Barkha Singhal

Non-ribosomal peptide synthetases (NRPS) are multi-modular enzymes that produce secondary metabolites. The diverse and functionally important biosurfactants derived from various microbes have numerous applications across multiple industries. Probiotic-derived biosurfactants, especially those from bacteria classified as Generally Recognized As Safe (GRAS), are emerging as eco-friendly alternatives to synthetic surfactants. Although probiotics have been studied for their potential to produce biosurfactants, the molecular mechanisms behind this process remain unclear. This work aims to link the NRPS structures of probiotics to their potential for biosurfactant production using computational methods, along with a recent review of their types, applications, and biotechnological strategies to enhance their production. For this purpose, commercial probiotic species have been selected for in silico screening of NRPS and its domains. The in silico structural modeling and validation of these domains were performed, and their similarity was compared with that of the commercial biosurfactant surfactin, derived from B. subtilis. We explored the potential for biosurfactant production in probiotics, paving the way for future genetic engineering and synthetic biology approaches to modify NRPS. This comprehensive review and research effort broaden our understanding of the NRPS system in probiotics and their potential for industrial biosurfactant development.

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.

The present study investigates the extraction and characterization of biosurfactants from two Indian originated Lactobacillus probiotics. Biosurfactants derived through Lactobacillus species have potential applications in food, pharmaceutical, and biomedical sectors. In the current study, Lactobacillus helveticus MTCC5463 and Lactobacillus rhamnosus MTCC5462 species were aimed to extract, purify, and characterize surface-active properties of produced biosurfactants. The species were cultured in their selective medium and biosurfactants derived are extracted and purified through acid precipitation and solvent extraction. The characterization was performed through measurement of surface tension reduction emulsification index (E24), Fourier transform infrared spectroscopy (FTIR), gas chromatography–mass spectrometry (GC-MS), and ¹ H and ¹³ C nuclear magnetic resonance (NMR) spectroscopy. The biosurfactants reduced surface tension from 72 mN/m (distilled water baseline) to 38.73 (± 0.05) mN/m and 41.30 (± 0.04) mN/m for L. helveticus MTCC5463 and L. rhamnosus MTCC5462, respectively. The emulsification activity was high (E24 > 70%) for various hydrophobic substrates such as petrol, diesel, mustard, and refined oil. FTIR and GC-MS revealed a glycolipid nature, with major fatty acid components such as butanoic acid. The ¹ H NMR data support an amphiphilic glycolipid structure characterized by long aliphatic fatty acid chains [δ 0.90–1.50 parts per million (ppm)] and esterified (or hydroxyl‐adjacent) methylene groups. The ¹³ C NMR data are consistent with a biosurfactant structure comprising long aliphatic chains (δ 14.11; 29.35–31.92 ppm), methylene carbons adjacent to ester or hydroxyl groups (δ 40.95 ppm), and oxygenated carbons (δ 76.70–77.34 ppm) indicative of glycerol or sugar headgroup backbone. These findings highlighted that Lactobacillus -derived biosurfactants have strong surface-active properties that can be explored for various biomedical applications in future studies.

The overwhelming use of PET plastic in various day-to-day activities led to the voluminous increase in PET waste and growing environmental hazards. A plethora of methods have been used that are associated with secondary pollutants. Therefore, microbial degradation of PET provides a sustainable approach due to its versatile metabolic diversity and capacity. The present work highlights the cutinase enzyme's role in PET degradation. This study focuses on the bacterial cutinases homologs screened from 43 reported phylum of bacteria. The reported bacterial cutinases for plastic degradation have been chosen as reference sequences, and 917 sequences have shown homology across the bacterial phyla. The dienelactone hydrolase (DLH) domain was identified for attaining specificity towards PET binding in 196 of 917 sequences. Various computational tools have been used for the physicochemical characterization of 196 sequences. The analysis revealed that most selected sequences are hydrophilic, extracellular, and thermally stable. Based on this analysis, 17 sequences have been further pursued for three-dimensional structure prediction and validation. The molecular docking studies of 17 selected sequences revealed efficient PET binding with the three sequences derived from the phylum Bacteroidota, the lowest binding energy of -5.9 kcal/mol, Armatimonadota, and Nitrososphaerota with -5.8 kcal/mol. The two enzyme sequences retrieved from the phylum Bacteroidota and Armatimonadota are metagenomically derived. Therefore, the present studies concluded that there is a high probability of finding cutinase homologs in various environmental resources that can be further explored for PET degradation. Graphical abstract