Robust oxidoreductases in non-aqueous system is promising to bridge the gap between research and industrial application. Malate dehydrogenases (MDHs) were select as model enzyme for redesign based on motif assembly study. Computationally inserted the robust motifs which response to salt concentration into the selected scaffold, and resulted novel MDHs. Top-scoring MDH with structure compatibility and dynamic harmony is expressed for experimental verification. Result indicated the MDH03 with enhanced thermostability, extended pH adaptation and ionic liquid tolerance. The activity of MDH03 was 1.78-fold of parent MDH in present of [EMIM]BF4. Further study of amino acid residues interaction network explain the robustness of MDH03 based on high-density salt bridges. Research also indicated the hydrophobic contacts and pi-pi contacts of interfacial interactions of motifs play key role for activity and stability of MDH03.This work promote an approach to design robust dehydrogenase with high ionic liquids tolerance and the further application in biosensors.

To design D-amino acid dehydrogenase (DAADH) for enhanced stability, the interactions of the subunit interfaces of DAADH were analyzed. Interaction network analysis of DAADH indicated that there are only weak interactions between the A and B subunits. Several co-evolved residue pairs were selected for mutation to enhance interfacial interactions of subunits, and 11 designed MDHs were obtained. DA06 and DA11 were selected for experimental verification for their salt bridges are 1.4 and 1.2-fold of that of DAwild, respectively. DA11 can maintain 93% activity in 80℃, while it was only 40% for DAwild. Thermostabiliy study indicated the half-life of DA11 was 2-fold of DAwild. Molecular dynamics simulations revealed that the extraordinary stability of the DA11 was due to the formation of extra interfacial salt bridges. The paper provided a strategy of mutations outside the active site of enzyme by co-evolutionary analysis which can reduce the effect of the activity-thermostability trade-off.