• Title/Summary/Keyword: Molecular characterization

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Purification and Characterization of Polyphenol Oxidase from Flammulina velutipes (팽나무버섯 polyphenol oxidase의 정제 및 특성)

  • Pyo, Han-Jong;Son, Dae-Yeul;Lee, Chan
    • Korean Journal of Food Science and Technology
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    • v.34 no.4
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    • pp.552-558
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    • 2002
  • Polyphenol oxidase from Flammulina velutipes was purified and characterized. Purification of polyphenol oxidase was achieved by ammonium sulfate precipitation, Superdex G-200 gel filtration chromatography, Phenyl superose affinity chromatography, Mono-Q anion exchange chromatography and Superdex S-200 gel filtration chromatography on FPLC. After these purification steps specific activity of purified polyphenol oxidase increased to 199.1 units/mg. Polyphenol oxidase from F. velutipes was composed of a single polypeptide with molecular weight of about 40 kDa. Optimum pH and temperature for the enzyme reaction were found to be 6.0 and $25^{\circ}C$, respectively. The activity of the enzyme gradually decreased at acidic pH between 3 and 5, and the enzyme lost its activity at alkaline pH between 8 and 10. This enzyme exhibited high substrate specificity to o-diphenols. Km-values for L-DOPA and caffeic acid were found to be 3.97 mM and 1.78 mM, respectively. 2-mercaptoethanol, L-ascorbic acid, sodium bisulfite, EDTA and $Mg^{2+}$ inhibited the activity of pholyphenol oxidase and $Cu^{2+}$, $Fe^{2+}$, $Zn^{2+}$ and $Ni^{2+}$ increased enzyme activity. The activity of enzyme was well maintained at $-70^{\circ}C$ for over 4 months, and at $-20^{\circ}C$ for 1 months.

Production of Cyclodextrin Glucanotransferase from Aspergillus sp. CC-2-1 and its Characterization (Aspergillus sp. CC-2-1에 의해 생산되는 Cyclodextrin Glucanotransferase의 생산 및 특성)

  • Cho, Young-Je;Kim, Myoung-Uk
    • Korean Journal of Food Science and Technology
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    • v.32 no.5
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    • pp.1158-1167
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    • 2000
  • To produce ${\beta}-cyclodextrin({\beta}-CD)$, a cyclodextrin glucanotransferase(CGTase) producing Aspergillus sp. CC-2-1 was isolated from soil. The enzyme was purified and its enzymological characteristics were investigated. It was found that production of CGTase reached to the maximum when the wheat bran medium containing 0.1% albumin, 2% $(NH_4)_2S_2O_8$, 2% soluble starch and 0.2% $KH_2PO_4$ was cultured for 5 days at $37^{\circ}C$. The purity of CGTase was increased by 13.14 folds after DEAE-cellulose ion exchange chromatography and Sephadex G-100, G-150 gel filtration and the specific activity was 172.14 unit/mg. Purified enzyme was confirmed as a single band by the polyacrylamide gel electrophoresis. The molecular weight of CGTase was estimated to be 27,800 by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the CGTase activity were 9.0 and $80^{\circ}C$, respectively. The enzyme was stable in pH $8.0{\sim}11.0$ at $60{\sim}80^{\circ}C$. The activity of purified enzyme was activated by $K^+,\;Cu^{2+}$ and $Zn^{2+}$. The activity of the CGTase was inhibited by the treatment with 2,4-dinitrophenol and iodine. The result suggests that the purified enzyme has phenolic hydroxyl group of tyrosine, histidine imidazole group and terminal amino group at active site. The reaction of this enzyme followed typical Michaelis-Menten kinetics with the $K_m$ value of 18.182 g/L with the $V_{max}$ of 188.68 ${\mu}mole/min$. The activation energy for the CGTase was calculated by Arrhenius equation was 1.548 kcal/mol.

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Extraction and Physicochemical Characterization of Barley Bran $\beta$-glucan (보리겨 $\beta$-glucan의 추출 및 이화학적 특성)

  • 김선영;유정희
    • Korean journal of food and cookery science
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    • v.19 no.5
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    • pp.616-623
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    • 2003
  • Waxy barley brans were collected during the pearling process. The extraction of $\beta$-glucan from barley bran was effected by the extraction conditions. The $\beta$-glucan content increased with temperature, but not with pH. The highest yield, 6.5%, was achieved at pH 7.0 and 55$^{\circ}C$. At pH 10 and 45$^{\circ}C$, 48.5% of the $\beta$-glucan in barley bran was recovered in the gum product, with 54.6% purity. The protein and starch contaminations were high, reaching 13.6 and 23.7%, respectively. The $\beta$-glucan content was greatest in the subaleurone and aleurone regions (bran fractions 1, 2, 3 and 4), and declined considerably toward the inner layers. A monosaccharide analysis of the purified, $\beta$-glucan, from bran fractions 1, 2, 3 and 4, indicated that glucose constituted the majority of the gum. The small amounts of the arabinose and xylose found in the gum may indicate the presence of arabinoxylans as minor constituents. The molecular weights of the $\beta$-glucans isolated from bran fractions 1,2 and 3 were found to be 4.09${\times}$10$^{5}$ ∼-4.41${\times}$10$^{5}$ . The major glycosidic linkages of the $\beta$-glucans demonstrated the presence of 2, 4, 6-Me-Glc and 2, 3, 6-Me-Glc. When flow behaviors of barley bran $\beta$-glucan were examined, $\beta$-glucan exhibited pseudoplastic fluid properties.

Isolation and Characterization of Helicobacter pylori Urease Inhibitor from Rubus coreanus Miquel (복분자(Rubus coreanus Miquel)로부터 Helicobacter pylori Urease Inhibitor의 분리 및 특성)

  • 양성우;호진녕;이유현;신동훈;홍범식;조홍연
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.33 no.5
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    • pp.769-777
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    • 2004
  • A Helicobacter pylori urease inhibitor from Rubus coreanus Miquel has been isolated and partially characterized for aiming to Prevent H. pylori growth and decrease harmful accumulation of ammonia in human gastric mucosa. We screened urease inhibitory activities in 519 extracts library prepared by solvent extraction from 173 kinds of edible plants, medicinal herbs, herbs and seaweeds using a colorimetric urease assay system. As results of primary and secondary screening, 70% acetone extract of Rubus coreanus Miquel was selected as potent candidate, showing about 24% inhibitory activity. The acetone extract was sequentially partitioned into RCE/RCWI and RCB/RCW2 layers with ethyl acetate and butanol. The major active component in RCW2, water layer from butanol fractionation was revealed to be peptidic or proteinous substance by inhibitory activity determination after pronase digestion and periodate oxidation. RCW2-IIIc a was isolated by sequential column chromatography on DEAE-Toyopearl 650C, Butrl-Toyopearl 650M and Sephadex LH-20. The isolated urease inhibitor RCW2-IIIc $\alpha$, was highly pure proteinous substance with molecular weight of 13kDa by high-performance gel permeation liquid chromatography. RCW2-IIIc$\alpha$ has about 5 times higher inhibitory activity than 70% acetone extract, showing high stability against heat treatment and peptic digestion.

Identification, sequence characterization and expression analysis of the arginine kinase gene in response to laminarin challenge from the Oriental land snail, Nesiohelix samarangae (동양달팽이(Nesiohelix samarangae)의 arginine kinase 유전자 분석 및 발현 패턴에 관한 연구)

  • Jeong, Ji Eun;Lee, Yong Seok
    • The Korean Journal of Malacology
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    • v.29 no.3
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    • pp.171-179
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    • 2013
  • Arginine kinase (ArK) is known to play an important role in most invertebrates the level of ATP by phosphorylation of phosphagens in cell and immuninty in living organisms. ArK has been identified in many kinds of organisms ranging from invertebrate to vertebrate. However, no ArK gene has been cloned and investigated from N. samarangae. This leads us to identify ArK cDNA (NsArK) from the expressed sequence tag (EST) sequencing of N. samarangae. Sequence analysis indicated that the coding region of 1,065 bp contains 355 amino acid residues. Molecular phylogenetic analysis shows that NsArK had very high similarities with mollusca and arthropoda. In an attempt to investigate a potential role of NsArK in the digestive gland of N. samarangae, expression patterns were analyzed. RT-PCR analsysis shows that NsArK mRNA is induced in the rane of 1.2 fold at 6 hr by laminarin when compared with the control. The immunnologial and physiological role of NsArK remains to be further investigated in N. samarangae.

Synthesis and Characterization of Polyethylenimine-conjugated Polydiacetylene Liposome as a Gene Delivery Carrier (폴리디아세틸렌 리포좀 표면에 저분자량의 폴리에틸렌이민을 연결한 새로운 유전자 전달체 합성 및 특징 연구)

  • Lee, Young Hwa;Yim, Kang Hyuck;Heo, Jungseok;Choi, Joon Sig
    • Polymer(Korea)
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    • v.38 no.1
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    • pp.43-48
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    • 2014
  • In this paper, we made a new polycationic polymeric liposome composed of low molecular weight polyethylenimine (PEI) and 10,12-pentacosadiynoic acid (PCDA). PCDA liposome was prepared by ultraviolet irradiation. PEI was further conjugated on the surface of the polymerized PCDA liposome using coupling reagents to make PCDA-PEI. The blue-to-red transition of PCDA liposome was observed during the coupling reaction. The size distribution of liposome and complexes with plasmid DNA was measured by dynamic light scattering (DLS). The complex formation was also identified by agarose gel electrophoresis and PicoGreen reagent assay. We confirmed the complex formation of the polymeric liposome with DNA and then performed transfection and cytotoxicity assay in HEK 293 and HeLa cells. As a result, PCDA-PEI showed significant gene transfection efficiency and low cytotoxicity. This study shows that PEI-conjugated PCDA liposome could be an efficient gene or drug delivery carrier.

Preparation and Characterization of Nylon 6,5 Copolymers from ε-Caprolactam and 2-Piperidone (ε-Caprolactam과 2-Piperidone으로부터 나일론 6,5 공중합체 제조 및 특성평가)

  • Kim, Hye Young;Goh, Jin-Seon;Ryu, Mi Hee;Kim, Dae Su;Song, Bong-Keun;Lee, Seung Hwan;Park, Si-Jae;Jegal, Jonggeon
    • Polymer(Korea)
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    • v.38 no.1
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    • pp.31-37
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    • 2014
  • To prepare biomass based nylon 6,5 copolymers, ${\varepsilon}$-caprolactam and 2-piperidone, the monomers of nylon 6,5 copolymers, were synthesized respectively from lysine and 5-aminovaleric acid which were produced from glucose by the fermentation process. The copolymers were then polymerized by the anionic ring opening polymerization of them at $40^{\circ}C$, using potassium tert-butoxide as a catalyst and acetyl-2-caprolactam and carbon dioxide as initiators. The prepared copolymers were characterized with various analytical methods: their viscosity molecular weight ($M_{\eta}$) was as high as 30000 g/mol and polymerization yield was over 50%, and it was found that they were semi-crystalline polymers having melting point at $165^{\circ}C$ which was much lower than its thermal degradation point, $250^{\circ}C$. These polymers were expected to have good thermal processability and application fields.

Manufacture and Characterization of Silkworm Gland Hydrolysate (누에 실샘 가수분해물의 제조 및 특성 규명)

  • Hwang, Jung Wook;Lee, Heui Sam;Kim, Hojin;Kim, Kyu-Oh;Choi, Yong-Soo
    • Journal of Sericultural and Entomological Science
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    • v.50 no.2
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    • pp.76-81
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    • 2012
  • Silk protein has been explored to be used for biomedical applications for several decades. However, it has not been used in this field cause to their irreversible crystallization after dissolving in water. The existing methods of silk protein hydrolysis using silkworm cocoon were used with harmful solvents and through a very complicated process. Therefore, we have developed novel methods for the production of water-soluble hydrolysate using silkworm gland. We manufactured two types of silkworm gland-derived hydrolysate (water-soluble SGH, SSGH; total SGH, TSGH) and compared the characteristics with commercial cocoon-derived sericin hydrolysate (CSH). The molecular weight of SGH ranged from 7 to 50 kDa (SSGH) and 5 to 15 kDa (TSGH) within glycine, alanine, and aspartic acid as a main amino acid composition. In contrast, CSH ranged from 15 to 50 kDa within serine and aspartic acid. The results of FTIR implied that SGH was more soluble form than CSH, as shown by the decrease in the ${\beta}$-sheet structure at $1630cm^{-1}$ on amide I peak. In comparison with 10% fetal bovine serum, 0.1% (1 mg/ml) SSGH had equivalent effect on the proliferation of human dermal fibroblasts and mesenchymal stem cells. All results of the SSGH made by novel manufacturing process indicate the SSGH is more preferable as a culture medium supplement than cocoon-derived sericin.

High Voltage β-Ga2O3 Power Metal-Oxide-Semiconductor Field-Effect Transistors (고전압 β-산화갈륨(β-Ga2O3) 전력 MOSFETs)

  • Mun, Jae-Kyoung;Cho, Kyujun;Chang, Woojin;Lee, Hyungseok;Bae, Sungbum;Kim, Jeongjin;Sung, Hokun
    • Journal of the Korean Institute of Electrical and Electronic Material Engineers
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    • v.32 no.3
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    • pp.201-206
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    • 2019
  • This report constitutes the first demonstration in Korea of single-crystal lateral gallium oxide ($Ga_2O_3$) as a metal-oxide-semiconductor field-effect-transistor (MOSFET), with a breakdown voltage in excess of 480 V. A Si-doped channel layer was grown on a Fe-doped semi-insulating ${\beta}-Ga_2O_3$ (010) substrate by molecular beam epitaxy. The single-crystal substrate was grown by the edge-defined film-fed growth method and wafered to a size of $10{\times}15mm^2$. Although we fabricated several types of power devices using the same process, we only report the characterization of a finger-type MOSFET with a gate length ($L_g$) of $2{\mu}m$ and a gate-drain spacing ($L_{gd}$) of $5{\mu}m$. The MOSFET showed a favorable drain current modulation according to the gate voltage swing. A complete drain current pinch-off feature was also obtained for $V_{gs}<-6V$, and the three-terminal off-state breakdown voltage was over 482 V in a $L_{gd}=5{\mu}m$ device measured in Fluorinert ambient at $V_{gs}=-10V$. A low drain leakage current of 4.7 nA at the off-state led to a high on/off drain current ratio of approximately $5.3{\times}10^5$. These device characteristics indicate the promising potential of $Ga_2O_3$-based electrical devices for next-generation high-power device applications, such as electrical autonomous vehicles, railroads, photovoltaics, renewable energy, and industry.

Five Newly Collected Turnip Mosaic Virus (TuMV) Isolates from Jeju Island, Korea are Closely Related to Previously Reported Korean TuMV Isolates but Show Distinctive Symptom Development

  • Hu, Wen-Xing;Kim, Byoung-Jo;Kwak, Younghwan;Seo, Eun-Young;Kim, Jung-Kyu;Han, Jae-Yeong;Kim, Ik-Hyun;Lim, Yong Pyo;Cho, In-Sook;Domier, Leslie L;Hammond, John;Lim, Hyoun-Sub
    • The Plant Pathology Journal
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    • v.35 no.4
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    • pp.381-388
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    • 2019
  • For several years, temperatures in the Korean peninsula have gradually increased due to climate change, resulting in a changing environment for growth of crops and vegetables. An associated consequence is that emerging species of insect vector have caused increased viral transmission. In Jeju Island, Korea, occurrences of viral disease have increased. Here, we report characterization of five newly collected turnip mosaic virus (TuMV) isolates named KBJ1, KBJ2, KBJ3, KBJ4 and KBJ5 from a survey on Jeju Island in 2017. Full-length cDNAs of each isolate were cloned into the pJY vector downstream of cauliflower mosaic virus 35S and bacteriophage T7 RNA polymerase promoters. Their fulllength sequences share 98.9-99.9% nucleotide sequence identity and were most closely related to previously reported Korean TuMV isolates. All isolates belonged to the BR group and infected both Chinese cabbage and radish. Four isolates induced very mild symptoms in Nicotiana benthamiana but KBJ5 induced a hypersensitive response. Symptom differences may result from three amino acid differences uniquely present in KBJ5; Gly(382)Asp, Ile(891)Val, and Lys(2522)Glu in P1, P3, and NIb, respectively.