• 미생물학회지
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• 제34권3호
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• pp.82-86
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• 1998

Yarrowia lipolytica 504D가 생산하는 alkaline proteinase를 정제한 결과, 분자량은 32,000으로 나타났고, pH 9.5와 $42^{\circ}C$에서 최적활성을 보였으며, pH 4-10의 범위와 $45^{\circ}C$까지 비교적 안정한 것으로 나타났다. PMSF를 비롯하여 EDTA, EGTA, phenanthrolin도 효소활성을 저해하여 정제효소가 serine proteinase인지 metal proteinase인지 불확실하였다. 그러나 28% 활성증가를 보인 $Cu^{2+}$ 외에 $Zn^{2+}$를 비롯한 대부분의 무기염들이 효소활성을 증가시키지 못하였고, 또한 EDTA의 첨가로 불활성화된 효소도 Ca 염의 첨가로 활성이 복원되었다. 따라서 정제효소는 serine proteinase(E.C. 3.4.21.14)로 추정되었다.

• 한국지하수토양환경학회지:지하수토양환경
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• 제16권4호
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• pp.53-61
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• 2011

This study examined evaluation on stabilization of major and trace elements in tailings by various surface water-preventing materials. Six columns were filled with tailings of the Sinlim mine, then covered with tailings only, compacted soils, clay, soil-bentonite mixture, pozzolan and bentonite mat. After injection of artificial rain water, the leachate was sampled with times (3, 6, 9 and 12 pore volume) and analysed for major (Ca, Na, Mg, K) and trace elements (As, Cd, Cu, Pb, Zn) by ICP-AES. With exception to pozzolan type, the pH values of leachate from the other types became stabilized from 5.5 to 7.5, and EC (electric conductivity) of leachate from them decreased with times. For the pozzolan type, however, the pH and EC of leachate increased with time due to its alkalinity producing system. Concentrations of most major and trace elements in leachate decreased and stabilized with time. Consequently, soil-bentonite mixed cover shows the best ability of water-preventing and reducing mobility of elements in tailings site.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.792-799
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• 2005

Two novel $\beta$-glucuronidases, which metabolize glycyrrhizin (GL) to 18$\beta$-glycyrrhetinic acid-3-O-$\beta$-D-glucuronide (GAMG), were purified from Streptococcus LJ-22 isolated from human intestinal microflora. $\beta$-Glucuronidases I and II were purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, butyl toyopearl, Q-Sepharose, hydroxyapatite Ultrogel, and GL-attached Sepharose column chromatographies, with the final specific activities of 137 and 190 nmole/min/mg, respectively. The molecular sizes of both $\beta$-glucuronidases were found to be 140 kDa by gel filtration, and they consisted of two identical subunits (M.W. 67 kDa by SDS-PAGE). $\beta$-Glucuronidases I and II showed optimal activity at pH 7.0 and pH 6.5, respectively. Both purified enzymes were potently inhibited by $Cu^{2+}$ and PCMS, and had maximum activity on glycyrrhizin, but did not hydrolyze p-nitrophenyl-$\beta$-glucuronides, baicalin, or GAMG These findings suggest that the biochemical properties and substrate specificities of these enzymes are different from those of the previously purified $\beta$-glucuronidases. This is the first reported purification of sugar (not aglycone)-recognizing $\beta$-glucuronidases from intestinal bacteria.

• Applied Biological Chemistry
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• 제36권2호
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• pp.86-92
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• 1993

무에서 DEAE Bio-Gel, Con-A 및 Superose-6 칼럼을 이용하여 myrosinase를 정제하고 그 효소학적 특성을 검토한 결과 myrosinase(II)는 2개의 subunits를 가졌으며, 이들의 분자량은 SDS-GAGE상에서 각각 53 및 39 KD였다. 정제된 무효소의 비활성도는 37,500 units/mg이었으며, 정제도는 44배였다. 최적 활성 pH는 phosphate 및 Tris-HCl 완충액에서 $6.5{\sim}7.0$이었으며, pH7.0에서 그 효소활성이 가장 안정하였다. 활성 최적온도는 $37{\sim}38^{\circ}C$였고, 열안정성은 $30^{\circ}C$ 이하였다. 무 myrosinase에 대한 무기염의 영향은 구리 및 수은 이온은 효소 활성을 매우 저해하며, ascorbic acid의 농도별 영향은 1 mM일 때 최대활성을 나타내었다. Ascorbic acid analogue에 대한 활성은 dehydroascorbic acid에 대해서는 거의 없었으며, 나머지 analogue들도 ascorbic acid보다 상당히 활성이 낮았다. 무 myrosinase에 대한 환원제의 영향은 2-mercaptoethanol과 dithiothreitol에 의해서는 활성을 나타내지 않았으나, 이들을 ascorbic acid 등 2-mercaptoethanol과 함께 첨가하면 활성을 나타내었다.

• 한국대기환경학회지
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• 제29권3호
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• pp.317-324
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• 2013

This research assessed the surface properties of modified activated carbons with three different acids and five different metals for ammonia gas removal. Raw bituminous coal-based activated carbon ($4{\times}8$ mesh) had low adsorption capacity of 0.72 mg $NH_3/g$ based on the analysis in the column adsorption experiment. Adsorption capacities of carbons modified with $CH_3COOH$, $H_3PO_4$, and $H_2SO_4$ increased up to 3.34, 21.00, and 35.21 mg $NH_3/g$, respectively. Those of carbons with Cu, Zn, Zr, Fe, and Sn were 9.63, 9.13, 7.09, 25.12 and 15.03 mg $NH_3/g$. Ammonia adsorption was enhanced by the presence of surface oxygen groups on carbon materials, which influenced pH of carbon surface. BET surface area of raw carbon was analyzed to be $1087m^2/g$, but it decreased by carbon surface modification. Fe-impregnated carbon showed $503.02m^2/g$ of surface area. These observations were mostly caused by chemical adsorption.

• Journal of Microbiology and Biotechnology
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• 제10권5호
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• pp.612-619
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• 2000

To investigate the biochemical properties of V. mimicus metalloprotease, whose gene was isolated previously from Vibrio mimicus ATCC33653, overexpression and purification were attempted. The 1.9 kb of open reading frame was amplified by PCR from pVMC193 plasmid which ligated the VMC gene with pUC19 and introduced into Escherichia coli BL21 (DE3) using the overexpression vector, pET22b (+). The overexpressed metalloprotease (VMC) was purified with Ni-NTA column chromatography and characterized with various protease inhibitors, pHs, temperatures, and substrates. The purified VMC showed the proteolytic activity against gelatin, soluble and insoluble collagens, and synthetic peptides. Unlike the observations made with all metalloproteases originated from other Vibrio sp., the VMC did not hydrolyze the casein. The proteolytic activity was critically decreased when the VMC was treated with metal chelating reagents, such as EDTA, 2,2-bipyridine, and 1, 10-phenanthroline. In particular, the 71 kDa VMC exhibited the hemagglutinating activity against human erythrocyte. As the purified VMC was treated with $CuCl_2$ and $NiCl_2$ for the chemical modification of metal binding, the proteolytic activity and hemagglutinating activity were profoundly influenced. The multialignment analysis made on the reported Vibrio metalloproteases showed the difference of amino acid sequence similarity between the two distinctive classes of Vibrio metalloproteases.

• Journal of Microbiology and Biotechnology
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• 제1권3호
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• pp.188-196
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• 1991

The ${\beta}-glucosidase$ was purified to homogeneity from the culture filtrate of P. verruculosum by column chromatography. The enzyme was a glycoprotein with a relative size of approximately 220 kDa with an isoelectric point of 4.8, which was composed of dimeric protein of 105 kDa. The enzyme was stable up to $60^{\circ}C$ and the presence of glycerol significantly increased its thermostability. The enzyme was found to hydrolyze both ${\beta}-aryl$ and ${\beta}-alkyl-glucosides$ in addition to ${\beta}-glucosyl$ glucose and catalyzed glucosyl transfer to cellobiose. The enzyme attacked laminarin in an exotype-like fashion. The apparent Km's of the enzyme toward cellobiose, laminaribiose, laminarin were 0.53 mM, 0.35 mM and 1.11 mM, respectively. Glucose and glucono-${\delta}-lactone$ were competitive inhibitors for the enzyme. Copper ($Cu^{2+}$), mercury ($Hg^{2+}$) and p-chloromercuribenzoate were strong inhibitors of the enzyme. The immunoblotting result revealed that one form of ${\beta}-glucosidase$ was biosynthesized, irrespective of carbon sources used. Polyacrylamide gel electrophoresis analysis of the in vitro translated product of total RNA from avicel grown mycelium established that the P. verruculosum ${\beta}-glucosidase$ precursor was approximately 95 kDa in size. The amino acid composition and N-terminal amino acid sequence are given.

• 한국미생물·생명공학회지
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• 제25권2호
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• pp.166-172
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• 1997

Aspergillus niger was selected as a strain producing the potent raw starch hydorlyzing enzyme. These experiments were conducted to investigate the conditions of the glucoa- mylase production, the purification of the enzyme, some characteristics of the purified enzyme and hydrolysis rate on various raw starches such as com, rice, potato, glutinous rice, sweet potato, wheat and barley. The optimum cultural temperature and time for the enzyme production on wheat bran medium were $30^{\circ}C$ and 96hrs, respectively. The respective addition of yeast extract and nutrient broth on wheat bran medium increased slightly the enzyme production. The enzyme was purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. The specific activity of the purified enzyme was 30.7u/mg-protein and the yield of enzyme activity was 25.8%. The purified enzyme showed a single band on polyacrylamide disc gel electrophoresis and its molecular weight was estimated to be 56,000 by SDS-polyacrylamide disc gel electrophoresis. The isoelectric point for the purified enzyme was pH3.7. The optimum temperature and pH were $65^{\circ}C$ and pH 4.0, respectively. The purified enzyme was stable in the pH range of pH 3.0-9.5 and below $45^{\circ}C$, and its thermal stability was slightly increased by the addition of $Ca^{2+}$. The purified enzyme was activated by $Co^{2+},\;Sr^{2+},\;Mn^{2+},\;Fe^{2+},\;Cu^{2+}$. Raw rice starch, raw corn starch, raw glutinous rice starch, raw sweet potato starch, raw wheat starch and raw barley starch showed more than 90% hydrolysis rate in 48hrs incubation. Even raw potato starch, most difficult to be hydrolyzed, showed 80% hydrolysis rate. The purified enzyme was identified as glucoamylase.

• Journal of Microbiology and Biotechnology
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• 제22권11호
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• pp.1540-1548
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• 2012

Manganese peroxidase (MnP) was isolated from the culture filtrate of the wood log mushroom Stereum ostrea (S. ostrea), grown on Koroljova medium, and then purified by ammonium sulfate [70% (w/v)] fractionation, DEAE-cellulose anion exchange chromatography, and Sephadex G-100 column chromatography, with an attainment of 88.6-fold purification and the recovery of 22.8% of initial activity. According to SDS-PAGE the molecular mass of the MnP was 40 kDa. The optimal pH and temperature were found to be 4.5 and $35^{\circ}C$, respectively. The enzyme was stable even after exposure to a pH range of 4.5 to 6.0, and at temperatures of up to $35^{\circ}C$ at a pH of 4.5 for 1h. The $K_m$ and $V_{max}$ values for the substrate phenol red were found to be $8{\mu}m$ and 111.14 U/mg of protein, respectively. The MnP also oxidized other substrates such as guaiacol, DMP, and veratryl alcohol. Sodium azide, EDTA, SDS, $Cu^{2+}$, and $Fe^{2+}$, at 1-5 mM, strongly inhibited enzyme activity, whereas $Ca^{2+}$ and $Zn^{2+}$ increased enzyme activity. The participation of the purified enzyme in the decolorization of dyes suggests that S. ostrea manganese peroxidase could be effectively employed in textile industries.

• ALGAE
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• 제22권3호
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• pp.247-252
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• 2007

Crude fucoidan was extracted from the sporophyll of Korean Undaria pinnatifida collected at a coastal area ofWando, Korea, mainly by dilute acid extraction, ethanol precipitation, CaCU Precipitation, with an yield of approxi-mately 3.9% in mass. It was further purified by DEAE-cellulose column chromatography and its chemical composi-don and in vitro anticoagulant activity was determined. The average molecular mass of the purified fucoidan wasestimated about 2.1 x 103 kDa by size-fractionation HPLC and it consisted of neutral sugar (52.34% in mass), uronicacid (26.2%), and sulfate esters (7.4%). From the HPAEC-PAD analysis, the monosaccharide composition of thepurified fucoidan was shown to be fucose, galactose, xylose, and mannose, with a molar ratio of 1, 0.2, 0.02, 0.15,respectively, demonstrating that major monosacd-iande was fucose (72.3% in mol percentage) and other sugars,xylose (1.5%), galactose (14.6%), and mannose (10.9%) were present as minor component. The results suggested thatthis fucoidan is a sulfated, U-type fucoidan. The activated partial thrombloplastin time (APTT) assay of the purifiedfucoidan showed that the purified fucoidan elicited anticoagulant activity in a dose-dependent manner. Five jUg ofsporophyll fucoidan delayed the blood clotting time up to 5 times than untreated control and also up to 1.5 timesthan the same amount of the commercial fucoidan, respectively. Although it is preliminary, these results suggestthat the fucoidan of Korean Undaria vinnatifida sporophyll would be promising candidates for the development ofan anticoaeulant.