• Title/Summary/Keyword: LC-MS-MS

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Identification of an ISR-Related Metabolite Produced by Pseudomonas chlororaphis O6 against the Wildfire Pathogen Pseudomonas syringae pv. tabaci in Tobacco

  • Park, Myung-Ryeol;Kim, Young-Cheol;Park, Ju-Yeon;Han, Song-Hee;Kim, Kil-Yong;Lee, Sun-Woo;Kim, In-Seon
    • Journal of Microbiology and Biotechnology
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    • v.18 no.10
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    • pp.1659-1662
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    • 2008
  • Pseudomonas chlororaphis O6 exhibits induced systemic resistance (ISR) against P. syringae pv. tabaci in tobacco. To identify one of the ISR metabolites, O6 cultures were extracted with organic solvents, and the organic extracts were subjected to column chromatography followed by spectroscopy analyses. The ISR bioassay-guided fractionation was carried out for isolation of the metabolite. High-resolution mass spectrometric analysis of the metabolite found $C_{9}H_{9}O_{3}N$ with an exact mass of 179.0582. LC/MS analysis in positive mode showed an $(M+H)^{+}$ peak at m/z 180. Nuclear magnetic resonance ($^{1}H,\;^{13}C$) analyses identified all protons and carbons of the metabolite. Based on the spectroscopy data, the metabolite was identified as 4-(aminocarbonyl) phenylacetate (4-ACPA). 4-ACPA applied at 68.0 mM exhibited ISR activity at a level similar to 1.0 mM salicylic acid. This is the first report to identify an ISR metabolite produced by P. chlororaphis O6 against the wildfire pathogen P. syringae pv. tabaci in tobacco.

Thiazinogeldanamycin, a New Geldanamycin Derivative Produced by Streptomyces hygroscopicus 17997

  • Ni, Siyang;Wu, Linzhuan;Wang, Hongyuan;Gan, Maoluo;Wang, Yucheng;He, Weiqing;Wang, Yiguang
    • Journal of Microbiology and Biotechnology
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    • v.21 no.6
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    • pp.599-603
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    • 2011
  • A new geldanamycin (GDM) derivative was discovered and isolated from the fermentation broth of Streptomyces hygroscopicus 17997. Its chemical structure was elucidated as thiazinogeldanamycin by LC-MS, sulfur analysis, and NMR. The addition of cysteine to the fermentation medium significantly stimulated the production level of thiazinogeldanamycin, suggesting cysteine as a precursor of thiazinogeldanamycin production. Although showing a decreased cytotoxicity against HepG2 cancer cells, thiazinogeldanamycin exhibited an improved water solubility and photostability. Thiazinogeldanamycin may represent the first natural GDM derivative characterized so far that uses GDM as its precursor. Its appearance also clearly indicates that an appropriate end-point of fermentation is of critical importance for the maximal production of GDM by Streptomyces hygroscopicus 17997.

Preparation and Antioxidant Activities In Vitro of a Designed Antioxidant Peptide from Pinctada fucata by Recombinant Escherichia coli

  • Wu, Yanyan;Ma, Yongkai;Li, Laihao;Yang, Xianqing
    • Journal of Microbiology and Biotechnology
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    • v.28 no.1
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    • pp.1-11
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    • 2018
  • An antioxidant peptide derived from Pinctada fucata meat using an Alcalase2.4L enzymatic hydrolysis method (named AOP) and identified by LC-TOF-MS has promising clinical potential for generating cosmetic products that protect skin from sunshine. To date, there have been few published studies investigating the structure-activity relationship in these peptides. To prepare antioxidant peptides better and improve their stability, the design and expression of an antioxidant peptide from Pinctada fucata (named DSAOP) was studied. The peptide contains a common precursor of an expression vector containing an ${\alpha}$-helix tandemly linked according to the BamHI restriction sites. The DNA fragments encoding DSAOP were synthesized and subcloned into the expression vector pET-30a (+), and the peptide was expressed mostly as soluble protein in recombinant Escherichia coli. Meanwhile, the DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity of DSAOP $IC_{50}$ values were $0.136{\pm}0.006$, $0.625{\pm}0.025$, and $0.306{\pm}0.015mg/ml$, respectively, with 2-fold higher DPPH radical scavenging activity compared with chemosynthesized AOP (p < 0.05), as well as higher superoxide radical scavenging activity compared with natural AOP (p < 0.05). This preparation method was at the international advanced level. Furthermore, pilot-scale production results showed that DSAOP was expressed successfully in fermenter cultures, which indicated that the design strategy and expression methods would be useful for obtaining substantial amounts of stable peptides at low costs. These results showed that DSAOP produced with recombinant Escherichia coli could be useful in cosmetic skin care products, health foods, and pharmaceuticals.

Purification and Identification of a Natural Antioxidant Protein from Fertilized Eggs

  • Yang, Shaohua;Wang, Lulu;Wang, Ying;Ou, Xiaoqian;Shi, Zhaoyuan;Lu, Chongchong;Wang, Wei;Liu, Guoqing
    • Food Science of Animal Resources
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    • v.37 no.5
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    • pp.764-772
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    • 2017
  • Fertilized hen eggs are rich in a variety of bioactive ingredients. In this study, we aimed to obtain an antioxidant protein from fertilized eggs and the radical scavenging abilities on 1, 1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl radical (${\bullet}OH$), superoxide anion ($O^{2-}{\bullet}$) were used to evaluate the antioxidant activity of the purified protein. During 20 d of incubation, the radical scavenging ability of protein extracted from fertilized eggs exhibited significantly differences and the protein on day 16 showed higher antioxidant capacity. Based on this, the antioxidant protein of the samples on day 16 were isolated for the follow-up study. With a molecular weight 43.22 kDa, the antioxidant protein was purified by Diethylaminoethyl cellulose -52 (DEAE-52) column and Sephadex G-100. The LC-MS analysis showed that the purified protein molecular weight was 43.22 kDa, named D2-S. The sequence of amino acids was highly similar to ovalbumin and the coverage reached to 84%. The purified protein showed a radical scavenging rate of $52.34{\pm}3.27%$ on DPPH and $63.49{\pm}0.25%$ on ${\bullet}OH$, respectively. Furthermore, the C-terminal amino acid sequence was NAVLFFGRCVSP, which was consistent with the sequence of ovabumin. These results here indicated that purified protein may be a potential resource as a natural antioxidant.

A comparative study on the degradation of methyl orange, methylene blue and congo red by atmospheric pressure jet

  • Park, Ji Hoon;Yusupov, Maksudbek;Lingamdinne, Lakshmi Prasanna;Koduru, Janardhan Reddy;Bogaerts, Annemie;Choi, Eun Ha;Attri, Pankaj
    • Proceedings of the Korean Vacuum Society Conference
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    • 2016.02a
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    • pp.190.1-190.1
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    • 2016
  • One of the most serious problems faced by billions of people today is the availability of fresh water. According to statistics, 15% of the world's total output of dye products is discharged into the environment as dye wastewater, which seriously pollutes groundwater resources. For the treatment of chemically and biologically contaminated water the advanced oxidation processes (AOPs) shows the promising action. The main advantage with AOPs is the ability to degrade the organic pollutants to $CO_2$ and $H_2O$. For this degradation process the AOPs generation of powerful and non-selective radicals that may oxidize majority of the organic pollutants present in the water body. To generate the various reactive chemical species such as radicals (${\bullet}OH$, ${\bullet}H$, ${\bullet}O$, ${\bullet}HO_2$) and molecular species ($H_2O_2$, $H_2$, $O_2$) in large amount in water, we have used the atmospheric pressure plasma. Among the reactive and non-reactive species, the hydroxyl radical (${\bullet}OH$) plays important role due to its higher oxidation potential (E0: 2.8 V). Therefore, in this work we have checked the degradation of various dyes such as methyl orange, methylene blue and congo red using different type of atmospheric pressure plasma sources (Indirect jet and direct jet). To check the degradation we have used the UV-visible spectroscopy, HPLC and LC-MS spectroscopy. Further, to estimate role of ${\bullet}OH$ on the degradation of dyes we have studied the molecular dynamic simulation.

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Biocontrol of Orchid-pathogenic Mold, Phytophthora palmivora, by Antifungal Proteins from Pseudomonas aeruginosa RS1

  • Sowanpreecha, Rapeewan;Rerngsamran, Panan
    • Mycobiology
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    • v.46 no.2
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    • pp.129-137
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    • 2018
  • Black rot disease in orchids is caused by the water mold Phytophthora palmivora. To gain better biocontrol performance, several factors affecting growth and antifungal substance production by Pseudomonas aeruginosa RS1 were verified. These factors include type and pH of media, temperature, and time for antifungal production. The results showed that the best conditions for P. aeruginosa RS1 to produce the active compounds was cultivating the bacteria in Luria-Bertani medium at pH 7.0 for 21 h at $37^{\circ}C$. The culture filtrate was subjected to stepwise ammonium sulfate precipitation. The precipitated proteins from the 40% to 80% fraction showed antifungal activity and were further purified by column chromatography. The eluted proteins from fractions 9-10 and 33-34 had the highest antifungal activity at about 75% and 82% inhibition, respectively. SDS-PAGE revealed that the 9-10 fraction contained mixed proteins with molecular weights of 54 kDa, 32 kDa, and 20 kDa, while the 33-34 fraction contained mixed proteins with molecular weights of 40 kDa, 32 kDa, and 29 kDa. Each band of the proteins was analyzed by LC/MS to identify the protein. The result from Spectrum Modeler indicated that these proteins were closed similarly to three groups of the following proteins; catalase, chitin binding protein, and protease. Morphological study under scanning electron microscopy demonstrated that the partially purified proteins from P. aeruginosa RS1 caused abnormal growth and hypha elongation in P. palmivora. The bacteria and/or these proteins may be useful for controlling black rot disease caused by P. palmivora in orchid orchards.

Genomics and LC-MS Reveal Diverse Active Secondary Metabolites in Bacillus amyloliquefaciens WS-8

  • Liu, Hongwei;Wang, Yana;Yang, Qingxia;Zhao, Wenya;Cui, Liting;Wang, Buqing;Zhang, Liping;Cheng, Huicai;Song, Shuishan;Zhang, Liping
    • Journal of Microbiology and Biotechnology
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    • v.30 no.3
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    • pp.417-426
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    • 2020
  • Bacillus amyloliquefaciens is an important plant disease-preventing and growth-promoting microorganism. B. amyloliquefaciens WS-8 can stimulate plant growth and has strong antifungal properties. In this study, we sequenced the complete genome of B. amyloliquefaciens WS-8 by Pacific Biosciences RSII (PacBio) Single Molecule Real-Time (SMRT) sequencing. The genome consists of one chromosome (3,929,787 bp) and no additional plasmids. The main bacteriostatic substances were determined by genome, transcriptome, and mass spectrometry data. We thereby laid a theoretical foundation for the utilization of the strain. By genomic analysis, we identified 19 putative biosynthetic gene clusters for secondary metabolites, most of which are potentially involved in the biosynthesis of numerous bioactive metabolites, including difficidin, fengycin, and surfactin. Furthermore, a potential class II lanthipeptide biosynthetic gene cluster and genes that are involved in auxin biosynthesis were found. Through the analysis of transcriptome data, we found that the key bacteriostatic genes, as predicted in the genome, exhibited different levels of mRNA expression. Through metabolite isolation, purification, and exposure experiments, we found that a variety of metabolites of WS-8 exert an inhibitory effect on the necrotrophic fungus Botrytis cinerea, which causes gray mold; by mass spectrometry, we found that the main substances are mainly iturins and fengycins. Therefore, this strain has the potential to be utilized as an antifungal agent in agriculture.

Action of atmospheric pressure non-thermal plasma on the biomolecules and bio-organism

  • Attri, Pankaj;Park, Ji Hoon;Kumar, Naresh;Ali, Anser;Kim, In Tae;Lee, Weontae;Choi, Eun Ha
    • Proceedings of the Korean Vacuum Society Conference
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    • 2015.08a
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    • pp.66.1-66.1
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    • 2015
  • Plasma medicine is an upcoming research area that has attracted the scientists to explore more deeply the utility of plasma. So, apart from the treating biomaterials and tissues with plasma, we have studied the effect of plasma with different feeding gases on modification of biomolecules. Additionally, we have checked the action of nanosecond pulsed plasma on the biomolecules. We have checked the plasma action on proteins ((Hemoglobin (Hb) Myoglobin (Mb) and lysoenzyme), calf thymus DNA and amino acids. The structural changes or structural modification of proteins and DNA have been studied using circular dichroism (CD), dynamic light scattering (DLS), gel electrophoresis, protein oxidation test, UV-vis spectroscopy and 1D NMR, while Liquid Chromatograph/Capillary Electrophoresis-Mass Spectrometer(LC/CE-MS) based qualitative bio-analysis have been used to study the modification of amino acids. We have also shown the effect of NaCl and ionic liquid on the formation of OH radicals using electron spin resonance and fluorescence techinques.

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The Chemical Constituents of the Stem Barks of Fraxinus rhynchophylla (물푸레나무(Fraxinus rhynchophylla) 수피의 추출성분)

  • Yang, Eun-Ju;Lee, Dong-Geun;Lee, Jong-Won;Kim, Yae-Sil;Lim, Sun-Ha;Song, Kyung-Sik
    • Applied Biological Chemistry
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    • v.50 no.4
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    • pp.348-351
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    • 2007
  • The stem barks of Fraxinus rhynchophylla was extracted with 95% EtOH, and the concentrated extract was successively partitioned with $CH_2Cl_2$, n-BuOH, and $H_2O$ in order to investigate the major phytochemicals. From the $CH_2Cl_2$ soluble fraction, a sterol (1) was isolated through the repeated silica gel column chromatographies. Three additional compounds (2-4) were isolated from the n-BuOH soluble fraction through silica gel, RP-18, and Sephadex LH-20 column chromatographies. Their chemical structures were elucidated as daucosterol $(1;{\beta}-sitosterol-3-O-{\beta}-D-glucopyranoside)$, caffeic acid (2), 6,8-dihydroxy-7-methoxycoumarin (3), and coniferaldehyde glucoside (4) by comparing their spectral data with those in the literatures. All isolates (1-4) were the first to be isolated from F. rhynchophylla.

Dyeabilities with Various Fabrics and Chemical Composition of Brown Colorants from Pine Bark (적송수피 색소 성분의 화학적 조성과 섬유 염색성)

  • Kim, Yong-Sook;Bae, Soon Ei
    • Fashion & Textile Research Journal
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    • v.15 no.1
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    • pp.138-146
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    • 2013
  • Chemical compositions and biological functions of brown colorants extracted from pine bark(Pinus densiflora) have been studied. Dyeing test using multifiber fabrics with extracted colorants were preliminary carried out. Dyeing conditions and fastness tests of selected fabrics have been also studied. The brown colorants were produced 1.5% concentrations by solvent extraction from milled pine bark using methanol. The colorants were extracted with 80% methanol as best choice by a criteria of solid quantity and dyeability on fabrics. The chemical compositions were identified as mixtures of taxifolin epicatechin and procyanidin by LC/MS analysis. The brown colorants could be dyed not only natural fibers such as cotton, silk and wool but also synthetic fiber as nylon and semi-synthetic fiber as viscose rayon. Maximum K/S values was shown at 400 nm according to different fiber with color appearance of redish brown. Optimum pH and temperature of dyeing conditions was 4 and above $80^{\circ}C$, respectively. The brown colorants had a strong antioxidant activity compared to Butylated hydroxyanisole as standard and weak antimicrobial activity against E. coli. compared to kanamycin. Washing, rubbing, perspiration, dry cleaning and light fastness for cotton, nylon and silk dyed with the brown colorants were carried out by KS K method. Most of color fastness such as washing, rubbing, perspiration, and dry cleaning were represented as 4-5 grade. However, light fastness was reported as 2-3 grade. From this studies, brown colorants produced pine bark have a high potentials for natural dyeing on fabrics with antioxidant activity.