• Title/Summary/Keyword: Protease

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Proteome Data Analysis of Hairy Root of Panax ginseng : Use of Expressed Sequence Tag Data of Ginseng for the Protein Identification (인삼 모상근 프로테옴 데이터 분석 : 인삼 EST database와의 통합 분석에 의한 단백질 동정)

  • Kwon, Kyung-Hoon;Kim, Seung-Il;Kim, Kyung-Wook;Kim, Eun-A;Cho, Kun;Kim, Jin-Young;Kim, Young-Hwan;Yang, Deok-Chun;Hur, Cheol-Goo;Yoo, Jong-Shin;Park, Young-Mok
    • Journal of Plant Biotechnology
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    • v.29 no.3
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    • pp.161-170
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    • 2002
  • For the hairy root of Panax ginseng, we have got mass spectrums from MALDI/TOF/MS analysis and Tandem mass spectrums from ESI/Q-TOF/MS analysis. While mass spectrum provides the molecular weights of peptide fragments digested by protease such as trypsin, tandem mass spectrum produces amino acid sequence of digested peptides. Each amino acid sequences can be a query sequence in BLAST search to identify proteins. For the specimens of animals or plants of which genome sequences were known, we can easily identify expressed proteins from mass spectrums with high accuracy. However, for the other specimens such as ginseng, it is difficult to identify proteins with accuracy since all the protein sequences are not available yet. Here we compared the mass spectrums and the peptide amino acid sequences with ginseng expressed sequence tag (EST) DB. The matched EST sequence was used as a query in BLAST search for protein identification. They could offer the correct protein information by the sequence alignment with EST sequences. 90% of peptide sequences of ESI/Q-TOF/MS are matched with EST sequences. Comparing 68% matches of the same sequences with the nr database of NCBI, we got more matches by 22% from ginseng EST sequence search. In case of peptide mass fingerprinting from MALDI/TOF/MS, only about 19% (9 proteins of 47 spots) among peptide matches from nr DB were correlated with ginseng EST DB. From these results, we suggest that amino acid sequencing using tandem mass spectrum analysis may be necessary for protein identification in ginseng proteome analysis.

Characterization of HtrA2-deficient Mouse Embryonic Fibroblast Cells Based on Morphology and Analysis of their Sensitivity in Response to Cell Death Stimuli. (HtrA2 유전자가 결손된 mouse embryonic fibroblast 세포주의 형태학적 특징 및 세포사멸 자극에 대한 감수성 조사)

  • Lee, Sang-Kyu;Nam, Min-Kyung;Kim, Goo-Young;Rhim, Hyang-Shuk
    • Journal of Life Science
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    • v.18 no.4
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    • pp.522-529
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    • 2008
  • High-temperature requirement A2(HtrA2) has been known as a human homologue of bacterial HtrA that has a molecular chaperone function. HtrA2 is mitochondrial serine protease that plays a significant role in regulating the apoptosis; however, the physiological function of HtrA2 still remains elusive. To establish experimental system for the investigation of new insights into the function of HtrA2 in mammalian cells, we first obtained $HtrA2^{+/+}$ and $HtrA2^{-/-}$ MEF cells lines and identified those cells based on the expression pattern and subcellular localization of HtrA2, using immunoblot and biochemical assays. Additionally, we observed that the morphological characteristics of $HtrA2^{-/-}$ MEF cells are different form those of $HtrA2^{+/+}$ MEF cells, showing a rounded shape instead of a typical fibroblast-like shape. Growth rate of $HtrA2^{-/-}$ MEF cells was also 1.4-fold higher than that of $HtrA2^{+/+}$ MEF cells at 36 hours. Furthermore, we verified both MEF cell lines induced caspsase-dependent cell death in response to apoptotic stimuli such as heat shock, staurosporine, and rotenone. The relationship between HtrA2 and heat shock-induced cell death is the first demonstration of the research field of HtrA2. Our study suggests that those MEF cell lines are suitable reagents to further investigate the molecular mechanism by which HtrA2 regulates the balance between cell death and survival.

Peptide Properties of Rapid Salted and Fermented Anchovy Sauce Using Various Pretenses 1. Hydrolysis of Anchovy Sauce and Actomyosin by Various Pretenses (단백질 분해효소를 이용하여 제조한 속성 멸치 액젓의 펩티드 특성 1. 단백질 분해효소에 의한 멸치 액젓 및 Actomyosin의 가수분해)

  • KIM In-Soo;CHOI Young-Joon;HEU Min-Soo;CHO Young-Je;IM Yeong-Sun;GU Yeun-Suk;YEO Saeng-Gyu;PARK Jae-Woon
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.32 no.4
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    • pp.481-487
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    • 1999
  • The optimal conditions of enzymatic hydrolysis for preparation of rapid salted and fermented anchovy sauce (SFAS) using various pretenses such as trypsin, chymotrypsin, crude enzyme from squid liver and viscera, Alcalase, Neutrase and Protamex were studied. SFAS prepared with squid viscera had higher level of VBN (173.6 mg/100 g) when stored for 70 days than other samples, and peroxide values were almost equal among all samples during fermentation period. Total amino acids and nonprotein nitrogenous compounds remarkably increased as SFAS treated with Alcalase or Protamex which exhibited higher the hydrolysis rate of $57\%$ at 60 day than others. The optimal pHs of trypsin, chymotryosin, Alcalase, Neutrase and Protamex on anchovy actomyosin were 7.5, 6.5, 6.5, 7.0 and 5.0, respectively. Optimal temperatures of trypsin, chymotryosin, Alcalase and Neutrase were 55, 45, 60 and $55^{\circ}C$, respectively. Otherwise, Protamex activity increased as temperature increased from 20 to $70^{\circ}C$. Protamex had higher $K_m$ (3.545) and $V_{max}$ value (2.688) than others. Protamex affected less by NaCl had $52.5\%$ activity at the fermentation condition of $20^{\circ}C$ and $25\%$ NaCl. Protamex appeared to be very effective for the hydrolysis of crude actomyosin from ancnovy.

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Effects of Silk Protein Hydrolysates on Blood Glucose in C57BL/KsJ db/db Mice (실크단백질 효소 가수분해물이 2형 당뇨 마우스 C57BL/KsJ db/db의 혈당에 미치는 영향)

  • Shin, Mi-Jin;Park, Min-Jeong;Youn, Myung-Sub;Lee, Young-Sook;Nam, Moon-Suk;Park, In-Sun;Jeong, Yoon-Hwa
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.35 no.9
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    • pp.1166-1171
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    • 2006
  • This study was performed to investigate the effect of silk protein hydrolysates on blood glucose in diabetic mice (C57BL/KsJ db/db). The silk protein hydrolysates hydrolyzed by protease contains 87.52% of peptides of which molecular weight was below 2,000 dalton. The content of free amino acids was 14.80 g/100 g silk protein hydrolysates and major free amino acids were Pro, Thr, Arg and Ala. Silk protein hydrolysates were administered to the animals for 9 weeks at doses of 0.2, 0.5% and 0.8% solution. The body weight increase in the 0.5 and 0.8% fed groups were higher than control group. Food and water intake in the silk protein hydrolysates fed groups were lower than control group. The weight of liver was not different among groups, while the weight of kidney in control group was higher than silk protein hydrolysates fed groups. The blood glucose level in silk protein hydrolysates fed groups was lower than control group. In the glucose tolerance test, the blood glucose level in control group was the highest at 15 minutes after glucose injection while those in silk protein hydrolysates fed groups were the highest at 30 minutes. Results in this study suggest that silk protein hydrolysates show hypoglycemic effect in C57BL/KsJ db/db mice.

Biological Control of Root-knot Nematode by Streptomyces sampsonii KK1024 (Streptomyces sampsonii KK1024를 이용한 뿌리혹선충 (Root-knot nematode)의 생물학적 방제)

  • Kim, Sang-Su;Kang, Seon-I;Kim, Jin-Si;Lee, Yong-Sung;Hong, Sung-Hyun;Naing, Kyaw Wai;Kim, Kil-Yong
    • Korean Journal of Soil Science and Fertilizer
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    • v.44 no.6
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    • pp.1150-1157
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    • 2011
  • Streptomyces sampsonii KK1024 having strong chitinolytic activity was isolated from crab-shell rich soil at Muan, Jeolanamdo. The KK1024 produced chitinase, protease, gelatinase and lipase. When 50% of KK1024 culture broth was treated to juveniles and eggs of root-knot nematode, juvenile mortality at 3 days was 81.67% and egg hatch rate at 5 days was 2.00%. When $183.7{\mu}g\;mL^{-1}$ of crude enzyme produced by KK1024 was treated, juvenile mortality at 3 days was 96.00% and egg hatch rate at 5 days was 5.33%. At 1% of butanol extract from KK1024, juvenile mortality was highest with 90.00% and egg hatch rate was lowest with 0%. The comparison of the effect of KK1024 culture broth with only medium, synthetic fertilizer, and commercial nematicide on tomato growth and nematode infection was examined in pot trials. KK1024 culture broth showed lower number of egg mass and gall in plant, and population of juveniles in soil compared with only medium and synthetic fertilizer treatment, but not in commercial nematicide. However, the highest shoot weight and length was discovered in KK1024 culture broth. These results suggest that Streptomyces sampsonii KK1024 producing lytic enzymes and nematicidal compounds can be one of candidates for biocontrol agents against root-knot nematodes.

Inhibitory Effects of Rosa rugosa Crude Extract and Solvent Fractions on Adipogenic Differentiation of 3T3-L1 Preadipocytes (해당화 추출물이 3T3-L1 지방세포 분화에 미치는 영향)

  • Lee, Hyunjung;Yang, Jiho;Choi, Mi-na;Jeon, Seongeun;Zhou, Xianrong;Oh, Jung Hwan;Karadeniz, Fatih;Seo, Youngwan;Kong, Chang-Suk
    • Journal of Life Science
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    • v.32 no.12
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    • pp.979-988
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    • 2022
  • Halophytes have been reported to possess a variety of physiological activities, such as anti-cancer, anti-oxidant, anti-diabetes, anti-inflammatory, and anti-obesity. Studies on the roots of the halophyte Rosa rugosa, in particular, have shown a variety of physiological activities and are known to be effective for nursing diabetic complications in traditional Korean medicine. In this study, the effect of R. rugosa on adipogenesis was investigated in 3T3-L1 pre-adipocytes treated with crude extract and solvent fractions (H2O, n-BuOH, 85% aq. MeOH, and n-Hex) obtained from R. rugosa roots. Treatment with extract and the solvent fractions inhibited the formation of intracellular lipid droplets in differentiated 3T3-L1 adipocytes compared to the untreated group. In particular, n-BuOH and 85% aq. MeOH fractions effectively decreased the expression of adipogenic transcription factors: peroxisome proliferator activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP1c) in both mRNA and protein levels. In conclusion, these results suggest that R. rugosa contains anti-adipogenic molecules that can be utilized as a nutraceutical against obesity. Further refining of n-BuOH and 85% aq. MeOH fractions and analysis of their action mechanisms could yield potential therapeutic agents with anti-adipogenic effects.

Studies on the physio-chemical properties and the cultivation of oyster mushroom(Pleurotus ostreatus) (느타리버섯의 생리화학적성질(生理化學的性質) 및 재배(栽培)에 관(關)한 연구(硏究))

  • Hong, Jai-Sik
    • Applied Biological Chemistry
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    • v.21 no.3
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    • pp.150-184
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    • 1978
  • Nutritional characteristics and physio-chemical properties of mycelial growth and fruitbody formation of oyster mushroom(Pleurotus ostreatus)in synthetic media, the curtural condition for the commerical production in the rice straw and poplar sawdust media, and the changes of the chemical components of the media and mushroom during the cultivation were investigated. The results can be summarized as follows: 1. Among the carbon sources mannitol and sucrose gave rapid mycelial growth and rapid formation of fruit-body with higher yield, while lactose and rhamnose gave no mycelial growth. Also, citric acid, succinic acid, ethyl alcohol and glycerol gave poor fruit-body formation, and acetic acid, formic acid, fumaric acid, n-butyl alcohol, n-propyl alcohol and iso-butyl alcohol inhibited mycelial growth. 2. Among the nitrogen sources peptone gave rapid mycelial growth and rapid formation of fruit-body with higher yield, while D,L-alanine, asparatic acid, glycine and serine gave very poor fruit-body formation, and nitrite nitrogens, L-tryptophan and L-tyrosine inhibited mycelial growth. Inorganic nitrogens and amino acids added to peptone were effective for fruit-body growth, and thus addition of ammonium sulfate, ammonium tartarate, D,L-alanine and L-leucine resulted in about 10% increase fruit-body yield. L-asparic acid about 15%, L-arginine about 20%, L-glutamic acid, and L-lysine about 25%. 3. At C/N ratio of 15.23 fruit-body formation was fast, but the yield decreased, and at C/N ratio of 11.42 fruit-body formation was slow, but the yield increased. Also, at the same C/N ratio the higher the concentration of mannitol and petone, the higher yield was produced. Thus, from the view point of both yield of fruit-body and time required for fruiting the optimum C/N ratio would be 30. 46. 4. Thiamine, potassium dihydrogen phosphate and magnecium sulfate at the concentration of $50{\mu}g%$. 0.2% and 0.02-0.03%, respectively, gave excellent mycelial and fruit-body growth. Among the micronutrients ferrous sulfate, zinc sulfate and manganese sulfate showed synergetic growth promoting effect but lack of manganese resulted in a little reduction in mycelial and fruit-body growth. The optimum concentrati on of each these nutrients was 0.02mg%. 5. Cytosine and indole acetic acid at 0.2-1mg% and 0.01mg%, respectively, increased amount of mycelia, but had no effect on yield of fruit-body. The other purine and pyrimidine bases and plant hormones also had no effect on mycelial and fruit-belly yield. 6. Illumination inhibited mycelial growth, but illumination during the latter part of vegetative growth induced primordia formation. The optimum light intensity and exposure time was 100 to 500 lux and 6-12 hours per day, respectively. Higher intensity of light was injurous, and in darkness only vegetative growth without primordia formation was continued. 7. The optimum temperature for mycelial growth was $25^{\circ}C$ and for fruit-body formation 10 to $15^{\circi}C$. The optimum pH range was from 5.0 to 6.5. The most excellent fry it-body formation were produced from the mycelium grown for 7 to 10 days. The lesser the volume of media, the more rapid the formation of fruit-body; and the lower the yield of fruit-body; and the more the volume of media, the slower the formation of fruit-body, and the higher the yield of fruit-body. The primordia formation was inhibited by $CO_2$. 8. The optimum moisture content for mycelial growth was over 70% in the bottle media of rice straw and poplar sawdust. 10% addition of rice bran to the media exhibited excellent mycelial growth and fruit-body formation, and the addition of calciumcarbonate alone was effective, but the addition of calcium carbonate was ineffective in the presence of rice bran. 9. In the cultivation experiments the total yield of mushroom from the rice straw media was $14.99kg/m^2$, and from the sawdust media $6.52kg/m^2$, 90% of which was produced from the first and second cropping period. The total yield from the rice straw media was about 2.3 times as high as that from the sawdust media. 10. Among the chemical components of the media little change was observed in the content of ash on the dry weight basis, and organic matter content decreased as the cultivation progressed. Moisture content, which was about 79% at the time of spawning, decreased a little during the period of mycelial propagation, after which no change was observed. 11. During the period from spawning to the fourth cropping about 16.7% of the dry matter, about 19.3% of organic matter, and about 40% of nitrogen were lost from the rice straw media; about 7.5% of dry mallet, about 7.6% of organic matter, and about 20% of nitrogen were lost from the sawdust media. For the production of 1kg of mushroom about 232g of organic matter and about 7.0g of nitrogen were consumed from the rice straw media; about 235g of organic matter and about 6.8g of nitrogen were consumed from the sawdust media, 1㎏ of mushroom from either of media contains 82.4 and 82.3g of organic matter and 5.6 and 5.4g of nitrogen, respectively. 12. Total nitrogen content of the two media decreased gradually as the cultivation progressed, and total loss of insoluble nitrogen was greater than that of soluble nitrogen. Content of amino nitrogen continued to increase up to the third cropping time, after which it decreased. 13. In the rice straw media 28.0 and 13.8% of the total pentosan and ${\alpha}$-cellulose, respectively, lost during the whole cultivation period was lost during the period of mycelial growth; in the sawdust media 24.1 and 11.9% of the total pentosan and ${\alpha}$-cellulose, respectively, was lost during the period of mycelial growth. Lignin content in the media began to decrease slightly from the second cropping time, while the content of reduced sugar, trehalose and mannitol continued to increase. C/N ratio of the rice straw media decreased from 33.2 at spawining to 30.0 at ending; that of the sawdust media decreased from 61.3 to 60.0. 14. In both media phosphorus, potassium, manganese and zinc decreased, at magnesium, calcium and copper showed irregular changes, and iron had a tendency to be increased. 15. Enzyme activities are much higher in the rice straw media than in the sawdust media. CMC saccharifying and liquefying activity gradually increased from after mycelial propagation to the second cropping, after which it decreased in both media. Xylanase activity rapidly and greatly increased during the second cropping period rather than the first period. At the start of the third cropping period the activity decreased rapidly in the rice straw media, which was not observed in the sawdust media. Protease activity was highest after mycelial propagation, after which it gradually decreased. The pH of the rice straw media decreased from 6.3 at spawning to 5.0 after fourth cropping; that of the sawdust media decreased from 5.7 to 4.9. 16. The contents of all the components except crude fibre of the mushroom from the rice straw media were higher than those from the sawdust media. Little change was observed in the content of the components of mushroom cropped from the first to the third period, but slight decrease was noticed at the fourth cropping.

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