• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1059-1065
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• 2012

Biocatalytic transfer of oxygen in isolated cytochrome P450 or whole microbial cells is an elegant and efficient way to achieve selective hydroxylation. Cytochrome P450 CYP105P2 was isolated from Streptomyces peucetius that showed a high degree of amino acid identity with hydroxylases. Previously performed homology modeling, and subsequent docking of the model with flavone, displayed a reasonable docked structure. Therefore, in this study, in a pursuit to hydroxylate the flavone ring, CYP105P2 was co-expressed in a two-vector system with putidaredoxin reductase (camA) and putidaredoxin (camB) from Pseudomonas putida for efficient electron transport. HPLC analysis of the isolated product, together with LC-MS analysis, showed a monohydroxylated flavone, which was further established by subsequent ESI/MS-MS. A successful 10.35% yield was achieved with the whole-cell bioconversion reaction in Escherichia coli. We verified that CYP105P2 is a potential bacterial hydroxylase.

• BMB Reports
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• v.45 no.12
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• pp.736-741
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• 2012

Certain members of the cytochromes P450 superfamily metabolize polyunsaturated long-chain fatty acids to several classes of oxygenated metabolites. An approach based on in silico analysis predicted that Streptomyces peucetius CYP107N3 might be a fatty acid-metabolizing enzyme, showing high homology with epoxidase enzymes. Homology modeling and docking studies of CYP107N3 showed that oleic acid can fit directly into the active site pocket of the double bond of oleic acid within optimum distance of $4.6{\AA}$ from the Fe. In order to confirm the epoxidation activity proposed by in silico analysis, a gene coding CYP107N3 was expressed in Escherichia coli. The purified CYP107N3 was shown to catalyze $C_9-C_{10}$ epoxidation of oleic acid in vitro to 9,10-epoxy stearic acid confirmed by ESI-MS, HPLC-MS and GC-MS spectral analysis.

• Molecules and Cells
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• v.28 no.4
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• pp.397-401
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• 2009

Flavonoids are a group of polyphenolic compounds that have been recognized as important due to their physiological and pharmacological roles and their health benefits. Glycosylation of flavonoids has a wide range of effects on flavonoid solubility, stability, and bioavailability. We previously generated the E. coli BL21 (DE3) ${\Delta}pgi$ host by deleting the glucose-phosphate isomerase (Pgi) gene in E. coli BL21 (DE3). This host was further engineered for whole-cell biotransformation by integration of galU from E. coli K12, and expression of calS8 (UDP-glucose dehydrogenase) and calS9 (UDP-glucuronic acid decarboxylase) from Micromonospora echinospora spp. calichensis and arGt-4 (7-O-glycosyltransferase) from Arabidopsis thaliana to form E. coli (US89Gt-4), which is expected to produce glycosylated flavonoids. To test the designed system, the engineered host was fed with naringenin as a substrate, and naringenin 7-O-xyloside, a glycosylated naringenin product, was detected. Product was verified by HPLC-LC/MS and ESI-MS/MS analyses. The reconstructed host can be applied for the production of various classes of glycosylated flavonoids.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.342-348
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• 2011

Bacillus subtilis EBS05, an endophytic bacteria strain isolated from a medicinal plant Cinnamomum camphor, can produce antagonistic compounds that effectively inhibit plant pathogenic fungi. The greenhouse experiments showed that wheat sharp eyespot disease (WSED) was reduced by 91.2%, 88.2% and 43.0% after the treatment with fermentation broth, bacteria-free filter and a fungicide fludioxonil, respectively. The culture broth of strain EBS05 can more effectively control WSED than can fludioxonil. The fermentation broth and bacteria-free filter ability to suppress WSED was not significantly different, suggesting that an active secreted substance played a major role in controlling WSED. Separation and purification of the active compounds was carried out by serial processes, including hydrochloric acid (pH 2.0) treatment, methanol extraction and Sephadex LH-20 column chromatography, silica gel column chromatography and reverse-phase high-pressure liquid chromatography (HPLC), respectively. The purified compounds, one of active peaks in the HPLC spectrum, were obtained from the collection. Analysis of the chemical structures by time-of-flight mass spectrometry (TOF-MS) and electrospray ionization mass spectrometry/mass spectrometry (ESI-MS/MS) showed that the active substances produced by the endophytic bacteria EBS05 are mixture of the ${\beta}$-hydroxy-C12~C15-$Leu^7$ surfactin A isomers with 1035.65 Da, 1021.64 Da, 1007.63 Da and 993.65 Da molecular weights, respectively.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2018.04a
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• pp.61-61
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• 2018

Buckwheat is a important vegetable in asia for long time. Recently, Buckwheat has attracted attention to its potential for health benefit and especially as a gluten-free food. Conventional buckwheat studies have focused on seeds and sprouts, but we studied the components of the aerial parts and roots of buckwheat after harvesting. Therefore, we hope that this research will be a basic study to expand the application range of buckwheat plants which are discarded after harvesting. The ethanol extracts of aerial part and roots of buckwheat (Daegwan, Yangjul) were analysed using high performance liquid chromatography (HPLC). Rutin was detected as the major compound in Daegwan, Yangjul aerial part and Daegwan root extracts, except yangjul root extract. So we analysed Yangjul root extract using liquid chromatography-mass spectrometry (LC-MS) and then obtained the informations about the components in Yangjul root extract. Yangjul root extract was analysed by $LC-MS^n$ in negative ESI mode within the range m/z 150-2000 amu. Totally, four components was found in the Yangjul root extract and obtained the fragments ion patterns of ones. Further study, we have to confirm the structure of components in Yangjul root extract and try to more biological activity test the components for development as useful food or cosmetic ingredients.

• The Korean Journal of Mycology
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• v.41 no.4
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• pp.236-242
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• 2013

It is known not only that antifungal compounds such as sparassol, methyl orsellinate (ScI) and methyl-dihydroxy-methoxy-methylbenzoate (ScII) were produced during submerged culture from Sparassis crispa, but also that ScI and ScII were appeared higher antifungal activity than sparassol. The aim of this study, antifungal compounds of Sparassis latifolia were purified from mycelial culture media and identified by using NMR and ESI-MS. Based on HPLC analysis, methyl orsellinate and sparassol were detected at 15 min and 31 min of retention time, respectively. The compounds derived from S. latifolia were classified into four production patterns according to their strains. The strains originated from host plant Larix kaempferi and Pinus koraiensis showed different patterns of compound production, whereas the strains originated from host plant P. densiflora and Abies holophylla showed almost same patterns. There was no correlation between mycelial biomass and compound production. KFRI 645 strain from L. kaempferi exhibited higher methyl orsellinate production (0.170 mg/ml). Sparassol was produced by KFRI 747 from P. densiflora (0.004 mg/ml). Thus, our result revealed the new fact that methyl orsellinate and sparassol have different patterns according to the strains originated from different host plants.

• The Korean Journal of Pesticide Science
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• v.16 no.3
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• pp.209-216
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• 2012

In the continued research on natural fungicides for control of plant diseases by using plant-derived products, we found that Spatholobus suberectus Dunn had a strong fungicidal activity against several plant pathogens. S. suberectus (1 kg) was extracted with 80% aqueous MeOH and then the concentrated extract was partitioned with n-hexane, EtOAc, n-BuOH and $H_2O$ successively. The four layers were tested their disease contron efficacies against 6 plant diseases such as rice blast (RCB), rice sheath blight (RSB), tomato grey mold (TGM), tomato late blight (TLB), wheat leaf rust (WLR), and barley powdery mildew (BPM). The EtOAc fraction was highly active showing over 80% control against RCB, TGM, TLB, and BPM. By using silica gel chromatography, preparative TLC and HPLC, six compounds that were expected to have antifungal activity were separated. Their chemical structures were identified as ethanone, hydroxytyrosol, epicatechin, procyanidin B2, dimethoxy daizein and formononetin by ESI-MS, $^1H$-NMR, $^{13}C$-NMR, and 2D-NMR spectroscopic analyses. The chemicals except epicatechin were first reported in S. suberectus. Study on in vitro and in vivo antifungal activities of the isolated compounds is in progress.

• Korean Journal of Food Science and Technology
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• v.34 no.1
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• pp.114-117
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• 2002

During the screening of inhibitors of caspase-3 induction in U937 human monocytic leukemia cells from natural sources, Forsythiae fructus, which showed a high level of inhibition, was selected. And then, the compound was purified from the methanol extract using silica gel column chromatography and HPLC. The inhibitor was identified as rengyolone, by spectroscophic methods of ESI-MS, $^1H-NMR$, $^{13}C-NMR$, DEPT, and HMBC. Rengyolone showed inhibitory activity of caspase-3 induction, a major protease of apoptosis cascade, with an $IC_{50}$ value of $6.25\;{\mu}g/mL$ after 7 h of treatment in U937 cells. It also showed inhibitory activity of caspace-1 induction, with an $IC_{50}$ value of $7.50\;{\mu}g/mL$ after 40 h of treatment in D10S cells. In addition, it showed protective effect against cell death with an $IC_{50}$ value of $11\;{\mu}g/mL$ on U937 cells induced by etoposide after 24 h of treatment, but did not show any cytotoxicity at the same condition without etoposide, a caspase 3 inducing agent.

• Journal of Korean Society on Water Environment
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• v.32 no.6
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• pp.670-699
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• 2016

In this study, 16 antibiotics were selected from among the top 30 veterinary antibiotics sold in South Korea in 2014, as well as from among the pharmaceuticals targeted by EPA method 1694, in order to review analytical methods for the detection of trace levels of antibiotics in environmental samples: surface water, soils, animal origin foods, and manures. LC-MS/MS was heavily used. In the chromatography for the detection of the selected antibiotics, the $C_{18}$ column was mostly used at the temperature of $30{\sim}40^{\circ}C$. Water and methanol/acetonitrile were commonly chosen as a nonpolar and a polar mobile phase, respectively. Gradient elution was applied to separate multiclass antibiotics. Volatile additives, such as formic acid, acetic acid, and ammonium acetate were mixed with the mobile phase to improve the ionization efficiency of analytes and the sensitivity in MS detection. Electrospray ionization (ESI) was widely used in the LC-MS/MS and positive ionization was preferred to determine the selected antibiotics. A protonated $[M+H]^+$ molecule was selected as a precursor ion, and its two transitions were analyzed, one for quantitative measurement and the other for confirmation. This study reviewed linearity of the calibration curve, recovery, repeatability, method detection limits (MDLs), and method quantification limits (MQLs) for each target compound used to validate the developed analytical methods.

• Fisheries and Aquatic Sciences
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• v.19 no.4
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• pp.22.1-22.7
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• 2016

One of the main compounds in Ishige okamurae, diphlorethohydroxycarmalol (DPHC), is known to exhibit antiviral and anti-inflammatory effects. However, it has not been investigated extensively. In this study, preparative centrifugal partition chromatography (CPC) coupled with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ($ABTS^+$) online HPLC was employed for effectively separating considerable amounts of antioxidant compounds from marine algae. Two main antioxidant compounds, DPHC and octaphlorethol A (OPA), respectively, were confirmed and isolated from the ethyl acetate (EtOAc) fraction of I. okamurae by $ABTS^+$ online HPLC and preparative CPC systems. The presence of DPHC and OPA was confirmed in the EtOAc fraction of I. okamurae by both liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI/MS) and $ABTS^+$ online HPLC systems: DPHC (39 mg) and OPA (23 mg) were successfully isolated from I. okamurae (500 mg) with optimum solvent composition (0.5:10:4:6; n-hexane/EtOAc/MeOH/water, v/v) with corresponding partition coefficients (K) of 1.62 and 2.71, respectively, by preparative CPC. Hence, CPC coupled with $ABTS^+$ online HPLC is convenient for the efficient and simple isolation of these antioxidant compounds from I. okamurae.