A Sequence-Based Approach Reveals a Novel Family IV Lipolytic Enzyme in Cereibacter sphaeroides 2.4.1

Bacterial lipases and esterases are central to lipid metabolism and biotechnology, yet they are underexplored in purple non-sulfur photosynthetic bacteria. To address this gap, we mined the genome of Cereibacter sphaeroides 2.4.1 by retrieving the sequences annotated by alpha beta hydrolase, esterase, and lipase from NCBI database. All sequences aligned to well-known of bacterial esterase or lipase and the higher identity was cloned and expressed in Escherichi coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed soluble expression of four proteins, with LipCs2 (26.4 kDa), LipCs3 (39.4 kDa), and LipCs9 (24.1 kDa) showing activity on tributyrin agar and preference for short-chain para-nitrophenyl esters. Although LipCs9 (314.11 ± 2.44 mM h^-1 mg^-1) showed the highest activity on pNP-butyrate, LipCs3 (25.57 ± 2.22 mM h^-1 mg^-1) was prioritized due to its novelty and strong performance rather than LipCs2 (17.00 ± 0.62 mM h^-1 mg^-1). Optimization of IPTG induction (0.25, 0.5, and 1 mM) for LipCs3 expression in E. coli revealed severe cytotoxicity in both cytoplasmic and periplasmic systems, prompting its heterologous expression in Bacillus subtilis WB800. Despite successful secretion, the intracellular (10.29 U) and extracellular (11.24 U) levels remained nearly equal, indicating inefficient signal peptide function. Phylogenetic analysis classified LipCs3 within bacterial family IV lipolytic enzymes, closely related to Brefeldin A esterase. Docking analysis revealed a close proximity between the ligand carbonyl group of pNP-butyrate and the hydroxyl of Ser153 within the binding pocket. Genomic analysis revealed a GntR binding motif overlapping the -10 promoter region of lipCs3 gene, suggesting transcriptional regulation. These findings broaden the current understanding of family IV bacterial lipolytic by establishing LipCs3 as a novel representative of this group.