Highly Selective Enzymatic Degradation of Undesirable Phenols in Brettanomyces and Smoke Taint Contaminated Wines.

This is the first-year progress report on a multi-year project divided into two-phases with the first (current) phase focusing on discovery and design of enzymes with exclusive degradation of 4-ethylguaiacol (4EG) and 4-ethylphenol (4EP), the main sensory-active components in wine contaminated with Brettanomyces 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 the current proposal—will use the base of both engineered laccases and the specific 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, and syringol.

Summary of Major Research Accomplishments and Results by Objective

Objective 1 – Library screening:  Library screening resulted in the discovery of 13 laccases with almost 100% 4EG degradation in a buffered pH 7 environment, with three also showing 90% 4EP degradation. When the environment is made more acidic, at pH 4, only laccases #33 and #36 degrade 80-90% 4EG with minimal 4EP activity. When testing in model wine with 4g/L tartaric acid, 13% ethanol, at pH 3.5, only laccases #15 and #16 completely degrade 4EG with small amounts of 4EP activity; with laccases #11 and #34 also degrading 60% 4EG. When the phenols are isolated, then #11, #15, and #36 degrade 80-90% 4EG with #36 degrading almost 30% 4EP after one week. A sequence similarity network that clustered laccases by 70% homology showed multiple clusters had activity on 4EG and 4EP at pH 7. Activity in both pH environments were limited to one cluster. High activity on 4EG in model wine was slightly spread out over three clusters, with additional low to mild activity on 4EP in two of them.

Objective 2 – Computational analysis & design:  Library screening resulted in two pairs of homologous laccases (#15 & #16; #33 & #36) representing two different paths for design towards an exclusively 4EG and 4EP degrading enzyme. However, structural analysis of the active site with the docked phenol revealed a wide-open site on all active laccases. These open active sites greatly increase the risk of off-target degradation of beneficial flavor aromatic compounds. This slowed down the next design steps with first needing to map out the active site landscape for residues required for desired activity by building and testing point mutations to alanine. Current plans include testing these point mutants for altered activities. Then, computational design with newly developed open-sourced AI-tools will be utilized to close the active site with increased specificity and exclusivity on 4EG and 4EP.