Purification, and characterization of detergent-compatible serine protease from Bacillussafensis strain PRN1: A sustainable alternative to hazardous chemicals in detergent industry.

Owing to vast therapeutic, commercial, and industrial applications of microbial proteases microorganisms from different sources are being explored. In this regard, the gut microbiota of Monopteruscuchia were isolated and examined for the production of protease. All the isolates were primarily and secondarily screened on skim milk and gelatin agar plates. The protease-positive isolates were characterized morphologically, biochemically, and molecularly. Out of the 20 isolated strains,6 belonging to five different genera viz.Bacillus,Priestia,Aeromonas,Staphylococcus, and Serratia demonstrated proteolytic activity. Bacillussafensis strain PRN1 demonstrated the highest protease production and, thus, the largest hydrolytic clear zones in both skim milk agar (15 ± 1 mm) and gelatin (16 ± 1 mm) plates. The optimized parameters (time, pH, temperature, carbon, nitrogen) for highest protease activity and microbial growth of B.safensis strain PRN1 includes 72 h (OD600 = 0.56,1303 U/mL), pH 8 (OD600 = 0.83, 403.29 U/mL), 40 °C (OD600 = 1.75, 1849.11 U/mL), fructose (OD600 = 1.22, 1502 U/mL), and gelatin (OD600 = 1.88, 1015.33 U/mL). The enzyme was purified to homogeneity using salt-precipitation and gel filtration chromatography. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified enzyme was a monomer of a molecular weight of ∼33 kDa. The protease demonstrated optimal activity at pH 8 and 60 °C. It was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), demonstrating that it belongs to the serine-proteases family. The compatibility of the enzyme with surfactants and commercial detergents demonstrates its potential use in the detergent industry. Furthermore, the purified enzyme showed antibacterial and blood-stain removal properties.

Prophylactic application of vaginal lactic acid bacteria against urogenital pathogens and its prospective use in sanitary suppositories / Aplicación profiláctica de bacterias ácido lácticas vaginales contra patógenos urogenitales y su uso prospectivo en supositorios sanitarios

Beneficial and pathogenic microbes coexist in the vaginal canal, where a diminishing population of lactic acid bacteria may cause recurring urogenital infections. Probiotic bacteria Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus vaginalis, and pathogenic microbes Enterococcus faecalis, Enterobacter cloacae, Shigella sp., Staphylococcus epidermidis, and Escherichia fergusonii were isolated from vaginal swabs. Lactobacillus sp. and their probiotic culture free supernatant (PCFS) inhibited the growth of the above-mentioned urogenital pathogens. L. crispatus produced both lactic acid and hydrogen peroxide, exhibiting the best antimicrobial potential against the studied pathogens. Lyophilized L. crispatus had a shelf life of 12 months and the lyophilized PCFS also retained its antibacterial property with a minimum inhibition concentration of 1 μg/μL. Carboxy-methyl cellulose-alginate, a green alternative to super-absorbent polymers, was encapsulated with L. crispatus cells. The probiotic in its encapsulated state retained its viability for 21 days, and the bead showed 30% solvent absorptive capacity. PCFS-laced non-woven fabric displayed antibacterial property with no change in its physicochemical properties. These probiotic and postbiotic formulations have excellent prophylactic potential for urogenital infections. Such formulations can be exploited as additives in sanitary suppositories to enhance vaginal health.(AU)

Inhibiting pathogenicity of vaginal Candida albicans by lactic acid bacteria and MS analysis of their extracellular compounds.

Maintaining healthy vaginal microflora post-puberty is critical. In this study we explore the potential of vaginal lactic acid bacteria (LAB) and their extracellular metabolites against the pathogenicity of Candida albicans. The probiotic culture free supernatant (PCFS) from Lactobacillus crispatus, L. gasseri, and L. vaginalis exhibit an inhibitory effect on budding, hyphae, and biofilm formation of C. albicans. LGPCFS manifested the best potential among the LAB PCFS, inhibiting budding for 24 h and restricting hyphae formation post-stimulation. LGPCFS also pre-eminently inhibited biofilm formation. Furthermore, L. gasseri itself grew under RPMI 1640 stimulation suppressing the biofilm formation of C. albicans. The PCFS from the LAB downregulated the hyphal genes of C. albicans, inhibiting the yeast transformation to fungi. Hyphal cell wall proteins HWP1, ALS3, ECE1, and HYR1 and transcription factors BCR1 and CPH1 were downregulated by the metabolites from LAB. Finally, the extracellular metabolome of the LAB was studied by LC-MS/MS analysis. L.gasseri produced the highest antifungal compounds and antibiotics, supporting its best activity against C. albicans. Vaginal LAB and their extracellular metabolites perpetuate C. albicans at an avirulent state. The metabolites produced by these LAB in vitro have been identified, and can be further exploited as a preventive measure against vaginal candidiasis.

Prophylactic application of vaginal lactic acid bacteria against urogenital pathogens and its prospective use in sanitary suppositories.

Beneficial and pathogenic microbes coexist in the vaginal canal, where a diminishing population of lactic acid bacteria may cause recurring urogenital infections. Probiotic bacteria Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus vaginalis, and pathogenic microbes Enterococcus faecalis, Enterobacter cloacae, Shigella sp., Staphylococcus epidermidis, and Escherichia fergusonii were isolated from vaginal swabs. Lactobacillus sp. and their probiotic culture free supernatant (PCFS) inhibited the growth of the above-mentioned urogenital pathogens. L. crispatus produced both lactic acid and hydrogen peroxide, exhibiting the best antimicrobial potential against the studied pathogens. Lyophilized L. crispatus had a shelf life of 12 months and the lyophilized PCFS also retained its antibacterial property with a minimum inhibition concentration of 1 µg/µL. Carboxy-methyl cellulose-alginate, a green alternative to super-absorbent polymers, was encapsulated with L. crispatus cells. The probiotic in its encapsulated state retained its viability for 21 days, and the bead showed 30% solvent absorptive capacity. PCFS-laced non-woven fabric displayed antibacterial property with no change in its physicochemical properties. These probiotic and postbiotic formulations have excellent prophylactic potential for urogenital infections. Such formulations can be exploited as additives in sanitary suppositories to enhance vaginal health.

Bioprospecting of indigenous biosurfactant-producing oleophilic bacteria for green remediation: an eco-sustainable approach for the management of petroleum contaminated soil.

In the present study, the efficiency of four different strains of Pseudomonas aeruginosa and their biosurfactants in the bioremediation process were investigated. The strains were found to be capable of metabolizing a wide range of hydrocarbons (HCs) with preference for high molecular weight aliphatic (ALP) over aromatic (ARO) compounds. After treating with individual bacteria and 11 different consortia, the residual crude oils were quantified and qualitatively analyzed. The bacterial strains degraded ALP, ARO, and nitrogen, sulphur, oxygen (NSO) containing fractions of the crude oil by 73-67.5, 31.8-12.3 and 14.7-7.3%, respectively. Additionally, the viscosity of the residual crude oil reduced from 48.7 to 34.6-39 mPa s. Further, consortium designated as 7 and 11 improved the degradation of ALP, ARO, and NSO HCs portions by 80.4-78.6, 42.7-42.4 and 21.6-19.2%, respectively. Moreover, addition of biosurfactant further increased the degradation performance of consortia by 81.6-80.7, 43.8-42.6 and 22.5-20.7%, respectively. Gas chromatographic analysis confirmed the ability of the individual strains and their consortium to degrade various fractions of crude oil. Experiments with biosurfactants revealed that polyaromatic hydrocarbons (PAHs) are more soluble in the presence of biosurfactants. Phenanthrene had the highest solubility among the tested PAHs, which further increased as biosurfactant doses raised above their respective critical micelle concentrations (CMC). Furthermore, biosurfactants were able to recover 73.5-63.4% of residual oil from the sludge within their respective CMCs. Hence, selected surfactant-producing bacteria and their consortium could be useful in developing a greener and eco-sustainable way for removing crude oil pollutants from soil.

Inhibition of Staphylococcus aureus and Pseudomonas aeruginosa Biofilm and Virulence by Active Fraction of Syzygium cumini (L.) Skeels Leaf Extract: In-Vitro and In Silico Studies.

Syzygium cumini L. Skeels (Myretacae family) is a native plant of the Indian subcontinent which has wide socio-economical importance and is well known for its ant diabetic activity. The present study aimed to investigate the antibiofilm activity of purified fraction (EA) from S. cumini leaf extract against P. aeruginosa and S. aureus. The EA did not show any effect on growth of P. aeruginosa and S. aureus at the concentration of 900 µg/ml. At this concentration EA showed biofilm inhibition up to 86 ± 1.19% (***P < 0.0001) and 86.40 ± 1.19% (***P < 0.0001) in P. aeruginosa and S. aureus respectively. SEM examination also confirmed the reduction in biofilm formation. Further EA also disrupted some virulence phenotypes in P. aeruginosa and S. aureus. Bioactive compounds detected by GC-MS showed their possible molecular interaction with RhlG/NADP active-site complex (PDB ID: 2B4Q), LasR-TP4 complex (PDB ID: 3JPU) and Pseudaminidase (PDB ID: 2W38) from P. aeruginosa. The in vitro biofilm inhibition, virulence factor inhibition and the mode of interaction of bioactive components in Syzygium cumini with QS proteins of bacteria reported in this study might be an affordable and effective alternative method of controlling quorum sensing/biofilm-associated infections.

Molecular docking simulation analysis of the interaction of dietary flavonols with heat shock protein 90.

Hsp90 is a major protein involved in the stabilization of various proteins in cancer cells. The present investigation focused on the molecular docking simulation studies of flavanols as inhibitors of Hsp90 at the high affinity adenosine triphosphate (ATP) binding site and analyzed absorption, distribution, metabolism, excretion and toxicity (ADME-toxicity). The molecular docking analysis revealed that the flavanols showed competitive inhibition with ATP molecule at the active site and enhanced pharmacological parameters.

Natural polyhydroxyalkanoate-gold nanocomposite based biosensor for detection of antimalarial drug artemisinin.

The worrisome trend of antimalarial resistance has already highlighted the importance of artemisinin as a potent antimalarial agent. The current investigation aimed at fabricating a biosensor based on natural polymer polyhydroxyalkanoate-gold nanoparticle composite mounting on an indium-tin oxide glass plate for the analysis of artemisinin. The biosensor was fabricated using an adsorbing horse-radish peroxidase enzyme on the electrode surface for which cyclic voltammetry was used to monitor the electro-catalytic reduction of artemisinin under diffusion controlled conditions. Electrochemical interfacial properties and immobilization of enzyme onto a polyhydroxyalkanoate-gold nanoparticle film were evaluated, and confirmed by cyclic voltammetry, electrochemical impedance spectroscopy and scanning electron microscopy. The differential pulse voltammetric peak current for artemisinin was increased linearly (concentration range of 0.01-0.08µg mL(-1)) with sensitivity of 0.26µAµg mL(-1). The greater sensitivity of the fabricated biosensor to artemisinin (optimum limits of detection were 0.0035µg mL(-1) and 0.0036µg mL(-1) in bulk and spiked human serum, respectively) could be of much aid in medical diagnosis.

Assessment of five soil DNA extraction methods and a rapid laboratory-developed method for quality soil DNA extraction for 16S rDNA-based amplification and library construction.

Extraction of DNA from soil samples using standard methods often results in low yield and poor quality making them unsuitable for community analysis through polymerase chain reaction (PCR) due to the formation of chimeric products with smaller template DNAs and the presence of humic substances. The present study focused on the assessment of five different methods for metagenomic DNA isolation from soil samples on the basis of processing time, purity, DNA yield, suitability for PCR, restriction digestion and mDNA library construction. A simple and rapid alkali lysis based on indirect DNA extraction from soil was developed which could remove 90% of humic substances without shearing the DNA and permits the rapid and efficient isolation of high quality DNA without the requirement of hexadecyltrimethylammonium bromide and phenol cleanup. The size of DNA fragment in the crude extracts was >23 kb and yield 0.5-5 µg/g of soil. mDNA purification using Sephadex G-50 resin yielded high concentration of DNA from soil samples, which has been successfully used for 16S rDNA based amplification of a 1500 bp DNA fragment with 27F and 1492R universal primers followed by restriction digestion and mDNA library construction.

Possible protection of silver nanoparticles against salt by using rhamnolipid.

Rhamnolipid (RL) from Pseudomonas aeruginosa was dissolved in distilled water with concentration equivalent to its critical micelle concentration (CMC). Silver nanoparticles (SNP) synthesized in the RL colloid were found to be stable for more than 1 month. Further, after 1 month when the SNP in RL colloid (SNPRL) were exposed to NaCl solution it took about 60 mg/ml as compared to 2 mg NaCl/ml in the case of SNP colloid for degrading silver nanoparticles. The inference suggests that the RL must undergo vesicle formation and to prevent silver nanoparticle exposure to NaCl.

Immobilizing silver nanoparticles (SNP) on Musa balbisiana cellulose.

Cellulose from Musa balbisiana was purified. A part of it was dispersed in distilled water using ultrasonication. The silver nanoparticles (SNP) were synthesized in the colloidal cellulose solution and stability of the nanoparticles was tested using UV-Vis spectrophotometer. Further characterization of the composite was done using spectral analysis by Fourier Transform Infrared Spectroscopy (FTIR) to reveal any bond formation between silver nanoparticles with M. balbisiana cellulose. Here we found that cellulose/silver nanoparticle colloid is stable for 29 days and there is no chemical interaction of cellulose with silver nanoparticles.

Isolation and immobilization of aroid polyphenol on magnetic nanoparticles: enhancement of potency on surface immobilization.

Five polyphenolic compounds were isolated from four aroid species [using High Performance Liquid Chromatography (HPLC)] and identified using Fourier Transform Infrared (FTIR) spectroscopy and Nuclear Magnetic Resonance (NMR) analysis. The compounds benzoic acid, caffeic acid, coumaric acid, ferulic acid and syringic acids were immobilized on magnetic nanoparticles (MNPs) using poly(ethylene glycol) (PEG) as linker polymer. The obtained PEG-MNP-polyphenol trios were characterized using transmission electron microscopy (TEM) and FTIR. Their biochemical characterization were done to find out the DPPH scavenging capacity and amount of polyphenol loaded (galic acid equivalent) in each type of MNP-polyphenol trio. To know the antioxidant activity the MNP-polyphenol trios were subjected to H(2)O(2) induced haemolysis prevention assay and syringic acid trio was found to most potent. To ensure that the MNP-polyphenol trios were magnetically active the hysteresis loop analysis was also done.

Optimization of Nutrient Requirements and Culture Conditions for the Production of Rhamnolipid from Pseudomonas aeruginosa (MTCC 7815) using Mesua ferrea Seed Oil.

Environmental awareness has led to a serious consideration for biological surfactants and hence non-edible vegetable oils may serve as a substitute carbon source for bio-surfactant production (rhamnolipid) which might be an alternative to complex synthetic surfactants. There are reports of rhamnolipid production from plant based oil giving higher production than that of glucose because of their hydrophobicity and high carbon content. Therefore the contribution of non-edible oil such as Mesua ferrea seed oil could serve as a good carbon source for rhamnolipid production. Moreover the use of rhamnolipid production from non-edible plant based seed oil has not been reported elsewhere. The present work focus on the optimal production of rhamnolipid by considering both micro and macro nutrients and culture conditions using response surface methodology. The study observes that micronutrients play a significant role in rhamnolipid production from Pseudomonas aeruginosa (MTCC 7815). The investigation results with the statistically optimize parameters able to produce a higher rhamnolipid production and this methodology could be used to optimize the nutrients requirements and culture conditions. The present findings would assist in bioremediation of crude oil contaminated ecosystems.

Molecular docking studies of quercetin and its analogues against human inducible nitric oxide synthase.

Nitric oxide synthases (NOS) catalyze to produce nitric oxide (NO) from L-arginine. The isoform of NOS i.e. inducible nitric oxide synthases (iNOS) expression is observed in various human malignant tumors such as breast, lung, prostate and bladder, colorectal cancer, and malignant melanoma. Also an increased level of iNOS expression and activity has been found in the tumor cells of gynecological malignancies, stroma of breast cancer and tumor cells of head and neck cancer. Because of its importance in causing tumors and cancer, iNOS enzyme has become a new target in finding novel inhibitors as anti cancer agents. The present work focuses on the molecular docking analysis of quercetin and its analogues against iNOS enzyme. Earlier there are reports of quercetin inhibiting iNOS enzyme in certain experiments as anti cancer agent. But the clinical use of quercetin is limited by its low oral bioavailability and therefore needed its molecular modification to improve its pharmacological properties. In the present study ten analogues of quercetin were found to be docked at the active site cavity with favorable ligand-protein molecular interaction and interestingly from the ADME-Toxicity analysis these analogues have enhanced pharmacological properties than quercetin.

Bio-plastic (P-3HB-co-3HV) from Bacillus circulans (MTCC 8167) and its biodegradation.

Polyhydroxyalkanoates (PHAs) are naturally occurring polyesters synthesized by bacteria for carbon and energy storage and it has commercial potential as bioplastic. The bacterial species Bacillus circulans MTCC 8167, isolated from crude oil contaminated soil, can efficiently produce medium chain length polyhydroxyalkanoates (P-3HB-co-3HV) from cheap carbon sources like dextrose. The molecular mass of P-3HB-co-3HV was reported as 5.1×10(4)Da with polydispersity index of 1.21 by gel permeation chromatography. In the present investigation different bacteria and fungi species were used for testing the biodegradability of the extracted polymer. The FTIR spectra of the biodegraded PHBV film showed a decrease in the peak from 1735 cm(-1) (untreated film) to 1675 cm(-1), and disappearance of a peak present in the control at 2922 cm(-1) indicating the breakdown of ester (>C=O) or O-R group and -C=H bond, respectively. From biodegradability testing, the tested microorganisms were found to have decisive contribution to the biodegradation of P-3HB-co-3HV polymer.

Enhancing the stability of colloidal silver nanoparticles using polyhydroxyalkanoates (PHA) from Bacillus circulans (MTCC 8167) isolated from crude oil contaminated soil.

Polyhydroxyalkanoate (PHA) was produced by growing Bacillus circulans (MTCC 8167) in the specific detection medium. The identification of the polymer as PHA was confirmed by fluorescence microscopy. The PHA was purified and characterized using FT-IR. The silver nanoparticles (SNP) were synthesized from AgNO3 in the dispersed colloids of PHA (0.085%) using NaBH4 (sodium borohydrate as reducing agent). The stability was tested using wave length scanning with a UV-Vis spectrophotometer and finally with transmission electron microscopy. The PHA stabilized solution was found to be stable for 30 days as against the low stability of silver nanoparticles (SNP) solution alone.

Production and physico-chemical characterization of a biosurfactant produced by Pseudomonas aeruginosa OBP1 isolated from petroleum sludge.

Pseudomonas aeruginosa strain OBP1, isolated from petroleum sludge, was used to produce biosurfactant from a modified mineral salt medium with 2% n-hexadecane as sole source of carbon. The crude biosurfactant was fractionated using TLC and HPLC. Using FTIR spectroscopy, ¹H NMR, and LC-MS analyses, the chemical structure of the purified fraction of crude biosurfactant was identified as rhamnolipid species. The LC-MS spectra show that monorhamnolipid (L-rhamnopyranosyl-ß-hydroxydecanoyl-ß- hydroxydecanoate, Rha-C10-C10) was produced in abundance with the predominant congener [M-H]⁻ ions for L-rhamnopyranosyl-L-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rha-Rha-C10-C10). Seven different carbon substrates and five nitrogen sources were examined for their effect on rhamnolipid production. Using n-hexadecane (20 g/l) as carbon substrate and urea along with (NH4)2SO4 (2 g/l each) as nitrogen source was found to be the best, with a maximum yield of 4.8 g/l. The biosurfactant reduced the surface tension of water to 31.1 mN m⁻¹ with a critical micelle concentration of 45 mg/l. The biosurfactant showed a better emulsifying activity against a variety of hydrocarbon and achieved a maximum emulsion index of 82% for diesel. The purified biosurfactant showed a significant antibacterial activity against Staphylococcus aureus at a minimum inhibitory concentration of 8 µg/ml.

'Poly(ethylene glycol)-magnetic nanoparticles-curcumin' trio: directed morphogenesis and synergistic free-radical scavenging.

Research on curcumin (polyphenol derived from the rhizome of herb Curcuma longa) has occupied a unique niche owing to its distinctive properties, unique molecular architecture and multi-potent efficacies, particularly in the bio-medical domain with recent interest in material science. In the present study, conjugation of curcumin onto biocompatible poly(ethylene glycol)--templated magnetic iron oxide nanoparticles (MNP) (2-5 nm), prepared through a simple wet chemical route is reported along with modulation of the activity of the partners. Statistical optimization, using response surface methodology, of sonication parameters (8 min, 0.4 cycle and 60% amplitude) for maximal curcumin loading (86%), has shown major morphology directing effect generating triads, tetrads, amongst others and chain-like arrangement (TEM imaging) of the bio-conjugated nanoparticles (80-90 nm). Intonation of the magnetometric parameters of the PEG assisted MNPs on bioconjugation was also evident. The synergistic potency of the partners, the polymer templated iron oxide nanoparticles and curcumin, was unmasked during scavenging of diphenyl picryl hydrazyl (DPPH). The system represents an architecturally appealing, magnetically responsive bio-conjugated system possessing synergistic participation of the partners, with prospective applications in medical domain.

Biocompatible novel starch/polyaniline composites: characterization, anti-cytotoxicity and antioxidant activity.

Starch/polyaniline composites have been synthesized using oxidative polymerization of polyaniline in an aqueous dispersion of starch isolated from Colocasia esculenta corm. Scanning electron micrographs reveals the growth of polyaniline over the surface of the starch granules. DPPH scavenging and haemolysis prevention assay have been performed to estimate the antioxidant activity and cytotoxicity of the composites. Formation of new properties of the composites as compared to starch and poloyaniline was evident from the X-ray diffraction analysis (XRD). Characterization done using UV-Vis, FTIR and DSC analysis provide evidence of composite formation. Composite possesses antioxidant nature which increases with the concentration of polyaniline. The haemolysis prevention activity of these novel composite materials is found to increase as compared to the pure polyaniline with minor compromise in the antioxidant activity. The materials show tremendous potential for biomedical applications.

Ultrasonication: enhances the antioxidant activity of metal oxide nanoparticles.

The present work is a modification for Serpen et al.'s method for antioxidant activity determination for insoluble materials with special reference to metal oxide nanoparticles. As reported in our former publications the mixing procedures were followed as mentioned by Serpen et al. A new mixing procedure was followed for enhancing the reaction between metal oxide nanoparticles and DPPH in the solution. The sonication inside a water bath enhances the DPPH scavenging capacity of metal oxide nanoparticles up to 13.94% and 18.96% in case of ferric oxide and nickel oxide nanoparticles respectively. The control DPPH solution without any nanoparticles does not show any degradation due to sonication.