Highly Selective Enzymatic Degradation of Undesirable Phenols in Brettanomyces and Smoke Taint Contaminated Wines. AVF Project #2024-2795.
Annual Progress Summary
We’ve completed the second year of a multi-year project with significant progress in developing enzymes capable of selectively degrading 4-ethylguaiacol (4EG) and 4-ethylphenol (4EP), the primary sensory-active compounds in Brettanomyces-contaminated wine and responsible for a portion of the ‘taint’ phenols found in smoke-tainted wine (Licker 1998, Mirabelli-Montan 2021). A second phase of this project—beyond the scope of this past year— will build upon engineered laccases and detailed knowledge of their active site modifications to create additional enzymes capable of fully and exclusively degrading the remaining smoke taint marker compounds, including guaiacol, 4-methylguaiacol, o-, p-, m-cresol, 4-methylsyringol, and syringol.
A library of 46 laccase enzymes from the Siegel lab was expressed and tested under various conditions, revealing multiple candidates with strong activity against these phenols. Further testing in model wine (13% ethanol, 4 g/L tartaric acid, pH 3.5) identified several laccases capable of rapidly and completely degrading 4EG, while 4EP degradation remained moderate. To date, our most effective enzymes have achieved 100% degradation of 4EG and 30% degradation of 4EP under industrially relevant conditions. To better understand the structure-function relationship governing specificity and activity, single-point mutations were introduced into two wild-type laccases, targeting residues in and around the active site. While both wild-type enzymes can fully degrade 4EG and a portion of 4EP, assay conditions were adjusted to approximately 50% 4EG degradation to facilitate comparisons across mutants. These mutations resulted in both increases and decreases in activity for both phenols, identifying key residues essential for catalysis and revealing ‘hot spot’ regions that are critical for AI-driven enzyme design. Structural modeling of enzyme-substrate complexes suggests that achieving higher specificity and efficiency will require significant modifications to the laccase active site, as current enzymes exhibit broad substrate tolerance that may lead to off-target degradation of beneficial wine aroma compounds. These findings provide a strong foundation for enzyme optimization and computational design aimed at improving specificity and catalytic efficiency.
Building on these insights, we are integrating state-of-the-art AI-driven tools to design and experimentally validate novel enzymes with enhanced activity and selectivity. Over the next year, we will computationally design and characterize approximately 100 novel enzyme variants, leveraging insights from the initial laccase screening and structural analysis. These enzymes will be tested in model wine conditions to assess activity and specificity, with a focus on minimizing off-target effects while enhancing 4EG and 4EP degradation. Once optimized laccases are established, this work will expand to other smoke taint marker compounds, such as guaiacol and cresols, paving the way for a broader enzymatic solution to mitigate wine taint. This project’s long-term vision is to develop highly selective, industrially viable laccases that efficiently remove phenolic contaminants while preserving the integrity of wine flavor and aroma.
