Pectinase from a Fish Gut Bacterium, Aeromonas guangheii (SS6): Production, Cloning and Characterization.

During the present research, 11 gut bacteria were isolated from the freshwater fish, Systomus sarana (General name: olive barb) and upon screening, the strains produced extracellular pectinase enzyme. Among them, the SS6 strain was found to produce a high quantity of 208.731 U/ml pectinase and through molecular characterization the SS6 strain was identified as Aeromonas guangheii. During the culture of SS6 strain, a set of parameters were optimized through the response surface methodology with a Box-Behnken design, for the production of the enzyme. The optimal conditions were found to be 2.11% of maltose, 2.20% of yeast extract, 6.5 of pH, and a temperature of 27.3 °C at 32-h incubation. Under the above conditions, the activity of pectinase production was enhanced to 371 U/ml. The purified pectinase's molecular weight was determined to be ~ 50 kDa (by 10% 2-D PAGE). Totally, nine peptides were identified from the purified pectinase enzyme through the MALDI-TOF-MS analysis and MASCOT tool was used to get the mass spectrum of the peak at 2211 of peptide that indicated the reference pectinase protein. The referenced gene primer (pectate lyases) was PCR amplified and its nucleotide sequence was analyzed. The exo-pelA gene was cloned in pREST vector, which was found to be over expressed in Escherichia coli BL21. The ORF encoded for a mature protein comprising of 425 amino acids (1236 nucleotides) with a predicted molecular weight of ~ 48.7 kDa. The present findings underline the potential of the fish-gut microbes as a source of biotechnologically important enzymes.

Isolation of a bacterial strain from the gut of the fish, Systomus sarana, identification of the isolated strain, optimized production of its protease, the enzyme purification, and partial structural characterization.

BACKGROUND: The present study focuses on the isolation of Bacillus thuringiensis bacterium from the gut of fresh water fish, Systomus sarana, the innovative optimization of culture parameters to produce maximum protease enzyme, by the isolated bacterium, and the elucidation of peptide profile of the protease. And the experimental data and results were authenticated through the response surface method (RSM) and Box-Behnken design (BBD) model. RESULTS: During the RSM optimization, the interaction of the highest concentrations (%) of 2.2 maltose, 2.2 beef extract, and 7.0 pH, at 37 °C incubation, yielded a maximum protease enzyme of 245 U/ml by the fish gut-isolated, B. thuringiensis. The spectral analysis of the obtained enzyme revealed the presence of major functional groups at the range of 610-3852 cm-1 viz., alkynes (-C≡C-H: C-H stretch), misc (P-H phosphine sharp), α, ß-unsaturated aldehydes, and through PAGE analysis, its molecular weight was determined as 27 kDa. The enzyme's MALDI-TOF/MS analysis revealed the presence of 15 peptides from which the R.YHTVCDPR.L peptide has been found to be a major one. CONCLUSIONS: The fish gut-isolated bacterium, B. thuringiensis, SS4 exhibited the potential for high protease production under the innovatively optimized culture conditions, and the obtained result provides scope for applications in food and pharmaceutical industries.

Pilot scale wastewater treatment, CO2 sequestration and lipid production using microalga, Neochloris aquatica RDS02.

In present investigation carried out large-scale treatment of tannery effluent by the cultivation of microalgae, Neochloris aquatica RDS02. The tannery effluent treatment revealed that significant reduction heavy metals were chromium-3.59, lead-2.85, nickel-1.9, cadmium-10.68, zinc-4.49, copper-0.95 and cobalt-1.86 mg/L on 15th day of treatment using N. aquatica RDS02. The microalgal biosorption capacity q max rate was Cr-88.66, Pb-75.87, Ni-87.61, Cd-60.44, Co-52.86, Zn-84.90 and Cu-54.39, and isotherm model emphasized that the higher R 2 value 0.99 by Langmuir and Freundlich kinetics model. The microalga utilized highest CO2 (90%) analyzed by CO2 biofixation and utilization kinetics, biomass (3.9 mg/mL), lipid (210 mg mL-1), carbohydrate (102.75 mg mL-1), biodiesel (4.9 mL g-1) and bioethanol (4.1 mL g-1). The microalgal-lipid content was analyzed through Nile red staining. Gas chromatography mass spectrometric (GCMS) analysis confirmed that the presence of a biodiesel and major fatty acid methyl ester (FAME) profiling viz., tridecanoic acid methyl ester, pentadecanoic acid methyl ester, octadecanoic acid methyl ester, myristic acid methyl ester, palmitic acid methyl ester and oleic acid methyl ester. Fourier transform infrared (FTIR) analysis confirmed that the presence of a functional groups viz., phenols, alcohols, alkynes, carboxylic acids, ketones, carbonyl and ester groups. The bioethanol production was confirmed by high-performance liquid chromatography (HPLC) analyze.

Evaluation of Salmonella bongori derived biosurfactants and its extracellular protein separation by SDS-PAGE using petridishes: A simply modified approach.

Presently, through the preliminary screening assays, the Salmonella bongori BH11 was found to be an effective biosurfactants (BSFs) producer. The secreted BSFs were extracted using methanol: chloroform and characterized through FTIR, TLC, HPLC and GCMS analyses. Further, the extracellular protein was extracted (TCA/acetone method), estimated (Lowry's method) and separated (standard and modified SDS-PAGE). Through the obtained characteristic FTIR peaks (1107.09cm-1), its content was presumed to be glycolipids and as rhamnose/rhamnolipids through the TLC-Rf value. GCMS revealed 6 compounds, in which Toluene (32%) and 5-(2-Thienyl) pentanoic acid (23%) are the major ones. The crude BSFs exhibited preponderant antibacterial effects on Staphylococcus aureus and Serratia marcescens. Also, it inhibited the biofilm formation of S. aureus, Pseudomonas aeruginosa, P. fluorescens and S. marcescens. Considerably, 76% mortality of IV instar larvae of Culex quinquefasciatus was recorded from BSFs, when compared to SDS. The presently followed protein separation technique using two petridishes might attract the attention of the researchers, as it would emerge as a standard procedure in future. This is the first report on the screening of BSFs from Salmonella bongori that showed antagonistic property, larvicidal potentials and the presently followed modified SDS-PAGE protein separation technique is a simple, reliable and cost effective one.

Investigation of antifungal activity of surfactin against mycotoxigenic phytopathogenic fungus Fusarium moniliforme and its impact in seed germination and mycotoxicosis.

The mycotoxigenic phytopathogenic fungus such as Fusarium moniliforme contamination in maize kernels may not only affect seed germination but also negatively cause mycotoxicosis in animals and humans. There is no effective fungicides to control the growth of F. moniliforme on maize kernels. Hence, effective bioactive compounds are needed to prevent plant and animal diseases associated with F. moniliforme contamination in cereals. Surfactin is an well-known antimicrobial lipopeptide has strong antifungal activities against several phytopathogenic fungi and may have potential uses in agriculture. So, in this present study the antifungal activity of surfactin extracted from Brevibacillus brevis KN8(2) was investigated against F. moniliforme, further its impact in seed germination and mycotoxicosis was also studied. Our results showed that surfactin inhibited and damaged the hyphae of F. moniliforme in in vitro. The agarose gel electrophoresis, SDS-PAGE analysis and biochemical assay presented that surfactin damaged the DNA, protein and reduced the GSH content in F. moniliforme. Furthermore, surfactin prevent maize seed germination problem and mycotoxicosis in animal model associated with F. moniliforme via prevention of F. moniliforme contamination on maize kernels. These findings revealed that surfactin could be an effective bio-fungicide in the plant disease management.

Effect of biogenic silver nanocubes on matrix metalloproteinases 2 and 9 expressions in hyperglycemic skin injury and its impact in early wound healing in streptozotocin-induced diabetic mice.

Microbial contamination along with over expressions of matrix metalloproteinases 2 and 9 impairs wound healing in diabetic patients. Silver-based antimicrobial agents have been successfully used for treating non-healing chronic wounds associated with infection. However, topical application of silver-ion compounds impairs wound healing process. Thus, usage of biogenic silver nanoparticles appears as a new means to reduce the toxicity of silver compounds in the wound care system. Here, following our previous method, AgNPs was synthesized using the culture filtrate of Brevibacillus brevis KN8(2) then characterized by UV-visible spectrophotometry, TEM, SAED, XRD and DLS measurements. The antibacterial activity of AgNPs was evaluated against the most common wound infecting pathogens Pseudomonas aeruginosa and Staphylococcus aureus by well diffusion assay. Further, the wound healing efficacy of biogenic AgNPs was examined in streptozotocin-induced diabetic mice by measuring wound area closure, histopathology, mRNA and protein expressions of MMP-2, MMP-9. Our results demonstrates that besides antimicrobial activity, biogenic AgNPs decreased the mRNA and protein expression of MMP-2 and MMP-9 in wounded granulation tissues leads to early wound healing in diabetic mice. These findings revealed that biogenic AgNPs synthesized from B. brevis KN8(2) could be an effective therapeutic agent in the management of diabetic foot ulcer with/without infection.

Anti-pseudomonal and anti-endotoxic effects of surfactin-stabilized biogenic silver nanocubes ameliorated wound repair in streptozotocin-induced diabetic mice.

Pseudomonas aeruginosa and its lipopolysaccharides play a key role in the pathogenesis of diabetic foot infection, for which, currently no effective therapeutic agents are available. Hence, newer forms of therapeutic agents are required for treating Pseudomonas aeruginosa infection. In this present study, nanocrystalline silver nanoparticles (AgNPs) were synthesized using culture filtrate of Brevibacillus brevis KN8(2) followed by an investigation of its in vivo anti-pseudomonal and anti-endotoxic properties. Biosynthesized AgNPs was predominantly cubical in shape with an average particle size of 15.40 nm as observed through field emission scanning electron microscopy (FESEM) and X-ray diffraction analysis. The LC-ESI-MS/MS analysis indicates the presence of surfactin in culture filtrate of B. brevis KN8(2). The MIC of surfactin-stabilized AgNPs against P. aeruginosa was 10 µg ml-1 and its wound repair activity was observed in P. aeruginosa-infected wounds of diabetic mice by measuring wound area closure, bacterial counts, mRNA expressions, and histopathology. Further, surfactin-stabilized AgNPs suppressed the transcription of LPS-triggered expression of the TNF-α in wounds that LPS-assisted extension of wound repair in diabetes mellitus conditions was circumvented quite well. Results gathered in this study established that surfactin-stabilized AgNPs could effectively offer to the novel treatment of Gram-negative bacilli infection in diabetic wounds.