Fish gut symbiotic bacterium Bacillus thuringiensis: RSM optimization for its extracellular lipase enzyme production,...

Lipase enzymes play a vital role in digestion and nutrient metabolism in host organisms, with symbiotic bacteria producing abundant enzymes, carbohydrates, vitamins, and other nutrients. This study aimed to isolate, identify, and screen lipase-producing bacteria from the gut of Systomus sarana, optimize enzyme production using Response Surface Methodology (RSM), and characterize the extracted lipase protein. A total of 11 bacterial strains were isolated and identified through 16S rRNA sequencing. Among these, Bacillus thuringiensis (SS5) exhibited the highest enzyme index (5.46 mm) and crude enzyme activity (109 U/mL). Using RSM optimization, growth conditions were refined to pH 7.5, temperature 35 °C, incubation time 30 h, with 2.3 % peptone and 2.34 % lactose, resulting in enhanced lipase production of 210 U/mL. The partially purified protein (~30 kDa) was characterized by SDS-PAGE and FTIR spectroscopy, revealed functional groups such as acids, aliphatic amines, and aromatics. MALDI-TOF/MS analysis identified eight peptides, with one major peptide sequence (IYVYYSDIMHVMNTMGQR). The modelled protein structure based on 259 amino acids was validated through homology modeling. Molecular docking studies demonstrated strong binding affinities (-7.36 to -8.95 kcal/mol) between the lipase protein and fatty acids (linoleic acid, linolenic acid, oleic acid, palmitic acid) as well as tripalmitin. These findings highlight the potential of fish gut-derived Bacillus thuringiensis as a valuable source of lipase enzymes for industrial applications such as bioremediation and biodiesel production. Further exploration of these bacterial enzymes within their native ecosystems is recommended to expand their biotechnological utility.