Abstract
Natural enzymes are able to function effectively under optimal physiological conditions, but the intrinsic performance often fails to meet the demands of industrial production. Existing strategies are based mainly on the evaluation and subsequent combination of single-point mutations; however, this approach often suffers from a limited number of designable residues and from low accuracy. Here, we propose a strategy (Co-MdVS) based on coevolutionary analysis and multidimensional virtual screening for precise design to improve enzyme robustness, employing nattokinase as a model. Using this strategy, we efficiently screened 8 dual mutants with enhanced thermostability from a virtual mutation library containing 7980 mutants. After further iterative combination, the optimal mutant M6 exhibited a 31-fold increase in half-life at 55 °C, significantly enhanced acid resistance, and improved catalytic efficiency with different substrates. Molecular dynamics simulations indicated that the reduced flexibility of thermal and acid-sensitive regions resulted in a significantly increased robustness of M6. Furthermore, the potential of multidimensional virtual screening in enhancing design precision has been validated on l-rhamnose isomerase and PETase. Therefore, the Co-MdVS strategy introduced in this research may offer a viable approach for developing enzymes with enhanced robustness.