Semi-rational engineering of Issatchenkia terricola acetaldehyde dehydrogenase for enhanced thermostability and catalytic efficiency in detoxification of aldehydes

Acetaldehyde dehydrogenase is crucial for detoxifying aldehydes in pharmaceutical, food, and biofuel industries. We enhanced Issatchenkia terricola ALDH (ist-ALDH) thermostability and catalytic activity toward aliphatic and aromatic aldehydes via molecular engineering. The combinatorial mutant H62G/D240P/F429Vshifted the optimal temperature from 35 °C to 55 °C. It had better thermal stability with a half-life (t 1/2 ) of 315.07 ± 4.24 min at 40 °C, 14.41-fold that of the wild-type (WT). Kinetic studies showed higher catalytic efficiency for both aliphatic and aromatic aldehydes. H62G/D240P/F429V had 11.96-fold higher catalytic activity for acetaldehyde than WT, with K m , V max , k cat , and k cat / K m improved by 3.44-, 10.99-, 10.98-, and 39.30-fold, respectively. For aromatic substrates like furfural and 5-hydroxymethylfurfural, catalytic efficiencies were 19.41- and 46.77-fold of the WT levels. Molecular dynamics and structural analysis indicated that enhanced hydrophobic packing and optimized electrostatic potential stabilized H62G/D240P/F429V rigid conformation. Substrate binding studies showed the hydrogen bond distance between Cys327 and acetaldehyde in the mutant decreased from 2.18 Å to 2.09 Å compared to the WT. Additionally, an additional hydrogen bond was formed between Cys328 and the aldehyde group, with a bond distance of 1.94 Å. These findings establish a basis for the property engineering of tailoring ALDH properties, with implications for developing aldehyde detoxification systems in industrial biocatalysis.