A deep-sea alkaline lipase, MyLip2, fromMoritella yayanosiiwas 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 (kcat/Km) toward p-nitrophenyl palmitate, with increasedVmax and kcat. 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 MyLip2 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.
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