Rational surface charge engineering of haloalkane dehalogenase for boosting the enzymatic performance in organic solvent solutions

• A computation-aided surface charge engineering strategy was developed to enhance the catalytic performance of haloalkane dehalogenase DhaA in organic solvents. • The four-substitution variant E16R/E93R/E121R/E257R exhibited a five-fold improvement in organic solvent resistance and a seven-fold increase in half-life in 40% dimethylsulfoxide. • The overall catalytic performance of the best variant was at least 26 times higher than the wild-type enzyme. • Mechanistic studies revealed that enhanced organic solvent resistance arises from improved structural stability, increased local hydrophobicity, substrate enrichment, and reduced solvent access to the catalytic triad.