The alkaline- or acid-stable enzymes are more advantageous for agricultural and industrial applications. Cytochrome P450BM3 is a biotechnologically important and versatile enzyme capable of producing various drug metabolites. However, the engineering of physicochemical properties of P450BM3 remains scarce. In this project, we first selected 4-nitrophenyl acetate and 4-nitrophenyl butyrate as the substrates of P450BM3. We then applied semi-rational directed evolution strategy to engineer P450BM3 for enhanced activities in acidic/alkaline conditions. We analyzed the possible residues on the surface that may be closely related to the optimum pH of P450BM3, and constructed a virtual of 380 saturated mutants. By using DynaMut website calculation, the top7 mutants with enhanced stabilities were selected and expressed in E. coli. The K24I and D422W variants were screened out to exhibit ~0.5-fold and 0.4-fold increased activities toward 4-nitrophenyl acetate in acidic condition (pH=6.0). Our study provides foundation for further engineering of P450BM3 to adapt acidic/alkaline conditions for practical use. Read More