• 미생물학회지
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• 제36권1호
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• pp.26-32
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• 2000

Toluene, phenyl 등의 분해균주인 Burkholderia cepacia G4로부터 tomB 유전자를 클로닝하여 얻은 재조합 균주 E. coli CNU312로부터 catechol 2,3-dioxygenase를 정제하여 효소학적 특성을 조사하였다. Catechol 2,3-dioxygenase는 native 분자량이 약 140.4 kDa이었으며 4개의 동일한 35 kDa subunit로 구성된 homotetramer로 생각된다. Catechol의 $K_(m)$값과 $V_(max)$값은 372.6 $\mu$M과 39.27 U/mg이었으며, 1.56 mM 이상의 기질 농도에서는 활성이 감소되었다. 효소 활성의 최적 pH는 8.0이었으며, pH 7.0-8.0 범위에서 안정하였다. 최적 활성온도는 $40^{\circ}C$였으며, $60^{\circ}C$이상에서 완전히 활성을 상실하였다. 또한 $Fe^(2+)$, $Fe^(3+)$ 를 비롯한 대부분의 금속 이온에 의해 활성이 감소되었으며, $Mg^(2+)$, $K^(+)$에는 영향을 받지 않았다. 효소 활성부위를 알아보기 위해 화학변형제를 처리한 결과, tryptophan과 histidine이 효소 활성부위에 존재하는 것으로 추정된다. 그리고 10%의 유기용매에 안정성을 보이지 않았으며, $H_(2)$$O_(2)$, EDTA, ο-phenanthroline에도 활성이 감소되었다. 또한 2-mercaptoethanol, dithiothreitol, 그리고 ascorbic acid와 같은 환원제에 대해서도 안정성을 보이지 않았다. 이 효소는 catechol에 대해 높은 기질 특이성을 보였으며, 3-methylcatechol, 4-methylcatechol, 그리고 4-chlorocatechol에 대해 약간의 활성을 보였다. 그러나 2,3-dihydroxybiphenyl에 대해서는 거의 활성을 보이지 않았다.

• 대한약침학회지
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• 제17권1호
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• pp.44-50
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• 2014

Objectives: This study was undertaken to isolate a fibrinolytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate its enzymatic characteristics and hemorrhagic activity as a potential pharmacopuncture agent. Methods: The fibrinolytic enzyme was isolated by using chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fibrin plate assay. The characteristics of the enzyme were investigated using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrinolytic enzyme with the molecular weight of 32kDa (FE-32kDa) from Gloydius blomhoffii siniticus showed a fibrin hydrolysis zone at the concentration of 0.2 mg/mL in the fibrin plate assay. The fibrin hydrolysis activity of the enzyme was inhibited completely by ethylenediaminetetraacetic acid (EDTA), ethyleneglycoltetraacetic acid (EGTA), and 1, 10-phenanthroline, thiothreitol and cysteine, and partially by phenylmethanesulfonylfluoride (PMSF). Metal ions such as $Fe^{2+}$ and $Hg^{2+}$ inhibited the fibrin hydrolysis completely, but $Zn^{2+}$ enhanced it. FE-32kDa hydrolyzed ${\alpha}$-chain but did not hydrolyze ${\beta}$-chain and ${\gamma}$-chain of fibrinogen. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into low-molecular-weight polypeptides, but the extent of hydrolysis was limited. FE-32kDa induced hemorrhage beneath back skin of mice at the dose of $2{\mu}g$. Conclusions: FE-32kDa is a ${\alpha}$-fibrin(ogen)olytic metalloprotease that requires $Zn^{2+}$ for fibrinolytic activity and causes hemorrhage, suggesting that the enzyme is not appropriate for use as a clinical pharmacopuncture.

• 화훼연구
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• 제17권3호
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• pp.158-164
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• 2009

본 연구는 불과 80% 에탄올 용매가 마가렛트, 큰금계국, 저먼캐모마일 및 알프스민들레 꽃 추출물의 항산화효과에 미치는 영향을 탐색하기 위해서 수행하였다. 추출수율은 물 추출물에서 27.15~40.25%, 80% 에탄올 추출물에서 24.92~42.84%로 나타났다. 총 폴리페놀 및 총 플라보노이드의 함량은 4종 모두 80% 에탄올 추출물에서 많게 나타났으며, 큰금계국 꽃의 80% 에탄올 추출물에서 함량이 가장 많았다. 4종 꽃 추출물의 DPPH radical 소거능, ABTS radical 소거능 및 $Fe^{2+}$ chelating 효과는 모두 80% 에탄올 추출물에서 높았다. DPPH radical 소거능은 알프스민들레 꽃의 80% 에탄올 추출물에서 기장 높았으며, 합성 항산화제인 BHT 보다 소거활성이 높았다. ABTS radical 소거능은 저먼캐모마일 꽃의 80% 에탄올 추출물에서 가장 높았다. 국화과 꽃 추출물은 DPPH radical 보다는 ABTS radical 소거능이 우수하였으며, 저먼캐모마일과 큰금계국 꽃의 80% 에탄올 추출물의 ABTS radical 소거능은 ascorbic acid와 BHT 보다 우수하였다. $Fe^{2+}$ chelating 효과는 알프스민들레 꽃의 80% 에탄올 추출물에서 기장 우수하였다. EDTA와 chelating 효과를 비교한 결과, 4종의 꽃 추출물은 EDTA 보다 chelating 효과가 극히 낮았다. 본 연구의 결과, 마가렛트, 큰금계국, 저먼캐모마일, 알프스민들레 등 국화과 4종의 꽃을 이용하여 천연 항산화제를 개발할 때에는 80% 에탄올을 용매로 추출하는 것이 시료의 항산화 물질 추출 효율 및 항산화 활성을 증가시킬 수 있는 효율적인 방법으로 생각된다.

• The Korean Journal of Physiology and Pharmacology
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• 제3권2호
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• pp.147-155
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• 1999

Antioxidant effects of serotonin and L-DOPA on neuronal tissues were examined by studying the oxidative damages of brain synaptosomal components. The study further explored the mechanism by which they exert protective actions. Serotonin and L-DOPA (1 ${\mu}M$ to 1 mM) significantly inhibited lipid peroxidation of brain tissues by either $Fe^{2+}$ and ascorbate or t-butyl hydroperoxide in a dose dependent fashion. Protective effect of serotonin on the peroxidative actions of both systems was greater than that of L-DOPA. Protein oxidation of synaptosomes caused by $Fe^{2+}$ and ascorbate was attenuated by serotonin and L-DOPA. Protein oxidation more sensitively responded to L-DOPA rather than serotonin. Serotonin and L-DOPA (100 ${\mu}M$) decreased effectively the oxidation of synaptosomal sulfhydryl groups caused by $Fe^{2+}$ and ascorbate. The production of hydroxyl radical caused by either $Fe^{3+},$ EDTA, H_2O_2$ and ascorbate or xanthine and xanthine oxidase was significantly decreased by serotonin and L-DOPA (1 mM). Equal concentrations of serotonin and L-DOPA restored synaptosomal $Ca^{2+}$ uptake decreased by $Fe^{2+}$ and ascorbate, which is responsible for SOD and catalase. Protective effects of serotonin and L-DOPA on brain synaptosomes may be attributed to their removing action on reactive oxidants, hydroxyl radicals and probably iron-oxygen complex, without chelating action on iron.

• The Korean Journal of Physiology and Pharmacology
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• 제3권6호
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• pp.631-640
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• 1999

The present study investigated the stimulatory effects of iron (or ascorbate) on cyclosporine-induced kidney mitochondrial damage. Damaging effect of $50\;{\mu}M$ cyclosporine plus $20\;{\mu}M\;Fe^{2+}$ on mitochondrial lipids and proteins of rat kidney and hyaluronic acid was greater than the summation of oxidizing action of each compound alone, except sulfhydryl oxidation. Cyclosporine and $100\;{\mu}M$ ascorbate showed an enhanced damaging effect on lipids but not on proteins. The peroxidative action of cyclosporine on lipids was enhanced with increasing concentrations of $Fe^{2+}.$ Ferric ion $(20\;{\mu}M)$ also interacted with cyclosporine to stimulate lipid peroxidation. Damaging action of cyclosporine on mitochondrial lipids was enhanced by ascorbate $(100\;{\mu}M\;and\;1\;mM)$. Iron chelators, DTPA and EDTA, attenuated carbonyl formation induced by cyclosporine plus ascorbate. Cyclosporine $(100\;{\mu}M)$ and $50\;{\mu}M\;Fe^{2+}$ $(or\;100\;{\mu}M\;ascorbate)$ synergistically stimulated degradation of $2-{\alpha}$ deoxyribose. Cyclosporine $(1\;to\;100\;{\mu}M)$ reduced ferric ion in a dose dependent manner, which is much less than ascorbate action. Addition of $Fe^{2+}$ caused a change in absorbance spectrum of cyclosporine in $230{\sim}350$ nm of wavelengths. The results show that cyclosporine plus iron (or ascorbate) exerts an enhanced damaging effect on kidney mitochondria. Iron and ascorbate appear to promote the nephrotoxicity induced by cyclosporine.

• 한국미생물·생명공학회지
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• 제24권3호
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• pp.282-289
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• 1996

2,3-DHBP dioxygenase was purified from E. coli CK1092 carrying the pcbC gene, which was cloned from 4-chlorobiphenyl-degrading Pseudomonas sp. P20. Purification of this enzyme was done by acetone precipitation, DEAE- Sephadex A-25 ion exchange chromatography, and preparative gel electrophoresis. The molecular weight of subunit was 34 kDa determined by SDS-PAGE, and that of native enzyme was about 270 kDa. It suggests that this enzyme consist of eight identical subunits. This enzyme was specifically active against only 2,3-DHBP as a substrate with 18 $\mu$M of Km value, but not catechol, 3-methylcatechol, 4-methylcatechol and 4-chlorocatechol. The optimal pH and temperature of 2,3-DHBP dioxygenase were pH 8.0 and 40-60$\circ$C. The enzyme was inhibited by Cu$^{2+}$, Fe$^{2+}$ and Fe$^{3+}$ ions, and was inactivated by H$_{2}$0$_{2}$2 and EDTA. The lower concentrations of some organic solvents such as acetone and ethanol don't stabilize the activity of 2,3-DHBP dioxygenase. The enzyme was completely inactivated by adding the reagents such as N-bromosuccinimide, iodine and p- diazobenzene sulfonic acid.

• 미생물학회지
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• 제24권3호
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• pp.302-307
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• 1986

내성 P.aeruginosa에 의해 생성되는 항생물질 파괴효소인 cephalosporinase는 cephaloridine을 기질로 하여 효소활성도는 $45^{\circ}C$와 pH 8.5에서 각기 최고의 활성도를 나타내었다. SDS. carbenicillin 빛 cephalosporin계 항생체에 의해 효소의 활성도는 현저하게 억제되었으며 효소의 cephaloridine에 대한 $V_{max}$ 값을 100으로 하였올때 cefo p perazone의 $V_{max}$ 값은 2.8로 나타났다. 측정된 효소의 분자량은 $37.500{\pm}3,000$이었다. 다른 Gram음성셰균이 분비하는 cephalosporinase와 비교해본 결과 균체 자체의 특성에 의해 cepha]osporinase는 서로 다름을 알 수 있였 으며 이로 인하여 각각의 균체가 cephalosporin계 항생제에 대한 내성의 차이를 나타내는 하나의 중요한 원인으로 판단되었다.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1004-1008
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• 2004

An extracellular acid phytase from Pseudomonas fragi Y9451 was purified to homogeneity from the culture supernatant by salting-out, DEAE-Sepharose column chromatography, CM-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 74 kDa on gel filtration and 54 kDa and 25 kDa on SDS-PAGE, suggesting that the native enzyme was a heterodimeric protein. The purified enzyme was most active at pH 4.5 and $70^{\circ}C$ and fairly stable from pH 4.0- 6.0. It was specific for phytate and exhibited a $K_{m}$ value of 27 mM (sodium phytate, pH 4.5, $50^{\circ}C$). The phytase activity was strongly inhibited (at maximum by 87%) by $Fe^{3+},\;Cu^{2+},\;Fe^{2+}$, and $Zn^{2+}$ at 5 mM concentration, and greatly inhibited by $Ca^{2+}$ at 10 mM concentration. However, EDTA notably stimulated the phytase activity at 10 mM concentration. With optimum pH and stability, Pseudomonas fragi phytase could be a potential candidate for animal feed applications.

• 환경위생공학
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• 제14권4호
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• pp.21-28
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• 1999

This study was carried out the simultaneous removal or organics and Cr(VI) in aqueous suspensions of phtocatalyst under circular type reactor and UV light illumination. In this experiment, comparison on the removal of Cr(VI) by photoreduction using UV light, photocatalyst adsorption using TiO2, ZnO, and FeCl3 as photocatalyst, and phtocatalysis using UV light with photocatalysts as well as the effect of experimental parameters such as phtocatalyst dosage, a kinds of organics and their concentration was examined. The major results of this study were as follows; 1. It was found that photocatalyst adsorption and phtocatalysis were applicable to the removal of Cr(VI), and Cr(VI) was more effectively eliminated by TiO2 than ZnO, and FeCl3. 2. phtocatalytic removal efficiency of Cr (VI) increased with increasing phtocatalyst dosage. However, over 1.0g/l of phtocatalyst dosage, the efficiency reached a plateau. 3. phtocatalytic removal of Cr(VI) was enhanced by addition of organics such as salicylic acid, mandelic acid, EDTA, and citric acid, and phtocatalytic oxidation of organics were also observed. 4. It was found that the simultaneous removal of organics and Cr(VI) using phtocatalysis was possible.

• 대한화학회지
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• 제15권2호
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• pp.49-54
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• 1971

It is shown that the impurities of Cu(II), Pb(II), Zn(II) and Ag(I) in Bismuth metal and the components of Pb(II), Zn(II) and Sn(IV) in Bismuth alloy are separated into their components from each other by elutions through $3.14cm^2{\times}10cm$ cation exchange resin, $Dowex\;50w\;{\times}\;8$ (100~200 mesh), column with the mixed solutions of HAc and NaAc as the eluents. The elution curve of Fe(III) has a long tailing and is not separated quantitatively from Bi(III). The eluents used for this separation are as follows; 1M HAc + 0.1M NaAc (pH 3.36) for Fe(III) and Bi (III). 0.3M HAc + 0.3M NaAc (pH 4.70) for Cu(II), Pb(II) and Zn(II). 0.5M HAc + 0.5M NaAc (pH4.70) for Ag(I) and Sn(IV). The analysis of cations eluted are carried out by spectrophotometry and EDTA titrimetry. Their recoveries are more than 99%.