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.

Molecular characterization and antioxidant assay of pigment producing bacteria, Sphingomonas paucimobilis and Microbacterium arborescens isolated from fresh water sediments.

This study focuses on isolation of pigment producing bacteria from fresh water sediment. The isolated bacteria were grown in nutrient broth and the maximum absorbance of 2.512 was obtained for the extracted pigment at 500 nm. The effective strains were optimized, pH 11 and temperature 30 °C was found to be more favorable for its maximum growth. The isolates were identified based on their molecular characterestics as Microbacterium arborescens and Sphingomonas paucimobilis, molecular size of the amplified 16S rRNA gene sequence was found to be approximately 1270 and 765 bp respectively. The antioxidant property of the pigment was analyzed using DPPH and ABTS assay. The IC50 value of Microbacterium arborescens was higher in all the three assays in comparison with Sphingomonas paucimobilis. The extracted pigment was characterized for the presence of compounds using GC-MS and FTIR analysis to determine the functional groups. As the pigment obtained from M. arborescens had shown better antioxidant activity it may be used as colorant in food industrial applications.

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.

Isolation and growth inhibition potential of entomopathogenic nematodes against three public health important mosquito vectors.

The prevalence of mosquito vector borne diseases and the resistance of mosquitoes to conventional pesticides have been of important public concern to the mosquito endemic countries. Present study was conducted to identify the native bio-larvicidal potential of the entomopathogenic nematodes; Steinernema siamkayai (KPR-4) Heterohabditis indica (KPR-8), Steinernema glaseri and Steinernema abbasi. The isolated nematodes were subsequently cultured and evaluated their larvicidal potential against the larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Among the tested four different nematode species, the S. abassi exerted the highest mortality against A. aegypti (97.33%), the H. indica (KPR-8) against A. stephensi (97.33%) and the S. siamkayai (KPR-4) against C. quinquefasciatus (98.67%). The maximal mosquito-larvicidal property of EPNs was found with the LC50 and LC90 values (IJs/larvae): S. abbasi = 12.47 & 54.35 on A. aegypti; H. indica KPR-8 = 19.88 & 66.81 on A. stephensi and S. siamkayai KPR-4 = 16.69 & 58.97 on C. quinquefasciatus, respectively. The presently generated data on the molecular and larvicidal characteristics of the entomopathogenic nematodes form an important baseline data that upon further research would lead to the development of eco-friendly mosquito-control agent.

Comparative extraction of Salmonella bongori derived metabolites and their toxicity on bacterial pathogens, mosquito-larvae, zebrafish-embryo and brine-shrimp: A modified approach.

The present study pertains to two different (standard and adapted) extraction-procedures to extract bacterial extracellular metabolites from the cell-free supernatant (CFS) of S. bongori. Metabolites were extracted with the different polarity solvents using lyophilized-CFS mediated procedure, which revealed more number of compounds than standard procedure. The crude-extracts (CEs) were characterized using FTIR, HPLC and GC-MS analyses. The commonly presented compounds in standard (ME, EA & HE) and lyophilization-mediated extracts (LME, LEA & LHE) were identified through Heat-map analysis. Antibacterial assay: all CEs showed considerable activity on tested MTCC-strains, in which, LME and LEA were found preponderant. Larvicidal bioassay: LME resulted maximum mortality than other CEs on Culex-larvae. Zebrafish embryo-toxicity assay: except HE, all CEs exhibited toxicity at 100 ppm after 96 hpf. Brine shrimp-toxicity assay: ME, LME, EA and LEA have shown significant mortality after 24 h. With these observations, the adapted-extraction-procedure could form significance in the drug development process.

Multipurpose efficacy of the lyophilized cell-free supernatant of Salmonella bongori isolated from the freshwater fish, Devario aequipinnatus: toxicity against microbial pathogens and mosquito vectors.

Presently, the discovery of effective drugs and pesticides from eco-friendly biological sources is an important challenge in the field of life sciences. The present research was aimed for standardizing an innovative approach in the evaluation of the biological potentiality of the metabolites of fish-associated bacteria. We have identified 17 skin-associated bacteria from the freshwater fish, giant danio, Devario aquipinnatus. They were screened through biofilm forming and extracellular enzyme producing ability. The results of preliminary antibacterial evaluation of the bacterial supernatants underlined the importance of three potential strains (BH8, BH10 and BH11) for further applied research. Hence, such strains were subsequently subjected to a novel extraction procedure to overcome the difficulties found in polar solvents mixed with the supernatant. The lyophilized cell-free supernatant (LCFS) of 3 isolates were individually extracted by using methanol. During the testing of LCFS's methanolic extract (LCFS-ME) of 3 isolates, only the extract of BH11-strain exhibited potent inhibitory activity against the pathogenic bacteria and fungi. Furthermore, the larvicidal and mosquitocidal assays on the filariasis vector, Culex quinquefasciatus also showed its potent toxicity on both the adults and developmental instars of mosquito. Through molecular and phylogenetic analyses, the BH11 strain was identified as Salmonella bongori (KR350635). The present finding emphasized that the S. bongori could be an important novel source of effective antimicrobials and mosquitocidal agents.

Bio-pesticidal effects of Trichoderma viride formulated titanium dioxide nanoparticle and their physiological and biochemical changes on Helicoverpa armigera (Hub.).

The control of agricultural pests through eco-friendly nanopesticides is a challenge of crucial environmental importance nowadays. The current study was aimed to discover a novel biopesticides through Trichoderma viride mediated synthesis of titanium dioxide nanoparticles (TDNPs). The main chemical and physical features of the TDNPs were assessed by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), energy dispersive X-ray (EDX) and size distribution and shape of the NPs studied through the scanning electron microscope (SEM), high-resolution transmission electron microscope (HR-TEM) and dynamic light scattering (DLS). The extracellular synthesized nanoparticles were evaluated for their larvicidal, antifeedant and pupicidal activities against Helicoverpa armigera. TDNPs exhibited highest mortality rate on first (100%), second (100%) and third (92.34%), instar larvae of H. armigera at 100 ppm. The detoxifying enzymes such as, ß-glucosidase and carboxylesterase were reduced whereas glutathione S-transferase increased during the treatment of TDNPs against H. armigera at 100 ppm. No toxic effects were found on Eudrilus eugeniae filter paper and artificial soil assays treated with TDNPs at 100 ppm. However, cypermethrin was toxic to earthworms after 72 h treatment. Therefore, TDNPs could act as significant inhibitors on the development of H. armigera, although, no adverse effect was found on earthworms.