Biocatalysis in organic solvents (OSs) has numerous important applications

but native enzymes in OSs often exhibit limited catalytic performance. Herein

we proposed a computation-aided surface charge engineering strategy to improve the catalytic performance of haloalkane dehalogenase DhaA in OSs based on the energetic analysis of substrate binding to the DhaA surface. Several variants with enhanced OS resistance were obtained by replacing negative charged residues on the surface with positive charged residue (Arg). Particularly

a four-substitution variant E16R/E93R/E121R/E257R exhibited the best catalytic performance (five-fold improvement in OS resistance and seven-fold half-life increase in 40% (vol) dimethylsulfoxide). As a result

the overall catalytic performance of the variant could be at least 26 times higher than the wild-type DhaA. Fluorescence spectroscopy and molecular dynamics simulation studies revealed that the residue substitution mainly enhanced OS resistance from four aspects:(a) improved the overall structural stability

(b) increased the hydrophobicity of the local microenvironment around the catalytic triad

(c) enriched the hydrophobic substrate around the enzyme molecule

and (d) lowered the contact frequency between OS molecules and the catalytic triad. Our findings validate that computationaided surface charge engineering is an effective and ingenious rational strategy for tailoring enzyme performance in OSs.