• Title/Summary/Keyword: Mono Q

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Expression of SARS-3CL Protease in a Cell-Free Protein Synthesis System (무세포 단백질 합성법을 이용한 활성형 SARS-3CL protease의 발현)

  • Park, Sun-Joo;Kim, Yong-Tae
    • Journal of Life Science
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    • v.22 no.4
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    • pp.552-558
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    • 2012
  • Severe acute respiratory syndrome (SARS) is a severe respiratory infectious disease caused by a novel human coronavirus, SARS-CoV. The 3CL protease is a key enzyme in the proteolytic processing of replicase polyprotein precursors, pp1a and pp1ab, which mediate all the functions required for viral genomic replication and transcription. Therefore, this enzyme is a target for the development of chemotherapeutic agents against SARS. A large quantity of active SARS-3CL protease is required for development of anti-SARS agents. Here we have constructed overexpression vector for the production of the SARS-3CL protease. The gene encoding SARS-3CL protease was amplified using polymerase chain reaction and cloned into the pET29a expression vector, resulting in pET29a/SARS-3CLP. Recombinant SARS-3CL protease was successfully synthesized by the dialysis mode of the cell-free protein expression system, and purified by three-step fast protein liquid chromatography using HighQ and MonoP column chromatographies and Sephacryl S-300 gel filtration. In addition, the produced SARS-3CL protease was found to be an active mature form. This study provides efficient methods not only for the development of anti-SARS materials from natural sources, but also for the study of basic properties of the SARS-3CL protease.

Removal of Nitrate-Nitrogen in Pickling Acid Wastewater from Stainless Steel Industry Using Electrodialysis and Ion Exchange Resin (전기투석과 이온교환수지를 이용한 스테인레스 산업의 산세폐수 내 질산성 질소의 제거)

  • Yun, Young-Ki;Park, Yeon-Jin;Oh, Sang-Hwa;Shin, Won-Sik;Choi, Sang-June;Ryu, Seung-Ki
    • Journal of Environmental Science International
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    • v.18 no.6
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    • pp.645-654
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    • 2009
  • Lab-scale Electrodialysis(ED) system with different membranes combined with before or after pyroma process were carried out to remove nitrate from two pickling acid wastewater containing high concentrations of $NO_3\;^-$(${\approx}$150,000 mg/L) and F($({\approx}$ 160,000 mg/L) and some heavy metals(Fe, Ti, and Cr). The ED system before Pyroma process(Sample A) was not successful in $NO_3\;^-$ removal due to cation membrane fouling by the heavy metals, whereas, in the ED system after Pyroma process(Sample B), about 98% of nitrate was removed because of relatively low $NO_3\;^-$ concentration (about 30,000 mg/L) and no heavy metals. Mono-selective membranes(CIMS/ACS) in ED system have no selectivity for nitrate compared to divalent-selective membranes(CMX/AMX). The operation time for nitrate removal time decreased with increasing the applied voltage from 10V to 15V with no difference in the nitrate removal rate between both voltages. Nitrate adsorption of a strong-base anion exchange resin of $Cl\;^-$ type was also conducted. The Freundlich model($R^2$ > 0.996) was fitted better than Langmuir mode($R^2$ > 0.984) to the adsorption data. The maximum adsorption capacity ($Q^0$) was 492 mg/g for Sample A and 111 mg/g for Sample B due to the difference in initial nitrate concentrations between the two wastewater samples. In the regeneration of ion exchange resins, the nitrate removal rate in the pickling acid wastewater decreased as the adsorption step was repeated because certain amount of adsorbed $NO_3\;^-$ remained in the resins in spite of several desorption steps for regeneration. In conclusion, the optimum system configuration to treat pickling acid wastewater from stainless-steel industry is the multi-processes of the Pyroma-Electrodialysis-Ion exchange.

Purification and Characterization of Endo-polygalacturonase Produced by Plant Pathogenic fungus, Botrytis cinerea (식물 병원진균 Botrytis cinerea가 생산하는 Endo-polygalacturonase의 순수정제와 특성)

  • Kim, Byung-Young;Lee, Tae-Ho;Rha, Eu-Gene;Chung, Young-Ryun;Lee, Chang-Won;Kim, Jae-Won
    • The Korean Journal of Mycology
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    • v.25 no.4 s.83
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    • pp.330-339
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    • 1997
  • Botrytis cinerea T91-1 has shown to produce at least four different polygalacturonases in a liquid medium containing citrus pectin as a carbon source. One of the enzymes, its molecular weight was estimated as 37 kDa by denatured polyacrylamide gel electrophoresis, was purified by a series of procedures including acetone precipitation, ion exchange, heparin affinity, and reverse phase column chromatographies. By viscometric analysis, the enzyme was revealed as an endo-polygalacturonase. The enzyme activity was inhibited by divalent cations such as $Ca^{2+}$, $Co^{2+}$, and $Cu^{2+}$. Km and Vmax for polygalacturonic acid hydrolysis were 0.33 mg/ml and 28.6 nM/min, respectively. The optimum temperature for enzymatic activity was $55^{\circ}C$ and the enzyme showed optimal pH values between 4.0 and 4.5. The enzyme was stable up to 12 hours in the range of pH 4 to 7 and at the temperature below $30^{\circ}C$. Amino acid sequence from N-terminal up to 6 amino acids determined by Edman degradation showed little homology with polygalacturonases from fungi and plants.

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Analysis of Inter-Species Association and Covariation in a Natural Deciduous Forest (천연활엽수림(天然闊葉樹林)에서의 수종간(樹種間) 상관관계(相關關係)와 공변이관계(共變異關係)의 분석(分析))

  • Kim, Ji Hong;Kuen, Kee Hyun
    • Journal of Korean Society of Forest Science
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    • v.80 no.4
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    • pp.360-368
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    • 1991
  • Utilizing chi-square test statistics, inter-species association and covariation were analyzed for the 37 woody plant species in a deciduous forest dominated by Quercus mongolica and Q. variabilis. within 50 temporarily established $20m{\times}20m$ square quadrats, the association for each pair of species was presented based on the presence-absence parameters. Acer palmatum had significant positive association with Acer mono and Kalopanax pictus, but negative association with Pinus densiflora. Other positively associated species pairs were Prunus sargentii-Macckia amurensis, Quercus serrata-Kalopanax pictus, Symplocos chinensis var. pilosa-Euonymus oxyphyllus, and Ulmus davidiana var. japonica-Lindera obtusiloba. The covariation far each pair of species was evaluated based on the quantitative measures, density and basal area. Overall results showed that the association and covariation values among species generally agreed with each other. Because covariation was calculated by density and basal area of the tallied species in the sample plots, the number of species pairs of covariation tended to be greater than those of association. Especially, Pinus densiflora, considered to be pioneer species in the successional stage, had negative covariation with most of climax species. These ecological information could be applied to silvicultural practices, such as ecosystem classification, establishment of mixed hardwood forest, and tending operations for marking crop trees and desirable species.

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Production of Recombinant Human Hyperglycosylated Erythropoietin Using Cell Culture Technology by Improving Sialylation. (Sialic Acid 함량 증가 배양기술에 의한 재조합 인간 다당쇄 에리스로포이에틴의 생산)

  • 박세철;이승오;박만식;김승훈;김준환;송무영;이병규;고인영;강희일
    • Microbiology and Biotechnology Letters
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    • v.32 no.2
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    • pp.142-148
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    • 2004
  • Erythropoietin is a main regulator of human erythropoiesis. Recombinant human erythropoietin (rhEPO) is one of the glycoproteins produced in animal cells, and it has oligo saccharides chains which comprise about 40% of its molecular mass. Because the content of sialic acid can extend circulatory lifetime, the high degree of sialylation is often a desirable feature of therapeutic glycoproteins. In this study, the sialylation of rhEPO produced by chinese hamster ovary cell culture was maximized by supplementing the culture medium with N-acetylm-annosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), a sialidase inhibitor. Feeding of 20 mM ManNAc/0.5 mM NeuAc2en into culture medium increased the sialic acid content by nearly tenfold compared with unsupplemented medium. This effect was achieved without affecting the cell growth or product yield. Six erythropoietin fractions differing in sialic acid content, ranging from 11∼15% of EPO, were identified from chinese hamster ovary cell-derived rhEPO by mono Q column chromatography. It was found that, at 20 mM ManNAc/0.5 mM NeuAc2en feeding, productivity of hyper-glycosylated EPO increased up to 50%, compared with the unsupplemented medium.

Secondary Dispersion of Several Broadleaved Tree Seeds by Wildlife in Mt. Jungwang, Pyeongchang-gun, Korea (중왕산 몇 활엽수 종자의 야생동물에 의한 2차 분산)

  • Kim, Gab-Tae;Kim, Hoi-Jin
    • Korean Journal of Environment and Ecology
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    • v.27 no.1
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    • pp.64-70
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    • 2013
  • To examine the proportion of secondary seed dispersion by wildlife and to identify the related wildlife, We measured seed removal rates and related wildlife at the natural mixed broadleaved forest in mountain Jungwang, Pyeungchang-gun in middle part of Korean peninsula, seed-tray contained four kind of tree seeds were setted from mid-september to late-October for three years. Mean seed removal rates(MSRR) of acorn(Q. mongolica)is shown the highest values 87.2%, next MSRR of samara(A. pseudosieboldianum) does 33.1%, MSRR of samara(A. pictum subsp. mono) does 29.2%. and then SRR of samara(A. mandshuricum) does only 13.6%. Acorns are removed more faster than samaras. Among the related wildlife, Siberian chipmunks most freqently visit seed tray, and ordered Korean squirrel, Wild boar, and Yellow-throated marten. Most secondary dispersed acorns might be consumed by Wild boar, and small portions does comsumed or scatter-hoarded by rodents. Most secondary dispersed samaras might be consumed by rodents, and small portions does scatter-hoarded by rodents. A few sound seeds are remained on the forest floor in late October owing to the seed consunption and seed hoarding by wildlife. We observe seedling establishment of four broadleaved tree species. From these results, We proved Wild boar is seed comsummer, and rodents are seed consummer and seed disperser. Further studies on secondary seed dispersion, seed consumption and hoarding and related wildlife should be needed.

Purification and Characterization of Mitochondrial Mg2+-Independent Sphingomyelinase from Rat Brain

  • Jong Min Choi;Yongwei Piao;Kyong Hoon Ahn;Seok Kyun Kim;Jong Hoon Won;Jae Hong Lee;Ji Min Jang;In Chul Shin;Zhicheng Fu;Sung Yun Jung;Eui Man Jeong;Dae Kyong Kim
    • Molecules and Cells
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    • v.46 no.9
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    • pp.545-557
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    • 2023
  • Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg2+-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg2+, Mn2+, Ni2+, Cu2+, Zn2+, Fe2+, and Fe3+ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg2+-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

A Study on Marketing of Cultured Laver Products (양식해태의 유통에 관한 조사 연구)

  • 유충열
    • The Journal of Fisheries Business Administration
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    • v.4 no.1_2
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    • pp.19-57
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    • 1973
  • Laver io one of the most necessary and seasonal items in Korean food from oldtimes. Laver is lagely eaten in dried form, and its supply depends entirely upon culture weeds. The history of laver culture in Korea about sixty or seventy years is older than in Japan. Significance of laver culture is divided into two aspects, one is food supply in the nation, and the other is export to other countries. Houses engaged in laver culture are about foully thousands, and laver production in 1972 is estimated as 1, 3 bitten sheets. (1 sheet is a dried laver of 20 cm sq, in the shape of paper) Especcially meaning of layer production is the concentration of labour input, and systematic management of labour. From around 1920, the method of laver culture was introduced by Japanese Imperialism for mono culture in shallow seas, and mass products of laver is provided to Japan market, DOMESTIC MARKET Fundamental consume function calculates at below, $D_{(68_71)}$=16354 $Y^{0.471}$ $P^{-1.0662}$ where D is total layer demand, Y income variable, P price variable. It means income elasticity is 476. in the whole country, and price elasticity is 1, 07. But generally income elasticity is higher in urban area than in rural area, as shown at 1, 3 in Seoul city. Expence of laver in house expenditure is mutually correlated with another expence, See Table 12 about the relative function. See Table 14 and 16 about the relation between the gathering and the changes of price in auction, wholesale and retail price support system is for two effects, one of which is constraint of the upper price, the other is rise of the lower price. Before the system control, the equation in three year average calculated as below, $Y_{b}$ =18, 907.7455+15435.9364 t (r=0.89) where the origin t=0 is the November and the units are month. Post the system control, $Y_{p}$ =30, 047.9636+1, 631.1721t (r=0.97) therefore, this system has an effect only on the rise of lower price, Average annual margins of laver products at four market levels according to the consumer spent is below. EXPORTING MARKET Japanese demand function of laver products is, Log D=5, 289+1, 108 Log Y-1, 395 Log P (r=0.987) where D is Japanese laver demand, Y income variable, P price variable. according to which income elasticity is 1. 1 and price elasticity is 1.4. Laver production in 1970 tile highest record till then, is estimated as six billion sheets. But the recent improvement of laver culture techniques, the production of seeds and freezing storage of seeds has been stabilized. Futher new culture farms have been developed by means of break- water fences or by floating culture method. These improvements have been backed up with increased demand of laver products. Import quantity and price of Korean laver products are restrained by three organizations, that is producer, distributor and consumer. This relationship calculated by regression equation shows that import is influenced only producer organization, at the sacrifice of consumer profit. For increase to export of laver products, we urgently require to open foreign trade of laver products for Japanese consumer, .and Japan has political responsibility to solve Korean laver structure. But with long run timeseries, as regards Japanese production and import quantity, importing function shows increasing trend as below, 250 million sheets <3, 947.1674+0.005 $L_{g}$ >) 600 million sheets where $L_{q}$ is relative production quantity of laver in Japan. (unit; 100 thousand sheets) Our Export effort should be put on the highly processed products whithin the restraind quote.ote.

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Identification and Characterization of Lactobacillus salivarius subsp. salivarius from Korean Feces

  • Bae, Hyoung-Churl
    • Proceedings of the Korean Society for Food Science of Animal Resources Conference
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    • 2004.05a
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    • pp.89-119
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    • 2004
  • This study was conducted to isolate lactobacilli having probiotic characteristics to be used as health adjuncts with fermented milk products. Acid tolerant strains were selected in Lactobacilli MRS broth adjusted to pH 4.0 from 80 healthy persons (infants, children and adults). And bile tolerant strains were examined in Lactobacilli MRS broth in which 1.0% bile salt was added. By estimation above characteristics, the strains No. 27, which was isolated from adult feces, was selected and identified as Lactobacillus salivarius subsp. salivarius based on carbohydrate fermentation and 16S rDNA sequencing. It was used as a probiotic strain in fermented milk products. The pH of fermented milk decreased from pH 6.7 to 5.0 and titratable acidity increased from 0.3% to 1.0% by L. salivarius subsp. salivarius (isolation strain 20, 35, and 37), when incubated for 36 h at $37^{\circ}C$. The number of viable cell counts of fermented milk was maximized at this incubation condition. The SDS-PAGE evidenced no significant change of casein but distinct changes of whey protein were observed by isolated L. salivarius subsp. salivarius for titratable acidity being incubated by $0.9{\sim}1.0%$ at $37^{\circ}C$. All of the strains produced 83.43 to 131.96 mM of lactic acid and 5.39 to 26.85 mM of isobutyric acid in fermented products. The in vitro culture experiment was performed to evaluate ability to reduce cholesterol levels and antimicrobial activity in the growth medium. The selected L. salivarius subsp. salivarius reduced $23{\sim}38%$ of cholesterol content in lactobacilli MRS broth during bacterial growth for 24 hours at $37^{\circ}C$. All of the isolated L. salivarius subsp. salivarius had an excellent antibacterial activity with $15{\sim}25$ mm of inhibition zone to E. coli KCTC1039, S. enteritidis KCCM3313, S. typhimurium M-15, and S. typhimurium KCCM40253 when its pH had not been adjusted. Also, all of the isolated L. salivarius subsp. salivarius had partial inhibition zone to E. coli KCTC1039, E. coli KCTC0115 and S. enteritidis KCCM3313 when it had been adjusted to pH 5.7. The selected strains were determined to have resistances of twelve antibiotic. Strains 27 and 35 among the L. salivarius subsp. salivarius showed the highest resistance to the antibiotics. Purified ${\alpha}$-galactosidase was obtained by DEAE-Sephadex A-50 ion exchange chromatography, Mono-Q ion exchange chromatography and HPLC column chromatography from L. salivarius subsp. salivarius 27. The specific activity of the purified enzyme was 8,994 units/mg protein, representing an 17.09 folds purification of the original cell crude extract. The molecular weight of enzyme was identified about 53,000 dalton by 12% SDS-PAGE. Optimal temperature and pH for activity of this enzyme were $40^{\circ}C$ and 7.0 respectively. The enzyme was found to be stable between 25 and $50^{\circ}C$. ${\alpha}$-galactosidase activity was lost rapidly below pH 5.0 and above pH 9.0. This enzyme was liberated galactose from melibiose, raffinose, and stachyose, and also the hydrolysis rate of substrate was compound by HPLC. These results indicated that some of the L. salivarius subsp. salivarius (strain 27 and 35) are considered as effective probiotic strains with a potential for industrial applications, but the further study is needed to establish their use as probiotics in vivo.

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