Amine transaminases (ATAs), belonging to the class III transaminases within the superfamily of pyridoxal-5 '-phosphate-dependent enzymes, catalyze transamination reactions between amino donors and amino acceptors. These enzymes are particularly appealing for their role in stereospecific synthesis of chiral amines. However, the stability of most ATAs is not satisfying, limiting their suitability for industrial applications. Among them, the amine transaminase from Silicibacter pomeroyi (Sp-ATA) has drawn attention due to its high activity and broad substrate scope under mild conditions and high pH. Nevertheless, maintaining the activity at higher temperatures is a challenge. Previous studies to enhance enzyme function through directed evolution have shown promising results, yet predicting the cooperative effects of individual stabilizing mutations remains challenging. An alternative strategy is ancestral sequence reconstruction (ASR), which is based on gene sequences to create a more or less artificial phylogenetic tree. This study aims to leverage ASR techniques to explore the thermostability, solvent tolerance, and substrate profile of Sp-ATA, to find more stable transaminases. By using Sp-ATA as a template and incorporating insights from ancestral sequences, this strategy offers a promising approach for developing robust biocatalysts suitable for industrial applications.

Protein-based absorbent materials exhibit significant limitations in water retention compared to synthetic superabsorbent polymers (SAPs), widely used in agriculture, hygiene, and biomedical applications. Recent investigations have focused on leveraging highly soluble charged proteins such as patatin (a glycoprotein derived from potatoes) as natural alternatives to synthetic SAPs, given their unique structural properties and the opportunity they provide as sustainable raw material alternatives. This study investigates how the intrinsic amino acid composition and charged residues of patatin can be modified through mutagenesis to tailor its superabsorbent properties. Here, patatin was expressed in Escherichia coli to improve the water absorption capacity by altering its amino acid composition. By increasing liquid accessibility and charge density, our method of altering the charged profile of the protein significantly enhances the protein's swelling capacity, doubling its absorption compared to native patatin. Additionally, molecular dynamics simulations reveal that protein variants enriched with lysine and aspartic acid facilitate increased hydrogen bonding interactions with water molecules, thereby enhancing hydration. These results provide a fundamental understanding of how to tailor the physicochemical nature of proteins to develop them as viable bio-based absorbents for advanced sanitary applications, combining material science and biotechnology.