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

doi:10.1016/j.cjche.2023.08.007

摘要/Abstract

摘要： 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.

关键词: Surface charge engineering, Organic solvent resistance, Molecular dynamics simulation, Haloalkane dehalogenase

Abstract: 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.

Key words: Surface charge engineering, Organic solvent resistance, Molecular dynamics simulation, Haloalkane dehalogenase

Yin Wu, Yan Sun. Rational surface charge engineering of haloalkane dehalogenase for boosting the enzymatic performance in organic solvent solutions[J]. 中国化学工程学报, 2024, 65(1): 276-285.

Yin Wu, Yan Sun. Rational surface charge engineering of haloalkane dehalogenase for boosting the enzymatic performance in organic solvent solutions[J]. Chinese Journal of Chemical Engineering, 2024, 65(1): 276-285.

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