Unlocking the role of chaperones on lipase from Photobacterium marinum J15

Lipase, a Family I lipolytic enzyme, catalyzes fatty acid synthesis and hydrolysis. It is classified into six subfamilies due to its conserved pentapeptide motif, Gly-Xaa-Ser-Xaa-Gly, and exhibits broad substrate specificity and regiospecificity, making it valuable for various applications. Photobacterium strain J15, a gram-negative bacterium isolated from Johor seawater, Malaysia, displays lipolytic activity. The gene encoding lipase (LipJ15) and its chaperone (Lif15) was codon-optimized, yielding proteins of 42.57 kDa and 33.24 kDa, respectively. Interestingly, LipJ15 shares only 56.14% amino acid sequence identity with Pseudomonas aeruginosa lactonizing lipase, suggesting unique structural features. Cloning and expression of LipJ15 were conducted using the recombinant pET- 32b(+)::LipJ15+pBB550 system in E. coli Origami (DE3). The pBB550 vector carried chaperone genes (dnaK, dnaJ, GroESL). Expression at 0.3 mM IPTG and 20°C resulted in abundant soluble protein, as confirmed by SDS-PAGE. However, lipase activity was low at 0.3 mM IPTG (3.55 U), with the highest activity at 0.6 mM IPTG (7.37 U). Structural analysis of LipJ15 revealed an α/β-hydrolase fold comprising 38.1% helices, 13.9% strands, and a catalytic triad (Ser92, Asp239, His261) within the conserved GXSXG motif. The structure also included a disulfide bridge and a Ca2+ binding site. Evaluation using the Ramachandran plot, Verify3D, and ERRAT confirmed the model's quality, with 89.8% of residues in favored regions. Protein-ligand docking analysis demonstrated LipJ15's high specificity toward short-chain substrates. Its structure could significantly contribute to structural biology by serving as a template for solving unsolvable protein structures with low sequence identity through molecular replacement, enhancing our understanding of related enzymes.

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