Abstract Description

This study reports the cloning, expression, and characterization of an extracellular lipase from Serratia liquefaciens AB1, isolated from petroleum-contaminated soil in Nigeria. The 1,800 bp lipase gene encoding a 599-amino acid protein, molecular weight of 64 kDa, was cloned into the pET28a vector and expressed in Escherichia coli BL21, with recombinant colonies exhibiting lipase activity on rhodamine B–olive oil agar. The crude enzyme showed 964.47 ± 0.42 U/ml activity, 425.02 ± 0.42 U/ml after His-tag purification, with a specific activity of 131.59 U/mg, substantially higher than the native enzyme. Structural modeling revealed a typical α/β-hydrolase fold with predicted catalytic triad and calcium-binding sites, while sequence alignment of two S. liquefaciens lipases (SlipB and SlipA) indicated conserved active sites and substitutions enhancing thermostability and solvent tolerance. The recombinant lipase exhibited optimal activity at 60 °C and pH 8, retained over 50 % activity at 80 °C for 2 hours, and showed the highest substrate specificity toward long-chain esters, particularly p-nitrophenyl palmitate (C16), confirming true lipase activity. Kinetic analysis showed Michaelis–Menten behavior with p-NPP as the preferred substrate (Km = 4.232 mM, Vmax = 42.456 µM·mg⁻¹·min⁻¹). Enzyme activity was influenced by metal ions, Ca²⁺, while Hg²⁺ inhibited activity. The lipase enhanced activity in water-immiscible solvents isopropanol, and tolerated chemical modifiers, including SDS and DTT. Among surfactants and additives tested, Triton X-100 elicited the highest activity. The recombinant S. liquefaciens lipase demonstrates high catalytic efficiency, substrate specificity for long-chain esters, thermostability, and chemical resilience, highlighting its potential in petroleum bioremediation and industrial biocatalysis.