The efficient immobilization and orientation of bilirubin oxidase (BOx) on different solid substrates are essential for its application in biotechnology. The T1 copper site within BOx is responsible for the electron transfer. In order to obtain quick direct electron transfer (DET), it is important to keep the distance between the T1 copper site and electrode surface small and to maintain the natural structure of BOx at the same time. In this work, the combined parallel tempering Monte Carlo simulation with the all-atom molecular dynamics simulation approach was adopted to reveal the adsorption mechanism, orientation, and conformational changes of BOx from Myrothecium verrucaria (MvBOx) adsorbed on charged self-assembled monolayers (SAMs), including COOH-SAM and NH-SAM with different surface charge densities (±0.05 and ±0.19 C·m). The results show that MvBOx adsorbs on negatively charged surfaces with a "back-on" orientation, whereas on positively charged surfaces, MvBOx binds with a "lying-on" orientation. The locations of the T1 copper site are closer to negatively charged surfaces. Furthermore, for negatively charged surfaces, the T1 copper site prefers to orient closer to the surface with lower surface charge density. Therefore, the negatively charged surface with low surface charge density is more suitable for the DET of MvBOx on electrodes. Besides, the structural changes primarily take place on the relatively flexible turns, coils, and α-helix. The native structure of MvBOx is well preserved when it adsorbs on both charged surfaces. This work sheds light on the controlling orientation and conformational information on MvBOx on charged surfaces at the atomistic level. This understanding would certainly promote our understanding of the mechanism of MvBOx immobilization and provide theoretical support for BOx-based bioelectrode design.

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The efficient immobilization of haloalkane dehalogenase (DhaA) on carriers with retaining of its catalytic activity is essential for its application in environmental remediation. In this work, adsorption orientation and conformation of DhaA on different functional surfaces were investigated by computer simulations; meanwhile, the mechanism of varying the catalytic activity was also probed. The corresponding experiments were then carried out to verify the simulation results. (The simulations of DhaA on SAMs provided parallel insights into DhaA adsorption in carriers. Then, the theory-guided experiments were carried out to screen the best surface functional groups for DhaA immobilization.) The electrostatic interaction was considered as the main impact factor for the regulation of enzyme orientation, conformation, and enzyme bioactivity during DhaA adsorption. The synergy of overall conformation, enzyme substrate tunnel structural parameters, and distance between catalytic active sites and surfaces codetermined the catalytic activity of DhaA. Specifically, it was found that the positively charged surface with suitable surface charge density was helpful for the adsorption of DhaA and retaining its conformation and catalytic activity and was favorable for higher enzymatic catalysis efficiency in haloalkane decomposition and environmental remediation. The neutral, negatively charged surfaces and positively charged surfaces with high surface charge density always caused relatively larger DhaA conformation change and decreased catalytic activity. This study develops a strategy using a combination of simulation and experiment, which can be essential for guiding the rational design of the functionalization of carriers for enzyme adsorption, and provides a practical tool to rationally screen functional groups for the optimization of adsorbed enzyme functions on carriers. More importantly, the strategy is general and can be applied to control behaviors of different enzymes on functional carrier materials.

Electrostatically driven enzyme orientation leads to different enzyme activities and electron transfer rates on different charged surfaces.