55 C-degrees for 2 h. In addition, fusion naringinases exhibited better catalytic performance than the mixed free enzyme systems, and the catalytic efficiency of DL-NGase was increased by 49.8%. Furthermore, DL-NGase whole-cell catalyst was applied for the high-efficient biosynthesis of naringenin, and the final naringenin yield was 13.5 mg/mL with a time-space yield of 2.25 mg/mL/h. These results demonstrate the great potential of artificial fusion naringinases for the efficient bioconversion of naringin to naringenin.