Exploring the influence of lid region residues on fatty acid selectivity in a lipase originating from <i>Rhizopus oryzae</i>

Lipases are vital in modifying lipid substrates across industries such as food, cosmetics, and pharmaceuticals. Among their features, fatty acid selectivity is particularly important for industrial applications. Rhizopus oryzae lipase (ROL) stands out for its high selectivity and broad applicability. In this study, we engineered single-residue variants of ROL by targeting Ala89 and Phe95 in its lid region. Additionally, a lid-swap chimera was created by replacing ROL's 15-residue lid with that of the homologous lipase from Rhizomucor miehei (RML). These variants were expressed and characterized to assess changes in substrate selectivity. Our results highlight the lid's key role in determining fatty acid preference. Notably, mutating Phe95 to smaller residues (Ile or Ala) significantly increased selectivity toward medium-chain fatty acid (MCFA) esters. In contrast, substituting Ala89 with bulkier residues (Phe or Trp) reduced activity-except in the lid-swap variant. Interestingly, although the lid-swap variant contains Trp89, the surrounding smaller, non-conserved residues may alleviate steric hindrance. This chimera retained high activity but shifted its preference from MCFAs to long-chain fatty acids (LCFAs), a novel observation. Overall, the engineered variants exhibited distinct substrate preferences without compromising thermostability, suggesting their potential for tailored applications in food, nutrition, and cosmetic industries.