• Title/Summary/Keyword: carboxylic acids

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Evaluation of Luminescent P450 Analysis for Directed Evolution of Human CYP4A11

  • Choi, Seunghye;Han, Songhee;Lee, Hwayoun;Chun, Young-Jin;Kim, Donghak
    • Biomolecules & Therapeutics
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    • v.21 no.6
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    • pp.487-492
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    • 2013
  • Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency ($k_{cat}/K_m$) for lauric acid hydroxylation mainly due to an increase in $K_m$. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in $k_{cat}$ and an increase in Km. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

Organic Compounds in the Nak Dong River and Its Toxicity (낙동강 수질중 유기물질과 독성)

  • 류병호;심종환;최진택;조현철;정종순
    • Journal of Environmental Health Sciences
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    • v.20 no.1
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    • pp.39-53
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    • 1994
  • This study aims to investigate organic compounds and its toxicity by Ames test and chromosomal aberration in the water of the Nak Dong River. Six sampling sites such as Goryung, Hagueun, Maelie, Duksan, Haedong and Myungiang were selected for these pur15oses. 200 l water samples were absorbed on XAD-2 resin columns (2.5X30cm), eluted with organic solvents mixture of acetone: cyclohexane and then dried under vacuum condition. The extracts from the XAD-2 resin was injected into GC/MS and 184 organic compounds were identified such as aldehydes, aromatic compounds, ketones, phenols, hydrocarbons, alcohols, carboxylic acids, alkanes and some unknowns. The US EPA priority pollutants such as naphthlene, bis(2-ethylhexyl)phthalate and other pollutants, 1,2-diethyl benzene, 1,2,3,4-tetrahydronaphthalene and cyclohexanol were detected in these samples. The concentration of chemical pollutants such as 1,2-diethyl benzene, nephthalene, 1,2,3,4-tetrahydronaphthalene, bis(2-ethylhexyl)phthalate and cyclohexanol were ranged into 1.228 $\mu$g/l, 298 $\mu$g/l, 30.191 $\mu$g/l, 1.147 $\mu$g/l and 2.839 $\mu$g/l, respectively. The mutagenic activity of XAD-2 extracts were tested on Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and then exhibited strong mutagenic activity against S. typhimurium TA 98 and TA 100 in the presence of S$_9$. Amon them, bis(2-ethylhexyl)phtalate and 1,2-diethyl benzene showed the most strongest mutagenic activity against S. typhimurium TA 98 and TA 100 in the presence of S$_9$. On the other hands, chromosomal aberration of XAD-2 extracts in the human blood cells were not occurred by the sampling water at Goryung, Hagueun, Maelie and Duksan, Chromosomal aberration were also not occurred by the each concentration of 0.05, 0.1 amd 0.3 mg/l of each 1,2-diethyl benzol, bis(2-ethylhexyl)phthalate, naphthalene, phenol, cyclohexanol and benzothiazol test solution.

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Identification of Essential Histidines in Cyclodextrin Glycosyltransferase Isoform 1 from Paenibacillus sp. A11

  • Kaulpiboon, Jarunee;Pongsawasdi, Piamsook
    • BMB Reports
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    • v.36 no.4
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    • pp.409-416
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    • 2003
  • The isoform 1 of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from Paenibacillus sp. A11 was purified by a preparative gel electrophoresis. The importance of histidine, tryptophan, tyrosine, and carboxylic amino acids for isoform 1 activity is suggested by the modification of the isoform 1 with various group-specific reagents. Activity loss, when incubated with diethylpyrocarbonate (DEP), a histidine modifying reagent, could be protected by adding 25 mM methyl-$\beta$-cyclodextrin substrate prior to the modification. Inactivation kinetics of isoform 1 with DEP resulted in second-order rate constants ($k_{inactivation}$) of $29.5\;M^{-1}s^{-1}$. The specificity of the DEP-modified reaction for the histidine residue was shown by the correlation between the loss of isoform activity and the increase in the absorbance at 246 nm of N-carbethoxyhistidine. The number of histidines that were modified by DEP in the absence and presence of a protective substrate was estimated from the increase in the absorbance using a specific extinction coefficient of N-carbethoxyhistidine of $3,200\;M^{-1}cm^{-1}$. It was discovered that methyl-$\beta$-CD protected per mole of isoform 1, two histidine residues from the modification by DEP. To localize essential histidines, the native, the DEP-modified, and the protected forms of isoform 1 were digested by trypsin. The resulting peptides were separated by HPLC. The peptides of interest were those with $R_t$ 11.34 and 40.93 min. The molecular masses of the two peptides were 5,732 and 2,540 daltons, respectively. When the data from the peptide analysis were checked with the sequence of CGTase, then His-140 and His-327 were identified as essential histidines in the active site of isoform 1.

Effects of Green Tea (Camellia sinensis) Waste Silage and Polyethylene Glycol on Ruminal Fermentation and Blood Components in Cattle

  • Nishida, T.;Eruden, B.;Hosoda, K.;Matsuyama, H.;Nakagawa, K.;Miyazawa, T.;Shioya, S.
    • Asian-Australasian Journal of Animal Sciences
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    • v.19 no.12
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    • pp.1728-1736
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    • 2006
  • The effects of green tea (Camellia sinensis) waste silage and supplemental polyethylene glycol (PEG) on rumen fermentation and blood components were studied in cattle. Six Holstein steers were fed three diets in a 3${\times}$3 Latin square design, replicated twice. One diet was a control with no added silage, and the other two diets were supplemented (20% of the dry matter) with green tea waste silage either with (PEG) or without PEG (tea). Most of the fermentation parameters including major volatile fatty acids (VFA) were not affected by the diet treatments. The concentrations of high density lipoprotein cholesterol in the PEG group and urea nitrogen in the tea and PEG groups were greater than those in the control before morning feeding. The plasma 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid equivalent concentration was not different before morning feeding, but 3 h after morning feeding, its concentrations in both the tea and PEG groups were higher than in the control. Although the concentration of plasma vitamin A in the animals was not affected by feeding green tea waste silage, the concentrations of plasma vitamin E were significantly higher in the tea and PEG groups than in the control, both before and 3 h after morning feeding. The results from the present study suggest that feeding diets containing 20% of the dietary dry matter as green tea waste silage to Holstein steers has no negative impact on their ruminal fermentation, and increases their plasma antioxidative activity and concentration of vitamin E.

Can Non-aqueous Solvent Desalinate?: Suggestion of the Screening Protocol for Selection of Potential Solvents (비수용성 용매를 이용한 탈염화 가능한가?: 적용 가능한 용매선정 기법 제안)

  • Choi, Oh Kyung;Seo, Jun Ho;Kim, Gyeong Soo;Kim, Dooil;Lee, Jae Woo
    • Journal of Korean Society on Water Environment
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    • v.36 no.1
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    • pp.48-54
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    • 2020
  • This paper presents a screening protocol for the selection of solvents available for the solvent extraction desalination process. The desalination solvents hypothetically and theoretically require the capability of (1) Forming hydrogen bonds with water, (2) Absorbing some water molecules into its non-polar solvent layer, (3) Changing solubility for water-solvent separation, and (4) Rejecting salt ions during absorption. Similar to carboxylic acids, amine solvents are solvent chemicals applicable for desalination. The key parameter for selecting the potential solvent was the octanol-water partitioning coefficient (Kow) of which preferable value for desalination was in the range of 1-3. Six of the 30 amine solvents can absorb water and have a variable, i.e., temperature swing solubility with water molecule for water-solvent separation. Also, the hydrogen bonding interaction between solvent and water must be stronger than the ion-dipole interaction between water and salt, which means that the salt ions must be broken from the water and only water molecules absorbed for the desalination. In the final step, three solvents were selected as desalination solvents to remove salt ions and recover water. The water recovery of these three solvents were 15.4 %, 2.8 %, 10.5 %, and salt rejection were 76 %, 98 %, 95 %, respectively. This study suggests a new screening protocol comprising the theoretical and experimental approaches for the selection of solvents for the desalination method which is a new and challenges the desalination process in the future.

Efficient Oxidative Scission of Alkenes or Alkynes with Heterogeneous Ruthenium Zirconia Catalyst (루테늄 지르코니아 불균일 촉매를 이용한 알켄 또는 알킨의 효과적인 산화절단반응)

  • Irshad, Mobina;Choi, Bong Gill;Kang, Onyu;Hong, Seok Bok;Hwang, Sung Yeon;Heo, Young Min;Kim, Jung Won
    • Applied Chemistry for Engineering
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    • v.27 no.6
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    • pp.659-663
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    • 2016
  • The efficiency of a heterogeneous ruthenium zirconia catalyst ($Ru(OH)_x/ZrO_2$) was demonstrated to the selective oxidative transformation of alkenes or alkynes. The scissions of C-C double bonds to aldehydes and triple bonds to diketones or carboxylic acids were carried out with (diacetoxyiodo)benzene as an oxidant under dichloromethane (5 mL)/water (0.5 mL) solvent system at $30^{\circ}C$ for wide range of substrates. The $Ru(OH)_x/ZrO_2$composite showed higher catalytic activity and selectivity than other ruthenium-based homogeneous or heterogeneous catalysts for the scission reaction. The catalyst exhibited a high mechanical stability, and no leaching of the metal was observed during the reaction. These features ensured the reusability of the catalyst for several times for the oxidative cleavage of unsaturated hydrocarbons.

Studies on the Synthesis of Pterdine Substituted Pyridonecarboxylic Acids as Potential Antibacterial Agents and their Antimicrobial Activities (항균제로서 Pteridine이 치환된 Pyridonecarboxylic Acids의 합성 및 항균 작용에 관한 연구)

  • Ryu, Seoung Ryuall;Choo, Dong Joon
    • Applied Chemistry for Engineering
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    • v.7 no.6
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    • pp.1096-1104
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    • 1996
  • In order to synthesize a new antibacterial and antitumor agents, we have prepared new analogues pteroic acid(13a, 13b), which means C-9 position of pteroic acid has been replaced by norfloxacin(8) or ciprofloxacin(9) and amino group of C-2 position by $CH_3$. These derivatives were synthesized coupling at N-4 piperazine of norfloxacin and ciprofloxacin with 2-amino-3-cyano-5-chloromethylpyrazine(20) provided 1-alkyl(ethyl, cyclopropyl)-6-fluoro-1,4-dihydro-4-oxo-7-[[4-N-(2-amino-3-cyanopyrazin-5-yl)methyl]piperazin-1-yl]-3-quinoline-carboxylic acid(12a, 12b). It was then cyclized with acetamidine. HCI to obtain new analogues of C-2 desaminomethylpteroic acid(13a, 13b) in yield of 76.2% and 82.8 % respectively. These compounds were tested in vitro on antibacterial activity against Gram-positive and Gram-negative bacteria including Pseudomonas aeruginosa ATCC9027. In general, these synthesized compounds(13a, 13b) showed less potent activities than those of norfloxacin.

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Hydrogen-Bonding Induced Alternating Thin Films of Dendrimer and Block Copolymer Micelle

  • Park, Chi-Young;Rhue, Mi-Kyo;Im, Min-Ju;Kim, Chul-Hee
    • Macromolecular Research
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    • v.15 no.7
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    • pp.688-692
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    • 2007
  • The hydrogen-bonding induced alternating multilayer thin films of dendrimers and block copolymer micelles were demonstrated. The block copolymer micelles derived from amphiphilic poly(2-ethyl-2-oxazoline)block-$poly({\varepsilon}-carprolactone)$ (PEtOz-PCL) in aqueous phase have a core-shell structure with a mean hydrodynamic diameter of 26 nm. The hydrogen bonding between the PEtOz outer shell of micelle and the carboxyl unit of poly(amidoamine) dendrimer of generation 4.5 (PAMAM-4.5G) at pH 3 was utilized as a driving force for the layerby-layer alternating deposition. The multilayer thin film was fabricated on the poly(methyl methacrylate) (PMMA) thin film spin-coated on silicon wafer or glass substrate by the alternate dipping of PEtOz-PCL micelles and PAMAM dendrimers in aqueous solution at pH 3. The formation of multilayer thin film was characterized by using ellipsometry, UV-vis spectroscopy, and atomic force microscopy. The PEtOz outer shell of PEtOz-PCL micelle provided the pH-responsive hydrogen bonding sites with peripheral carboxylic acids of PAM AM dendrimer. The multilayer thin film was reversibly removed after dipping in aqueous solution at $pH{\geq}5.6$ due to dissociation of the hydrogen bonding between PEtOz shell of PEtOz-PCL micelle and peripheral carboxyl units of PAMAM dendrimer.

Reaction of Organic Halogen Compounds with Metals (Part Ⅰ) A Formation of Organometallic Complex in Dimethyl Formamide Solvent (有機할로겐 化合物과 金屬과의 反應 (第1報) 디메칠호름아마이드 溶媒存在下에서의 有機金屬콤프렉스 生成에 關한 硏究)

  • Yon Sun Kim
    • Journal of the Korean Chemical Society
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    • v.7 no.3
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    • pp.216-224
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    • 1963
  • Reaction of organic chlorine containing ester, alcohol, and acids with metallic tin and zinc in dimethyl formamide solvent gave a good yield of organo metallic complex. The same reaction under a mixed U.V. irradiation could not give an appreciable yield of the complex except in the case of an elevated reaction temperature. The solvation effect of dimethyl formamide of the metallic complex formation was markedetly increased as compared to the reaction in toluene and cyclohexane. In case of chlorine containing carboxylic acid, the formation of organo chloro zinc complex of the salt was observed. The reaction of organo zinc complex with a carbonyl precursor gave the addition product together with a dimerized product. Especially the aldehyde species enhanced the formation of zinc complex. The addition reaction was simple and convenient, but the yield was not high.(30-40% for the acid, 73% for the ester, 14.6% for alcohol). The result was discussed on basis of solvent effect and the procedures were described.

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The Effect of Enhancers on the Penetration of Albuterol through Hairless Mouse Skin

  • Choi, Han-Gon;Rhee, Jong-Dal;Yu, Bong-Kyu;Kim, Jung-Ae;Kwak, Mi-Kyung;Woo, Jong-Soo;Oh, Dong-Hun;Han, Myo-Jung;Choi, Jun-Young;Piao, Mingguan;Yong, Chul-Soon
    • Journal of Pharmaceutical Investigation
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    • v.36 no.5
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    • pp.321-329
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    • 2006
  • Albuterol, a selective ${\beta}_2$-adrenergic receptor stimulant, has been introduced as a potent bronchodilator for patients with bronchial asthma, chronic obstructive bronchial disease, chronic bronchitis and pulmonary emphysema. The percutaneous permeation of albuterol sulfate was investigated in hairless mouse skin in vitro with and without pretreatment with enhancers. The enhancing effects of ethanol and various penetration enhancers such as terpenes, non-ionic surfactants, pyrrolidones, and fatty acids on the permeation of albuterol sulfate were evaluated using Franz diffusion cells. Among terpenes studied, 1,8-cineole was the most effective enhancer, which increased the permeability of albuterol sulfate approximately 33-fold compared with the control without enhancer pretrement, followed by d-limonene with enhancement ratio of 21.79. 2-Pyrrolidone-5-carboxylic acid increased the permeability of albuterol sulfate approximately 5.5-fold compared with the control. Other pyrrolidones tested showed only slight permeability enhancing effect with enhancement ratio less than 2.8. Nonionic surfactants showed moderate enhancing effects. Lauric acid increased the permeability of albuterol sulfate approximately 30-fold with decreasing the lag time from 2.85 to 0.64 hr. Oleic acid and linoleic acid showed enhancement ratio of 24.55 and 22.91, respectively. These findings would allow a more rational approach for designing formulations for the transdermal delivery of albuterol sulfate and similar drugs.