• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.450-456
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• 1999

A high molecular-weight water-soluble fraction(PO) obtained by the ethanol precipitation of 0.1 N NaOH extracts of the mushroom Pleurotus ostreatus showed 82% anti-complementary activity for complement consumption hemolysis. The PO consisted of 42% carbohydrate (w/w), 50% protein (w/w), and 3% uronic acid (w/w). Fifty-eight percent of the anti-complementary activity decreased by periodate oxidation and 22% by protease digestion, suggesting that the sugar and protein moieties are essential for this activity. Two polysaccharide fractions, PO-IIIa-1 and PO-IIIa-2, with anti-complementary activity were isolated from the PO using DEAE-Sepharose FF followed by Sephadex G-75 and Sepharose CL-6B gel permeation chromatographies. The PO-IIIa-2 was found by HPLC to be nearly homogeneous, with the molecular mass of 531 kDa, and showed 96% $ITCH_{50}$ (inhibition against the total complement hemolysis of deionized water as the control) at a concentration of 1 mg/ml. This fraction contained galactose, mannose, fucose, and glucose with molar ratios of 1.75:1:0.65 and 0.59, respectively. The majority of galactose and mannose units in the PO-IIIa-2 were composed of TGalp1 ->, ->6Galp1->, ->2,6Galp1->, and ->Manp1->. The PO-IIIa-1 (molecular mass of 2000 kDa), exhibiting higher activity than the PO-IIIa-2, was further purified into two fractions, unbound proteoglycan (PO-IIIa-1A) and bound glucomannan (PO-IIIa-lB), by affinity chromatography using ConA-Sepharose CL-4B. The anti-complementary activity of each affinity purified fraction decreased as compared to that of the native PO-IIIa-1 fraction, indicating that the formation of complex between both polysaccharide fractions was necessary for full anti-complementary activity.

• BMB Reports
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• 제28권5호
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• pp.375-381
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• 1995

Two forms of glucoamylase (GI and GII) from starch-grown Lipomyces kononenkoae CBS 5608 mutant were purified to apparent homogeneity by means of ultrafiltration, Sephacryl S-200 gel filtration and DEAE Sephadex A-50 chromatography. The apparent molecular weight was calculated as ca. 150 kDa for GI and ca. 128 kDa for GII, respectively. Both enzymes were glycoproteins with isoelectric points of 5.6 (GI) and 5.4 (GII). They had a pH optimun of 4.5 and were stable from pH 5 to 8. The temperature optimum for both enzymes was $60^{\circ}C$, but they were rapidly inactivated above $70^{\circ}C$. The $K_m$ values toward starch were estimated to be 6.57 mg per ml for GI and 4.52 mg per ml for GII, and the $V_{max}$ values were 16.28 ${\mu}M$ per mg for GI and 32.25 ${\mu}M$ per mg for GII, respectively. The $K_m$ and $V_{max}$ values of GII for ${\alpha}-$ or ${\beta}-cyclodextrin$ were estimated to be 0.15 mg per ml and 2.0 mg per ml, respectively ($K_m$) and 1.02 ${\mu}M$ per mg or 1.02 ${\mu}M$ per mg, respectively ($V_{max}$). Neither enzyme exhibited pullulanase activity but they released only glucose from starch or cyclodextrin. Amino acid analysis indicated that both glucoamylases were enriched in proline and acid amino acids. Glucoamylase GII strongly cross-reacted with a monoclonal antibody raised against GI enzymes, and the two enzymes shared very similar amino acid composition. Western blot analysis indicated that L. kononenkoae CBS 5608 mutant produced two forms of glucoamylase on starch, and that synthesis of them was subject to glucose repression.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.897-905
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• 2003

Phospholipase D (PLD) and ADP-ribosylation factor (ARF) were partially purified on a series of column chromatography, and their biochemical properties were characterized to understand the regulatory mechanism of PLD activation by ARF protein in the antigen-induced immune responses in guinea pigs. Heparin Sepharose and high-Q Sepharose column chromatographies were used for the purification of PLD, and Sephadex G-25, DEAE Sephacel, Source 15 PHE (HIC), Superdex-75, and Uno-Q column chromatographies were used for the purification of ARF. The purified PLD and ARF proteins were identified with anti-rabbit PLD- or ARF-specific antibodies, showing about 64 or 85 kDa for the molecular mass of PLD and 29 or 35 kDa for the sizes of ARF. Partial cDNA of ARF3 was cloned by RT-PCR in guinea pig lung tissue and its nucleotides and amino acids were sequenced. Guinea pig ARF3 showed 92% of nucleotides sequence identity and 100% of amino acid sequence homology with human ARF3. The ARF-regulated PLD activity was measured in the oleate or ARFs-containing mixed lipid vesicles. The purified and recombinant ARF (rARF) activities were assessed with the $GTP{\gamma}S$ binding assay. The PLD activity was induced by oleate in a dose-dependent manner. The purified ARF and recombinant ARF3 increased PLD activity in guinea pig lung tissues. These data show that the activity of membrane-bound PLD can be regulated by the cytosolic ARF proteins, suggesting that ARF proteins in guinea pig lung can act as a regulatory factor in controlling the PLD activity in allergic reaction.

• Applied Biological Chemistry
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• 제33권1호
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• pp.73-78
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• 1990

한국산 토마토로부터 endo-polygalacturonase를 gel filtration과 이온교환크로마토그라피에 의하여 약 24배 정제할 수 있었고, 최대 효소활성을 위한 PH는 5.0, 최적온도는 $50^{\circ}C$ 였으며 pH안정범위는 $4.0{\sim}5.0$, 열안정성은 $50^{\circ}C$에서 1시간 열처리 하였을 때 약 45 % 실활 되었다. 정제된 이 효소는 SDS-polyacrylamide gel 전기영동에 의하여 단일밴드로 확인되었으며, 그 분자량은 50,000정도였고, Km값은 $1.43{\times}10^{-1}\;mol/l이었다. 금속이온중 $Ag^+$, $Zn^{++}$이온이 효소의 활성을 촉진시켰으며, $Na^+$, $K^+$등의 이온에 의해서는 활성이 저해되었다.

• 한국수산과학회지
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• 제46권4호
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• pp.351-358
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• 2013

A protease inhibitor from fish eggs was fractionated using chromatographic methods. The fractionation efficiency was evaluated in terms of specific inhibitory activity (SIA, U/mg), purity (fold), total inhibitory activity (TIA, U), and recovery (%). The protease inhibitor (PI) from egg extracts of skipjack tuna (ST Katsuwonus pelamis), yellowfin tuna (YT Thunnus albacares) and Alaska pollock (AP Theragra chalcogramma) was fractionated using Sephadex G-50 gel filtration and DEAE-Sepharose CL-6B anion exchange chromatography based on protein size exclusion and net charge, respectively. Fractions exhibiting strong inhibitory activity were contained in the 30-50 kDa fraction on gel filtration and in the range of 0.4-0.7 M NaCl gradient fraction on anion exchange chromatography. The respective TIA and percent recovery of the fraction obtained with gel filtration toward trypsin and $N{\alpha}$-benzoyl-L-arginine-p-nitroanilide (BAPNA) were 2,758.7 U and 29.6% for ST, 1,005.5 U and 25.6% for YT, and 1,267.5 U and 26.0% for AP. Gel filtration chromatography was more effective at fractionating PI than using ion exchange chromatography. These results suggest that fish eggs act as serine protease inhibitors and might be useful for protease inhibition in foodstuffs.

• 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.

• 미생물학회지
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• 제14권3호
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• pp.117-127
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• 1976

A cellulase fraction (F IV-1) purified to about 8-folds was obtained from crude cellulase prepared from the wheat bran culture of S.atra. The partial purification of the enzyme was made by DEAE Sephadex and Sephdex cloumn chromatography in conjuction with ammonium sulfate precipitation. After stading at various pH's for 22 hours at $20^{\circ}C$, F IV-1 was most stable at pH 5.0 but when the enzyme fraction was stood for 74 hours, the point of pH stability was raised to around pH 6.0-7.0. After heating at various temperatures for 1 hour, F IV-1 was most stable at $20^{\circ}C$. The optimal enzyme activities of F IV-1 were seen at pH 6.0 and $50^{\circ}C$. The optimal concentrations of $Zn^{++}\;and\;Ca^{++}$ for the activities of crude cellulase were 6 and 4 mM respectively, but $Ca^{++}$ inhibited the enzyme activity at concentrations below 2 mM and above 6mM. Both $Cu^{++}\;and\;Mn^{++}$ ions inhibited cellulase activities and a ocmplete inactivation of crude cellulase was achieved at concentratioins of 5 and 2 mM of ions respectively. When Na-CMC was used as substrate, the Km values of crude cellulase and F IV-1 were calculated to be $5{\times}10^{-4}\;and\;2{\times}10^{-5}mM$, and V values 32 and 1.35 mmoles/hour, respectively. The Ki values of $Mn^{++}$ for crude cellulase and F IV-1 were found to be $8{\times}10^{-2}\;and\;3{\times}10^{-2}\;mM\;while\;those\;of\;Cu^{++}\;were\;at\;2{\times}10^{-1}\;and\;1{\times}10^{-1}\;mM\;respectively.\;Both\;Mn^{++}\;and\;Cu^{++}$ showed competitive inhibition with substrate.

• 생명과학회지
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• 제15권1호
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• pp.94-99
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• 2005

충청남도 목천과 충청북도 오창 근교의 토양으로부터 망간을 산화하는 64 집락을 분리하고 이 중 망간 산화능이 가장 우수한 한 균주를 최종 선별하여 생리, 생화학적 특성을 조사하고, 16S rRNA 염기 서열분석 등을 통하여 동정한 결과 최종 선별된 균주는 Aeromonas sp. MN44로 확인되었다. 최종 선별된 Aeromenas sp. MN44는 lactose를 제외한 여러 당들은 이용하지 못하였으며, 중금속내성은 lithium과 manganese에 대해서는 mg/ml 단위의 높은 농도까지 중금속 내성을 가지고 있었지만 cadmium에는 전혀 내성을 나타내지 않았다. 또 kanamycin, chloramphenicol, ampicillin, tetracycline, spectinomycin등 조사한 모든 항생제에 대해 전혀 내성을 갖지 않았다. Aeromonas sp. MN44가 생성하는 망간산화물질의 최적 pH는 pH 7.4로 확인되었으며, 이 균이 생성하는 망간 산화 factor는 proteinase K와 가열처리에 의해 저해되는 단백질이고, ammonium sulfate 침전과 ion exchange chromatography 그리고 gel filtration의 단계를 통해 부분 정제한 망간 산화 factor의 분자량은 약 113 kDa로 확인되었다.

• Journal of the Korean Wood Science and Technology
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• 제20권4호
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• pp.73-84
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• 1992

It was observed that the hot-water extract of the bark of Kalopanax pictus N. had the highest anti-complementary activity among the 11 kinds of forest materials. Methanol-and ethanol-soluble portions had low anti-complementary activities, but crude polysaccharide. HKP-0 had a high activity of 80%. HKP-0 contained 54.8% of total sugar and 27.9% of protein. The neutral sugars of HKP-0 consisted of mainly arabinose, galactose and glucose. HKP-4 fraction obtained by cetavlon treatment of HKP-0 showed the highest anti-complementary activity of 90%. The activity was not changed by pronase digestion bu decreased greatly by periodate oxidation. HKP-4 consisted of mainly arabinose and glucose with molar ratio of 1.0 : 22.4, HKP-4-I, an unabsorbed fraction from HKP-4 on DEAE Sepharose CL-6B column showed higher yield and activity than those of absorbed fractions. HKP-4-I was homogeneous, and its molecular weight was about 25,000. HKP-4-I contained 84.0% of neutral sugar and consisted of arabinose and glucose with molar ratio of 1.0 : 11.2. The anti-complementary activity of HKP-4-I was not decreased by the treatment of polymyxin B, and the polysaccharide activated both classical and alternative pathway in complement system. Void volume fraction obtained from HKP-4-I hydrolyzed with ${\alpha}$-amylase on Sephadex G-25 column only had a high anti-complementary activity.

• Journal of Microbiology and Biotechnology
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• 제11권5호
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• pp.845-852
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• 2001

A microorganism producing fibrinolytic enzyme was isolated from Korean traditional soybean paste and identified as Bacillus sp. KDO-13. The fibrinolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-celluose, and gel chromatography on Sephadex G-100 of the culture supernatant of Bacillus sp. KDO-13. The molecular weight of the purified enzyme was estimated to be 44,000 by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 8.0 and $50{\circ}C$, respectively. The enzyme activity was relatively stable at pH 7.0-9.0 and temperature below $50{\circ}C$. the activity of the enzyme was inhibited by $AI^{3+}$ and $Hg^{2+}$, but activated by $Co^{2+}$\;and\;Ni^{2+}. In addition, the enzyme activity was potently inhibited by EDTA and 0-phenanthroline. The purified enzyme could completely hydrolyze a fibrin substrate within 6 h in vitro, and had a low $K_m$ value for fibrin hydrolysis. It was concluded that the purified enzyme was a metalloprotease with relatively high specificity for fibrinolysis, and thus, could be applied as an effective thrombolytic agent.