Response surface methodology based optimization of keratinase from Bacillus velezensis strain ZBE1 and nanoparticle synthesis, biological and molecular characterization.

The increasing demands of keratinases for biodegradation of recalcitrant keratinaceous waste like chicken feathers has lead to research on newer potential bacterial keratinases to produce high-value products with biological activities. The present study reports a novel keratinolytic bacterium Bacillus velezensis strain ZBE1 isolated from deep forest soil of Western Ghats of Karnataka, which possessed efficient feather keratin degradation capability and induced keratinase production. Production kinetics depicts maximum keratinase production (11.65 U/mL) on 4th day with protein concentration of 0.61 mg/mL. Effect of various physico-chemical factors such as, inoculum size, metal ions, carbon and nitrogen sources, pH and temperature influencing keratinase production were optimized and 3.74 folds enhancement was evidenced through response surface methodology. Silver (AgNP) and zinc oxide (ZnONP) nanoparticles with keratin hydrolysate produced from chicken feathers by the action of keratinase were synthesized and verified with UV-Visible spectroscopy that revealed biological activities like, antibacterial action against Bacillus cereus and Escherichia coli. AgNP and ZnONP also showed potential antioxidant activities through radical scavenging activities by ABTS and DPPH. AgNP and ZnONP revealed cytotoxic effect against MCF-7 breast cancer cell lines with IC50 of 5.47 µg/ml and 62.26 µg/ml respectively. Characterizations of nanoparticles were carried out by Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive X-ray, X-ray diffraction, thermogravimetric analysis and atomic force microscopy analysis to elucidate the thermostability, structure and surface attributes. The study suggests the prospective applications of keratinase to trigger the production of bioactive value-added products and significant application in nanotechnology in biomedicine.

In-vitro and computational analysis of Urolithin-A for anti-inflammatory activity on Cyclooxygenase 2 (COX-2).

Cyclooxygenase 2 (COX-2) participates in the inflammation process by converting arachidonic acid into prostaglandin G2 which increases inflammation, pain and fever. COX-2 has an active site and a heme pocket and blocking these sites stops the inflammation. Urolithin A is metabolite of ellagitannin produced from humans and animals gut microbes. In the current study, Urolithin A showed good pharmacokinetic properties. Molecular docking of the complex of Urolithin A and COX-2 revealed the ligand affinity of -7.97 kcal/mol with the ligand binding sites at TYR355, PHE518, ILE517 and GLN192 with the 4-H bonds at a distance of 2.8 Å, 2.3 Å, 2.5 Å and 1.9 Å. The RMSD plot for Urolithin A and COX-2 complex was observed to be constant throughout the duration of dynamics. A total of 3 pair of hydrogen bonds was largely observed on average of 3 simulation positions for dynamics duration of 500 ns. The MMPBSA analysis showed that active site amino acids had a binding energy of -22.0368 kJ/mol indicating that throughout the simulation the protein of target was bounded by Urolithin A. In-silico results were validated by biological assays. Urolithin A strongly revealed to exhibit anti-inflammatory effect on COX-2 with an IC50 value of 44.04 µg/mL. The anti-inflammatory capability was also depicted through reduction of protein denaturation that showed 37.6 ± 0.1 % and 43.2 ± 0.07 % reduction of protein denaturation for BSA and egg albumin respectively at 500 µg/mL. The present study, suggests Urolithin A to be an effective anti-inflammatory compound for therapeutic use.

Molecular docking and simulation studies against nucleoside diphosphate kinase (NDK) of Pseudomonas aeruginosa with secondary metabolite identified by genome mining from paenibacillusehimensis.

Pseudomonas aeruginosa is one of the leading opportunistic pathogens that causes nosocomial pneumonia and mostly in people with cystic fibrosis. In the present study, an in-silicoapproach was adopted to identify the novel drug target against Pseudomonas aeruginosa by employing subtractive genomics and molecular docking studies. Each step in the subtractive genomics scrutinized the bacterial proteome and determined a potential drug target against Pseudomonas aeruginosa. 71 essential proteins were obtained from the subcellular localization method that resides in the extracellular region. Metabolic pathways were studied to elucidate the unique pathways where the involvement of proteins present in the pathogen was predicted and a total of 6 unique pathways were determined. By, Genome mining of the source organism Paenibacillusehimensis, 9 ligands were obtained. The molecular docking analysis between the binding site of target protein NDK and ligands was carried out by employing the AutoDock Vina tool. Based on the highest binding affinity, Paenibactin, AnabaenopeptinNZ857 and Nostamide A complex with NDK protein with a lower binding energy of -7.5 kcal/mol, -7.4and -7.2 kcal/molrespectively were considered for the simulation studies. Molecular dynamics simulation studies showed the ligand in complex with protein was highly stable and rigid for a duration of 150 ns. For Paenibactin, AnabaenopeptinNZ857 and Nostamide Acomplex with protein, RMSD plot showed a deviation of ∼0.2-0.3 nm till ∼30ns/50 ns-110ns and further stabilized. The radius of the gyration plot clearly showed that the values stayed at ∼1.45 nm- 1.55 nm showing compactness and stability. The SASA stayed at the range ∼80nm2 and at least one total number of hydrogen bonds was shown throughout the 150 ns simulation for all three possible ligand-protein complexes. In the RMSF plot, the maximum fluctuation was ranged from ∼0.4-0.42 nm at the range between ∼57ns-60ns.The Paenibactin, AnabaenopeptinNZ857 and Nostamide A complex with NDK protein showed a stable, rigid and compact interaction throughout the simulation of duration 150 ns.Communicated by Ramaswamy H. Sarma.