A Combination of Two‐Enzyme System and Enzyme Engineering Improved the Activity of a New PET Hydrolase Identified from Soil Bacterial Genome

Abstract We here report a novel PET hydrolase originating from a soil microbial genome sequence. This enzyme, bbPET0069, exhibits characteristics resembling a cutinase‐like Type I PET‐degrading enzyme but lacks disulfide bonds. Notably, bbPET0069 displayed remarkable synergy with Candida antarctica lipase B (CALB), demonstrating rapid and efficient PET degradation. To improve the PET degradation activity of bbPET0069, we employed a 3D structural modeling to identify mutation sites around its substrate binding domain combined with a protein language model for effective mutation prediction. Through three initial rounds of directed evolution, we achieved a significant enhancement in PET degradation with CALB, resulting in a 12.6‐fold increase compared to wild‐type bbPET0069 without CALB. We confirmed its PET degradation activity in PET nanoparticles and films, and our proposed approach enabled efficient PET degradation to terephthalic acid monomers up to 95.5%. Our approach, which integrates a two‐enzyme system with protein engineering, demonstrates the potential for enhancing the activity of emerging PET‐degradation enzymes, which may possess unique attributes.