Enhancing Solanum tuberosum EpoxideHydrolase 1 for Bioremediation: DehalogenaseActivity of Variants with Desyl Bromide andChloride

Halogenated organic compounds (HOCs) are environmental pollutants with several significantecological and health impacts due to their persistent chemical stability and bioaccumulation.Fortunately, organisms have evolved enzymes capable of degrading such compounds. This studyexplores the dehalogenase activity and substrate versatility of Solanum tuberosum epoxidehydrolase 1 (StEH1) variants with desyl halides, building on prior identification of the Y235Ivariant with enhanced activity with 2-phenylethyl chloride and -bromide. For desyl chloride, thenewly expressed Y235I variant slightly outperformed the wild type at 22 h and 2 h, while theolder Y235I showed marginally higher activity. For desyl bromide, the newly expressed Y235Ivariant achieved the highest benzoin yields across both time points, with Y235I variantsexhibiting greater dehalogenase activity than the wild type. The wild type also demonstratedsignificant activity, likely attributed to the structural similarity of desyl halides to trans-stilbeneoxide, which is a preferred StEH1 substrate. However, benzoin oxidation to benzil, observed as amerged peak at 5 minutes, complicated product quantification, especially for desyl bromidesamples, reducing 22 h yields and suggesting shorter reaction times for accurate benzoinquantification. These finding highlight the Y235I variant’s enhanced catalytic efficiency and theimportant role of substrate structure in StEH1 activity, though results require cautionsinterpretation due to benzil formation. Second-generation variants derived from the Y235I parentstructure, particularly GQMVII, faced significant hurdles in molecular cloning and proteinexpression/purification. Despite successful ligation, only GQMVII yielded correct sequence, butpurification failed likely due to protein degradation/absence or an excessively high imidazoleconcentration during purification.