Structural Insights into the Stability and Recognition Mechanism of the Antiquinalphos Nanobody for the Detection of Quinalphos in Foods.

Nanobodies (Nbs) have great potential in immunoassays due to their exceptional physicochemical properties. With the immortal nature of Nbs and the ability to manipulate their structures using protein engineering, it will become increasingly valuable to understand what structural features of Nbs drive high stability, affinity, and selectivity. Here, we employed an anti-quinalphos Nb as a model to illustrate the structural basis of Nbs' distinctive physicochemical properties and the recognition mechanism. The results indicated that the Nb-11A-ligand complexes exhibit a "tunnel" binding mode formed by CDR1, CDR2, and FR3. The orientation and hydrophobicity of small ligands are the primary determinants of their diverse affinities to Nb-11A. In addition, the primary factors contributing to Nb-11A's limited stability at high temperatures and in organic solvents are the rearrangement of the hydrogen bonding network and the enlargement of the binding cavity. Importantly, Ala 97 and Ala 34 at the active cavity's bottom and Arg 29 and Leu 73 at its entrance play vital roles in hapten recognition, which were further confirmed by mutant Nb-F3. Thus, our findings contribute to a deeper understanding of the recognition and stability mechanisms of anti-hapten Nbs and shed new light on the rational design of novel haptens and directed evolution to produce high-performance antibodies.

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 on enzyme activity and protection against thermal-inactivation and long-term storage of rBmAChE.

Preparation of Specific Nanobodies and Their Application in the Rapid Detection of Nodularin-R in Water Samples.

Nanobodies have several advantages, including great stability, sensibility, and ease of production; therefore, they have become important tools in immunoassays for chemical contaminants. In this manuscript, nanobodies for the detection of the toxin Nodularin-r (NOD-R), a secondary metabolite of cyanobacteria that could cause a safety risk for drinks and food for its strong hepatotoxicity, were for the first time selected from an immunized Bactrian camel VHH phage display library. Then, a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for NOD-R, based on the nanobody N56 with great thermostability and organic solvent tolerance, was established under optimized conditions. The results showed that the limit of detection for NOD-R was 0.67 µg/L, and the average spike recovery rate was between 84.0 and 118.3%. Moreover, the ic-ELISA method was validated with spiked water sample and confirmed by UPLC-MS/MS, which indicated that the ic-ELISA established in this work is a reproducible detection assay for nodularin residues in water samples.

Bio-production of Baccatin III, an Important Precursor of Paclitaxel by a Cost-Effective Approach.

Natural production of anti-cancer drug taxol from Taxus has proved to be environmentally unsustainable and economically unfeasible. Currently, bioengineering the biosynthetic pathway of taxol is an attractive alternative production approach. 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) was previously characterized as an acyltransferase, using 10-deacetylbaccatin III (10-DAB) and acetyl CoA as natural substrates, to form baccatin III in the taxol biosynthesis. Here, we report that other than the natural acetyl CoA (Ac-CoA) substrate, DBAT can also utilize vinyl acetate (VA), which is commercially available at very low cost, acylate quickly and irreversibly, as acetyl donor in the acyl transfer reaction to produce baccatin III. Furthermore, mutants were prepared via a semi-rational design in this work. A double mutant, I43S/D390R was constructed to combine the positive effects of the different single mutations on catalytic activity, and its catalytic efficiency towards 10-DAB and VA was successfully improved by 3.30-fold, compared to that of wild-type DBAT, while 2.99-fold higher than the catalytic efficiency of WT DBAT towards 10-DAB and Ac-CoA. These findings can provide a promising economically and environmentally friendly method for exploring novel acyl donors to engineer natural product pathways.