Unlocking Lignin's Potential: Engineered Bacterial Laccases to Produce Biologically Active Molecules.

Laccases are biocatalysts with immense potential in lignocellulose biorefineries to valorize emerging lignin monomers for sustainable chemicals. Despite reduced costs over the past two decades, enzymes remain a major expense in biorefining. Protein engineering can enhance enzyme properties and lower costs further. In this study, we used enzyme engineering tools to improve >400-fold the catalytic efficiency (k/K) of a hyperthermostable bacterial laccase for 2,6-dimethoxyphenol, a lignin-related phenolic compound. Furthermore, this evolved variant showed improved activity at neutral to alkaline pH for hydroxycinnamyl alcohols, hydrocinnamic acids, phenylpropanoid and vanillyl derivatives. We optimized conditions for the synthesis of syringaresinol, dehydrodiconiferyl alcohol, thomasidioic acid, biseugenol, dehydrodiisoeugenol, and diapocynin, detailing the pH, catalyst concentration, reaction time, temperature, and oxygenation of the reaction mixtures. Our biocatalytic system offers several advantages, including being free of organic solvents, achieving faster reaction times, using lower amounts of enzymes and delivering excellent yields (up to 100 %) than reported methods. Additionally, we provide insights that advance the state-of-the-art in lignin combinatory chemistry. This progress marks a significant step forward in valorizing the lignin chemicals platform, enabling high yields of dimeric compounds with structural scaffolds that can be exploited in various biotechnological areas, such as medicinal chemistry and polymer synthesis.