• 제목/요약/키워드: SDS-G-PAGE

검색결과 482건 처리시간 0.031초

Purification and Characterization

  • Nam, Suk-Woo;Seo, Dong-Wan;Sung, Dae-Seok;Han, Jeung-Whan;Hong, Sung-Youl;Lee, Hyang-Woo
    • Archives of Pharmacal Research
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    • 제21권2호
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    • pp.128-134
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    • 1998
  • Nitric oxide synthase, NOS (EC.1.14.13.39), was purified from bovine pancreas over 5,500-fold with a 7.6% yield using 30% ammonium sulfate precipitation, and $2^1$,$5^1$-ADP-agarose and calmodulin-agarose affinity chromatography. The purified bovine pancreatic NOS (bpNOS) showed a single band on SDS-PAGE corresponding to an apparent molecular mass of 160 kDa, whereas it was 320 kDa on non-denaturating gel-filtration. This indicated a homodimeric nature of the enzyme. The specific activity of the purified bpNOS was 31.67 nmol L-citrulline fored/mtn/mg protein and an apparent $K\textrm{m}$ for L-arginine was 15.72 $\mu\textrm{M}$, The enzyme activity was dependent on $Ca^{2+}$ and calmodulin, and to a lesser extent on NADPH, FAD and FMN. $H_4B$ was not required as a cofactor for the activity. In an inhibition experiment with L-arginine analogues, $N^G$-nitro-L-arginine (NNA) had the most potent inhibitory effect on bpNOS, and $N^{G}$, $N^{G1}$-dimethyl-L-arginine (symmetric; sDMA) did not have any inhibitory effect. Immunohistochemical analysis of the bovine pancreas using brain type NOS antibody (anti-bNOS antibody) revealed that acinar cells showed strong immunoreactivity against the antibody.

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Aspergillus sp. CC-29 생성하는 Alkaline Protease의 정제 및 특성 (Purification and Biological Characteristics of Alkaline Protease from Aspergillus sp. CC-29)

  • 최청;김두기;조영제;성태수
    • 한국식품영양과학회지
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    • 제19권5호
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    • pp.434-442
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    • 1990
  • Aspergillus sp. CC-29 ws selected for its strong protease activity among various stains of molds found in soil. It was found that the production of alkaline protease reached to maximum when the wheat bran medium containing glucose as carbon source had been cultured for 4 days. Alkaline proteased was purified 36.10 fold from Aspergillus sp. CC-29 The purification procedures included ammonium sulfate fractunation gel filteration on Sepha-dex G-75 G-150 and DEAE-cellulose ion-exchange chromatography, The yield of the purified enzyme was 22.40% The purified enzyme was confirmed as a single band by the polyacryla-mide. When the purified enzyme was applied to SDS-PAGE the molecular weight was estima-ted 24000. The optimum pH for the enzyme activity was 9.0 and the optimum temperature was 4$0^{\circ}C$ The reaction of this enzyme followed typical Michaelis-Menten kinetics with the Km value of 2.10$\times$10-4M with the Vmax of 29.41 $\mu$g/min. The enzyme was reactively stable in alkalic condition and unstable by heat treatment. The activity of alkaline protease was increased by the addition of Ca2+ whereas it was inhibited by Hg2+ Zn2+ at concentration of 1$\times$10-3M.

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폐포상피세포, 대식세포를 비롯한 각종 세포주에서 H2O2에 의한 Peroxiredoxin 동위효소들의 산화에 따른 불활성화와 재생 (Oxidative Inactivation of Peroxiredoxin Isoforms by H2O2 in Pulmonary Epithelial, Macrophage, and other Cell Lines with their Subsequent Regeneration)

  • 오윤정;김영선;최영인;신승수;박주헌;최영화;박광주;박래웅;황성철
    • Tuberculosis and Respiratory Diseases
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    • 제58권1호
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    • pp.31-42
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    • 2005
  • 배 경 : peroxiredoxins는 거의 모든 생명체에 공통적으로 보존되어 있으며, 최근에 발견된, 특이한 peroxidases로 인체에서 6가지 동위효소가 알려져 있으며, 산화스트레스에 대한 방어역할을 담당하고, $H_2O_2$신호전달 과정에서 중요한 조절 역할을 한다. peroxiredoxin은 $H_2O_2$ 처리 과정 중에서 자신이 산화되어 불활성화 되는데, 산화된 후 다시 재생되는 것으로 보고되나 그 생리적은 의미는 분명하지 않다. 이에 저자들을 폐상 피세포주, 대식세포주, 폐포모세혈관 내피세포주 및 기타 섬유모세포주 들에서 $H_2O_2$ 에 의한 Prx의 산화과정과 재생을 알아보고자 하였다. 방 법 : 수술 환자에서 적출한 정상 폐조직과, 세포주로는 평상시 산화 스트레스에 노출이 많을 것으로 예상되는 세포들로써, 폐포상피세포의 I 형 및 II 형 세포에서 기원한 A549, WI 26, Raw 264.7, Rat2,및 폐포 모세혈관 내피세포주 등을 이용하여 이를 $50{\mu}M$. $100{\mu}M$, $500{\mu}M$$H_2O_2$로 산화시켜 불활성화 한 후, 추적관찰 하였으며, 시간대 별로(0. 10, 30, 60, 120, 240, 480 분) 수확하여, 이를 1차원 non-reducing SDS-PAGE 및 2차원 전기영동로 분리 후, silver stain 과 Western blot으로 분석 하였다. 결 과 : 1. 실험에 사용된 모든 세포주에서, $H_2O_2$ 농도에 비례하여 peroxiredoxin I, II, III 의 불활성화를 관찰할 수 있었고, 10분에 최고로 불활성화되었다. 2. 산화된 이후, 30분경부터 peroxiredoxin 의 재생이 관찰되기 시작 하였으며, 2시간 이후부터 확연하였다. 3. 다시 재생된 peroxiredoxin은 $H_2O_2$투여로서, 다시 불활성화되어, 재생된 Prx 가 활성을 지닌 단백질임을 알 수 있었다. 4. 재생의 속도는 사용된 세포주마다 차이가 있었으며 (A549 >Raw 264.7 >$Rat_2$ >WI26), 단백질 합성억제제인 cycloheximide ($10{\mu}g/ml$) 존재 하에서도 변함 없이 관찰되었다. 결 론 : 세포 내에는 산화되어 불활성화된 peroxiredoxin을 재생하는 체계가 존재 하며, 이는 활성부위 cysteine을 갖는 다른 단백질에도 공통적으로 적용될 수 있는 분자 스위치일 가능성이 높으며, 산화에 의한 신호전달과정이나, 질병 모델에서 Prx 단백의 재생 체계의 이상과 병인에 관한 추가적인 연구가 필요할 것으로 사료된다.

저밀도 지방단백질의 산화에 대한 곰취 추출물의 항산화 효과 (Effects of Ligularia fischeri Extracts on Oxidation of Low Density Lipoprotein)

  • 정성원;김은정;황보현주;함승시
    • 한국식품과학회지
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    • 제30권5호
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    • pp.1214-1221
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    • 1998
  • 지질 산화에 의해 생성하는 MDA (malondialdehyde)를 thiobarbituric acid와 반응시켜 측정한 MDA 값은 곰취 추출물의 농도가 증가함에 따라 감소하므로서 추출물들에 의해 산화가 억제됨을 알 수 있었다. 메탄올 추출물의 경우 $15\;{\mu}g/mL$농도로 첨가시 71.7% (13.36 nmol/㎎)의 산화억제율을 나타내었으며 분획물의 경우 ethyl acetate 분획물이 동일농도에서 97% (1.35 nmol/mg)의 억제율을 나타내었다. 산화시간에 따른 MDA 값의 변화는 $25\;{\mu}g/mL$의 농도를 첨가시 에탄올, 메탄올 및 물 추출물의 경우 4시간까지 산화를 억제하였으며 분획물의 경우에는 $20\;{\mu}g/mL$의 농도를 첨가시 ethyl acetate 분획물이 8시간까지 강한 항산화효과를 나타내었다. Agarose gel electrophoresis에서는 에탄올, 메탄올 그리고 물 추출물 모두 항산화 효과가 인정되었다. 지질산화에 의해 생성되는 conjugated diene은 곰취 추출물의 경우 $Cu^{2+}$로 산화시켰을 때 대조에 비해 약 1.1배에서 2.8배까지 감소시키는 효과를 나타내었고 분획물의 경우 약 2.2배에서 3.2배까지의 감소효과를 나타냄으로서 강한 산화 억제효과를 나타내었다. SDS-PAGE를 이용하여 LDL내부에 존재하는 apo B-100부분에 대하여 산화에 따른 분해정도를 측정한 결과 에탄올, 메탄올 및 물 추출물이 천연 LDL의 band와 유사한 band를 나타내었다. Densitometer를 이용한 apo B-100함량은 천연 LDL의 함량을 100%로 하였을 때 에탄올, 메탄올 및 물 추출물의 경우 각각 77.8, 92.5 그리고 82.3%의 함량을 나타내었다. 분획물의 경우 헥산, ethyl acetate 및 물 분획물에서 각각 38.8, 94.5% 그리고 65.5%의 함량을 나타냄으로서 ethyl acetate 분획물이 강하게 산화를 억제함으로서 apo B-100의 분해를 막아주고 있음을 알 수 있었다.

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대두(大豆) 및 Aspergillus niger α-galactosidase의 효소학적(酵素學的) 연구(硏究) (Enzymatic Studies on the α-Galactosidases from Soybean and Aspergillus niger)

  • 금종화;오만진
    • 농업과학연구
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    • 제18권1호
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    • pp.49-73
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    • 1991
  • 발아대두 $\alpha$-galactosidase와 Aspergillus niger가 생산하는 $\alpha$-galactosidase의 효소학적 성질을 비교하기 위하여 대두 발아 중의 $\alpha$-galactosidase활성 및 소당류의 함량변화를 측정하였고, 대두 발아 과정 중 활성이 가장 강할때에 추출한 $\alpha$-galactosidase 및 Aspergillus niger를 밀기울 배양하였을 때 생성되는 $\alpha$-galactosidase를 염석, 이온교환 크로마토그래피 및 겔여과 등을 사용하여 정제한 후 정제효소의 이화학적 및 효소학적 성질을 측정, 비교하여 다음과 같은 결과를 얻었다. 1. 대두 $\alpha$-galactosidase의 활성은 대두를 $25^{\circ}C$에서 120시간 발아시켰을 때 가장 높았으며, 대두 중의 raffinose는 96시간, stachyose는 120시간 발아시켰을 때 완전히 분해되었다. 2. Aspergillus niger를 밀기울배지에서 $30^{\circ}C$, 4일간 배양했을 때 $\alpha$-galactosidase 활성이 최고에 달하였다. 3. 대두 $\alpha$-galactosidase는 황산암모늄 염석, DEAE-Cellulose 및 DEAE-Sephadex A-50 이온교환 크로마토그래피, Sephadex G-l50 겔여과 등에 의하여 6.6배까지 정제되었으며 그의 비활성이 825U/mg protein, 수율 2.5%에 달하였고, Aspergillus niger의 $\alpha$ -galactosidase는 23.7배까지 정제되었으며 그의 비활성이 1,229U/mg protein, 수율 14%에 달하였다. 4. 정제된 대두 및 Aspergillus niger의 $\alpha$-galactosidase는 HPLC, PAGE 및 SDS-PAGE에 의해서 순도가 확인되었다. 5. 정제효소의 이화학적 성질 1) Aspergillus niger의 $\alpha$-galactosidase는 periodic acid schiff 염색에 의하여 당단백질임이 확인되었다. 2) 대두 $\alpha$-galactosidase의 등전점은 pH4.8이었고, 분자량이 30,000인 monomer이었으나, Aspergillus niger의 $\alpha$-galactosidase는 등전점이 pH4.6이었고 분자량은 112,000이었으며 분자량 28,000인 monomer 4개로 구성된 tetramer이었다. 3) 대두 및 Aspergillus niger $\alpha$-galactosidase의 활성에 관여하는 아미노산은 diethyl pyrocarbonate에 의한 화학 수식에 의하여 histidine임이 확인되었다. 4) 대두 $\alpha$-galactosidase의 활성은 2-mercaptoethanol과 L-cysteine에 의하여 약간 저해되었다. 6. 정제효소의 효소학적 성질 1) 대두 $\alpha$-galactosidase의 최적 작용 pH는 pH6.0, 최적 작용온도는 $40^{\circ}C$이었고, Aspergillus niger $\alpha$-galactosidase 각각 pH6.5 및 $40^{\circ}C$이었다. 2) 대두 및 Aspergillus niger의 $\alpha$-galactosidase는 $45^{\circ}C$이하에서 비교적 안정하였으나 $60^{\circ}C$에서 10분 처리시 대두 $\alpha$-galactosidase는 25%, Aspergillus niger $\alpha$-galactosidase는 46%의 잔존활성을 나타내었다. 3) 대두 $\alpha$-galactosidase는 pH5.5~6.5, Aspergillus niger의 $\alpha$-galactosidase는 pH6.0~7.0에서 매우 안정하였다. 4) 대두 및 Aspergillus niger의 $\alpha$-galactosidase간에 기질 특이성상의 차이가 없었으며, stachyose보다 raffinose를 잘 분해하였고, gaIactose는 양효소의 활성을 저해하였다. 5) 대두 $\alpha$-galactosidase의 P-nitrophenyl-$\alpha$-gaIactopyranoside, raffinose 및 stachyose에 대한 Km값은 각각 5.3mM, 50.0mM 및 55.5mM이 었고, Aspergillus niger $\alpha$-galactosidase에 있어서는 각각 5.0mM, 37.0mM 및 55.5mM이었다. 6) 대두 $\alpha$-galactosidase의 p-nitrophenyl-$\alpha$-d-gaIactopyranoside에 대한 활성화 에너지는 13.024Kcal/mole, $Q_{10}$값은 2.0이 었으며, Aspergillus niger의 $\alpha$-galactosidase는 각각 8.515Kcal/mole 및 1.38이었다.

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Evaluation of Arabinofuranosidase and Xylanase Activities of Geobacillus spp. Isolated from Some Hot Springs in Turkey

  • Sabriye, Canakci;Inan, Kadriye;Murat, Kacagan;Belduz, Ali Osman
    • Journal of Microbiology and Biotechnology
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    • 제17권8호
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    • pp.1262-1270
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    • 2007
  • Some hot springs located in the west of Turkey were investigated with respect to the presence of thermophilic microorganisms. Based on phenotyping characteristics and 16S rRNA gene sequence analysis, 16 of the isolates belonged to the genus Geobacillus and grew optimally at about $60^{\circ}C$ on nutrient agar. 16S rRNA gene sequence analysis showed that these isolates resembled Geobacillus species by ${\ge}97%$, but SDS-PAGE profiles of these 16 isolates differ from some of the other species of the genus Geobacillus. However, it is also known that analysis of 16S rRNA gene sequences may be insufficient to distinguish between some species. It is proposed that recN sequence comparisons could accurately measure genome similarities for the Geobacillus genus. Based on recN sequence analysis, isolates 11, IT3, and 12 are strains of G stearothermophilus; isolate 14.3 is a strain of G thermodenitrificans; isolates 9.1, IT4.1, and 4.5 are uncertain and it is required to make further analysis. The presence of xylanase and arabinofuranosidase activities, and their optimum temperature and pH were also investigated. These results showed that 7 of the strains have both xylanase and arabinofuranosidase activities, 4 of them has only xylanase, and the remaning 5 strains have neither of these activities. The isolates 9.1, 7.1, and 3.3 have the highest temperature optima ($80^{\circ}C$), and 7.2, 9.1, AO4, 9.2, and AO17 have the highest pH optima (pH 8) of xylanase. Isolates 7.2, AO4, AC15, and 12 have optimum arabinofuranosidase activities at $75^{\circ}C$, and only isolate AC15 has the lowest pH of 5.5.

Inconsistency in the Improvements of Gel Strength in Chicken and Pork Sausages Induced by Microbial Transglutaminase

  • Kawahara, S.;Ahhmed, A.M.;Ohta, K.;Nakade, K.;Muguruma, M.
    • Asian-Australasian Journal of Animal Sciences
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    • 제20권8호
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    • pp.1285-1291
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    • 2007
  • This research investigated variation in the improvement of the texture of chicken and pork sausages induced by microbial transglutaminase (MTG). The extractability of myofibrillar proteins from these sausages as well as the ${\varepsilon}-({\gamma}-glutamyl)$lysine (G-L) content were also investigated. MTG treatment of sausages significantly increased the breaking strength values in both meat types, especially for samples incubated at $40^{\circ}C$. However, values of the breaking strength in both meat types were increased differently. The variation in protein extractability of samples incubated at $40^{\circ}C$ for both meat types could lead to some consideration of the mechanisms and the high accessions of myosin heavy chain (MHC) to MTG. SDS-PAGE analysis showed significant changes in the density of the bands after adding MTG, especially for the pork samples in which the bands disappeared totally. The G-L content in the presence of MTG was double that in control samples of both meat types. This study suggests that the binding ability of myofibrillar proteins with MTG is strong. This leads us to suggest that MTG functions positively with different improvements in the texture of chicken and pork products that are treated mechanically, such as sausages. Variability in gel improvement level between chicken and pork sausages was observed; this resulted from the variation in meat proteins in response to MTG, as well as to the original glutamyl and lysine content.

Characterization and Enhanced Production of Enterocin HJ35 by Enterococcus faecium HJ35 Isolated from Human Skin

  • Yoon Yoh Chang;Park Hye Jung;Lee Na-Kyoung;Paik Hyun-Dong
    • Biotechnology and Bioprocess Engineering:BBE
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    • 제10권4호
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    • pp.296-303
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    • 2005
  • A strain named as HJ35 was isolated from the skin of sixty-five men and fourteen women for acne therapy, in order to find an effective antimicrobial agent against Propionibacterium acnes. Isolate HJ35 was identified as Enterococcus faecium based on 16 rDNA sequence and produced enterocin HJ35 having antimicrobial activities against most lactic acid bacteria, En­terococcus spp., Staphylococcus aureus, S. epidermidis, Clostridium perfringens, some bacilli, Mi­crococcus flavus, Listeria monocytogenes, L. ivanovii, Escherichia coli, Pseudomonas fluorescens and Propionibacterium acnes, in the modified well diffusion method. Especially, enterocin HJ35 showed a bactericidal activity against Propionibacterium acnes P1. The antimicrobial activity of enterocin HJ35 was disappeared completely with the use of protease XIV. But enterocin HJ35 activity is very stable at high temperature (up to $100^{\circ}C$ for 30 min), in wide range of pH (3.0${\~}$9.0), and by treatment with organic solvents. The apparent molecular mass of enterocin HJ35 was estimated to be approximately 4${\~}$4.5 kDa on detection of its bactericidal activity after SDS-PAGE. In batch fermentation of E. faecium HJ35, enterocin HJ35 was produced at the mid­log growth phase, and its maximum production was obtained up to 2,300 AU/mL at the late stationary phase. By employing fed-batch fermentation, the enhanced production of enterocin HJ35 was achieved up to 12,800 AU/mL by feeding with 10 g/L glucose or 6 g/L lactate.

A Second Thioltransferase from Chinese Cabbage: Purification and Characterization

  • Cho, Young-Wook;Park, Eun-Hee;Lim, Chang-Jin
    • BMB Reports
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    • 제32권2호
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    • pp.133-139
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    • 1999
  • Thioltransferase, also known as glutaredoxin, was previously purified and characterized from Chinese cabbage (Brassica campestris ssp. napus var. pekinensis). However, in the process of gel filtration on Sephadex G-75, there were two activity peaks. In this study, a second thioltransferase (TTase CC-2) in the minor peak of the Sephadex G-75 elution profile was further purified using affinity chromatography on an S-hexylglutathione-agarose column by eluting with buffer solution containing 2.5 mM S-hexylglutathione. It showed a single band on SDS-PAGE indicating that TTase CC-2 is electrophoretically homogeneous. The molecular weight of TTase CC-2 was estimated to be about 22,000 daltons, and its isoelectric point was determined to be 6.73. Its size appears to be atypical and much larger than that of the first thioltransferase (TTase CC-1) from Chinese cabbage, and it can utilize 2-hydroxyethyl disulfide, S-sulfocysteine, and insulin as substrates. S-sulfocysteine was found to be a superior substrate for TTase CC-2. TTase CC-2 also displayed the reducing activity for non-disulfides such as dehydroascorbic acid. Its optimum pH was 8.5, which was consistent with that of TTase CC-1. TTase CC-2 activity was greatly activated by L-cysteine and reduced glutathione, and was found to be less heat-stable compared with TTase CC-1. Molecular and physiological differences between TTase CC-1 and TTase CC-2 remain to be elucidated. Chinese cabbage is the first plant which is known to contain two kinds of thioltransferases.

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Purification and Characterization of a Novel Antifungal Protein from Paenibacillus macerans PM1 Antagonistic to Rice Blast Fungus, Pyricularia oryzae

  • Bae, Dong-Won;Kawk, Weon-Sik;Lee, Joon-Taek;Son, Dae-Young;Chun, Sung-Sik;Kim, Hee-Kyu
    • Journal of Microbiology and Biotechnology
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    • 제10권6호
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    • pp.805-810
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    • 2000
  • An antifungal protein antagonistic to the rice blast fungus, Pyricularia oryzae was purified from Paenibacillus macerans PM-1 by ammonium sulfate fractionation, Q Sepharose Fast Flow column chromatography, Phenyl Sepharose CL-4B column chromatography and Superose 12 gen filtration. An apparent molecular mass of the purified antifungal protein was determined as 8 kDa by SDS-PAGE and 9 kDa by analytical gel filtration, respectively, suggesting that the purified protein is a monomer. The antifungal protein was stable at pH range from 7-12 and up to $100^{\circ}C$. The protein was also stable at 0.1-1% Tween 20 and Triton X-100. The N-terminal amino acid sequence of the antifungal protein was Thr-Glu-Leu-Pro-Leu-Gly-Ile-Val-Met-Asp-Lys-Tyr-Thr-Asp-Ala-Phe-Lys-Phe-Asp-Met-Phe. Comparison of the determined sequence with other peptide and DNA sequences did not reveal homology at all. Therefore, the purified antifungal protein was speculated to be a novel protein. The condidial germination in vitro of P. oryzae KJ301:93-39 by the purified protein ($5.9{\mu} g/ml$) was limited to $9{\pm}3.2%$ only, compared with $69{\pm}2.4%$ of the control. Ungerminated conidia were swollen at basa and mid cell by the purified protein. In vivo bioassay for inhibition of conidial germination of P. oryzae KJ 301, one of the most predominating racesin Korea. the purified protein ($5.9{\mu} g/ml$)strongly inhibited the conidial germination. The conidia, even though germinated, could not develop any further to produce appressoria efficiently.

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