Discovery to Engineering of Mycotoxin Deoxynivalenol Degrading Enzymes Based on the Specialized Glyoxalase I

Deoxynivalenol (DON) is a mycotoxin that is omnipresent in food and feed. Therefore, this study has focused on discovery, molecular characterization, and engineering of DON degrading enzymes, based on a DON isomerizing enzyme (e.g., the specialized glyoxalase I from Gossypium raimondii (Gr-SPG)). A molecular phylogeny-based sequence and structure analysis elucidated the evolutionary trajectory of the DON degrading enzymes. Ancestral sequence reconstruction led to the generation of thermostable evolutionary intermediates of SPG (e.g., Anc216). Molecular modeling and consensus protein design allowed to understand the structure and function relationships and also identify the key conserved mutations that influence catalytic activity and thermostability. Ultimately, a highly active and thermostable SPG (e.g., a quintuple mutant of Anc216 (Anc216_M5)) was constructed from a newly discovered extant SPG enzyme (OR9). The Anc216_M5 exhibited a T5010$T_{50}^{10}$ of 68 °C, which is 16.3 °C higher than that of the wild-type enzyme. Furthermore, the engineered enzyme showed 40% greater DON degrading activity than OR9, which is significantly higher than that of Gr-SPG. Therefore, it is assumed that Anc216_M5 is promising as a DON-detoxifying biocatalyst.