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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 &gt;400\u2010fold the catalytic efficiency (<jats:italic>k<\/jats:italic><jats:sub>cat<\/jats:sub>\/K<jats:sub>m<\/jats:sub>) of a hyperthermostable bacterial laccase for 2,6\u2010dimethoxyphenol, a lignin\u2010related 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\u2009%) than reported methods. Additionally, we provide insights that advance the state\u2010of\u2010the\u2010art 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.<\/jats:p>","DOI":"10.1002\/cssc.202401386","type":"journal-article","created":{"date-parts":[[2024,9,11]],"date-time":"2024-09-11T09:35:45Z","timestamp":1726047345000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Unlocking Lignin\u2032s Potential: Engineered Bacterial Laccases to Produce Biologically Active Molecules"],"prefix":"10.1002","volume":"18","author":[{"given":"V\u00e2nia","family":"Brissos","sequence":"first","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier Universidade Nova de Lisboa, Av da Rep\u00fablica  Oeiras 2780-157 Portugal"}]},{"given":"M\u00e1rcia","family":"R\u00e9nio","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier Universidade Nova de Lisboa, Av da Rep\u00fablica  Oeiras 2780-157 Portugal"}]},{"given":"Magdalena A.","family":"Lejmel","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier Universidade Nova de Lisboa, Av da Rep\u00fablica  Oeiras 2780-157 Portugal"}]},{"given":"Ricardo","family":"Estevinho","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier Universidade Nova de Lisboa, Av da Rep\u00fablica  Oeiras 2780-157 Portugal"}]},{"given":"M. Paula","family":"Robalo","sequence":"additional","affiliation":[{"name":"Departamento de Engenharia Qu\u00edmica Instituto Superior de Engenharia de Lisboa Instituto Polit\u00e9cnico de Lisboa  R. Conselheiro Em\u00eddio Navarro 1 Lisboa 1959-007 Portugal"},{"name":"Centro de Qu\u00edmica Estrutural Institute of Molecular Sciences, Complexo I Instituto Superior T\u00e9cnico Universidade de Lisboa  Av. Rovisco Pais Lisboa 1049-001 Portugal"}]},{"given":"M. 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