Construction and application of a bifunctional enzyme for the simultaneous enzymatic transformation of zearalenone and deoxynivalenol

Zearalenone (ZEN) and deoxynivalenol (DON) are two common toxins that often coexist and observed simultaneously in foods. In this study, we constructed and characterized the fusion enzymes consisting of ZEN lactonase (ZENM) from Monosporascus sp. GIB2. and DON dehydrogenase ( Pe DDH) from Pelagibacterium sp. SCN 63–126. The arrangement of protein domains, as well as the type and length of linker peptides were optimized for the fusion protein. When Pe DDH was positioned at the N-terminus of the fusion enzyme, the utilization of rigid linker peptides (EAAAK) 2 and (EAAAK) 3 led to a 70 % enhancement in the transformation activity of DON compared to flexible linkers (GGGGS) 2 and (GGGGS) 3 . Additionally, the longer linkers (EAAAK) 2 and (EAAAK) 3 enhanced DON transformation by approximately 70 % compared to EAAAK alone. Among all fusion constructed with different enzyme orders, the enzymatic transformation rate of ZEN exceeded 75 %, while positioning Pe DDH at the forefront of the fusion protein increased DON transformation from 68 % to 98 %. Following exploration of the reaction conditions, the fusion protein Pe DDH-(EAAAK) 3 -ZENM achieved an enzymatic transformation rate of 100 % for DON (30 μg/mL) within 16 h and 100 % for ZEN (80 μg/mL) within 30 min, indicating a great potential for the simultaneous transformation of ZEN and DON. • A fusion protein with zearalenone lactonase (ZENM) and deoxynivalenol dehydrogenase ( Pe DDH) was constructed. • The use of rigid linker peptides resulted in a 70% improvement in the degradation of DON compared to flexibleone. • Positioning Pe DDH at the forefront of the fused protein increased DON degradation from 68% to 98%. • Fusion proteindegraded 100% of zearalenone within 30 min and 100% of deoxynivalenol within 16 hours.