Characterization and molecular dynamics simulation of Lk2 lipase expressed in Pichia pastoris

Lipase is a versatile enzyme that serves as a biocatalyst in various industries. lk2 was successfully isolated from household waste compost through a metagenomic approach.

lk2 from plasmid pPICZαA- lk2 was integrated into chromosomes of Pichia. pastoris GS115 using the electroporation method. Lk2 was expressed on Pichia. pastoris by methanol induction. The enzyme was purified through Ion Metal Affinity Chromatography Ni-NTA. The purified enzyme was characterized based on hydrolytic activity and in silico analysis.

Lk2 was successfully expressed as an extracellular protein in Pichia pastoris. The cell-free supernatant exhibited hydrolysis activity to para-nitro phenyl laurate. The purified protein showed 15 times activity compared to cell-free supernatant and the size at around 35 kDa following gel electrophoresis. The enzyme showed optimum activity at 60^oC and pH 8. Lk2 preferred para nitro phenyl laurate as substrate. The enzyme's preference for medium-long carbon chains was corroborated by in silico analysis, which revealed favorable interactions between the enzyme and substrate, including affinity binding energy and optimal orientation of catalytic pocket to the substrate. Furthermore, the radius of gyration analysis of the Lk2 showed that the best structural compactness of Lk2 was at 60^oC. This is in line with the optimal temperature of Lk2 activity. In addition, docking analysis found important substrate binding residues, including Tyr30, Ser85, Leu121, Leu163, Leu166, Leu 233, and Leu254 beside Ser85, Asp231, and His253 as triad catalytic.

Lk2 belongs to a thermotolerant and alkaline lipase, prefers a medium-length carbon chain as substrate and is confirmed by in silico analysis. Several amino acid residues were probed to be important for substrate binding residues. The data give valuable information to develop the possibility of Lk2 as an industry's enzyme.