• Journal of Microbiology
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• v.33 no.3
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• pp.244-251
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• 1995

An extracellular protease of Salmonella schottmulleri was purified from culture filtrate by using 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, Ultrogel HA chromatography and Sephacryl S-200 HR molecular sieve chromatography. To measure enzyme activity, synthetic dipeptide substrate (CBZ-arg-arg-AFC) with low molecular weight was employed as substrate. The molecular weight of the purified enzyme was approximately 80 kDa when determined by gel filtration on Sephacryl S-200 HR and 73 kDa when estimated by SDS-PAGE. The isoelectric point was 5.45. The activity of the purified enzyme was inhibited by metal chelating agesnts such as EDTA and 1.10-phenanthroline. The divalent cations, such as Ca$\^$2+/, Zn$\^$2+/, Fe$\^$2+/, Mg$\^$2+/ enhanced its activity. These results suggested that it was a metalloprotease. It had a narrow pH optimum of 6.5-7.5 with a maximum at pH 7.0 and a temperature optimum of 40.deg.C. It was stable at least for 1 week at 40.deg.C and maintained its activity for 24 hours at 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium dodecyl sulfate (SDS) and was inactivated in a dose-dependent manner. However, it was resistant to Triton X-100 and the activity was enhanced to 32.3% with treatment of 0.025% Triton X-100.

• The Korean Journal of Pharmacology
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• v.32 no.3
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• pp.425-431
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• 1996

Human neutrophil elastases (HNElastase, EC 3.4.21.37), a causative factor of inflammatory diseases, are regulated by plasma proteinase inhibitors, alpha-proteinase inhibitor and ${\alpha}_2-macroglobulin$. Under certain pathological conditions, however, released enzymes or abnormal function of inhibitors may cause various inflammatory disease. NSAIDs have been clinically applied for treatment of inflammatory diseases. Inhibition of cyclooxygenase is a known mechanism of action of NSAIDs in the treatment of inflammatory disease. In in vitro experiments, HNElastase was inhibited by naproxen, phenylbutazone, and oxyphenbutazone, but ibuprofen, ketoprofen, aspirin, salicylic acid, and tolmetin did not inhibit elastase. HNElastase was also inhibited by chelating agents, EDTA & EGTA, and tetracyclines. Removal of divalent metal ions by EDTA caused inhibition of elastase, and reconstitution of the metal ions recovered the enzyme activity to a certain level. Frequencies and contours in the Raman spectra of various conditions of human neutrophil elastase undergo drastic changes upon partial removal and/or reconstitution of calcium and zinc ions. The metal ion content dependent activities and change of the contour of the Raman spectrogram suggest us that the mechanism of action of a chelator or chelator-like agents on neutrophil elastase may be related to the conformational change at/or near the active site, especially -C=O radical or -COOH radical.

• Microbiology and Biotechnology Letters
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• v.34 no.1
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• pp.28-34
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• 2006

A native extracellular acid phosphatase, phytase (EC 3.1.3.8), from Bacillus coagulans IDCC 1201 (commercially known as Lactobacillus sporogenes) used as probiotics, was characterized. Though some strains of B. coagulans have been evaluated with regard to several health-promoting effects, it has not been reported to produce phytase. Partially purified phytase front the strain IDCC 1201 had a pH optimum of 4.0 and a temperature optimum of $50^{\circ}C$, respectively. The requirement for divalent cations was studied and cobalt ion remarkably increased the enzyme activity. The removal of metal ions from the enzyme by EDTA decreased activity below 50%. The enzyme activity depleted restored when the assay was performed in the presence of $Co^{2+}$. Also, $Co^{2+}$ is the most active stimulator and has unique activation effect at high temperature. The phytase was specific for sodium phytate and p-nitrophenylphosphate, which is different from other known Bacilli phytases. The putative amino acid sequences of the phytase from B. coagulans IDCC 1201 were very similar to that of the phytase from B. subtilis strain 168. Based on these data, we concluded that the phytase from B. coagulans IDCC 1201 is a $Co^{2+}$-dependent acid phosphatase. Therefore, the strain B. coagulans IDCC 1201 is thought to be a valuable addititive for livestocks as well as a beneficial probiotics for human.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.38 no.4
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• pp.271-276
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• 2012

In this study, the dispersion ability of powder in low viscosity solubilization system that depends on variety and amount of additives and powders was investigated. A PMMA powder shows outstanding dispersion ability because of its repulsive force of partially charged chain and low density of porous structure. A sample, which contains salts, showed better dispersion tendency than a sample without any additives. The dispersion ability was quantity of salts dependent. Furthermore, a sample with divalent ion salts, like $MgSO_4$, showed better dispersion tendency than that of monovalent ion salts, like NaCl or KCl. The reason for the better dispersion tendency was due to the existence of ionized salts around the powders which significantly improves repulsive force between powders and consequently reduces powder aggregation. The sample with chelating agent, like EDTA as an additive, had improved dispersion ability. EDTA chelates and blocks metal cation therefore anion's character is maximized and repulsive force between powders is improved. As a result, salts and EDTA help to improve the powder dispersion ability and the stability of product.

• Journal of the Mineralogical Society of Korea
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• v.23 no.3
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• pp.235-242
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• 2010

Froth flotation using the residual water in the end of flotation process has been performed through controlling of pH. IEP (isoelectric point) of molybdenite and quartz in distilled water was below pH 3 and pH 2.7, respectively and the stabilized range was pH 5~10. In case of a suspension in reusing water, zeta potential of molybdenite decreased to below -10 mV or less at over pH 4 due to residual flocculants. As result of pH control, flotation efficiency in the alkaline conditions was deteriorated by flocculation, resulting from expanded polymer chain, ion bridge of the divalent metal cations ($Ca^{2+}$), and hydrophobic interactions between the nonpolar site of polymer/the hydrophobic areas of the particle surfaces. However, the weak acid conditions (pH 5.5~6) improved the efficiency of flotation as hydrogen ions neutralize polymer chains and then weakened its function. In cleans after rougher flotation, the Mo grade of 52.7% and recovery of 90.1% could be successfully obtained under the conditions of 20 g/t kerosene, 50 g/t AF65, 300 g/t $Na_2SiO_3$, pH 5.5 and 2 cleaning times. Hence, we developed a technique which can continuously supply waste water filtered from tailings into the grinding-rougher-cleaning processes.

• 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.

• Korean Journal of Microbiology
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• v.55 no.2
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• pp.123-130
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• 2019

Chelatase catalyzes the insertion of divalent metal into tetrapyrrole and plays a key role in the biosynthesis of metallated tetrapyrroles, such as cobalamin, siroheme, heme, and chlorophyll. SirB is a sirohydrochlorin (SHC) chelatase that generates cobalt-SHC or iron-SHC by inserting cobalt or iron into the center of sirohydrochlorin tetrapyrrole. To provide structural insights into the metal-binding and SHC-recognition mechanisms of SirB, we determined the crystal structure of SirB from Bacillus subtilis subsp. spizizenii (bssSirB) in complex with cobalt ions. bssSirB forms a monomeric ${\alpha}/{\beta}$ structure that consists of two domains, an N-terminal domain (NTD) and a C-terminal domain (CTD). The NTD and CTD of bssSirB adopt similar structures with a four-stranded ${\beta}-sheet$ that is decorated by ${\alpha}-helices$. bssSirB presents a highly conserved cavity that is generated between the NTD and CTD and interacts with a cobalt ion on top of the cavity using two histidine residues of the NTD. Moreover, our comparative structural analysis suggests that bssSirB would accommodate an SHC molecule into the interdomain cavity. Based on these structural findings, we propose that the cavity of bssSirB functions as the active site where cobalt insertion into SHC occurs.

• Development and Reproduction
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• v.7 no.2
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• pp.113-118
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• 2003

Follicular fluid(FF) of mammalian Graafian follicles contains various kinds of proteins and proteinases that are believed to play important roles during follicular growth oocyte maturation and ovulation of mature oocytes. Previous studies of human FF(hFF) demonstrated the presence of many serine/threonine proteinases and matrix metalloproteinases such as gelatinases, however, little is known about the caseinases. Present study was aimed to examine the presence and the property of caseinolytic enzyme in hFF. Using casein zymographic method, it was found that hFF, human adult serum and cord serum exhibited one intense 80 kDa and another weak 78 kDa bands having caseinolytic activity. When inhibitors were added to the zymographic substrate buffer, caseinolytic activity of both 80 kDa and 78 kDa proteins were inhibited by othylenediarnine tetraacetic acid(EDTA) or soybean trypsin inhibitor(SBTI), but not by E-64, phenylmethylsulfonyl fluoride(PMSF) or 1,10-phenanthroline. Thus both enzymes appear to belong to a family of trypsin-like enzyme. Addition of EDTA to the zymographic substrate buffer almost abolished the caseinolytic activity of both enzymes. However, further addition of a divalent metal ion such as CaC $l_2$, MgC $l_2$, MnC $l_2$ or ZnC $l_2$ to the same buffer fully restored the enzyme activity at 5 mM concentration despite the presence of EDTA. Based upon these observations, 80 kDa and 78 kDa caseinolytic enzymes are present in human follicular fluid and they appear to be trypsin-like enzymes of which caseinolytic activity needs the presence of $Ca^{++}$, aM $g^{++}$, M $n^{++}$ or Z $n^{++}$././././.

• Applied Microscopy
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• v.30 no.4
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• pp.347-355
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• 2000

Zinc is one of the most abundant oligoelements in the living cell. It appears tightly bound to some metalloproteins and nucleic acids, loosely bound to some metallothioneins or even as free ion. Small amounts of zinc ions (in the nanomolar range) regulate a plentitude of enzymatic proteins, receptors and transcription factors, thus rolls need accurate homeostasis of zinc ions. Zinc is an essential catalytic or structural element of many proteins, and a signaling messenger that is released by neural activity at many central excitatory synapses. Growing evidences suggest that zinc may also be a key mediator and modulator of the neuronal death associated with transient global ischemia and sustained seizures, as well as perhaps other neurological disease stoles. Some neurons have developed mechanisms to accumulate zinc in specific membrane compartment ('vesicular zinc') which can be evidenced using histochemical techniques. Substances giving a bright colour or emitting fluorescence when in contact with divalent metal ions are currently used to detect them inside cells; their use leads to the so called 'direct' methods. The fixation and precipitation of metal ions as insoluble salt precipitates, their maintenance along the histological process and, finally, their demonstration after autometallographic development are essential steps for other methods, the so called 'indirect methods'. This study is a short report on the autometallograhical approaches for zinc detection in the central nervous system (CNS) by means of a modified selenium method.