• Title/Summary/Keyword: isolation and purification

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Isolation of High-Quality mRNA from Tannin-Rich Persimmon Fruit (고 Tannin 함유 감과실로 부터 mRNA의 분리)

  • ;Dav
    • Food Science and Preservation
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    • v.4 no.1
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    • pp.45-51
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    • 1997
  • In our studies on the role of $\beta$-galactosidase in fruit softening, significant difficulty, was encountered in our attempts to extract RNA from persimmon(Diospyros kaki L. cv. Fuyu) fruit due to astringency and tannin content. Initial, unsuccessful RNA extractions involved methods using guanidinium isothiocyanate/CsCl with and without polyvinylpyrrolidone(PVP), phenol/sodium lauryl sulfate(SDS), guanidinium hydrochloride, as well as polysomal RNA purification method that used 0.2 M Tris-HCI (pH 9.0) containing KCI, Mg-acetate, EDTA, $\beta$-mercaptoethanol, and sucrose. A method was devised which employed treatment of fruit with CO2 gas to diminish astringency prior to RNA extraction, followed by extraction of tissue powders with Proteinase K extraction buffer containing PVP and ascorbate at an alkaline pH. This procedure resulted in the removal of tannins and other polyphenolics and extraction of relatively large amount of high-quality RNA suitable for cDNA library construction and polymerase chain reaction(PCR). Futhermore, the procedure does not use the toxic and corrosive chemical guanidinium isothiocyanate or require ultracentrifugation.

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Isolation and Characterization of Dark Brownish Pigments from Fruit Body of Auricularia auricula (흑목이 버섯 자실체의 흑갈색 색소 동정 및 특성)

  • Kim, Hyeon-Min;Hur, Won;Lee, Shin-Young
    • Food Engineering Progress
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    • v.13 no.4
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    • pp.282-288
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    • 2009
  • A dark brownish pigment from fruit body of Auricularia auricula was isolated and characterized in this report. The pigment was obtained with a yield of 0.61%(w/w) by alkaline extraction and subsequent purification steps. It showed the positive $FeCl_3$ test which was the indication of phenolic compounds. A synthetic melanin showed a similar spectrometric characteristics to the pigment extract regarding a characteristic UV absorption between 200-250 nm and infrared absorptions profiles in the finger print region including absorption peaks at 1701 and 1624 $cm^{-1}$. Its element analysis indicated that its atomic copmposition is close to that of DOPA melanin (eumelanin). With the result of its antioxidant activity in the TNBT (5-thio-2-nitrobenzoic acid) assay, we concluded that the dark brownish pigment from A. auricula is a melanin-like compound having a powerful antioxidative activity.

Isolation and Evaluation of an Antitumor Constituent from Pyrolae Herba (녹수초의 항암활성물질의 분리 및 항암력 평가)

  • Bae, Ki-Hwan;Kim, Hwan-Mook;Lee, Sang-Myung
    • YAKHAK HOEJI
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    • v.40 no.2
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    • pp.225-229
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    • 1996
  • The cytotoixic effect of Pylorae Herba (Pyrola japonica Klenze) against L1210 and K-562 cells was studied in vitro. The methanolic extract of Pylorae Radix was add ed to the culture of L1210 cells and K-562 cells for the cytotoxic activity and the ED50 values of hexane, ethylacetate, buthanol and water fractions from methanolic extract were determined using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) assay. The active constituent isolated by bioassay guided fractionation followed by purification gave rise to a yellow needle crystal and was clarified to be chimaphilline by the comparison with the published data. The average life spans with it were not prolonged significantly on tumor growth in hybrid female mouse (BDF1-KIST) inoculated subcutaneously with P388 cells.

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Isolation of a Pseudomonas aeruginosa Strain Producing an Extracellular Alkaline Lipase Catabolitely Regulated by Glucose, and Purification of the Lipase (포도당에 의해 합성억제되는 알카리성 Lipase를 생성분비하는 Pseudomonas aeruginosa 균주의 분리 및 이 효소의 정제)

  • 이정미;김란숙;김병오;박영덕;진익렬
    • Microbiology and Biotechnology Letters
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    • v.21 no.3
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    • pp.239-246
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    • 1993
  • Producing an extracellular alkaline lipase, this isolate JM123 was identified as a Pseudomonas aeruginosa strain from the results of the analyses of its morphological, biochemical and physiological properties. This strain showed the highest productivity of alkaline lipase when grown at pH 9.0 and 30C for 13-20 hours in the medium of 2% starch, 1% soytone, 0.5% peptone and 1% MgSO4.7H2O. However, this enzyme was greatly repressed when grown in the glucose containing medium. The culture broth was fractionated by the order of the ammonium sulfate precipitation, Sephadex G-200 gel filtration, DEAE-cellulose column chromatography, and Sephadex G-150 gel filtration.

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Isolation of Lipoxyeenase Isozymes from Soybean Seeds (대두 Lipoxygenase 이성효소의 분리)

  • Song, Young-Sun
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.17 no.3
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    • pp.185-190
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    • 1988
  • Soybean lipoxyeenase isozymes were isolated from acetone-defatted soybean seeds(Glycine max [L.] Merr. variety AmSoy) by ammonium sulfate fractionation, eel filtration, and ion exchange chromatoeraphy. The final preparation of lipoxygenase-1 and -2 obtained was 19- and 32-fold purified, respectively, to the crude extract. But a considerable loss of total enzyme activity occurred during purification. On 7% polyacrylamide gel electrophosis at pH 9.0, employing lipoxigenase specific staining technique, lipoxyeenase-1, -2, and -3 showed distinctive Rf values of 0.38, 0.29, and 0.33, respectively.

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Analysis of Basic Constituents in Korean Earthworm About Lamnodrilus gotai Hatai (한국산 토룡의 기초성분 분석 -Lamnodrilus gotai Hatai를 대상으로-)

  • Chung, Yong;Lee, Yun-Sil;Lee, Hyo-Min
    • YAKHAK HOEJI
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    • v.31 no.4
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    • pp.224-229
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    • 1987
  • The earthworm(Annelid) is a herbicine which has traditionally been used in the treatment of infectious fever, jaundice, infection of middle ear, laryngitis, pharyngitis, nephritis, headache, toothache and certain urinary tract infections from the olden times. Before the isolation and purification of biologically active components we analyzed the basic constituents(proteins, amino acids, mineral, etc.) with lyophilized powder of Lamnodrilus gotai Hatai. The results were as follows: Minerals detected and quantitatively analyzed were $Ca^#$, $Mg^#$, Fe, Zn, Mn, Cu, Co, Cr, Ni, Ge and Se. Amino acids detected were alanine, phenylalanine, leucine, glutamic acid, tyrosine, threonine, arginine, aspartic acid, methionine, lysine, serine, histidine, isoleucine, glycine, proline and etc. The constituents of proteins, fat, fiber, ash and phosphorous were measured. These constituents were compared and discussed with those of other investigations.

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Isolation and Partial Chemical Characterization of the Yolk Proteins from Drosophila sp. (robusta species group) (Drosophila sp.(robusta species group)의 난황 단백질의 분리 및 부분적 화학적 특성)

  • Kim, Se-Jae;Gi
    • The Korean Journal of Zoology
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    • v.35 no.1
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    • pp.17-22
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    • 1992
  • The three yolk polypeptides have been isolated and partially characterized. Their molecular weights of YPI, YP2, and YP3 were 48, 000, 47, 000, and 46, 000, respectivelv, as judged by SDS-polyacrvlamide gel electrophoresis. They have different digestion products upon in situ peptide mapping by limited proteolysis. Two-dimensional gel electrophoresis showed that their isoelectric points were heterogeneous from 5.92 to 6.54. And thew showed three different antigen-antibody reactions when each polvpeptides is reacted with antisera made to a mixture of all of three. These data reported here indicate that the yolk proteins are consisted of distinctive polypeptides in Drosophlla sp. (robusta species group).

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Studies on Factors Affecting Isolation and Fusion of Protoplasts of Quercus Species (참나무류(類)의 원형질체(原形質體) 분리(分離) 및 융합(融合)에 영향(影響)을 끼치는 요인(要因)에 관한 연구(硏究))

  • Hyun, Jung Oh;Kim, Jae Hun;Chang, Suk So
    • Journal of Korean Society of Forest Science
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    • v.71 no.1
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    • pp.66-73
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    • 1985
  • Factors affecting isolation and fusion of protoplasts of three Quercus species were investigated and procedures for isolation, purification and fusion of protoplasts of the three species were also established. Unhardened leaves and rapidly growing callus cultures were good source of viable protoplasts. The optimum composition of enzyme mixture for rapid isolation of protoplasts from leaf mesophyll tissues and calli was Cellulase Onozuka R-10 (20g/l, Macerozyme R-10(10g/l), Pectinase(250 units/l, $CaCl_2$, $2H_2O$(14mM), $MgSO_4{\cdot}7H_2O$(1.8mM), $KNO_3$(1.0mM), $H_3BO_3$(1.0mM), $KH_2PO_4$(0.2mM), KI($1.0{\mu}M$), 1,4-dithiothreitol (0.1mM) and mannitol (0.6M). Optimum density of protoplasts for maximum fusion was $2{\times}10^5/ml$ which was the highest protoplast density given in this study. Optimum concentration and duration of PEG 1450 treatment for inducing fusion appeared to be 29%(W/V) final PEG 1450 concentration and 5-10 minutes, respectively.

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Studies on Lectins From Marine Shells (V) - Isolation and Purification of Letin from Tapes philippinarum. (해양 패류 렉틴성분 연구 (V) - 반지락조개의 렉틴성분 분리정제에 관한 연구)

  • 정시련;김장환;전경희
    • YAKHAK HOEJI
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    • v.31 no.2
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    • pp.52-59
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    • 1987
  • The result of the screening of lectins on 28 species of marine shell fishes(mollusks) showed that 10 species (Tapes philippinarum, Cyclosunetta menstrualis, Neptunea arthritica cumingii, Omphalius pfeifferi carpenteri, Chlorostoma argyrostoma turbinatum, Chiorostoma argyrostoma lischkei, Semisulcosira corea, Neptunea polycosta, Babylonica japonica, Noverita didyma) were present hemagglutinating properties to human A, B, and O group, and animal blood erythrocytes. A new lectin from Tapes philippinarum. was isolated and partially characterized. The lectin was purified by (NH$_4$)$_2$SO$_4$ precipitation and ion exchange chromatography on DE 53 column. Six fractions were obtained from DE 53 column by salt gradient elution but only 0.3M, and 0.4M NaCl fraction had strong lectin activity. On its 0.3M NaCl fraction, purity was identified by polyacrylamide gel electrophoresis. This lectin was inhibited by N-acetyl-D-galactosamine. It seems that two kinds of lectin react as antigen by immunochemical studies.

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Purification and cDNA Cloning of Insect Defensin from Lepidopteran Lavae, Galleria mellonella

  • Jeong, Woo-Hyuk;Yun, Eun-Kyung;Lee, Young-Shin;Kim, Iksoo;Ryu, Kang-Sun;Lee, In-Hee
    • Proceedings of the Korean Society of Sericultural Science Conference
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    • 2003.04a
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    • pp.76-76
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    • 2003
  • Here we report an antifungal peptide isolation from G. mellonella larvae. The peptide shows a high degree of sequence homology to an insect defensin, named heliomicin, first reported in Lepidoptera. The peptide was purified by a three-step procedure consisting of acid extraction, gel permeation chromatography and reversed-phase HPLC. First the N-terminal amino acid sequence of the purified peptide was determined by automated Edman degradation. (omitted)

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