MSc

The study entitled " Development of bioactive polygalactans and polyketides from seaweed associated Bacillus and assessment of therapeutic potential" was conducted at the Marine Biotechnology Fish Nutrition and Health Division, ICAR- Central Marine Fisheries Research Institute, Kochi, during 2022-23. In this study, eighty-one bacterial isolates were obtained through culture-dependent isolation of seaweed symbionts from India's southern coast. Bacillus velezensis accounted for 54% of the isolates, followed by Bacillus siamensis SUB12205505 at 33%, Bacillus subtilis SUB12455553 at 8%, and Bacillus filamentous SUB12455666 at 5% (GenBank accession numbers: OP715891, OP715894, OQ096500, and OQ096501, respectively).
B. siamensis SUB12205505 and B. velezensis SUB12205577, isolated from seaweeds Turbinaria conoides and Dictyota cervicornis, respectively, demonstrated clearance zones over 25 mm on spot-over-lawn assay against various clinical pathogens, including MRSA and VRSA. The selected isolates were sensitive to commercial antibiotics, and no pathogenicity was observed. These isolates did not amplify the hbl and nhe genes, and their non-pathogenic nature was confirmed through absence of hemolysis on blood agar. The bacterial extracts showed potent antibacterial activity against MRSA and VRSA, with MIC ranging from 6.25-12.5 µg/mL. The 700 bp pks genes (OQ657454 and OQ709135) were amplified from B. siamensis OP715894 and B. velezensis MTCC13303, showing 99% resemblance in the BLAST search.
Crude extracts from B. velezensis SUB12205577 exhibited superior ACE-1 inhibition (IC50 659.6 µg/mL) compared to other species. The α-amylase inhibitory activity of the solvent extract from B. velezensis SUB12205577 (IC50 3.15-3.81 μg/mL) surpassed that of acarbose (IC50 3.21 µg/mL). The extracts were also found to significantly inhibit pro-inflammatory enzymes, which are crucial in the metabolic process of inflammation. The solvent extract from B. siamensis OP715894 and B. velezensis MTCC13303 selectively inhibited COX-2 and 5-LOX enzymes. In addition, the ethyl acetate extract showed significant activity in a dose-response in vitro study of ACE-1 inhibition (IC50 5.77-6.18 µg/mL). Bacillus velezensis MTCC13303 had G+C contents of 46.09% with genome size of 4,176,473 bp. Functional pks genes (OQ657454, OQ709135) were found in the active strain.
An in-house pipeline mapped gene terms for molecular function, biological activity, and cellular component. AntiSMASH genome mining revealed 17 SMBGCs in Bacillus velezensis MTCC13303 involved in secondary metabolite biosynthesis. The SMBGCs in the heterotrophic bacteria encoded nrps for fengycin, bacillibactin, and surfactin synthesis, as well as pks for macrolactin, difficidin, and bacillaene synthesis. Additionally, they contained the RiPP LAP cluster, bacilysin, and one saccharide cluster. A sulfated polyglucurono-glucopyranose (EPBv-
3) characterized as [→4)-α-Glcp-{(3→1)-β-GlcAp}-α-3,6-O-di-SO3-Glcp-(1→] from Bacillus velezensis MTCC13303 was assessed for pharmacological properties employing different in vitro models.
The isolated exopolysaccharide EPBv-3 displayed potential free radical quenching activities (IC50 < 2.6 mM), whereas it exhibited potentially greater attenuation against the inducible pro-inflammatory enzyme cyclooxygenase -2 (COX-2, IC50 2.15 mM) with a greater selectivity index (1.33) than the synthetic non-steroidal anti-inflammatory drug ibuprofen (0.43). In silico molecular docking studies exhibited lesser docking score and binding energy of EPBv-3 with the aminoacid residues of COX-2 enzyme (−11.88 and −12.32 kcal/mol, respectively) recognized its prospective inhibitory property against the enzyme catalyzing the rate-limiting step to form the inflammatory causing prostaglandins (PGs). The results specified that the studied compound was composed of [→4-α-Glcp-(1→4)-α-3,6-O-di-SO3-Glcp-(1→] disaccharide repeating unit as backbone, with the β-GlcAp residues branching as C-3 substituent of the α-Glcp. The bioactive heterotrophic Bacillus velezensis MTCC13303 holds significant potential as a promising pharmaceutical agent.

B.SC-M.SC

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