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Find video protocols related to scientific articles indexed in Pubmed.
Draft Genome Sequence of Brucella abortus S99: Designated Antigenic Smooth Reference Strain Used in Diagnostic Tests in India.
Genome Announc
PUBLISHED: 08-21-2014
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Brucella abortus strain S99 is widely used for the preparation of colored, plain, recombinant and smooth lipopolysaccharide antigens for the preparation of Brucella diagnostic kits. The genome of this strain was sequenced and the length of the genome was 3,253,175 bp, with 57.2% G+C content. A total of 3,365 protein coding genes and 53 RNA genes were predicted.
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Elevated levels of circulating DNA in cardiovascular disease patients: metagenomic profiling of microbiome in the circulation.
PLoS ONE
PUBLISHED: 08-18-2014
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Cardiovascular diseases (CVDs) are the leading cause of death worldwide. An expanding body of evidence supports the role of human microbiome in the establishment of CVDs and, this has gained much attention recently. This work was aimed to study the circulating human microbiome in CVD patients and healthy subjects. The levels of circulating cell free DNA (circDNA) was higher in CVD patients (n?=?80) than in healthy controls (n?=?40). More specifically, the relative levels of circulating bacterial DNA and the ratio of 16S rRNA/?-globin gene copy numbers were higher in the circulation of CVD patients than healthy individuals. In addition, we found a higher circulating microbial diversity in CVD patients (n?=?3) in comparison to healthy individuals (n?=?3) by deep shotgun sequencing. At the phylum level, we observed a dominance of Actinobacteria in CVD patients, followed by Proteobacteria, in contrast to that in healthy controls, where Proteobacteria was predominantly enriched, followed by Actinobacteria. The circulating virome in CVD patients was enriched with bacteriophages with a preponderance of Propionibacterium phages, followed by Pseudomonas phages and Rhizobium phages in contrast to that in healthy individuals, where a relatively greater abundance of eukaryotic viruses dominated by Lymphocystis virus (LCV) and Torque Teno viruses (TTV) was observed. Thus, the release of bacterial and viral DNA elements in the circulation could play a major role leading to elevated circDNA levels in CVD patients. The increased circDNA levels could be either the cause or consequence of CVD incidence, which needs to be explored further.
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Genome Sequencing of a Mung Bean Plant Growth Promoting Strain of P. aeruginosa with Biocontrol Ability.
Int J Genomics
PUBLISHED: 08-12-2014
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Pseudomonas aeruginosa PGPR2 is a mung bean rhizosphere strain that produces secondary metabolites and hydrolytic enzymes contributing to excellent antifungal activity against Macrophomina phaseolina, one of the prevalent fungal pathogens of mung bean. Genome sequencing was performed using the Ion Torrent Personal Genome Machine generating 1,354,732 reads (6,772,433 sequenced bases) achieving ~25-fold coverage of the genome. Reference genome assembly using MIRA 3.4.0 yielded 198 contigs. The draft genome of PGPR2 encoded 6803 open reading frames, of which 5314 were genes with predicted functions, 1489 were genes of known functions, and 80 were RNA-coding genes. Strain specific and core genes of P. aeruginosa PGPR2 that are relevant to rhizospheric habitat were identified by pangenome analysis. Genes involved in plant growth promoting function such as synthesis of ACC deaminase, indole-3-acetic acid, trehalose, mineral scavenging siderophores, hydrogen cyanide, chitinases, acyl homoserine lactones, acetoin, 2,3-butanediol, and phytases were identified. In addition, niche-specific genes such as phosphate solubilising 3-phytase, adhesins, pathway-specific transcriptional regulators, a diguanylate cyclase involved in cellulose synthesis, a receptor for ferrienterochelin, a DEAD/DEAH-box helicase involved in stress tolerance, chemotaxis/motility determinants, an HtpX protease, and enzymes involved in the production of a chromanone derivative with potent antifungal activity were identified.
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Identification and characterization of bile salt hydrolase genes from the genome of Lactobacillus fermentum MTCC 8711.
Appl. Biochem. Biotechnol.
PUBLISHED: 05-05-2014
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Lactobacillus fermentum is a lactic acid bacterium of probiotic importance, which is found ubiquitously in fermented milk products. Bile salt hydrolase (BSH) has a significant role in affording probiotic properties to lactobacilli. In the present study, two bsh genes encoding BSH1 and BSH2 were identified from the draft genome sequence of L. fermentum MTCC 8711. Nucleotide comparison revealed no significant similarity between bsh1 and bsh2 genes, whereas the deduced amino acid sequences showed 26 % sequence similarity between both BSH1 and BSH2. Pfam analysis revealed the presence of cys-2 active site residues in the catalytic pocket of both BSH1 and BSH2 highly essential for catalysis. Phylogentic analysis of BSH1 and BSH2 revealed the possible independent origin of these proteins in Lactobacillus. We cloned these genes in pSLp111.3, a Lactobacillus expression vector with signal peptide A (slpA) and expressed in the native L. fermentum strain for overexpression and extracellular secretion. The bsh1 gene failed to express and to produce promising BSH activity. However, bsh2 gene was overexpressed and the recombinant strain showed improved BSH activity. Induction of the recombinant strain with an optimal 2 % xylose concentration secreted 0.5 U/ml of the BSH into extracellular medium. Furthermore, the recombinant strain was able to completely assimilate the 100-?g/ml cholesterol within 24 h, whereas the native strain took 72 h for the complete assimilation of cholesterol.
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Insect gut microbiome - An unexploited reserve for biotechnological application.
Asian Pac J Trop Biomed
PUBLISHED: 02-25-2014
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Metagenomics research has been developed over the past decade to elucidate the genomes of the uncultured microorganisms with an aim of understanding microbial ecology. On the other hand, it has also been provoked by the increasing biotechnological demands for novel enzymes, antibiotic and signal mimics. The gut microbiota of insects plays crucial roles in the growth, development and environmental adaptation to the host insects. Very recently, the insect microbiota and their genomes (microbiome), isolated from insects were recognized as a major genetic resources for bio-processing industry. Consequently, the exploitation of insect gut microbiome using metagenomic approaches will enable us to find novel biocatalysts and to develop innovative strategies for identifying smart molecules for biotechnological applications. In this review, we discuss the critical footstep in extraction and purification of metagenomic DNA from insect gut, construction of metagenomic libraries and screening procedure for novel gene identification. Recent innovations and potential applications in bioprocess industries are highlighted.
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Biological real-time reaction calorimeter studies for the production of penicillin G acylase from Bacillus badius.
Appl. Biochem. Biotechnol.
PUBLISHED: 02-10-2014
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Penicillin G acylase (PGA) is a commercially important enzyme that cleaves penicillin G to 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PAA). The strain Bacillus badius has been identified as potential producer of PGA. A detailed calorimetric investigation on PGA production was carried out to enable generation of thermokinetic data possible for commercial application. Reaction calorimetric studies coupled with respirometric studies suggested that enzyme activity of the species B. badius was calorimetrically traceable. Three phases of growth were distinctly noticeable in the metabolic heat-time curve. Increase in enzymatic activity with restricted growth confirmed intracellular nature of the production process. The estimated heat yields due to biomass growth, 10.026 kJ/g, substrate consumption 22.761 kJ/g, and oxygen uptake 383 ± 10 kJ/mol helped to understand the energetic of the organism under study. Low oxycalorific coefficient confirmed the existence of fermentation-coupled metabolism of B. badius.
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Cloning, expression and characterization of a lipase encoding gene from human oral metagenome.
Indian J. Microbiol.
PUBLISHED: 02-01-2014
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The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6-7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.
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Draft Genome Sequence of Brucella melitensis Strain ADMAS-G1, Isolated from Placental Fluids of an Aborted Goat.
Genome Announc
PUBLISHED: 10-12-2013
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Here, we report the draft genome sequence and annotation of the Brucella melitensis strain designated ADMAS-G1, isolated from placental fluids of an aborted goat. The length of the genome is 3,284,982 bp, with a 57.3% GC content. A total of 3,325 protein-coding genes and 63 RNA genes were predicted.
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Genome Sequence of Lactobacillus fermentum Strain MTCC 8711, a Probiotic Bacterium Isolated from Yogurt.
Genome Announc
PUBLISHED: 09-28-2013
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Lactobacillus fermentum strain MTCC 8711 is a lactic acid bacterium isolated from yogurt. Here, we describe the draft genome sequence and annotation of this strain. The 2,566,297-bp-long genome consisted of a single chromosome and seven plasmids. The genome contains 2,609 protein-coding and 74 RNA genes.
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Identification and Structure Elucidation of a Novel Antifungal Compound Produced by Pseudomonas aeruginosa PGPR2 Against Macrophomina phaseolina.
Appl. Biochem. Biotechnol.
PUBLISHED: 06-10-2013
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Pseudomonas aeruginosa PGPR2 was found to protect mungbean plants from charcoal rot disease caused by Macrophomina phaseolina. Secondary metabolites from the culture supernatant of P. aeruginosa PGPR2 were extracted with ethyl acetate and the antifungal compound was purified by preparative HPLC using reverse phase chromatography. The purified compound showed antifungal activity against M. phaseolina and other phytopathogenic fungi (Fusarium sp., Rhizoctonia sp. Alternaria sp., and Aspergillus sp.). The structure of the purified compound was determined using (1)H, (13)C, 2D NMR spectra and liquid chromatography-mass spectrometry (LC-MS). Spectral data suggest that the antifungal compound is 3,4-dihydroxy-N-methyl-4-(4-oxochroman-2-yl)butanamide, with the chemical formula C14H17NO5 and a molecular mass of 279. Though chemically synthesized chromanone derivatives have been shown to have antifungal activity, we report for the first time, the microbial production of a chromanone derivative with antifungal activity. This ability of P. aeruginosa PGPR2 makes it a suitable strain for biocontrol.
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Strategies for enhancing the production of penicillin G acylase from Bacillus badius: influence of phenyl acetic acid dosage.
Appl. Biochem. Biotechnol.
PUBLISHED: 05-31-2013
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Bacillus badius isolated from soil has been identified as potential producer of penicillin G acylase (PGA). In the present study, batch experiments performed at optimized inoculum size, temperature, pH, and agitation yielded a maximum PGA of 9.5 U/ml in shake flask. The experiments conducted in bioreactor with different oxygen flow rates revealed that 0.66 vvm oxygen flow rate could be sufficient for the maximum PGA activity of 12.7 U/ml. From a detailed investigation on the strategies of the addition of phenyl acetic acid (PAA) for increasing the production of PGA, it was found that the controlled addition of 10 ml of 0.1 % (w/v) PAA once in every 2 h from 6th hour of growth showed the maximum PGA activity of 32 U/ml. Thus, our studies for the first time showed that at concentration above 0.1 % (w/v) PAA, the PGA production decreased. This selective condition paves the way for less costly bioprocess for the production of PGA.
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Polyethylene glycol-modified gelatin/polylactic acid nanoparticles for enhanced photodynamic efficacy of a hypocrellin derivative in vitro.
J Biomed Nanotechnol
PUBLISHED: 05-01-2013
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The present study focused on the development of a novel biodegradable nanoparticle system based on polyethyleneglycol-modified gelatin (PEG-GEL) and polylactic acid (PLA) biopolymers for improving the photodynamic efficacy of cyclohexane-1,2-diamino hypocrellin B (CHA2HB), a potent photodynamic therapeutic (PDT) agent. The CHA2HB-loaded PEG-GEL/PLA nanoparticles showed near-spherical morphology with an average size of 190 nm at a PLA to PEG-GEL ratio of 1:3. The drug loading was sufficient enough to produce potentially toxic reactive oxygen species (ROS) needed for photodynamic therapy (PDT). Slow and controlled drug release was observed in normal conditions, whereas enzyme assistance resulted in a relatively fast release due to partial disintegration of CHA2HB-loaded PEG-GEL/PLA nanoparticles. In vitro experiments indicated that CHA2HB-loaded PEG-GEL-PLA nanoparticles are efficiently taken up by cancer cells such as human breast adenocarcinoma (MCF-7), human gastric sarcoma (AGS) and mice specific Daltons lymphoma (DLA) in a time dependent manner. Further, CHA2HB-loaded PEG-GEL/PLA nanoparticles evoked superior phototoxicity compared to free-CHA2HB towards all the three cell lines investigated. Interestingly, PDT effectiveness was different for the different cell type studied. CHA2HB-loaded PEG-GEL/PLA nanoparticles induced both apoptotic and necrotic cell death as a result of photoirradiation. Thus, our data suggest that PEG-GEL/PLA nanoparticles are highly effective in delivery and phototoxic enhancement of CHA2HB against model cancer cells in vitro.
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Computational small RNA prediction in bacteria.
Bioinform Biol Insights
PUBLISHED: 03-07-2013
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Bacterial, small RNAs were once regarded as potent regulators of gene expression and are now being considered as essential for their diversified roles. Many small RNAs are now reported to have a wide array of regulatory functions, ranging from environmental sensing to pathogenesis. Traditionally, noncoding transcripts were rarely detected by means of genetic screens. However, the availability of approximately 2200 prokaryotic genome sequences in public databases facilitates the efficient computational search of those molecules, followed by experimental validation. In principle, the following four major computational methods were applied for the prediction of sRNA locations from bacterial genome sequences: (1) comparative genomics, (2) secondary structure and thermodynamic stability, (3) Orphan transcriptional signals and (4) ab initio methods regardless of sequence or structure similarity; most of these tools were applied to locate the putative genomic sRNA locations followed by experimental validation of those transcripts. Therefore, computational screening has simplified the sRNA identification process in bacteria. In this review, a plethora of small RNA prediction methods and tools that have been reported in the past decade are discussed comprehensively and assessed based on their attributes, compatibility, and their prediction accuracy.
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Antimicrobial peptides: versatile biological properties.
Int J Pept
PUBLISHED: 03-06-2013
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Antimicrobial peptides are diverse group of biologically active molecules with multidimensional properties. In recent past, a wide variety of AMPs with diverse structures have been reported from different sources such as plants, animals, mammals, and microorganisms. The presence of unusual amino acids and structural motifs in AMPs confers unique structural properties to the peptide that attribute for their specific mode of action. The ability of these active AMPs to act as multifunctional effector molecules such as signalling molecule, immune modulators, mitogen, antitumor, and contraceptive agent makes it an interesting candidate to study every aspect of their structural and biological properties for prophylactic and therapeutic applications. In addition, easy cloning and recombinant expression of AMPs in heterologous plant host systems provided a pipeline for production of disease resistant transgenic plants. Besides these properties, AMPs were also used as drug delivery vectors to deliver cell impermeable drugs to cell interior. The present review focuses on the diversity and broad spectrum antimicrobial activity of AMPs along with its multidimensional properties that could be exploited for the application of these bioactive peptides as a potential and promising drug candidate in pharmaceutical industries.
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Influence of periplasmic oxidation of glucose on pyoverdine synthesis in Pseudomonas putida S11.
Appl. Microbiol. Biotechnol.
PUBLISHED: 01-23-2013
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Fluorescent pseudomonads catabolize glucose simultaneously by two different pathways, namely, the oxidative pathway in periplasm and the phosphorylative pathway in cytoplasm. This study provides evidence for the role of glucose metabolism in the regulation of pyoverdine synthesis in Pseudomonas putida S11. We have characterized the influence of direct oxidation of glucose in periplasm on pyoverdine synthesis in P. putida S11. We identified a Tn5 transposon mutant of P. putida S11 showing increased pyoverdine production in minimal glucose medium (MGM). This mutant designated as IST1 had Tn5 insertion in glucose dehydrogenase (gcd) gene. To verify the role of periplasmic oxidation of glucose on pyoverdine synthesis, we constructed mutants S11 Gcd(-) and S11 PqqF(-) by antibiotic cassette mutagenesis. These mutants of P. putida S11 with loss of glucose dehydrogenase gene (gcd) or cofactor pyrroloquinoline quinone biosynthesis gene (pqqF) showed increased pyoverdine synthesis and impaired acid production in MGM. In minimal gluconate medium, the pyoverdine production of wild-type strain S11 and mutants S11 Gcd(-) and S11 PqqF(-) was higher than in MGM indicating that gluconate did not affect pyoverdine synthesis. In MGM containing PIPES-NaOH (pH?7.5) buffer which prevent pH changes due to gluconic acid production, strain S11 produced higher amount of pyoverdine similar to mutants S11 Gcd(-) and S11 PqqF(-). Therefore, it is proposed that periplasmic oxidation of glucose to gluconic acid decreases the pH of MGM and thereby influences pyoverdine synthesis of strain S11. The increased pyoverdine synthesis enhanced biotic and abiotic surface colonization of the strain S11.
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Mechanisms of the antifungal action of marine metagenome-derived peptide, MMGP1, against Candida albicans.
PLoS ONE
PUBLISHED: 01-01-2013
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Development of resistant variants to existing antifungal drugs continues to be the serious problem in Candida albicans-induced fungal pathogenesis, which has a considerable impact on animal and human health. Identification and characterization of newer drugs against C. albicans is, therefore, essential. MMGP1 is a direct cell-penetrating peptide recently identified from marine metagenome, which was found to possess potent antifungal activity against C. albicans.
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Global transcriptome analysis of Bacillus cereus ATCC 14579 in response to silver nitrate stress.
J Nanobiotechnology
PUBLISHED: 07-26-2011
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Silver nanoparticles (AgNPs) were synthesized using Bacillus cereus strains. Earlier, we had synthesized monodispersive crystalline silver nanoparticles using B. cereus PGN1 and ATCC14579 strains. These strains have showed high level of resistance to silver nitrate (1 mM) but their global transcriptomic response has not been studied earlier. In this study, we investigated the cellular and metabolic response of B. cereus ATCC14579 treated with 1 mM silver nitrate for 30 & 60 min. Global expression profiling using genomic DNA microarray indicated that 10% (n = 524) of the total genes (n = 5234) represented on the microarray were up-regulated in the cells treated with silver nitrate. The majority of genes encoding for chaperones (GroEL), nutrient transporters, DNA replication, membrane proteins, etc. were up-regulated. A substantial number of the genes encoding chemotaxis and flagellar proteins were observed to be down-regulated. Motility assay of the silver nitrate treated cells revealed reduction in their chemotactic activity compared to the control cells. In addition, 14 distinct transcripts overexpressed from the empty intergenic regions were also identified and proposed as stress-responsive non-coding small RNAs.
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Functional characterization of a putative ?-lactamase gene in the genome of Zymomonas mobilis.
Biotechnol. Lett.
PUBLISHED: 06-18-2011
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Zymomonas mobilis ZM4 is resistant to ?-lactam antibiotics but there are no reports of a ?-lactam resistance gene and its regulation. A putative ?-lactamase gene sequence (ZMO0103) in the genome of Z. mobilis showed a 55% amino acid sequence identity with class C ?-lactamase genes. qPCR analysis of the ?-lactamase transcript indicated a higher level expression of the ?-lactamase compared to the relative transcript quantities in antibiotic-susceptible bacteria. The putative ?-lactamase gene was cloned, expressed in Escherichia coli BL21 and the product, AmpC, was purified to homogeneity. Its optimal activity was at pH 6 and 30 °C. Further, the ?-lactamase had a higher affinity towards penicillins than cephalosporin antibiotics.
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Identification and characterization of alkaline serine protease from goat skin surface metagenome.
AMB Express
PUBLISHED: 03-28-2011
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Metagenomic DNA isolated from goat skin surface was used to construct plasmid DNA library in Escherichia coli DH10B. Recombinant clones were screened for functional protease activity on skim milk agar plates. Upon screening 70,000 clones, a clone carrying recombinant plasmid pSP1 exhibited protease activity. In vitro transposon mutagenesis and sequencing of the insert DNA in this clone revealed an ORF of 1890 bp encoding a protein with 630 amino acids which showed significant sequence homology to the peptidase S8 and S53 subtilisin kexin sedolisin of Shewanella sp. This ORF was cloned in pET30b and expressed in E. coli BL21 (DE3). Although the cloned Alkaline Serine protease (AS-protease) was overexpressed, it was inactive as a result of forming inclusion bodies. After solubilisation, the protease was purified using Ni-NTA chromatography and then refolded properly to retain protease activity. The purified AS-protease with a molecular mass of ~63 kDa required a divalent cation (Co2+ or Mn2+) for its improved activity. The pH and temperature optima for this protease were 10.5 and 42°C respectively.
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Root colonization of a rice growth promoting strain of Enterobacter cloacae.
J. Basic Microbiol.
PUBLISHED: 02-22-2011
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Enterobacter cloacae GS1 was isolated by in-planta enrichment of a rice rhizoplane bacterial community. It displayed strong seed adherence ability (2.5 × 10(5) cfu/seed) and colonized rice roots reaching up to 1.65 × 10(9) cfu/g of fresh root weight in a gnotobiotic root colonization system. E. cloacae GS1 was motile, able to solubilize tricalcium phosphate, and produced indole acetic acid like substances (15 ?g/ml). As an introduced bioinoculant in non-sterile soil, E. cloacae GS1 colonized rice roots and significantly improved the fresh weight, root length, shoot length, and nitrogen content in inoculated rice seedlings as compared to uninoculated controls. This isolate was tagged with green fluorescent protein and various stages of root colonization in gnotobiotic hydroponic environment and non-sterile soil environment were followed by fluorescence microscopy. Owing to its effective root colonizing ability and growth promoting potential, Enterobacter cloacae GS1 is a promising symbiotic bioinoculant for rice.
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Junker: an intergenic explorer for bacterial genomes.
Genomics Proteomics Bioinformatics
PUBLISHED: 02-01-2011
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In the past few decades, scientists from all over the world have taken a keen interest in novel functional units such as small regulatory RNAs, small open reading frames, pseudogenes, transposons, integrase binding attB/attP sites, repeat elements within the bacterial intergenic regions (IGRs) and in the analysis of those "junk" regions for genomic complexity. Here we have developed a web server, named Junker, to facilitate the in-depth analysis of IGRs for examining their length distribution, four-quadrant plots, GC percentage and repeat details. Upon selection of a particular bacterial genome, the physical genome map is displayed as a multiple loci with options to view any loci of interest in detail. In addition, an IGR statistics module has been created and implemented in the web server to analyze the length distribution of the IGRs and to understand the disordered grouping of IGRs across the genome by generating the four-quadrant plots. The proposed web server is freely available at the URL http://pranag.physics.iisc.ernet.in/junker/.
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Repeated Random Mutagenesis of alpha-Amylase from Bacillus licheniformis for Improved pH Performance.
J. Microbiol. Biotechnol.
PUBLISHED: 11-17-2010
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The alpha-amylases activity was improved by random mutagenesis and screening. A region comprising residues from the position 34-281 was randomly mutated in B. licheniformis alpha-amylase (AmyL), and the library with mutations ranging from low, medium, and high frequencies was generated. The library was screened using an effective liquid-phase screening method to isolate mutants with an altered pH profile. The sequencing of improved variants indicated 2-5 amino acid changes. Among them, mutant TP8H5 showed an altered pH profile as compared with that of wild type. The sequencing of variant TP8H5 indicated 2 amino acid changes, Ile157Ser and Trp193Arg, which were located in the solvent accessible flexible loop region in domain B.
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Functional characterization of a new holin-like antibacterial protein coding gene tmp1 from goat skin surface metagenome.
Appl. Microbiol. Biotechnol.
PUBLISHED: 04-30-2010
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We have identified a holin-like gene from a goat skin surface metagenome. The ORF designated tmp1 coding for 34 amino acids shared sequence similarity with putative holin-like toxin genes. To analyze the antibacterial activity of tmp1 encoded protein, this ORF was cloned and expressed in Escherichia coli BL21(DE3). The expressed gene product Tmp1 exhibited antibacterial activity against Gram-positive bacteria but not to Gram-negative bacteria. A single transmembrane domain (TMD) was identified within Tmp1 and deletion analysis of the N-terminal region and TMD indicated TMD to be responsible for antibacterial activity. The TMD-dependent antibacterial activity was validated using a synthetic peptide with the amino acid sequence of TMD. Besides antibacterial activity, Tmp1 also complemented the function of holin in a lysis-defective bacteriophage lambda. To broaden the spectrum of antibacterial activity, a mutant library of tmp1 was generated by random mutagenesis. Four mutants with amino acid substitutions at the N-terminus of Tmp1 exhibited increased antibacterial activity against Gram-positive and Gram-negative bacteria and were not hemolytic. An improved activity of these mutant proteins is attributed to their increased hydrophobicity.
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Functional analysis of a putative holin-like peptide-coding gene in the genome of Bacillus licheniformis AnBa9.
Arch. Microbiol.
PUBLISHED: 09-22-2009
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BhlA, a putative holin-like protein of Bacillus licheniformis AnBa9 expressed in Escherichia coli BL21(DE3) showed antibacterial activity against several gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and Micrococcus luteus. Deletion analysis of bhlA suggests that a hydrophobic transmembrane domain, BhlATM is essential for antibacterial activity. Though the minimum inhibitory concentration (MIC) of BhlA was sevenfold lower than BhlATM, transmembrane domain deleted construct (BhlATM) had no antibacterial activity. The expression of BhlA in E. coli was found to be toxic to cells. Therefore, the bhlA was cloned in yeast surface display vector pYD1 and expressed in Saccharomyces cerevisiae. The surface displayed yeast showed inhibition of several gram-positive bacteria. This recombinant yeast expressing BhlA may be used as biodrug for efficient control of multiple drug-resistant bacterial infections.
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Influence of medium components and fermentation conditions on the production of bacteriocin(s) by Bacillus licheniformis AnBa9.
Bioresour. Technol.
PUBLISHED: 07-31-2009
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Recently, antibacterial peptides are gaining more attention as an alternative therapeutics and food and other products from spoilage and deterioration. Antibacterial peptide producing strains were isolated from sediments of slaughterhouse sewage wastes. One among them, identified as Bacillus licheniformis inhibited the growth of several gram positive bacteria. Response surface methodology with central composite rotary design was used for optimization of fermentation medium and conditions for antibacterial peptide production. Lactose, NH(4)NO(3), yeast extract and NaCl and environmental factors such as pH, temperature and incubation period were selected as variables. Among ingredients, high concentration of yeast extract and NaCl had a positive effect on antibacterial peptide production and specific activity, respectively. Alkaline pH and high temperature favoured the production of antibacterial peptide by B. licheniformis AnBa9. Under optimized condition, B. licheniformis AnBa9 produced 25-fold higher production of antibacterial peptide than the un-optimized condition. Biochemical characteristics of the antibacterial peptides of B. licheniformis AnBa9 revealed that they are of bacteriocin type.
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Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.
Biotechnol. Lett.
PUBLISHED: 03-23-2009
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An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.
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Purification and characterization of keratinase from recombinant Pichia and Bacillus strains.
Protein Expr. Purif.
PUBLISHED: 02-13-2009
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The keratinase gene from Bacillus licheniformis MKU3 was cloned and successfully expressed in Bacillus megaterium MS941 as well as in Pichia pastoris X33. Compared with parent strain, the recombinant B. megaterium produced 3-fold increased level of keratinase while the recombinant P. pastoris strain had produced 2.9-fold increased level of keratinase. The keratinases from recombinant P. pastoris (pPZK3) and B. megaterium MS941 (pWAK3) were purified to 67.7- and 85.1-folds, respectively, through affinity chromatography. The purified keratinases had the specific activity of 365.7 and 1277.7 U/mg, respectively. Recombinant keratinase from B. megaterium was a monomeric protein with an apparent molecular mass of 30 kDa which was appropriately glycosylated in P. pastoris to have a molecular mass of 39 kDa. The keratinases from both recombinant strains had similar properties such as temperature and pH optimum for activity, and sensitivity to various metal ions, additives and inhibitors. There was considerable enzyme stability due to its glycosylation in yeast system. At pH 11 the glycosylated keratinase retained 95% of activity and 75% of its activity at 80 degrees C. The purified keratinase hydrolyzed a broad range of substrates and displayed effective degradation of keratin substrates. The K(m) and V(max) of the keratinase for the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA was found to be 0.201 mM and 61.09 U/s, respectively. Stability in the presence of detergents, surfactants, metal ions and solvents make this keratinase suitable for industrial processes.
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Cloning and expression of GH11 xylanase gene from Aspergillus fumigatus MKU1 in Pichia pastoris.
J. Biosci. Bioeng.
PUBLISHED: 01-20-2009
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A xylanase gene, xynf11a of Aspergillus fumigatus MKU1 was cloned and expressed in Pichia pastoris X33. Two exons of the xynf11a gene were amplified separately and fused by overlap extension PCR. The fused product was cloned in yeast expression vector pPICZB and expressed in P. pastoris under the control of the AOX1 promoter. P. pastoris transformants expressing recombinant xylanases were selected on xylan agar plate and their ability to produce the xylanase was evaluated in flask cultures. P. pastoris X33 (pZBxynf11aFP) efficiently secreted the recombinant xylanase into the medium and produced the high level of xylanase activity (14 U/ml) after 96 h of growth. The recombinant xylanase produced by P. pastoris showed maximum activity at pH 6.0 and temperature 60 degrees C. The recombinant xylanase did not exhibit any cellulase activity and hence it could be potentially used for pretreatment of paper pulp before bleaching.
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Isolation of a novel high erythritol-producing Pseudozyma tsukubaensis and scale-up of erythritol fermentation to industrial level.
Appl. Microbiol. Biotechnol.
PUBLISHED: 01-11-2009
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This study isolated a novel erythritol-producing yeast strain, which is capable of growth at high osmolarity. Characteristics of the strain include asexual reproduction by multilateral budding, absence of extracellular starch-like compounds, and a negative Diazonium blue B color reaction. Phylogenetic analysis based on the 26S rDNA sequence and physiological analysis indicated that the strain belongs to the species Pseudozyma tsukubaensis and has been named P. tsukubaensis KN75. When P. tsukubaensis KN75 was cultured aerobically in a fed-batch culture with glucose as a carbon source, it produced 245 g/L of erythritol, corresponding to 2.86 g/L/h productivity and 61% yield, the highest erythritol yield ever reported by an erythritol-producing microorganism. Erythritol production was scaled up from a laboratory scale (7 L fermenter) to pilot (300 L) and plant (50,000 L) scales using the dissolved oxygen as a scale-up parameter. Erythritol production at the pilot and plant scales was similar to that at the laboratory scale, indicating that the production of erythritol by P. tsukubaensis KN75 holds commercial potential.
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Purification and characterization of a beta-1,4-glucosidase from a newly isolated strain of Fomitopsis pinicola.
Appl. Microbiol. Biotechnol.
PUBLISHED: 01-06-2009
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An efficient beta-1,4-glucosidase (BGL) producing strain, Fomitopsis pinicola KMJ812, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular BGL was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The relative molecular weight of F. pinicola BGL was determined to be 105 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, or 110 kDa by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 4.5 and a temperature optimum of 50 degrees C. The enzyme showed high substrate specificity and high catalytic efficiency (kcat=2,990 s-1, Km=1.76mM, kcat/Km=1,700 mM-1 s-1) for p-nitrophenyl-beta-d-glucopyranoside. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 3, indicating that the F. pinicola BGL is a member of glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, F. pinicola BGL is distinguished from other BGLs by its high catalytic efficiency and strict substrate specificity.
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Assessment of Microbial Richness in Pelagic Sediment of Andaman Sea by Bacterial Tag Encoded FLX Titanium Amplicon Pyrosequencing (bTEFAP).
Indian J. Microbiol.
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Microbial diversity of 1,000 m deep pelagic sediment from off Coast of Andaman Sea was analyzed by a culture independent technique, bacterial tag encoded FLX titanium amplicon pyrosequencing. The hypervariable region of small subunit ribosomal rRNA gene covering V6-V9, was amplified from the metagenomic DNA and sequenced. We obtained 19,271 reads, of which 18,206 high quality sequences were subjected to diversity analysis. A total of 305 operational taxonomic units (OTUs) were obtained corresponding to the members of firmicutes, proteobacteria, plantomycetes, actinobacteria, chloroflexi, bacteroidetes, and verucomicrobium. Firmicutes was the predominant phylum, which was largely represented with the family bacillaceae. More than 44 % of sequence reads could not be classified up to the species level and more than 14 % of the reads could not be assigned to any genus. Thus, the data indicates the possibility for the presence of uncultivable or unidentified novel bacterial species. In addition, the community structure identified in this study significantly differs with other reports from marine sediments.
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Comparative genomics reveals novel Fur regulated sRNAs and coding genes in diverse proteobacteria.
Gene
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Ferric uptake regulator (Fur) is a transcriptional regulator controlling the expression of genes involved in iron homeostasis and plays an important role in pathogenesis. Fur-regulated sRNAs/CDSs were found to have upstream Fur Binding Sites (FBS). We have constructed a Positional Weight Matrix from 100 known FBS (19 nt) and tracked the Orphan FBSs. Possible Fur regulated sRNAs and CDSs were identified by comparing their genomic locations with the Orphan FBSs identified. Thirty-eight novel and all known Fur regulated sRNAs in nine proteobacteria were identified. In addition, we identified high scoring FBSs in the promoter regions of the 304 CDSs and 68 of them were involved in siderophore biosynthesis, iron-transporters, two-component system, starch/sugar metabolism, sulphur/methane metabolism, etc. The present study shows that the Fur regulator controls the expression of genes involved in diverse metabolic activities and it is not limited to iron metabolism alone.
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Genome sequence of Staphylococcus arlettae strain CVD059, isolated from the blood of a cardiovascular disease patient.
J. Bacteriol.
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We have isolated a Staphylococcus arlettae strain, strain CVD059, from the blood of a rheumatic mitral stenosis patient. Here, we report the genome sequence and potential virulence factors of this clinical isolate. The draft genome of S. arlettae CVD059 is 2,565,675 bp long with a G+C content of 33.5%.
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Inactivation of the transcriptional regulator-encoding gene sdiA enhances rice root colonization and biofilm formation in Enterobacter cloacae GS1.
J. Bacteriol.
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Enterobacter cloacae GS1 is a plant growth-promoting bacterium which colonizes rice roots. In the rhizosphere environment, N-acyl homoserine lactone (NAHL)-like quorum-sensing signals are known to be produced by host plants and other microbial inhabitants. E. cloacae GS1 was unable to synthesize NAHL quorum-sensing signals but had the NAHL-dependent transcriptional regulator-encoding gene sdiA. This study was aimed at understanding the effects of SdiA and NAHL-dependent cross talk in rice root colonization by E. cloacae GS1. Pleiotropic effects of sdiA inactivation included substantial increases in root colonization and biofilm formation, suggesting a negative role for SdiA in bacterial adhesion. We provide evidence that sdiA inactivation leads to elevated levels of biosynthesis of curli, which is involved in cellular adhesion. Extraneous addition of NAHLs had a negative effect on root colonization and biofilm formation. However, the sdiA mutant of E. cloacae GS1 was insensitive to NAHLs, suggesting that this NAHL-induced inhibition of root colonization and biofilm formation is SdiA dependent. Therefore, it is proposed that NAHLs produced by both plant and microbes in the rice rhizosphere act as cross-kingdom and interspecies signals to negatively impact cellular adhesion and, thereby, root colonization in E. cloacae GS1.
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Direct cell penetration of the antifungal peptide, MMGP1, in Candida albicans.
J. Pept. Sci.
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An antifungal peptide, MMGP1, was recently identified from marine metagenome. The mechanism of cellular internalization of this peptide in Candida albicans was studied using fluorescein 5-isothiocynate (Sigma, California, USA) labeling followed by fluorescence microscopy and flow cytometry analyses. The peptide could enter C.?albicans cells even at 4?°C, where all energy-dependent transport mechanisms are blocked. In addition, the peptide internalization was not affected by the endocytic inhibitor, sodium azide. The kinetic study has shown that the peptide was initially localized on cell membrane and subsequently internalized into cytosol. The MMGP1 treatment exhibited time-dependent cytotoxicity in C.?albicans as evidenced by SYTOX Green (Molecular Probes Inc., Eugene, Oreg) uptake.
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Genome sequence of the plant growth-promoting rhizobacterium Pseudomonas putida S11.
J. Bacteriol.
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Here we report the genome sequence of a plant growth-promoting rhizobacterium, Pseudomonas putida S11. The length of the draft genome sequence is approximately 5,970,799 bp, with a G+C content of 62.4%. The genome contains 6,076 protein-coding sequences.
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Genome sequence of the plant growth-promoting bacterium Enterobacter cloacae GS1.
J. Bacteriol.
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Here, we present the genome sequence of Enterobacter cloacae GS1. This strain proficiently colonizes rice roots and promotes plant growth by improving plant nutrition. Analyses of the E. cloacae GS1 genome will throw light on the genetic factors involved in root colonization, growth promotion, and ecological success of this rhizobacterium.
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Influence of siderophore pyoverdine synthesis and iron-uptake on abiotic and biotic surface colonization of Pseudomonas putida S11.
Biometals
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Fluorescent pseudomonads produce a characteristic fluorescent pigment, pyoverdines as their primary siderophore for iron acquisition under iron-limiting conditions. Here, we report the identification of a random transposon mutant IST3 of Pseudomonas putida S11 showing tolerance to iron starvation stress condition and increased pyoverdine production. The insertion of the Tn5 transposon was found to be in pstS gene of pstSR operon encoding sensor histidine kinase protein of the two-component signal transduction system. A pyoverdine negative derivative of IST3 mutant constructed was sensitive to iron stress condition. It indicated that increased survival of IST3 under iron-limiting condition was due to higher pyoverdine production. The iron starvation tolerant mutant (IST3) exhibited enhanced pyoverdine-mediated iron uptake in minimal medium which significantly improved its biofilm formation, seed adhesion and competitive root colonization.
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Identification of a novel antifungal peptide with chitin-binding property from marine metagenome.
Protein Pept. Lett.
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A novel antifungal peptide with 36 amino acids was identified by functional screening of a marine metagenomic library. The peptide did not show similarity with any existing antimicrobial peptide sequences in the databank. The108 bp ORF designated as mmgp1 was cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. Mass spectrometry analysis of the purified recombinant peptide revealed a molecular mass of 5026.9 Da. The purified recombinant peptide inhibited the growth of Candida albicans and Aspergillus niger. The peptide was predicted to adopt ?- helical conformation with an extended coil containing a ligand binding site for N-acetyl-D-glucosamine. The ?- helicity of the peptide was demonstrated by circular dichroism spectroscopy in the presence of chitin or membrane mimicking solvent, trifluoroethanol. The chitin binding property of the peptide was also confirmed by fast performance liquid chromatography.
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Pseudomonas sp. as a Source of Medium Chain Length Polyhydroxyalkanoates for Controlled Drug Delivery: Perspective.
Int J Microbiol
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Controlled drug delivery technology represents one of the most rapidly advancing areas of science. They offer numerous advantages compared to conventional dosage forms including improved efficacy, reduced toxicity, improved patient compliance and convenience. Over the past several decades, many delivery tools or methods were developed such as viral vector, liposome-based delivery system, polymer-based delivery system, and intelligent delivery system. Recently, nonviral vectors, especially those based on biodegradable polymers, have been widely investigated as vectors. Unlike the other polymers tested, polyhydroxyalkanoates (PHAs) have been intensively investigated as a family of biodegradable and biocompatible materials for in vivo applications as implantable tissue engineering material as well as release vectors for various drugs. On the other hand, the direct use of these polyesters has been hampered by their hydrophobic character and some physical shortcomings, while its random copolymers fulfilled the expectation of biomedical researchers by exhibiting significant mechanical and thermal properties. This paper reviews the strategies adapted to make functional polymer to be utilized as delivery system.
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Gelatin nanocarrier enables efficient delivery and phototoxicity of hypocrellin B against a mice tumour model.
J Biomed Nanotechnol
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Nanoparticles formulated from biodegradable and natural polymer gelatin, were investigated for their potential to enable efficient delivery and enhanced efficacy of a well-known photodynamic agent, Hypocrellin B (HB). The HB-loaded poly(ethylene glycol) modified gelatin nanoparticles (HB-PEG-GNP) possessed near-spherical shape, with particle size in the range of 292 +/- 42 nm, and demonstrated characteristic optical properties for photodynamic therapy (PDT). Photophysical studies of the HB-PEG-GNP demonstrated photogeneration of reactive oxygen species (ROS). The nanoparticles were tested for cellular uptake in vitro, on Daltons Lymphoma Ascites (DLA) cells and demonstrated dose dependent phototoxicity upon visible light treatment. HB-PEG-GNP induced mitochondrial damage, as investigated by JC-1 staining, leading to apoptotic cell death. Biodistribution measurements revealed that nanoformulation reduces liver uptake of HB-PEG-GNP and increases tumour uptake with time. In vivo PDT studies in solid tumour bearing mice showed markedly significant regression (38.5 +/- 2.2%, p < 0.05) for HB-PEG-GNP treated mice in contrast to those treated with free HB (29.36 +/- 1.62%). The present study reveals gelatin nanocarrier to be an effective drug delivery system for enhancement of therapeutic efficacy of the PDT agent, HB.
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Prevalence of bacteria in the circulation of cardiovascular disease patients, Madurai, India.
Heart Lung Circ
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Cardiovascular diseases (CVDs) have a complex aetiology determined by risk factors, which include genetic and environmental factors. Chronic infection and inflammation is reported to be a pathogenic determinant for the development of CVDs. Here, we report the prevalence of bacterial pathogens in the circulation of CVD patients in Madurai, India. Blood culturing was performed using BD BACTEC automated culture system and organisms were identified by16S rRNA gene sequence analysis. From a total of 133 samples screened, 47 samples showed culture positive which indicates a high level of bacteraemia in CVD patients. From the 47 samples that showed growth, we have identified 57 bacterial isolates comprising 35 different species. Coagulase negative Staphylococci (CoNS) was the most predominant group of bacteria and other notable bacterial species isolated in this study are discussed.
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Production, purification, and characterization of a ?-glucosidase of Penicillium funiculosum NCL1.
Appl. Biochem. Biotechnol.
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Penicillium funiculosum NCL1, a filamentous fungus, produced significantly higher levels of ?-glucosidase. The effect of initial pH, incubation temperature, and different carbon sources on extracellular ?-glucosidase production was studied in submerged fermentation. At 30 °C with initial pH 5.0, enzyme production was increased by 48-fold upon induction with paper mill waste, as compared to commercial cellulose powder. In zymogram analysis, four isoforms of ?-glucosidases were observed with wheat bran whereas a minimum of one isoform was observed with other carbon sources. A major ?-glucosidase (Bgl3A) with the apparent molecular weight of ~120 kDa, induced by paper mill waste, was purified 19-fold to homogeneity, with a specific activity of 1,796 U/mg. Bgl3A was a monomeric glycoprotein with 29% of neutral carbohydrate content. It showed optimum activity at pH 4.0 and 5.0, optimum temperature at 60 °C, and exhibited a half-life of 1 h at 60 °C. K(m) of Bgl3A was found to be 0.057 mM with p-nitrophenyl ?-D-glucoside and V(max) was 1,920 U/mg. The purified enzyme exhibited glucose tolerance with a K(i) of 1.5 mM. Bgl3A readily hydrolyzed glucosides with ?-linkage. Bgl3A activity was enhanced (156%) by Zn²? and was not affected by other metal cations and reagents. The supplementation of Bgl3A (5 U/mg) with Trichoderma reesei cellulase complex (5 FPU/mg) resulted in about 70% of enhanced glucose production, which emphasizes the industrial importance of Bgl3A.
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Single-step purification and immobilization of MBP-phytase fusion on starch agar beads: application in dephytination of soy milk.
Appl. Biochem. Biotechnol.
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Periplasmic phytase, appA from E. coli has been noticed as a superior feed and food additive owing to its high specific activity, acidic pH optimum and resistance to gastric proteases. E. coli phytase was expressed as a fusion protein with maltose-binding protein, affinity-purified to homogeneity and, subsequently, immobilized in one step using a cost-effective matrix prepared from starch agar bead. Immobilized enzyme revealed an activity optimum at pH 6, while that of free enzyme was observed at pH 4. Both the immobilized and free enzyme showed a temperature optimum at 60 °C. Cleavage of 87 kDa fusion protein using factor Xa released 45 kDa appA. Hydrolysis of soy milk using immobilized enzyme led to 10% increase in release of inorganic phosphate at 50 °C relative to free fusion protein. This study suggests the usability of MBP as an immobilizing linker to other food enzymes for economical use in industry.
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Genotypic and phenotypic diversity of PGPR fluorescent pseudomonads isolated from the rhizosphere of sugarcane (Saccharum officinarum L.).
J. Microbiol. Biotechnol.
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The genetic diversity of plant growth-promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with the sugarcane (Saccharum officinarum L.) rhizosphere was analyzed. Selected isolates were screened for plant growthpromoting properties including production of indole acetic acid, phosphate solubilization, denitrification ability, and production of antifungal metabolites. Furthermore, 16S rDNA sequence analysis was performed to identify and differentiate these isolates. Based on 16S rDNA sequence similarity, the isolates were designated as Pseudomonas plecoglossicida, P. fluorescens, P. libaniensis, and P. aeruginosa. Differentiation of isolates belonging to the same group was achieved through different genomic DNA fingerprinting techniques, including randomly amplified polymorphic DNA (RAPD), amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and bacterial repetitive BOX elements (BOX) analyses. The genetic diversity observed among the isolates and rep-PCR-generated fingerprinting patterns revealed that PGPR fluorescent pseudomonads are associated with the rhizosphere of sugarcane and that P. plecoglossicida is a dominant species. The knowledge obtained herein regarding the genetic and functional diversity of fluorescent pseudomonads associated with the sugarcane rhizosphere is useful for understanding their ecological role and potential utilization in sustainable agriculture.
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Superantigen profiles of emm and emm-like typeable and nontypeable pharyngeal streptococcal isolates of South India.
Ann. Clin. Microbiol. Antimicrob.
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The major virulence factors determining the pathogenicity of streptococcal strains include M protein encoded by emm and emm-like (emmL) genes and superantigens. In this study, the distribution of emm, emmL and superantigen genes was analyzed among the streptococcal strains isolated from the patients of acute pharyngitis.
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