Streptomyces sp. YJB-599가 생산하는 Genistein의 분리 및 정제

함병권,배동훈,유주현 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.3

토양으로부터 세포독성물질을 생산하는 strain No. 5-99 균주를 분리하였다. 본 균주의 세포벽 성분의 diaminopimelic acid(DAP)와 아미노산을 분석하여 본 결과, LL-DAP만을 함유하고 있었고, glycine이 검출된 것으로 보아 glycine으로 연결된 peptide를 가지는 peptidoglycan type A3이고 cell wall chemotype Ⅰ의 방선균인 Streptomyces sp.로 확인되었다. 따라서 본 균주를 Streptomyces sp. YJB-599라 명명하였다. 본 균주가 생산하는 활성물질은 용매추출 및 silica gel column chromatography를 이용하여 정제되었으며, 최종적으로 침상의 백색결정으로 분리하였다. 기기분석과 database로 구조분석을 행한 결과, 본 물질은 genistein으로 밝혀졌다. A cytotoxic material was produced by strain No. 5-99 which was isolated from soil. Analyzing the cell wall components, LL-diaminopimelic acid was identified. From the existance of glycine in the cell wall, this strain was identified to Streptomyces sp. which has cell wall chemotype I and peptidoglycan type A3 connected by glycine. So, we named this strain to Streptomyces sp. YJB-599. The Active material was purified through solvent extraction, silica gel column chromatography and crystallized to needle-shaped white crystal. Analyzing the structure of this crytal by instrumental analysis and database, it was determined to genistein.

Metarizium anisopliae (Metschn.) Sorok이 생산하는 Lactobacillus plantarum 용균효소의 분리, 정제 및 특성

류기철,함병권,백운하,유주현,배동훈 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.6

김치저장성을 향상시키기 위하여, Lactobacillus plantarum 용균효소 생산균주를 토양으로부터 분리한 후 그 효소를 정제하고 특성을 살펴보았다. 본 균주의 배양학적 및 형태학적 특성 등을 검토한 결과 Metarrisium anisopliae (Metschn). Sorok으로 동정되었으며, 본 효소는 affinity adsorption, CM-Sephadex C-50 column chromatography를 통하여 40%의 수율과 75배 정제도로 정제되었다. 효소활성의 최적 pH와 온도는 각각 4.0, 40℃였으며, pH 3.0~9.0, 60℃까지 효소의 안정성이 유지되었다. SDS-PAGE 및 gel filtration으로부터 본 효소의 분자량은 40,000 daltons인 monomeric protein임을 확인하였다. 본 효소는 Lactobacillus plantarum 세포벽 peptidoglycan의 peptide linkage를 절단 하는 endopeptidase였으며, 용균 spectrum으로부터 김치발효시 유용균인 Leuconostoc mesenteroides에 대해서는 활성을 거의 나타내지 않음을 확인할 수 있었다. 본 효소의 활성은 Ag^+, N-bromosuccinimide (NBS)에 의해서 완전히 저해되었으며, NBS에 의해서 활성이 저해된다는 사실로부터 본 효소는 활성에 tryptophan residue가 관여하는 것으로 확인되었다. 본 효소는 산성 아미노산의 함량이 염기성 아미노산의 함량에 비하여 높았으며, alanine, glycine, proline, tyrosine의 함량 또한 매우 높았다. To improve the preservation of Kimchi, we isolated Lactobacillus plantarum lytic enzyme-producing strain from soil, and the enzyme was purified and characterized. From the observation of cultural and morphological characteristics, the isolated strain was identified as Metarrisium anisopliae (Metschn). Sorok. The enzyme was purified to 75-folds with 40% yields through affinity adsorption and CM-Sephadex C-50 column chromatography. The optimum pH and temperature for lytic activity are 4.0 and 40℃, respectively, and the enzyme acitivity is stable between pH 3.0 and 9.0 and up to 50℃. The enzyme is a monomeric protein with molecular weight of 40,000 daltons by SDS-FAGE and gel filtration. The enzyme is endopeptidase which breaks the peptide linkage of Lactobacillus plantarum peptidoglycan. The lytic action spectra confirmed that Leuconostoc mesenteroides, a useful strain for the fermentation of Kimchi, is not lysed by the enzyme. The enzyme activity is inhibited by N-bromosuccinimide (NBS), which probably indicates the involvement of tryptophan residue in active site of the enzyme, and also inhibited by Ag^+. The amino acid composition analysis showed that the enzyme contains more acidic amino acids than basic ones, and composition of alanine, glycine, proline and tyrosines was very high.

Dicyma sp. YCH-37이 생산하는 효모세포벽 용해효소 : 1. 생산균주의 분리 및 효소의 정제 l. Isolation of the Strain and Purification of the Enzyme

정희철,함병권,배동훈,하세가와토루,유주현 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.4

효모세포벽 용해효소를 생산하는 균주를 토양으로부터 분리하였다. 본 균주에 대한 현미경 관찰 및 형태학적, 배양상의 특징을 검토한 결과 Dicyma sp.로 동정되어, 본 균주를 Dicyma sp YCH-37로 명명하였다. 세포에 아무런 처리도 가하지 않은 생균상태의 세균, 효모, 곰팡이에 대한 용균 spectrum을 측정한 결과, 세균 중에서는 Salmonella typhimurium을, 효모 중에서는 Torulopsis, Hansenula, Zygosaccharomyces rouxii 등을 잘 용해시켰으며, 가압증기살균 처리된 효모는 모두 용해시킬 수 있었다. 배양상등액을 yeast glucan을 이용한 affinity adsorption 및 DEAE-cellulose column chromatography로 정제한 결과, 13%의 수율과 204배의 정제도로 정제되었다. SDS-PAGE, gel filtration을 통해 분자량은 약 25,000 daltons임을 확인하였으며, 용균활성의 최적 pH와 온도는 각각 8.0과 50℃이고, pH 4.0~pH 10.0, 40℃까지 안정하였다. The strain YCH-37, which produces yeast cell wall lytic enzyme, was isolated from soil. From the microscopic observation, morphological and cultural characteristics, this strain was identified to fungus, Dicyma sp. So, we named this strain as Dicyma sp. YCH-37. The lytic enzyme effectvely lysed Salmonella typhimurium among intact living bacteria and Torulopsis, Hansenula, Zygosaccharomyces among intact living yeast, as well as autoclaved yeast strains. The yeast cell wall lytic enzyme was succesively purified to 204 folds with 13% yields through yeast glucan affinity adsorption and DEAE-cellulose column chromatography. The enzyme was identified to monomeric protein with molecular weight of 25,000 daltons from the results of SDS-PAGE and gel filtration. The optimum pH and temperature for the yeast lytic activity were 8.0 and 50℃, respectively. The enzyme was stale up to 40℃, and between pH 4.0~pH 10.0.

토양으로부터 Cellulose 분해효소를 생산하는 미생물의 분리,동정 및 최적배양조건의 결정

함병권(Byoung-Kwon Hahm),김윤근(Yoon-Keun Kim),유주현(Ju-Hyun Yu),배동훈(Dong-Hoon Bai) 한국식품과학회 1997 한국식품과학회지 Vol.29 No.5

Streptomyces sp. YBE-316이 생산하는 항암성 항생물질의 정제 및 특성

박재홍,함병권,배동훈,유주현 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.3

미생물로부터의 새로운 항암성 항생물질 개발을 목적으로 토양으로부터 분리한 Streptomyces sp. YBE316이 생산하는 항암성 항생물질은 오직 균체 내에만 존재하였고 pH 5.0∼10.0까지 안정하였으며 중성에서 활성이 가장 높았고 열에 대한 안전성은 낮았다 본 항암성 항생물질의 유기용매에 대한 용해도와 안정성을 검토한 결과 methanol에서 가장 좋았으며, 정제는 균체로부터 acetone 추출, butanol 추출, silica gel column chromatography 등을 이용하여 순차적으로 행하였다. 본 물질의 항암활성은 낮았으며, 항균활성은 항세균보다는 항진균 활성이 강하였다. 본 항암성 항생물질은, 기초적인 기기분석을 행한 결과, polyene계의 pentaene계 항생물질중 eurocidin이나 capacidin type의물질로 추측되었으며, 그 분자량은 최소한 683.0 dalton 이상이었고, mycosamine이나 perosamine 등의 amino sugar가 아닌 당으로 glycosylation 되어 있는 것으로 추측되었다. For the development of new antitumor antibiotics produced by microorganisms, Streptomyces sp. YBE-316 was isolated from soil. The productivity of the antitumor antibiotic from Streptomyces sp. YBE-316 gradually increased after 60 hours, and was maximum after 100 hours after inoculation in growth medium (2.0% sucrose, 1.0% soybean meal, 0.1% K_2HPO_4, pH 7.0) at 30℃, 150 rpm, 5 NL/min by 30 ℓ jar fermentor. This antitumor antibiotic was present only in mycelium, and stable in pH 5.0∼10.0 for 20 minutes at 100℃. Antitumor and antibiotic activities were maintained at neutral pH, and heat stability was low. This antitumor antibiotic was soluble in methanol and ethanol, and insoluble in water, ethyl acetate, chloroform, and n-hexane. This antitumor antibiotic was sequentially purified by acetone extraction from mycelium, butanol extraction, and silica gel column chromatography. Antitumor activity was low against most tested cell lines, but antibiotic activity was high and low against yeasts and bacteria, respectively. The visualization test showed that this antitumor antibiotic had higher hydroxyl, ketone, amino, carboxyl groups, and sugar(s) in its structure. Instrumental analyses showed that this antitumor antibiotic was a pentaene in polyene class antibiotics. In pentaene class antibiotics, this was considered as an eurocidin or capacidin type antibiotics. The molecular weight of this antitumor antibiotic was higher than 683.0 daltons, and this antitumor antibiotic might be glycosylated by other sugar(s), instead of mycosamine or perosamine, an amino sugar.

유전자 조작 , 균주분리 알칼리 내성 Bacillus sp . YA - 14 유래의 중복 Promoter 를 이용한 재조합 Plasmid 로부터의 Pectate lyase 의 발현

박희경,함병권,유주현,배동훈 ( Hee Kyoung Park,Byoung Kwon Hahm,Ju Hyun Yu,Dong Hoon Bai ) 한국미생물생명공학회 1997 한국미생물·생명공학회지 Vol.25 No.6

An Automated Fiber-optic Biosensor Based Binding Inhibition Assay for the Detection of Listeria Monocytogenes

Giyoung Kim,Mark Morgan,Daniel Ess,Byoung-Kwon Hahm,Aparna Kothapalli,Arun Bhunia 한국식품과학회 2007 Food Science and Biotechnology Vol.16 No.3

Conventional methods for pathogen detection and identification are labor-intensive and take days to complete. Biosensors have shown great potential for the rapid detection of foodborne pathogens. Fiber-optic biosensors have been used to rapidly detect pathogens because they can be very sensitive and are simple to operate. However, many fiber-optic biosensors rely on manual sensor handling and the sandwich assay, which require more effort and are less sensitive. To increase the simplicity of operation and detection sensitivity, a binding inhibition assay method for detecting Listeria monocytogenes in food samples was developed using an automated, fiber-optic-based immunosensor; RAPTOR (Research International, Monroe, WA, USA). For the assay, fiber-optic biosensorswere developed by the immobilization of Listeria antibodies on polystyrene fiber waveguides through a biotin-avidin reaction. Developed fiber-optic biosensors were incorporated into the RAPTOR to evaluate the detection of L. monocytogenes in frankfurter samples. The binding inhibition method combined with RAPTOR was sensitive enough to detect L. monocytogenes (5.4ⅹ10? CFU/mL) in a frankfurter sample.

실리마린의 간보호 작용에 미치는 쿠쿠마 추출물의 영향

이기명(Ki Myung Lee),함기백(Ki Baik Hahm),조성원(Sung Won Cho),오태영(Tae Young Oh),최설민(Seul Min Choi),김정훈(Jung Hoon Kim),안병욱(Byoung Ok Ahn),권종원(Jong Won Kwon),김원배(Won Bae Kim) 한국응용약물학회 2002 Biomolecules & Therapeutics(구 응용약물학회지) Vol.10 No.1

N/A Silymarin and curcumin have been used for supportive treatment of liver disease of different etiology due to their hepatoprotective activities. The present study was carried out to investigate the hepatoprotective effects of silymarin and/or curcuma extract against hepatotoxins induced liver injury. To investigate hepatoprotective effects, the silymarin and/or curcuma extract were pre-treated orally to experimental animals. And thereafter a single dose of hepatotoxin, carbon tetrachloride (CCl_4) and acetaminophen were administered through oral or intraperitoneal route, respectively. Chronic liver damage was induced by subcutaneous injection of CCl_4 for 3 weeks (2 times/week). Hepatoprotective and therapeutic effects were monitored by estimating serum ALT and AST levels and by measuring hepatic glutathione (GSH) and malondialdehyde (MDA) levels. Collagen type 1 was detected with immunostaining to assess fibrosis. The results showed that the mixture of silymarin and curcuma extract significantly reduced serum biochemistry levels and MDA levels compared with those of control group in both acute and chronic animal models. In antifibrotic effect, the relative hepatic collagen content was significantly decreased by silymarin and/or curcuma extract treatment. It was concluded that the complex of silymarin and curcuma extract have a both hepatoprotective and therapeutic effect synergically in rat liver injury induced by heptotoxins.

Purification, Characterization and Chemical Modification of the Xylanase from Alkali-tolerant Bacillus sp. YA-14

Park, Young Seo,Yum, Do Young,Hahm, Byoung Kwon,Bai, Dong Hoon,Yu, Ju Hyun 한국미생물 · 생명공학회 1994 Journal of microbiology and biotechnology Vol.4 No.1

The xylanase from alkali-tolerant Bacillus sp. YA-14 was purified to homogeneity by CM-cellulose, Sephadex G-50, and hydroxyapatite column chromatographies. The molecular weight of the purified enzyme was estimated to be 20,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme slightly hydrolyzed carboxymethyl cellulose and Avicel, but did not hydrolyze soluble starch, dextran, pullulan, and p-nitrophenyl-β-D-xylopyranoside. The maximum degree of hydrolysis by enzyme for birchwood xylan and oat spelts xylan were 47 and 40%, respectively. The Michaelis constants for birchwood xylan and oat spelts xylan were calculated to be 3.03 ㎎/㎖ and 5.0 ㎎/㎖, respectively. The activity of the xylanase was inhibited reversibly by HgCl_2, and showed competitive inhibition by N-bromosuccinimide, which probably indicates the involvement of tryptophan residue in the active center of the enzyme. The Xylanase was identified to be xylose-producing endo-type xylanase and did not show the enzymatic activities which cleave the branch point of the xylan structure.

Sclerotinia sclerotiorum에 의한 바질 균핵병

함수상,김병련,한광섭,권미경,박인희,Hahm, Soo Sang,Kim, Byoung Ryun,Han, Kwang Seop,Kwon, Mi Kyung,Park, In Hee 한국식물병리학회 2017 식물병연구 Vol.23 No.1

2011년부터 2013년까지 예산 지역의 농가포장에서 재배 중인 바질에서 균핵병으로 의심되는 증상이 지속적으로 관찰되었다. 초기에는 잎과 줄기가 갈색으로 변하기 시작하여 반점이 커지면서 점차 위쪽으로 진전, 위조하여 결국 전체적으로 썩어 죽는다. 잎과 줄기의 병반부에는 다량의 흰색 균사가 발생하며 $30-100{\mu}m$ 직경의 균핵이 형성하였다. PDA상에 균체는 흰색과 옅은 초콜릿 흑색으로 다양한 색을 나타내었고, 균핵은 검은색으로 대부분 불규칙한 구형이며 크기는 $5-50{\mu}m$였다. 병원균을 인위적으로 접종한 바질의 잎과 줄기에서는 자연 발생된 바질과 동일한 괴사와 위조증상을 보이며, 병반에서 역시 같은 균을 분리하였다. 이상과 같이 병원균의 균학적 특징, ITS 염기서열 분석, 병원성 검정 결과 본 병해는 Sclerotinia sclerotiorum에 의한 바질균핵병으로 명명할 것을 제안한다. During growing season of 2011 to 2013, Sclerotinia rot symptoms consistently have been observed on basil in Yesan-gun, Chungcheongnam-do in Korea. The typical symptom formed initially brownish spot on leaf and stem, and then advancing margins, wilting the whole plant and blighting, eventually died. On the surface of diseased lesions was observed cottony, white, dense mat of mycelial growth, and sclerotia ($30-100{\mu}m$ diameter) formed on stem and leaf. Morphological and cultural characteristic on potato dextrose agar, color of colony was white and colorless chocolate, sclerotium of irregular shape of the oval was black and $5-50{\mu}m$ diameter in size. In pathogenicity test, necrosis and wilt of the inoculated stem were observed in all plants and the pathogen was reisolated from stems. On the basis of mycological characteristics, pathogenicity, and internal transcribed spacer rDNA sequence analysis, this fungus was identified as Sclerotinia sclerotiorum. This is the first report of Sclerotinia rot on basil caused by S. sclerotiorum in Korea.