In silico and experimental characterization of NRPS adenylation domain-mediated biosurfactant production in Lactobacillus helveticus MTCC 5463

Abstract

Biosurfactants are amphiphilic biomolecules that are produced by microorganisms and have important industrial, environmental, and biomedical applications because of their biodegradability, low toxicity, and high surface activity. In the present study, the potential of biosurfactant production by a Lactobacillus helveticus indigenous strain (MTCC 5463) was investigated using integrated in silico , molecular, biochemical, and analytical approaches focused on the conserved non-ribosomal peptide synthetase (NRPS) adenylation domain. Comparative sequence and domain analysis of the Bacillus velezensis FZB42 reference identified domains for adenylation, condensation, and peptidyl carrier proteins (PCP) that are shared by biosurfactant biosynthetic genes. The AMP-binding domain was amplified, and then the resulting fragment was directionally cloned into the pET-28a expression vector and expressed in Escherichia coli BL21(DE3). A recombinant 14-kDa AMP-binding protein, which was associated with the production of a biosurfactant-like compound, was produced. The recombinant biosurfactant was extracted using chloroform:methanol solvent systems and found to have significant emulsification activity against petrol, diesel, mustard oil, and refined oil, and also its E24 value was found to be stable for 1 week. Thin-layer chromatography confirmed the presence of lipidic and glycolipid components, while FTIR analysis indicated the presence of hydroxyl, amide, aliphatic, and carbohydrate-associated functional groups, which are characteristics of a glycolipopeptide structure. The hydrophobic lipid architecture of the biosurfactant was supported by GC–MS profiling, which identified the presence of significant amounts of C16/C18 long-chain fatty acid derivatives and amides. Moreover, ¹H NMR and ¹³C NMR analyses indicated the presence of peptide bonds, glycerol/carbohydrate groups, and long hydrocarbon chains, giving further confirmation of the amphiphilic glycolipopeptide nature of the recombinant biosurfactant. In conclusion, the study shows that conserved NRPS adenylation domains can be utilized to produce biosurfactants in probiotic lactic acid bacteria and suggests that the recombinant glycolipopeptide biosurfactants produced by L. helveticus MTCC 5463 have potential for use in the industry.