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Pseudomonas putida 의 Protocatechuate 경로에 관여하는 초기 효소들의 유전자의 클로닝 및 염기서열
김재호(Jae Ho Kim),홍범식(Bum Shik Hong),신동훈(Dong Hoon Shin) 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.39 No.6
The major portions of two DNA fragments, one from degradative plasmid, pRA4000 from Pseudomonas putida NCIMB 9866, and the other from degradative plasmid, pRA500 from P. putida NCIMB 9869, which harbor the structural genes for the flavoprotein (pchF) and cytochrome (pchC) subunits of p-cresol methylhydroxylase (PCMH), have been sequenced. The DNA and deduced amino acid sequences for pchC and pchF have been published. In these fragments, a coding region (dhal) for an aldehyde dehydrogenase has been identified. It is proposed that this gene encodes for the aldehyde dehydrogenase which converts p-hydroxybenzyaldehyde to p-hydroxybenzoate. p-Hydroxybezealdehyde is the product of oxidation of p-cresol by PCMH. The fragment from P. putida 9869 also harbors the genes for the α (pcaG) and β (pcaH) subunits of protocatechuate 3,4-dioxigenase. The fragment from 9866 does not have any portion of these genes in the corresponding region A possible open reading frame (ORF) between pchC and pchF is seen for both clones, and a second putative open reading frame (ORF`) also exists in the 9866 clone. The gene organizations are dhal-pchC-ORF-pchF-pcaGH for the DNA fragment from 9869, and ORF`-dhal-pchC-ORF-pchF for the DNA fragment from 9866.
감잎(Diospyos kaki L.)으로부터 정제한 보체계 활성화 다당류
정영주,전혁,김경임,안정희,신동훈,홍범식,조홍연,양한철,Jung, Yung-Joo,Chun, Hyug,Kim, Kyung-Im,An, Jeung-Hee,Shin, Dong-Hoon,Hong, Bum-Shik,Cho, Hong-Yon,Yang, Han-Chul 한국식품과학회 2002 한국식품과학회지 Vol.34 No.5
감잎으로부터 항보체 활성물질을 분리정제하기 위해 감잎 (250 g)을 $100^{\circ}C$에서 3시간 동안 열수 추출하고 분자량 10 kDa membrane을 사용하여 농축한 후 ethanol 침전과 methanol 추출을 통해 조다당류(DKC)를 얻었다. 조다당류의 정제는 DEAE-Toyopearl 650C와 Bio-gel P60을 사용하여 실시하였다. 정제된 DKC-1c는 $1000\;{\mu}g/mL$의 농도에서 고전경로를 통해서는 85.4% 활성화시켰고 부경로에서는 65.1% 활성화시켰다. 정제 다당류 DKC-1c는 분자량은 66.6 kDa이고 정제도가 높은 중성다당류로써 주요 구성당은 glucose(29.0 mol.%), arabinose(24.3 mol.%), galactose(16.2 mol.%) 순으로 검출되었다. 면역전기영동을 통하여 확인한 결과 DKC-1c는 C3를 부경로에서도 C3a와 C3b로 활성화시키는 complement activator임이 확인되었다. Cold and hot water fractions of Diospyros kaki were screened to determine its anti-complementary activity. Flour of Diospyros kaki leaf (250 g) was boiled at $100^{\circ}C$ for 3 h and passed through a membrane of 10 kDa molecular weight (DK-0). DK-0 was precipitated with ethanol and refluxed with methanol to obtain the crude polysaccharide (DKC). DKC-1 was isolated by ion exchange chromatography on DEAE-Toyopearl 650C, and DKC-1c was purified from DKC-1 by size exclusion chromatography on Bio gel P-60. The anti-complementary activities of DKC-1c at $1000\;{\mu}g/mL$ were 85.4 and 61.1% via whole and alternative pathways, respectively. DKC-1c was determined as a neutral polysaccharide composed of glucose (29.0 mol.%), arabinose (24.3 mol.%), and galactose (16.2 mol.%) with the molecular weight of 66.6 kDa. Results of agarose gel immunoelectrophoresis revealed DKC-1c, as a complement activator, cleaved C3 into C3a and C3b via both pathways.
김으로부터 분리한 Angiotensin - I Converting Enzyme 저해제의 정제 및 특성
최수진(Soo-Jin Choi),전우진(Woo-Jin Jun),유광원(Kwang-Won Yu),신동훈(Dong-Hoon Shin),홍범식(Bum-Shik Hong),조홍연(Hong-Yon Cho),양한철(Han-Chul Yang) 한국식품영양과학회 2000 한국식품영양과학회지 Vol.29 No.4
본 연구는 70 여종의 국내산 해조류중 가장 높은 ACE저해 활성을 보였던 김(Porphyra yezoensis, 서천)의 산가수분해물로부터 ACE 활성 저해 펩타이드를 분리하여 그 특성을 조사하였다. ACE 저해물질의 분획은 균일하게 파쇄한 김을 2.5 N HCl로 산 가수분해한 후 중화하여 한외여과로 분자크기 3 kDa이하의 물질로 분리하였다. 분자크기 3 kDa이하의 물질에 대하여 column chromatography(Amberlite XAD 8, DEAE-Toyopearl, Sephadex LH-20)와 reverse phase HPLC(C_(18))를 순차적으로 수행하여 ACE 저해제인 PY30-II-b-h5물질을 분리하였다. PY30-II-b-h5는 분자크기는 약 580 dalton으로 glycine(24.5%), arginine(56.8%), proline(18.8%)의 아미노산 조성을 갖는 저분자 펩타이드였으며, ACE의 저해양상은 경쟁적 저해작용을 하였고, IC50 값은 10.6 μg/mL이었다. This study focused on the purification and characterization of ACE inhibitor from Porphyra yezoensis. The dried Porphyra yezoensis was ground and hydrolyzed with 2.5 N HCl, followed by neutralization and centrifugation. Then, the subsequential purification of ACE inhibitor was carried out by Amberlite XAD 8, DEAE-Toyopearl 650C, Sephadex LH-20 column chromatography and reverse phase HPLC with C_(18) column. The purified ACE inhibitor was peptide which consisted of glycine (24.5%), arginine (56.8%) and proline (18.8%). Also, it showed the competitive inhibition pattern to ACE. The apparent molecular mass of purified peptide was 580 dalton, and an IC50 value of ACE inhibitor was 10.6 μg.
복분자(Rubus coreanus Miquel)로부터 Helicobacter pylori Urease Inhibitor의 분리 및 특성
양성우(Sung Woo Yang),호진녕(Jin Nyoung Ho),이유현(Yoo Hyun Lee),신동훈(Dong Hoon Shin),홍범식(Bum Shik Hong),조홍연(Hong Yon Cho) 한국식품영양과학회 2004 한국식품영양과학회지 Vol.33 No.5
위염과 위궤양의 일차적 발병인자로 알려진 Helicobacter pylori의 생육을 억제하고 urease 산물인 암모니아의 축적에 의한 위점막 손상을 완화시킬 목적으로 복분자로부터 H.pylori urease 저해물질을 분리정제하고 소재화와 관련된 일부 성질을 규명하였다. 양념채소류, 차류, 죽류, 건강채소류 등의 식용식물, 약용식물, 허브 및 해조류, 총 173종으로부터 극성도에 따라 계통추출한 수용성인 냉수추출물(Fr. 1), methanol 추출물(Fr. 4), 열수추출물(Fr. 5) 519점을 대상으로 H.pylori urease 저해활성을 검색하였다. 1차 및 2차 저해활성 검색 결과 복분자의 70% acetone추출물이 약 24%의 가장 높은 저해활성을 나타내었으며, 이 추출물을 ethyl acetate와 butanol을 사용하여 ethyl acetate/DW layer(RCE/RCW1)와 butanol/DW layer(RCB/RCW2)로 순차 분획한 후 활성획분인 RCW2 내의 활성본체를 확인할 목적으로 periodate oxidation과 pronase digestion을 실시한 결과 펩타이드 또는 단백질성 물질로 판명되었다. 저해활성물질은 DEAE-Toyopearl 650C, Butyl-Toyopearl 650M 및 Sephadex LH-20 순의 column chromatography에 의해 분리정제되었다. 분리urease 저해물질, RCW2-Ⅲcα는 HPLC의 gel permeation chromatography에 의해 비교적 순도 높은 분자량 약 13 kDa의 단일 물질임이 확인되었다. 저해활성물질은 열에 안정성을 보이는 내열성 단백질임을 알 수 있었고 위내 단백분해효소인 pepsin에도 가수분해 저항성을 나타냄으로써 기능성식품의 소재로 높은 소재화 적성을 보였다. 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/RCW1 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 α was isolated by sequential column chromatography on DEAE-Toyopearl 650C, Butyl-Toyopearl 650M and Sephadex LH-20. The isolated urease inhibitor, RCW2-Ⅲcα, was highly pure proteinous substance with molecular weight of 13 kDa by high-performance gel permeation liquid chromatography. RCW2-Ⅲcα has about 5 times higher inhibitory activity than 70% acetone extract, showing high stability against heat treatment and peptic digestion.