Synergistic Pairwise Residues Identification for Efficient Evolution of Candida antarctica Lipase B for Synthesis of γ- and δ-Lactones

Abstract The lipase enzyme shows great potential for catalyzing the synthesis of valuable food flavor compounds, such as γ- and δ-lactones, but natural lipases are limited by low conversion rates. In this study, we developed a computational-guided strategy named Synergistic Pairwise Residues Evolution for the efficient directed evolution of Candida antarctica lipase B (CalB). This approach identifies synergistic hotspot residues by combining substrate-contact information with evolutionarily coupled functional sectors. E188Q/I189F, the top-performing mutant with 2.75-fold improved activity, achieved over 99.5% conversion within 5 h at 37 °C, compared with only 49% for wild-type CalB in the synthesis of δ-dodecalactone. This study presents an efficient strategy for building functional enzyme toolkits and demonstrates their high catalytic performance in lactone synthesis.