• BMB Reports
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• v.29 no.3
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• pp.253-260
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• 1996

An enzyme that hydrolyzes flavin-adenine dinucleotide (FAD) was found to be present in rat liver lysosomal membrane prepared from Triton WR-1339 filled lysosomes (tritosomes) purified by flotation on sucrose. This FAD phosphohydrolase (FADase) exhibited optimal activity at pH 8.5 and had an apparent Km of approximately 3.3 mM. The activity was decreased 50~70% by dialysis against EDTA and this was restored by $Zn^{2+}$, $Mg^{+2}$, $Hg^{+2}$, and $Ca^{+2}$ ions inhibited the enzyme, but $F^-$ and molybdate had no effect. The enzyme was also inhibited by p-chloromercuribenzoate (pCMB), reduced glutathione and other thiols, cyanide, and ascorbate. The presence of ATP, ADP, AMP. ${\alpha}-{\beta}-methylene$ ATP, AMP-p-nitrophenyl phosphate (PNP), GMP, and coenzyme A (CoA) decreased the activity on FAD, but pyrimidine nucleotides, adenosine, adenine, or $NAD^+$ were without effect. Phosphate stimulated the activity slightly. FAD phosphohydrolase activity was separated from ATPase and inorganic pyrophosphatase activities by solubilization with detergents and polyacrylamide gel electrophoresis and by linear sucrose density gradient centrifugation suggesting that the enzyme is different from ATPase, inorganic pyrophosphatase, and soluble lysosomal FAD pyrophosphatase. Paper chromatography showed that FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP which were further hydrolyzed to riboflavin and AMP by phosphatases known to be present in lysosomal membranes. Incubation of the intact Iysosomes with pronase showed that the active site of FAD phosphohydrolase must be oriented to the cytosol. The FAD hydrolyzing activity was detected in Golgi, microsome, and plasma membrane, but not in mitochondria or soluble lysosomal preparations.

• Journal of Pharmacopuncture
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• v.16 no.2
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• pp.46-54
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• 2013

Objective: This study was undertaken to isolate a fibrin(ogen)olytic enzyme from the snake venom of Gloydius blomhoffii siniticus and to investigate the enzymatic characteristics and hemorrhagic activity of the isolated enzyme 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 determined by using fibrin plate assay, protein hydrolysis analysis, and hemorrhage assay. Its amino acid composition was determined. Results: The fibrin(ogen)olytic enzyme with the molecular weight of 27 kDa (FE-27kDa) isolated from G. b. siniticus venom consisted of two heterogenous disulfide bond-linked polypeptides with the molecular weights of 15 kDa and 18 kDa. When more than $20{\mu}g$ of FE-27kDa was applied on the fibrin plate, fibrinolysis zone was formed as indicating its fibrinolytic activity. The fibrinolytic activity was inhibited completely by phenylmethanesulfonylfluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA) and partially by thiothreitol and cysteine. Metal ions such as $Hg^{2+}$ and $Fe^{2+}$ inhibited the fibrinolytic activity completely, but $Mn^{2+}$ did not. FE-27kDa preferentially hydrolyzed ${\alpha}$-chain of fibrinogen and slowly hydrolyzed ${\beta}$-chain, but did not hydrolyze ${\gamma}$-chain. High-molecular-weight polypeptides of gelatin were hydrolyzed partially into polypeptides with molecular weights of more than 45 kDa. A dosage of more than $10{\mu}g$ of FE-27kDa per mouse was required to induce hemorrhage beneath the skin. Conclusion: FE-27kDa was a serine proteinase consisting of two heterogeneous polypeptides, hydrolyzed fibrin, fibrinogen, and gelatin, and caused hemorrhage beneath the skin of mouse. This study suggests that the potential of FE-27kDa as pharmacopuncture agent should be limited due to low fibrinolytic activity and a possible side effect of hemorrhage.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.97-104
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• 2004

Water-sludge bacteria were screened to find a lipase enantioselectively hydrolyzing itraconazole precursor, which is well known as the starting material of antifungal drug agents. A bacterial strain was isolated and identified as Acinetobacter junii SY-01. After the strain was cultivated, the enzyme was purified 39.4-fold using ultrafiltration and gel filtration through a Sephadex G-100 chromatographic column and the activity yield was 34.9%. The molecular weight of the enzyme was about 40 kDa, as measured by SDS-PAGE, and the optimum pH was 7.0- 9.0 and stable at pH 6.0- 9.0. The optimum temperature was 45- $5^{\circ}C$, and 73% of the enzymes activity remained after incubation at 70% for 1 h. Enzyme activity was enhanced by gall powder, sodium deoxycholate, a cationic detergent Tween 80, and a non-ionic detergent Triton X-100, but was markedly inhibited by metal ions such as $Hg^{2＋},Cu^{2＋},Ni^{2＋}/,Ca^{2＋}$, and an anionic-surfactant sodium dodecylsulfate. The $K_{m}$ values for (R)- and (S)-enantiomers of the itraconazole precursor were 0.385 and 21.83 mM, respectively, and the $V_{max} values ($\mu$Mㆍmin^{-1}.)$ were 6.73 and 6.49, respectively. The acetyl group among the different acyl moieties of itraconazole precursor showed the highest enantioselectivity for the hydrolysis by the Acinetobacter junii SY-01 lipase, and the lipase from Acinetobacter junii SY-01 displayed better enantioselectivity than that of commercially available lipases and esterases.

• Microbiology and Biotechnology Letters
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• v.4 no.4
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• pp.131-137
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• 1976

Aspergillus niger S-1 was proved to be a good hesperidinase producer which have been selected for naringinase utilization. Enzyme of this strain had good characteristics and purified relative high degree with good recovery by ammonium sulfate or aceton treatment. Results obtained were summarized as follows (1) The enzyme was most active at 60$^{\circ}C$, when the reaction was performed in the pH 4.0 for 30min. Optimum pH for enzyme activity was 5.0 and activity was retained 78％ at pH value 3.5. (2) Hesperidinase activity retained 95％ of its full activity after treatment at 60$^{\circ}C$ for 30min at pH value 4.0., 70％ at 70$^{\circ}C$ and 65％ at 80$^{\circ}C$. Most stable pH of this enzyme was showed 5.0 after treatment for 24hr at 4$^{\circ}C$ (3) Only Magnesium ion activated enzyme reaction, while other metallic ions, Cu$\^$＋＋/, Mn$\^$＋＋/, Pb$\^$＋＋/, Mo$\^$＋＋/, Ag$\^$＋＋/, Hg$\^$＋＋/ inhibited. (4) Eleven fold purification with 35％ recovery was obtained in the case of 60％ aceton treatment and 10-fold purification with 5.6％ recovery was showed with 40％ aceton comparing to the crude extract Enzyme. (5) Crude enzyme precipitated with 0.4-0.6 saturated ammonium sulfate contained 13f6 of the original enzyme activity with 48-fold increase in specific activity and enzyme has been purified 25 fold with a yield 19％ by 0.6-5.8 saturation. (6) Hesperidinase formation was noticeably increased by addition of small amount of orange-peel extraction on the wheat bran medium.

• Korean Journal of Microbiology
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• v.32 no.1
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• pp.84-90
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• 1994

Kinetic parameters of various substrates and inhibitors were measured to elucidate the binding requirements of the active site of intracellular adenosine deaminase (ADA) in Aspergillus oryzae. 3'-Deoxyadenosine was the best substrate according to the value of relative kcat/$K_m$. Purine riboside was found to be the strongest inhibitor with the $K_i$ value of $3.7{\times}10^{-5}$M. Adenine acted neither as a substrate nor as an inhibitor, suggesting the presence of ribose at N-9 of adenosine was crucial to binding. ADA also catalyzed the dechlorination of 6-chloropurine riboside, generating inosine and chloride ions. Substrate specificity of 6-chloropurine riboside was 0.86% of adenosine. Purine riboside, a competitive inhibitor of ADA, inhibit the dechlorination with similar $K_i$ value, suggesting that the same binding site was involved in deamination and dechlorination. Among the sulfhydryl group reagents, mercurials, pchloromercuribenzoate (PCMB), mersalyl acid and $HgCl_2$ inactivated the enzyme. Mersalyl acid-inactivated ADA was reactivated by thiol reagents, but PCMB-inactivated enzyme was not. When ADA was treated with the mercurial reagents, the inhibition constants and inhibition patterns were determined. Each inhibition was competitive with substrate. The $K_i$ values of these mercurial reagents were lower in 10 mM phosphate buffer than in 100 mM phosphate buffer, showing phosphate dependency.

• Clean Technology
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• v.28 no.4
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• pp.323-330
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• 2022

Microfluidic paper-based analytical devices (μPADs) have recently been in the spotlight for their applicability in point-of-care diagnostics and environmental material detection. This study presents a double-sided printing method for fabricating 3D-μPADs, providing simple and cost effective metal ion detection. The design of the 3D-μPAD was made into an acryl stamp by laser cutting and then coating it with a thin layer of PDMS using the spin-coating method. This fabricated stamp was used to form the 3D structure of the hydrophobic barrier through a double-sided contact printing method. The fabrication of the 3D hydrophobic barrier within a single sheet was optimized by controlling the spin-coating rate, reagent ratio and contacting time. The optimal conditions were found by analyzing the area change of the PDMS hydrophobic barrier and hydrophilic channel using ink with chromatography paper. Using the fabricated 3D-μPAD under optimized conditions, Ni²⁺, Cu²⁺, Hg²⁺, and pH were detected at different concentrations and displayed with color intensity in grayscale for quantitative analysis using ImageJ. This study demonstrated that a 3D-μPAD biosensor can be applied to detect metal ions without special analysis equipment. This 3D-μPAD provides a highly portable and rapid on-site monitoring platform for detecting multiple heavy metal ions with extremely high repeatability, which is useful for resource-limited areas and developing countries.

• Bulletin of the Korean Chemical Society
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• v.34 no.12
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• pp.3681-3689
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• 2013

This study explored a direct SET-photochemical strategy to construct a new family of thioene conjugated-naphthalamide fluorophore based lariat-crown ethers which show strong binding properties towards heavy metal ions. Irradiations of designed nitrogen branched (trimethylsilyl)methylthio-terminated polyethylenoxy-tethered naphthalimides in acidic methanol solutions have led to highly efficient photocyclization reactions to generate naphthalamide based lariat type thiadiazacrown ethers directly in chemo- and regio-selective manners which undergo very facile secondary dehydration reactions during separation processes to produce their corresponding amidoenethio ether cyclic products tethered with electron donating diethyleneoxy- and diethyenethio-side arm chains. Fluorescence and metal cation binding properties of the lariat type enamidothio products were examined. The photocyclized amidoenethio products, thioene conjugated naphthalamide fluorophore containing lariat-thiadiazacrowns exhibited strong fluorescence emissions in region of 330-450 nm along with intramolecular exciplex emissions in region of 450-560 nm with their maxima at 508 nm. Divalent cation $Hg^{2+}$ and $Pb^{2+}$ showed strong binding to sulfur atom(s) in side arm chain and atoms in enethiadiazacrown ether rings which led to significant enhancement of fluorescence from its chromophore singlet excited state and concomitant quenching of exciplex emission. The dual fluorescence emission responses towards divalent cations might provide a new guide for design and development of fluorescence sensors for detecting those metals.

• Korean Journal of Microbiology
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• v.40 no.4
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• pp.275-281
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• 2004

Our previous research has demonstrated that the bacterium, Pseudomonas sp. NFQ-l capable of utilizing quin­oline (2,3-benzopyridine) as the sole source of carbon, nitrogen, and energy was isolated and characterized [Yoon et ai. (2003) Kor. J. Biotechnol. Bioeng. 18(3):174-179]. In this study, we have found that Pseudomonas sp. NFQ-l could degrade quinoline as well as benzoate, and extended this work to characterize the catechol 1,2­dioxygenase (C1,2O) purified from the bacterium cultured in benzoate media. Initially, C1,2O has been purified by ammonium sulfate precipitation, gel permeation chromatography, and Source 15Q. After Source 15Q, puri­fication fold was increased to approximately 14.21 unit/mg. Molecular weight of C1,2O was about 33 kDa. Physicochemical characteristics (e.g., substrate specificity, Km, Vmax, pH, temperature and effect of inhibitors) of purified C1,2O were examined. C1,2O demonstrated the activity for catechol, 4-methylcatechol and 3-meth­ylcatechol as a substrate, respectively. The Km and Vmax value of C1,2O for catechol was 38.54 ${\mu}M$ and $25.10\;{\mu}mol{\cdot}min^{-1}{\cdot}mg^{-1}.$ The optimal temperature of C1,2O was $30^{\circ}C$ and the optimal pH was approximately 8.5. Metal ions such as $Ag^+,\;Hg^+,\;Ca^{2+},\;and\;Cu^{2+}$ show the inhibitory effect on the activity of C1,2O. N-terminal amino sequence of C1,2O was analyzed as ^1TVKISQSASIQKFFEEA^{17}.$ In this work, we found that the amino acid sequence of NFQ-l showed the sequence homology of 82, 71, 59 and $53\%$ compared with C1,2O from Pseudomonas aeruginosa PA0l, Pseudomonas arvilla C-1., P. putida KT2440 and Pseudomonas sp. CA10, respectively.

• Journal of the Korean Chemical Society
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• v.35 no.2
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• pp.119-127
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• 1991

The protonation and the metal ion complexation of 15 to 18 membered diaza crown ether such as 1,12-diaza-3, 4 : 9, 10-dibenzo-5, 8-dioxacyclopentadecane(NtnOenH$_4$), 1,13-diaza-3,4 : 10,11-dibenzo-hydroxy-5,9-dioxacyclohexadecane(NtnOtnH$_4$), 1,13-diaza-3,4 : 10,11-dibenzo-15-hydroxy-5,9-dioxacyclohexadecane(Ntn(OH)OtnH$_4$), 1,15-diaza-3,4 : 12,13-dibenzo-5,8,11-trioxacycloheptadecane (NenOdienH$_4$) and 1,15-diaza-3,4 : 12,13-dibenzo-5,8,11-trioxacyclooctadecane(NtnOdienH$_4$) were studlied by potentiometry and NMR spectroscopy. The protonation constants were used to predict basicity of crown ethers. The sequence of the basicity was NenOdienH$_4$ < Ntn(OH)OtnH$_4$ < NtnOenH$_4$ < NtnOtnH$_4$ < NtnOdienH$_4$. Changes on the basicity were explained in terms of the effects of substituents and the degree of twistness of the macrocyclic ring. The sequence of the complex stabilities were Co(II) < Ni(II) < Cu(II) < Zn(II) for the transition metal complexes and Cd(II) < pb(II) < Hg(II) for the post-transition metal complexes. These changes on the stabilities were dependent on the basicity of the ligand and cavity size of the ring. For the heavy post-transiton metal complexes and Zn(Ⅱ) complex, the former factor was predominent and for the other transition metal complexes, the latter was affected on the stabilities. $^1$H and $^{13}$C-NMR studies for heavy post-transition metal complexes indicated that the nitrogen atom has greater affinity on metal ions than oxygen atom and the planarity of the rings was losed by the complexation with metal ions.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.327-333
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• 2005

Dihydroxy-acid dehydratase (DHAD, 2,3-dihydroxy-acid hydrolyase, EC 4.2.1.9) is one of the key enzymes involved in the biosynthetic pathway of the branched chain amino acid isoleucine and valine. Although the enzyme have been purified and characterized in various mesophiles including bacteria and eukarya, the biochemical properties of DHAD has bee not yet reported from hyperthermophilic archaea. In this study, we cloned, expressed, and purified a DHAD homologue from the thermoacidophilic archaeon Sulfolobus solfataricus P2, which grows optimally at $80\;^{\circ}C$ and pH 3, in E. coli. Characterization of the recombinant S. solfataricus DHAD (rSso_DHAD) revealed that it is the dimeric protein with a subunit molecular weight of 64,000 Da in native structure. rDHAD showed the highest activity toward 2,3-dihydroxyisovaleric acid among 17 aldonic acid substrates Interestingly, this enzyme also displayed 50 % activities toward some pentonic acids and hexonic acids when compared with the activity of this enzyme to the natural substrate. Moreover, rSso_DHAD indicated relatively higher activity toward D-gluconate than any other hexonic acids tested in substrates. $K_m$ and $V_{max}$ values of rSso_DHAD were calculated as $0.54\;{\pm}\;0.04\;mM$ toward 2,3dihydroxyisovalerate and $2.42\;{\pm}\;0.19\;mM$ toward D-gluconate, and as $21.6\;{\pm}\;0.4\;U/mg$ toward 2,3-dihydroxyisovalerate and $13.8\;{\pm}\;0.4\;U/mg$ toward D-gluconate, respectively. In the study for biochemical properties, the enzyme shows maximal activity between $70^{\circ}C$ and $80^{\circ}C$, and the pH range of pH 7.5 to 8.5. The half life time at $80^{\circ}C$ was 30 min. A divalent metal ion, $Mn^{2+}$, was only powerful activators, whereas other metal ions made the enzyme activity reduced. $Hg^{2+}$, organic mercury, and EDTA also strongly inhibited enzyme activities. Particularly, the rSso_DHAD activity was very stable under aerobic condition although the counterparts reported from mesophiles had been deactivated by oxygen.