Extracellular biosynthesis,characterization, optimization of silver nanoparticles (AgNPs) using Bacillus mojavensis BTCB15 and its antimicrobial activity against multidrug resistant pathogens

Abstract

Biosynthesis of metal nanoparticles is an area of interest among researchers because of its eco-friendly approach. Current study focuses at biosynthesis of silver nanoparticles (AgNPs) and optimization of physico-chemical conditions to obtain mono-dispersed and stable AgNPs having antimicrobial activity. Initially Bacillus mojavensis BTCB15 produced silver nanoparticles (AgNPs) of 105?nm. Silver nanoparticles (AgNPs) were characterized by particle size analyzer, UV-Vis Spectroscopy, Fourier transforms infrared spectroscopy (FTIR), Atomic force microscopy (AFM), and X-ray diffraction (XRD). Whereas, under optimal conditions of temperature 55?°C, pH 8, addition of surfactant Tween 20, and metal ion K₂SO₄, about 104% size reduction was achieved with average size of 2.3nm. Molecular characterization revealed 98% sequence homology with Bacillus mojavensis. AgNPs exhibited antibacterial activity at concentrations ranging from 0.5 to 2.5?µg/µl against Escherichia coli BTCB03, Klebsiella pneumonia BTCB04, Acinetobacter sp. BTCB05, and Pseudomonas aeruginosa BTCB01 but none against Staphylococcus aureus BTCB02. Highest antibacterial activity was observed at 0.27?µg/µl and lowest at 0.05?µg/µl of AgNPs indicated by zone of inhibition. Conclusively, under optimum conditions, Bacillus mojavensis BTCB15 was able to produce AgNPs of 2.3?nm size and had antibacterial activity against multi drug resistant pathogens.     

Purification and kinetics of a protease-resistant,neutral, and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 ameliorating food nutrition

Abstract

A novel protease-resistant and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 was purified 36-fold to homogeneity with a combination of ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-50 chromatographic techniques. The estimated molecular mass of the purified phytase was 46?kDa by electrophoresis with optimal activity at pH 7.0 and 70?°C. About 19% of original activity was maintained at 80?°C for 10?min. Phytase activity was stimulated in presence of surfactants like Tween-20, Tween-80, and Triton X-100 and metal ions like Ca⁺², K⁺, and Co⁺² and it was inhibited by SDS and Mg⁺², Al⁺², and Fe⁺². Purified enzyme showed specificity to different salts of phytic acid and values of K_m and V_max were 0.293?mM and 11.49 nmoles s^?1, respectively for sodium phytate. The purified enzyme was resistant to proteases (trypsin and pepsin) that resulted in amelioration of food nutrition with simultaneous release of inorganic phosphate, reducing sugars, and soluble protein.     

Biosynthesis,characterization and antimicrobial activity of silver nanoparticles by a halotolerant Bacillus endophyticus SCU-L

Abstract

We have conducted a thorough study on extracellular biosynthesis of silver nanoparticles (AgNPs) by a halotolerant bacterium Bacillus endophyticus SCU-L, which was identified by 16S rRNA gene sequencing analysis. This strain was selected during an ongoing research programme aimed at finding a novel biological method for green nanosynthetic routes using the extremophiles in unexplored hypersaline habitats. The biosynthesized AgNPs were characterized and analyzed with UV–vis spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, atomic force microscopy and X-ray diffraction. Further, the AgNPs were found to be spherical in shape with an average particle size of about 5.1?nm, and it was stable in aqueous solution for three months period of storage at room temperature under dark condition. Also, the synthesized AgNPs significantly presented antimicrobial activity against Candida albicans, Escherichia coli, Salmonella typhi and Staphylococcus aureus. The above results suggested that the present work may provide a valuable reference and theoretical basis for further exploration on microbial biosynthesis of AgNPs by halotolerant bacteria.     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

Virtual screening of LPXTG competitive SrtA inhibitors targeting signal transduction mechanism in Bacillus anthracis: a combined experimental and theoretical study

Abstract

Members of the sortase enzyme super family decorate the surfaces of Bacillus anthracis cell wall with proteins that play key roles in microbial pathogenesis and its biofilm formation. Bacillus anthracis Sortase-A (Ba-SrtA) is a potential target for new therapeutics as it is required for B. anthracis survival and replication within macrophages. An understanding of the binding site pocket and substrate recognition mechanism by SrtA enzymes may serve to be beneficial in the rational development of sortase inhibitors. Here, the LPXTG signal peptide-based competitive inhibitors are screened against the Ba-SrtA and compounds with reasonable inhibition, specificity, and mechanisms of inactivation of SrtA have been covered. The screened compounds are experimentally validated against the phylogenetically similar Gram-positive pathogen B. cereus. In situ microscopic visualizations suggest that these screened compounds showed the microbial and biofilm inhibitory activity against B. cereus. It facilitates the further development of these molecules into useful anti-infective agents to treat infections caused by B. anthracis and other Gram-positive pathogens. These results provide insight into basic design principles for generating new clinically relevant lead molecules. It also provides an alternative strategy where a screened ligand molecule can be used in combination to battle increasingly against the Gram-positive pathogens.     

An improved Method for Isolation of Rat Testis Golgi Apparatus H. H. Mollenhauer

The preparation of Golgi apparatus fractions from rat testis germ cells free from contamination by residual body fragments was accomplished by the use of the Yeda press as the homogenization device. The Golgi apparatus thus prepared retained excellent stuctural intactness. This method also allows for isolation of Golgi apparatus from single cell suspensions.     

An Extracellullar Nuclease from Bacillus Firmus VKPACU-1: Specificity and Mode of Action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5′UMP > 5′AMP > 5′GMP where as in hydrolysis of DNA the mononucleotides in the order of 5′dAMP > 5′dGMP > 5′dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3’ hydroxyl and 5’ phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

Characterization of polyhydroxyalkanoates accumulated by a moderately halophilic salt pan isolate Bacillus megaterium strain H16

Aim

Characterization of polyhydroxyalkanoates (PHA) accumulated by halophilic bacteria isolated from solar salterns.

Methods and Results

Twenty‐six halophilic isolates were obtained from solar salterns of Goa, India. They were screened for accumulation of PHA by Sudan black B, Nile blue A and Nile red stains. Strains H15, H16 and H26 were selected based on their intensity of Nile blue A/Nile red fluorescence. On the basis of phenotypic and genotypic characterization, the three isolates were identified as Bacillus megaterium. Growth kinetics and polymer accumulating capacity of strain H16 were studied in E2 mineral media with 2% glucose with/without NaCl. In the absence of NaCl, strain H16 accumulated PHA to 40·0% (w/w) of cell dry weight (CDW) at 42 h of growth, whereas in presence of 5% w/v NaCl, the culture showed longer lag phase of up to 24 h and accumulated a maximum PHA of 39% (w/w) CDW at 54 h of growth. The infrared spectra of both the polymers exhibited peaks at 1733·9 cm^?1 characteristic of C=O. Scans of ¹H nuclear magnetic resonance (NMR) showed a doublet at 2·5 ppm corresponding to methylene group (‐CH₂), the signal at 5·3 ppm corresponded to methine group (‐CH‐), and another signal at 1·3 ppm corresponded to the methyl group (‐CH₃). Scans of ¹³C NMR showed prominent peaks at 20, 40, 67–68 and 170 ppm, indicating the polymer to be homopolymer of 3‐hydroxybutyrates. The polymer is stable up to a temperature of 160°C.

Conclusion

Three moderately halophilic isolates (strain H15, H16 and H26) capable of accumulating PHA were isolated from solar salterns of Ribandar Goa, India, and identified as B. megaterium based on phenotypic and genotypic characterization. Strain H16 accumulated polyhydroxybutyrate in the presence and absence of NaCl up to 40% of its CDW.

Significance and Impact of the Study

This strain would be better suited for production of PHA at industrial level due to its tolerance to high concentration of NaCl.