Mary Riley,* Justin Siegel, Anita Oberholster, and David Mills
*University of California, Davis, 1 Shields Ave, Davis, CA, 95616 (myriley@ucdavis.edu)

This is the first year of a multi-year project to discover and design 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 (Figure 1). A library of 46 laccase enzymes were produced and tested in different buffered environments for activities on 4EG and 4EP, with multiple laccases degrading both phenols. All laccases were tested further in a model wine environment with 13% ethanol and tartaric acid, at pH 3.5. Multiple laccases quickly and completely degraded 4EG, along with mild activity on 4EP. Sequence analysis of the 4EG- and 4EP-active enzymes revealed two distinct clusters, each with a unique laccase that can degrade 100% of 4EG and 30% of 4EP in industrially relevant times, matrices, and concentrations. 4EP and 4EG docking interactions generated via computational modeling revealed the top-degrading laccases all display open active sites and likely contribute to activity on other, non-target, wine phenols, thereby reducing possible selective degradation of 4EG and 4EP. Currently, novel enzyme structures are being generated computationally to address this deficiency using open-source AI-tools, including RFDiffusion and LigandMPNN, with unprecedented abilities to design proteins and model their structures. The next phase of this project will use the knowledge of how laccases degrade 4EG and 4EP to create additional enzymes capable of fully, and exclusively, degrading the remaining smoke taint marker compounds including guaiacol, 4-methylguaiacol, o-, p-, and m-cresol, and syringol. The larger goal of this effort is to create a suite of novel enzymes that beneficially modulate wine flavor.

Funding Support: American Vineyard Foundation