Nanobodies as binding-chaperones stabilize the recombinant Bombyx mori acetylcholinesterase and protect the enzyme activity in pesticide detection

In our previous study, the recombinant type II acetylcholinesterase from Bombyx mori (rBmAChE) presented outstanding sensitivity to pesticides, which exhibited great potential in pesticides detection. However, the poor stability of rBmAChE and also the unclear mechanism of its sensitivity hindered the applications in on-site testing of pesticides residues. In this study, we constructed an immune nanobody library, in which we obtained 48 rBmAChE-specific nanobodies. Among them, Nb4 and Nb9 were verified as the most prominent enhancers of the enzyme activity and stabilizers under thermal stress, which indicated their usage as protective reagents for rBmAChE. The simultaneously addition of the two Nbs enhanced the thermal-stability of rBmAChE against exposure to 50-70 °C, and also remained 100% residual activity after 30 days storage at - 20 °C or 4 °C, whereas 80% and 62% at - 80 °C and 25 °C. The homologous modeling and docking of Nb4 and Nb9 to rBmAChE indicated the stabilization of Nb4 to the peripheral anion site (PAS) of rBmAChE while Nb9 protected the C-terminal structure. Substrate docking demonstrated the importance of electrostatic attraction during catalytic process, that might be enhanced by Nbs. As a result, Nb4 and Nb9 were proved to have great potential on rBmAChE applications due to their regulation