Engineered lipase enables selective hydrolysis of palmitic over oleic acyl chains at a two‑phase microfluidic...

The fatty acid chain length of lipids plays a pivotal role in determining their nutritional value and industrial applicability. However, the synthesis of functional lipids with defined fatty acid compositions is often limited by the insufficient chain-length specificity of conventional lipases. In this study, a chain-length-selective lipase targeting palmitic (C16) acyl chains was engineered using an integrated computational-experimental strategy, and its performance was evaluated at a two-phase microfluidic interface. The engineered variants exhibited a 0.79 to 28.48-fold enhancement in hydrolytic activity toward p-nitrophenyl palmitate (p-NP C16:0), along with improved thermal stability (t_1/2 of mutant I291Y increased from 0.40 to 2.04 h). Notably, the dominant mutant I291Y showed markedly enhanced selectivity, with hydrolytic activity toward p-NP C16:0 being 41.65-fold that toward p-nitrophenyl oleate (p-NP C18:1). Consistently, the V_max of I291Y for p-NP C16:0 was 284.19% higher than that for p-NP C18:1. Molecular docking and molecular dynamics simulations lead to a mechanistic hypothesis in which the substitution of isoleucine 291 with tyrosine is suggested to strengthen the affinity of the substrate binding tunnel for C16 acyl chains while generating steric hindrance against oleic (C18) acyl chains, thereby potentially conferring C16 selectivity. This structural modification also enabled I291Y to selectively hydrolyze C16 acyl chains in glyceryl tripalmitate rather than the C18 acyl chains in glycerol trioleate at the microfluidic two-phase interface. This work demonstrates a computationally guided engineering approach for developing a lipase with enhanced C16 preference, which may facilitate targeted lipid modification and the tailored production of nutritional lipids.