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Exploring the Nucleophilic N- and S-Glycosylation Capacity of Bacillus licheniformis YjiC Enzyme

  • Bashyal, Puspalata;Thapa, Samir Bahadur;Kim, Tae-Su;Pandey, Ramesh Prasad;Sohng, Jae Kyung
    • Journal of Microbiology and Biotechnology
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    • v.30 no.7
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    • pp.1092-1096
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    • 2020
  • YjiC, a glycosyltransferase from Bacillus licheniformis, is a well-known versatile enzyme for glycosylation of diverse substrates. Although a number of O-glycosylated products have been produced using YjiC, no report has been updated for nucleophilic N-, S-, and C- glycosylation. Here, we report the additional functional capacity of YjiC for nucleophilic N- and S- glycosylation using a broad substrate spectrum including UDP-α-D-glucose, UDP-N-acetyl glucosamine, UDP-N-acetylgalactosamine, UDP-α-D-glucuronic acid, TDP-α-L-rhamnose, TDP-α-D-viosamine, and GDP-α-L-fucose as donor and various amine and thiol groups containing natural products as acceptor substrates. The results revealed YjiC as a promiscuous enzyme for conjugating diverse sugars at amine and thiol functional groups of small molecules applicable for generating glycofunctionalized chemical diversity libraries. The glycosylated products were analyzed using HPLC and LC/MS and compared with previous reports.

In Vitro Glycosylation of Peptide (RKDVY) and RNase A by PNGase F

  • Park, Su-Jin;Lee, Ji-Youn;Park, Tai-Hyun
    • Journal of Microbiology and Biotechnology
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    • v.13 no.2
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    • pp.191-195
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    • 2003
  • The in vitro glycosylation of pentapeptide (Arg-Lys-Asp-Val-Tyr; RKDVY) and RNase A was carried out using PNGase F (peptide-N-glycosidase F), and the results were analyzed using MALDI-TOF-MS. Aminated N,N-diretyl chitobiose was used as the sugar in the glycosylation reaction, and the amination yield of N,N'-diacetyl chitobiose was about $60\%$. To reduce the water activity and shift the reaction equilibrium to a reverse reaction, 1,4-dioxane or ethylene glycol was used as the organic solvent in the enzymatic glycosylation. A certain extent of nonenzymatic glycosylaton, known as the Maillard reaction, was also observed, which occurs on an arginine or lysine residue when the length of tie sugar residue is one or two. However, the extent of glycosylation was much higher in the enzymatic reaction, indicating that PNGase F can be effectively used to produce glycopeptides and glycoproteins in vitro.

Monitoring of Hazardous Chemicals for Effluents of STPs and WWTP in the Nakdong River Basin (낙동강수계 주요 하·폐수처리장 방류수내 미량유해물질 모니터링)

  • Kim, Gyung-A;Seo, Chang-Dong;Lee, Sang-Won;Ryu, Dong-Choon;Kwon, Ki-Won
    • Journal of Environmental Science International
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    • v.23 no.7
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    • pp.1253-1268
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    • 2014
  • This study was investigated twenty two hazardous chemicals compounds for effluents of nine sewage treatment plants (STPs) and one waste water treatment plant (WWTP) in the Nakdong Ri-ver Basin. They are eleven phthalates(DMP, DEP, DIBP, DBP, BEEP, DNPP, DHP, DCP, DEHP, DNOP, Dinonyl phthalate, seven aliphatic hydrocarbons(n-Tridecane, n-Tetradecane, n-Pentadecan-e, n-Hexadecane, n-Heptadecane, n-Octadecane, n-Nonadecane, Isoquinoline, 2-Chloropyridine, 2-N-itrophenol, and Benzophenone. The twenty two compounds were analyzed by gas chromatograp-hy mass spectrometry (GC/MS) with liquid-liquid extraction (LLE). Twenteen of twenty two subs-tances were detected. They were DMP, DEP, DIBP, DBP, DEHP, n-Tetradecane, n-Pentadecane, n-Heptadecane, n-Octadecane, n-Nonadecane, Isoquinoline and Benzophenone. Among these, DEHP, DEP and Benzophenone were most frequently observed. They were obtained as $ND{\sim}36.881{\mu}g/L$, $ND{\sim}0.950{\mu}g/L$, $ND{\sim}2.019{\mu}g/L$, respectively. When the substances were calculated the average concentration at 10 points, the maximum average detection concentration was investigated at the Dalseocheon STP.

Inhibition of glutathion-S-transferase and amidase by impurities in technical grade benfuracarb (Benfuracarb 원제에 함유된 불순물들의 glutathione-S-transferase와 amidase 저해 특성)

  • Yum, Chang-Sub;Kim, Song-Mun;Yu, Ji-Sook;Hur, Jang-Hyun
    • The Korean Journal of Pesticide Science
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    • v.6 no.1
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    • pp.31-35
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    • 2002
  • Objectives of this study were to determine if impurities in technical grade benfuracarb inhibit glutathione-S-transferase and amidase and to identify structures of impurities in technical grade benfuracarb. Technical grade benfuracarb, active ingredient, and impurity inhibited glutathione-S-transferase, and their $I_{50}$ were $9.7{\times}10^{-4}M,\;>1.0{\times}10^{-3}M,\;1.8{\times}10^{-4}M$, respectively. Such inhibition, however, was not higher than that by ethacrynic acid, a selective inhibitor to GST. Technical grade benfuracarb, active ingredient, and impurity also inhibited amidase, and their $I_{50}$ were $6.0{\times}10^{-5}M,\;4.3{\times}10^{-4}M,\;7.6{\times}10^{-5}M$, respectively. Our results show that the inhibition of both detoxifying enzymes by impurities in benfuracarb was 10-fold lower than that by active ingredient, suggesting that both active ingredient and impurities are involved in the inhibition of both detoxifying enzymes. Of four impurities (IM $1{\sim}4$) that were separated from technical grade benfuracarb, IM 2 and IM 3 inhibited GST and amidase. Based on data from IR, $^1H$-NMR, $^{13}C$-NMR and MS, it was determined that IM 2 is ethyl-N-isopropylamino propionate and IM 3 is ethyl-N-isopropyl-N(chlorosulfenyl)aminopropionate.

Method Development and Validation for Analysis of Isopyrazam Residues in Agricultural Products (농산물 중 살균제 Isopyrazam의 개별 잔류분석법 확립)

  • Kim, Ji-Yoon;Kim, Ja-Young;Ham, Hun-Ju;Do, Jung-Ah;Oh, Jae-Ho;Lee, Young-Deuk;Hur, Jang-Hyun
    • The Korean Journal of Pesticide Science
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    • v.17 no.2
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    • pp.84-93
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    • 2013
  • Validated analytical methods for isopyrazam are meager or lacking. In the present study, a single residual analytical method was developed for isopyrazam in selected commodities. Isopyrazam was analyzed in brown rice, soybean, green pepper, mandarin, cucumber, and Korean melon. We tried different solvents and methods through extraction, partition and purification steps to obtain best analytical results. For isopyrazam samples were extracted with acetonitrile, concentrated and partitioned with n-hexane, clean-up using florisil with n-hexane/ethylacetate (70/30) and analyzed with HPLC/UVD. The limit of quantitation (LOQ) for isopyrazam was 1.0 ng (S/N > 10) and method LOQ (MLOQ) was 0.04 mg $kg^{-1}$. Recovery ranged through 81.0~105.3% (syn-isomer) and 80.8~105.6% (anti-isomer) at fortification level of 0.04 (MLOQ), 0.4 (10 ${\times}$ MLOQ), and 2.0 (50 ${\times}$ MLOQ). The coefficient of variation (CV) for isopyrazam was less than 10% regardless of sample types. These results were further confirmed with LC/MS, respectively. The proposed method is highly reproducible and sensitive and is suitable for routine analysis.

Synthesis of Chloroacetamide Compounds and their Herbicidal Activities (Chloroacetamide형(型) 화합물(化合物)의 합성(合成)과 제초활성(除草活性))

  • Hong, Moo-Ki;Jeong, Young-Ho;Oh, Se-Mun
    • Applied Biological Chemistry
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    • v.31 no.3
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    • pp.234-240
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    • 1988
  • Some chloroacetamide derivatives were synthesized from 2,6-dialkyl, aniline 4-chloroaniline, or 3,4-dichloroaniline with alkyl 2-bromopropionate and chloroacetyl chloride and identified by elemental analyses, NMR, and GC/MS spectra as N-(1'-methoxycarbonylethyl)-N-chloroacetyl-2,6-dimethylaniline(ACRI-S-8609), etc. These compounds synthesized were subjected to the test for pre-emergence herbicidal effecs on some grass weeds(Digitaria adscendens, Setaria viridis, Echinochloa crus-galli) and broad leaf weeds(Portulaca oleracea, Amaranthus lividus, Chenopodium album) in pots applied as wettable powder formulations. It was found that N-(1'-ethoxycarbonylethyl)-N-chloroacetyl-2,6-dimethylaniline(ACRI-S-8701) has the highest herbicidal effect on grass weeds, which corresponds to a 95% control effect at an application of 200g a.i/10a. Whereas, some chloroacetamide derivatives derived from 4-chloroaniline or 3,4-dichloroaniline had very weak herbicidal effects on grass and broad leaf weeds.

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Development of analytical method for determination of spinetoram residues in livestock using LC-MS/MS (LC-MS/MS를 이용한 축산물 중 Spinetoram 공정시험법 개발 및 검증)

  • Ko, Ah-Young;Kim, Heejung;Do, Jung Ah;Jang, Jin;Lee, Eun Hyang;Ju, Yun Ji;Kim, Ji Young;Chang, Moon-Ik;Rhee, Gyu-Seek
    • Analytical Science and Technology
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    • v.29 no.2
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    • pp.94-103
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    • 2016
  • An analytical method was developed to determine the amount of spinetoram (spinetoram J and spinetoram L) in livestock samples. The spinetoram was extracted with acetonitrile and purified through a primary secondary amine (PSA) sorbent. The spinetoram residues were then quantified and confirmed using a liquid chromatography–tandem mass spectrometer (LC-MS/MS) in the positive ion mode using multiple reactions monitoring (MRM). Matrix-matched calibration curves were linear over the calibration ranges (0.005-0.5 mg/kg) into a blank extract with r2 > 0.994. The limits of detection and quantification were 0.002 and 0.01 mg/kg, respectively. The recovery results of spinetram ranged between 81.9-106.4% at different concentration levels (LOQ, 10LOQ, 50LOQ, n=5) with relative standard deviations (RSDs) less than 10%. All values were consistent with the criteria ranges requested in the Codex guidelines (CAC/GL40, 2003). An interlaboratory study was conducted to validate the method. The proposed analytical method proved to be accurate, effective, and sensitive for spinetoram determination. The method will be used as an official analytical method in Korea.

Development of Simultaneous Analytical Method for Determination of Isoxaflutole and its Metabolite (Diketonitrile) residues in Agricultural Commodities Using LC-MS/MS (LC-MS/MS를 이용한 농산물 중 Isoxaflutole과 대사산물(Diketonitrile)의 동시시험법 개발)

  • Ko, Ah-Young;Kim, Heejung;Do, Jung Ah;Jang, Jin;Lee, Eun-Hyang;Ju, Yunji;Kim, Ji Young;Chang, Moon-Ik;Rhee, Gyu-Seek
    • The Korean Journal of Pesticide Science
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    • v.20 no.2
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    • pp.93-103
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    • 2016
  • A simultaneous analytical method was developed for the determination of isoxaflutole and metabolite (diketonitrile) in agricultural commodities. Samples were extracted with 0.1% acetic acid in water/acetonitrile (2/8, v/v) and partitioned with dichloromethane to remove the interference obtained from sample extracts, adjusting pH to 2 by 1 N hydrochloric acid. The analytes were quantified and confirmed via liquid chromatograph-tandem mass spectrometer (LC-MS/MS) in positive-ion mode using multiple reaction monitoring (MRM). Matrix matched calibration curves were linear over the calibration ranges ($0.02-2.0{\mu}g/mL$) for all the analytes into blank extract with $r^2$ > 0.997. For validation purposes, recovery studies were carried out at three different concentration levels (LOQ, 10LOQ, and 50LOQ) performing five replicates at each level. The recoveries were ranged between 72.9 to 107.3%, with relative standard deviations (RSDs) less than 10% for all analytes. All values were consistent with the criteria ranges requested in the Codex guideline (CAC/GL40, 2003). Furthermore, inter-laboratory study was conducted to validate the method. The proposed analytical method was accurate, effective, and sensitive for isoxaflutole and diketonitrile determination in agricultural commodities.

A New coumarin from the seeds of Jute (Corchorus olitorius L.)

  • Mukherjee, K.K.;Mitra, S.K.;Ganguly, S.N.
    • Natural Product Sciences
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    • v.4 no.1
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    • pp.51-52
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    • 1998
  • From the chloroform extract of the defatted seeds of jute a new coumarin $C_9H_6O_4$, $m.p.\;178-179^{\circ}C$, was isolated. The structure of the compound was established as 4,7-dihydroxy coumarin on the basis of physical methods viz. $^1H\;NMR,\;^{13}C\;NMR$ and Ms.

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The Aroma Components of Commercial Green Tea Picked in August (수확시기가 늦은 시판녹차의 향기성분)

  • Choi, Sung-Hee
    • Journal of Life Science
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    • v.5 no.2
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    • pp.20-20
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    • 1995
  • The aroma components of commercial green teas picked inAugust were collected and identified. The extract of aroma compounds in green tea were accoimplished by a modified rotary evaporation. The concentrated wxtracts were analyzed and identified by GC and GC-MS. In GC analysis, T$_{R}$ value of GC represented bt KI value which standardized. The most abundant components of green teas picked in August were 1-Penten-3-ol, trans, trans-2, 4-heptadienal, linalool, $\beta$-ionone and nerolidol.