Rational design of soluble expressed human aldehyde dehydrogenase 2 with high stability and activity in pepsin and trypsin

Abstract

Acetaldehyde dehydrogenase 2 (ALDH2) is a crucial enzyme in alcohol metabolism, and oral administration of ALDH2 is a promising method for alcohol detoxification. However, recombinant ALDH2 is susceptible to hydrolysis by digestive enzymes in the gastrointestinal tract and is expressed as inactive inclusion bodies in E. coli. In this study, we performed three rounds of rational design to address these issues. Specifically, the surface digestive sites of pepsin and trypsin were replaced with other polar amino acids, while hydrophobic amino acids were incorporated to reshape the catalytic cavity of ALDH2. The resulting mutant DE2–852 exhibited a 45-fold increase in soluble expression levels, while its stability against trypsin and pepsin increased by eightfold and twofold, respectively. Its catalytic efficiency (kcat/Km) at pH 7.2 and 3.2 improved by more than four and five times, respectively, with increased Vmax and decreased Km values. The enhanced properties of DE2–852 were attributed to the D457Y mutation, which created a more compact protein structure and facilitated a faster collision between the substrate and catalytic residues. These results laid the foundation for the oral administration and mass preparation of highly active ALDH2 and offered insights into the oral application of other proteins.