Enhancing catalytic efficiency of a deep-sea alkaline lipase through integrated engineering of lid-associated dynamics

A lipase with strong alkaline preference and broad tolerance was identified. • Catalytic efficiency increased by 360-fold via an integrated engineering strategy. • Lid dynamics modulation improves activity through mutations outside the lid region. A deep-sea alkaline lipase, My Lip2, from   Moritella yayanosii   was identified from a metagenomic library of 1,048,576 genes. The wild-type enzyme preferred medium- to long-chain p-nitrophenyl esters, with optimal activity at pH 10.5 and 40 °C, but its specific activity was only 2.93 U/mg toward p -nitrophenyl palmitate. To improve performance, we used a structure- and sequence-guided strategy targeting noncatalytic residues around the catalytic center and lid region. Combinatorial engineering produced triple A271F/V250L/L231P and quadruple A271F/V250L/L231P/T300K (4 M), with comparable specific activities of 743.4 and 745.4 U/mg; 4 M was chosen for its high activity and improved thermal tolerance. This variant showed ∼ 196–fold higher catalytic efficiency ( k cat / K m ) toward p –nitrophenyl palmitate, with increased   V max and k cat . Molecular docking, kinetics, and simulations indicated that the substitutions support a more open and catalytically accessible lid conformation, facilitating substrate access and turnover. Comparison with reported lipases indicated that My Lip2 and 4 M combine alkaline preference, medium– to long–chain activity, and improved performance. This work provides a high–performance deep–sea alkaline lipase and suggests that catalytic efficiency can be improved by tuning noncatalytic residues that influence the catalytic–center microenvironment and lid dynamics, without mutating the catalytic triad or redesigning the lid. Download: Download high-res image (190KB) Download: Download full-size image