세포응집물질을 생산하는 Aspergillus sp. LAM 94-142의 특성 및 물질생산 조건

이동희,이노운,이현우 건국대학교 산업기술연구원 1996 건국기술연구논문지 Vol.21 No.-

토양에서 분리한 세포응집물질을 생산하는 균주 LAM 94-142는 형태, 배양 및 생리학적 특성을 조사한 결과 Aspergillus parasiticus 또는 그 근연균으로 동정되었으며 물질을 생산하기 위한 배양최적 조건을 검토한 결과는 다음과 같다. 최적 탄소원과 질소원은 각각 1.5% glucose와 0.35% yeast extract였으며 Ca²+을 첨가하였을 때 물질생산이 현저히 증가되었으며 K+, Cu²+, Fe²+ 등의 금속이온은 물질 생성을 저해하였다. 또 인산의 K염은 물질 생합성을 촉진하였으나 ammonium염은 크게 저해하였다. 그리고 최적 초발 pH와 온도는 pH7과 30℃였으며 4일간 배양하였을 때 물질 생산량이 최대였다. A strain of mold, LAM 94-142, capable of producing cell aggregation factor was isolated from soil and identified as Aspergillus parasiticus or its related strain on the morphological, cultural and physiological characteristics. For production of the cell aggregation factor, glucose and yeast extract was favorable as carbon and nitrogen source, respectively. The factor production was increased by the addition of Ca²+ and potassium phosphates but inhibited by K+, Cu²+, Fe²+ and ammonium phosphates. Optimal initial pH and temperature for the factor production was pH 7 and 30℃, respectively. The production of the factor reached at maximum after 4 days in the medium containing 1.5% glucose, 0.35% yeast extract, 0.07% CaCl₂, and 0.05% KH₂PO₄.

Streptomyces sp. LAM 90-331이 생산하는 세포 응집물질

이동희 건국대학교 1994 學術誌 Vol.38 No.2

An Actinomycete, strain LAM 90-331, capable of producing microbial cell aggregation factor was isolated from soil, and identified as Streptomyces phaeoviridis or its related organism by cultural, morphological and physiological characteristics. For production of the agregation factor soluble starch and glycerol mixture(1 : 1), and corn steep powder was favorable as carbon and nitrogen source, respectively. The aggregation factor production was increased by the addition of Ca2+ and Mg2+, but depressed severely by other metal ions used. Optimal temperature and initial pH for production was around 28℃ and pH 7∼8, respectively. Accumulation of the aggregation factor in the culture broth reached at maximum level after 4 days cultivation under aeration rate 1vvm and impeller speed 200rpm in 5L-fermenter with 2.5L of medium containing soluble starch 1%, glycerol 1%, corn steep powder 1%, K2HPO4 0.05%, CaCl2 0.05%, MgSO4 · 7H2O 0.05% and Neorin 302 0.01%. The aggregation factor was isolated from culture broth by methanol precipitation, ultrafiltration and Sephadex G-100 column chromatography. It was stable in pH range 5∼ 10 at RT, and activity was increased remarkably by Ca2+ ion. The substance aggragated Bacillus subitilis, alcaligenes faecalis, Pesudomonas aeruginosa, P. fluorescens, P. putida, Staphylococcus aureus and Sacchromyces cerevisiae, but not acted to animal cells such as mouse red blood cell, mouse lymphocyte, sheep red blood cell and B16 melanoma.

Streptomyces속 균주 YS-87-8에 의한 항산화성 물질의 생산조건

이동희 건국대학교 1989 學術誌 Vol.33 No.2

One Streptomyces sp. capable of producing antioxidant was isolated from soil and named as YS-87-8, and the cultural conditions were studied. For the antioxidant production, sucrose and peptone was favorable as carbon and nitrogen source, respectively. The antioxidant production was slightly increased by the addition of Ca2+, but depressed markedly by Ag+, Co2+, Hg2+, Zn2+, Fe2+ and Fe3+. And the addition of amino acid such as histidine, proline, leucine and glycine, and vitamin such as hesperidin, folic acid and cyanocobalamin increased the production of the antioxidant remarkably. Optimal initial pH and temperature for the antioxidant production was pH 8 and 25℃, respectively. The production of the antioxidant reached at maximum after 7-8 days cultivation in the medium containing 1.5% sucrose, 0.4% peptone, 0.1% K2HPO4, 0.05% CaCl2ㆍ2H2O, 0.05% MgSO4ㆍ7H2O, 0.05% NaCl, 0.005% histidine and 10ppm hesperidin.

Bacillus subtilis가 생산하는 항진균성 항생물질 LAM-44B의 정제 및 특성

이동희,권태종 건국대학교 산업기술연구원 2005 건국기술연구논문지 Vol.30 No.-

A novel antifungal antibiotic for medically isolated azole-resistant Candida albicans was purified from the culture broth of Bacillus subtilis LAM 97-44 by butanol extraction. Diaion HP-20 and Dowex-50 adsorption chromatography, silica gel flash chromatography followed by 1st and 2nd HPLC and designated LAM-44B. LAM-44B was stable for 60min at 100℃, and pH range from 2 to 12. MIC value were observed at 1-6㎍/mL against various Candida albicans strains. The antibiotic showed no acute toxicity to rat, and no cytotoxicity to S180, MKN-45 P388, HeLa and 3T3 cell lines. LAM-44B was colorless powder soluble in water, methanol, butanol and positive to ninhydrin, Molish and Anthrone reaction. The antibiotic had maximum absorption at 281, 292, 305 and 320nm in methanol, and melting point was 232℃. The molecular weight and formula were determined to be 696 and C35H54NO13 by 'H-NMR, 13C-NMR, IR, ESI mass spectrum and elemental analysis.

Bacillus subtilis LAM 97-44가 생산하는 항진균성 항생물질의 정제 및 특성

이동희,이노운,권태종 한국농화학회 2003 Applied Biological Chemistry (Appl Biol Chem) Vol.46 No.2

병원에서 분리한 azole계 항진균성 항생물질에 대한 내성을 가지고 있는 Candida albicans에 대해 강한 활성을 가지는 항진균성 물질을 Bacillus subtilis LAM 97-44의 배양액으로부터 분리 정제한 후 그 특성을 조사하였다. 원심분리한 배양상등액을 butanol 추출, Diaion HP-20 과 Dowex-50 adsorption chromatography, silica gel flash chromatography와 HPLC로 정제하였고 TLC와 HPLC로 확인하여 그 물질을 LAM-44A라 명명하였다. LAM-44A는 pH와 열에 매우 안정하였으며 Candida sp., Cryptococcus sp. 등에 대해 강한 활성을 나타낸 반면에 독성은 매우 적었다. 분리한 물질은 273 nm에서 최대흡광도를 가진 융점 202˚C의 무색분말이었으며 ninhydrin 반응결과 음성이었고 ^1H-NMR, ^13C-NMR, IR spectrum, 원소분석 등의 결과로 볼 때 분자량 282의 C_14H_34O_5의 화학식을 가진 물질로 동정되었다. A novel antifungal antibiotic for azole-resistant Candida albicans was purified from the culture broth of Bacillus subtilis LAM 97-44 by butanol extraction, Diaion HP-20 and Dowex-50 adsorption chromatography, silica gel flash chromatography followed by HPLC and designated LAM-44A. LAM-44A was stable for 60 min at l00˚C, and pH range from 2 to 10. MIC values were observed at 0.5-3.5 ㎍/㎖ against various Candida albicans strains. The antibiotic showed no cytotoxicity for S180, MKN-45, P388, HeLa and 3T3 at the concentration of 1 ㎎/㎖. LAM-44A was colorless powder soluble in water, methanol, ethanol, butanol and negative to ninhydrin reaction. The antibiotic had maximum absorption at 273 nm in methanol, and melting point was 202˚C. The molecular weight and formula were determined to be 282 and C_14H_34O_5 by ^1H-NMR, ^13C-NMR, IR spectrum and elemental analysis.

메치실린 내성 황색 포도상구균에 유효한 항생물질을 생산하는 방선균의 선별 및 물질생산조건

이동희,홍환석 건국대학교 산업기술연구원 1999 건국기술연구논문지 Vol.24 No.-

토양시료로부터 다제내성 MRSA에 유효한 항생물질을 생산하는 Actinomycetes 균주 LAM 98-90을 분리하여 동정한 결과 Streptomyces 속 균주였으며 spore chain은 spiral, spore surface는 smooth type이었고, 세포벽은 LL-DAP를 함유하였다. Aerial mass color는 회색이었으며 melanoid와 가용성 색소는 생성하지 않았다. 그리고 LAM 98-90에 의한 항생물질의 생산조건을 검토한 결과 탄소원으로는 soluble starch가, 질소원으로는 yeast extract, beef extract, peptone이 가장 우수하였으며 Mg²+ 첨가로 물질생산량이 많이 증가하였다. 생산 최적온도와 초발 pH는 각각 35℃와 7이었으며, soluble starch 2%, yeast extract 0.1%, beef extract 0.1%, peptone 0.1%, MgSO₄7H₂O 0.05%, K₂HPO₄ 0.05%, NaCl 0.05% 조성의 배지로 5일간 배양하였을 때 배양액내의 항생물질 축적량이 최대에 달하였다. LAM 98-90 균주가 생산하는 항생물질은 Gram 양성세균에 대해서는 항균활성이 우수하였으나 Gram 음성세균에는 비교적 약하였으며 진균류에는 전혀 작용하지 못하였다. An antibiotic producing strain LAM 98-90, effective to the multidrug-resistant MRSA, was isolated from soil. The spore chain of LAM 98-90 was spiral, and the spore sulface was smooth. LAM 98-90 has LL-diaminopimelic acid in the cell wall. The aerial mass color of the strain was gray, and the reverse side color was brown. The strain did not produce melanoid and other soluble pigments. And according to the Taxon program, LAM 98-90 was identified to one strain of Streptomyces. For the antibiotic production, soluble starch, soluble starch, yeast extract, beef extract and peptone were favorable as carbon and nitrogen sources. The antibiotic production was stimulated by the addition of Mg²+, but inhibited by Ag+, Co²+ and Fe²+ completely. Optimal temperature and initial pH for production of the antibiotic was 35℃ and pH 7, respectively. Accumulation of the antibiotic in the culture broth reached at maximal level after 5 days cultivation in the medium containing soluble starch 2%, yeast extract 0.1%, beef extract 0.1%, peptone 0.1%, K₂HPO₄ 0.05%, MgSO₄·7H₂O 0.05%, and NaCl 0.05%, and NaCl 0.05%. The antibiotic inhibited the growth of Gram positives well, however moderated for Gram negatives, and did not inhibit yeast and mold.

수용성 항진균성 항생물질 생산을 위한 Bacillus sp. LAM 97-44의 배양조건

이동희,이노운 건국대학교 산업기술연구원 2000 건국기술연구논문지 Vol.25 No.-

A strain of Bacillus sp. LAM 97-44, capable of producing antifungal antibiotic was isolated and its cultural conditions for production of the substance was investigated. For production of the water-soluble antifungal antibiotic, glucose, glycerol, malt extract and ammonium phosphate were favorable as carbon and nitrogen source. The antibiotic production was increased by the addition of Mg2+ and Fe2+. Optimal initial pH and temperature was pH 7.0 and 30℃, respectively. The production of the antibiotic reached at maximum after 5 days in the medium containing 1.3% glucose, 0.4% glycerol, 0.1% malt extract, 0.2% NH4H2PO4, 0.1% K2HPO4, 0.03% MgSO4·7H2O and 0.03% FeSO4·7H2O.

Methanol 자화성 효모에 의한 균체 단백질 생산

권태종,이동희,정호권,최태부 건국대학교 1992 學術誌 Vol.36 No.2

SCP producing yeast JAM -3032 that has high productivity potential with methanol as carbon and energy source was isolated from soil, and conditions for SCP production, nutritional values, and alcohol oxidase of the microorganism were investigated. Isolated strain JAM-3032 was identified as Pichina on the morphological, cultural, physiological and biochemical characteristics. The production of cell mass reached at maximal level after 54 hrs cultivation at 28℃, pH 6.0 in the medium containing methanol 3.0%, yeast extract 0.8%, K2HPO4 0.1%, KH2PO4 0.1%. MgSO4ㆍ7H2O 0.05%, alanine 0.01%, nicotinic acid 0.04%, and biotin 0.04%. Dry cells of Pichia sp. JAM -3032 was composed of proteins 53.3%, carbohydrates 31. 5%, nucleic acids 5.6%, lipids 2.9%, ash 6.4% and amino acid composition of the proteins was satisfied in FAO reference. Biosynthesis of the alcohol oxidase of the strain JAM -3032 was induced by methanol. The enzyme was purified by treatment of ammonium sulfate fractionation, DEAE-Sephadex A-50 ion exchange chromatography and Sepharose 6B gel nitration. The purified enzyme was homogeneous on SDS-PAGE, and molecular weight was estimated to be about 520,000 D that was consisted of 8 subunits containing FAD as coenzyme. The optimal pH and temperature for the enzyme reaction was 7.5 and 37℃, respectively. Activation energy was 17.1Kcal/mol and half inactivating temperature was 40℃. The enzyme activity was inhibited by Ag+, Cu2+ and Hg2+ ions remarkably. The enzyme acted on methanol specifically, and its Km value was 0.89 mM.

Streptomyces 속 균주 HA-40이 생산하는 내열성 α-Amylase의 효소학적 성질

신운섭,이동희,이현우,권태종,정호권 建國大學校 附設 産業技術硏究所 1988 논문집 Vol.13 No.-

토양으로부터 분리한 Streptomyces 속 균주 HA-40이 생산하는 내열성 α-amylase의 효소학적 성질을 조사한 결과는 다음과 같다. 효소의 작용 최적 온도는 65℃였으며 activation energy 는 16.9Kcal/mole이었고 60℃에서는 안정하였으나 70℃에서는 30분 처리로 약 50%, 1시간 처리로 약 75%가 실활하였다. 그러나 ??를 0.1mM농도로 첨가한 경우엔 70℃ 에서 1시간 처리하여도 잔존활성도가 55%이상으로 열변성에 대한 ??의 보호효과가 있었다. 본 효소는 pH7에서 활성이 가장 강하였으며 안정성도 가장 높았다. 그리고 금속이온 중에서 ??의 첨가로 효소활성이 증대되었으며 ??, ??, ??등에 의해서 활성이 저해되었다. 각종 효소저해제는 대부분 영향을 미치지 못하였으나 EDTA가 반응액 중에 공존하면 효소활성이 강하게 저해되었다 본 효소는 maltotriose이하의 당류에 대해서는 분해력이 없었으며 amylose에 대한 환원당 생성력을 100으로 하였을때 soluble starch 108, amylopectin 94, glycogen 87, dextrin 48, potato starch 25, corn starch 18로 생전분에 대한 분해력은 soluble starch에 비해서 1/4∼1/5 정도밖에 되지 않았다. 그리고 soluble starch에 대한 Km 치는 1.9㎎/㎖였다. The properties of a thermostale α-amylase produced from Streptomyces sp. strain HA-40, isolated from soil, were investigated. The optimal temperature of the enzyme was 65℃ and the enzyme was stable below 60℃. However, at 70℃ about 75% of the activity was lost after 1hr treatment, but by the addition of ?? in the concentration of 0.1mM only 45% of the activity was lost. The enzyme was most active and stable at around pH 7. The activity of the enzyme was increased by the addition of ??, but depressed by ??, ??, and EDTA. The enzyme could be hydrolyze soluble starch, amylose, amylopectin and glycogen well, and could also digest dextrin and raw starch. The Km value for soluble starch was 1.9㎎/㎖.

하폐수중의 아닐린의 생물학적 분해를 위한 Pseudomonas속 균주 MIDR-79의 분리 및 특성

이재완,이동희,방병호 한국상하수도학회 1989 한국상하수도학회지 Vol.2 No.1

A bacterium, MIDR-79. was isolated from river bed which utilizes aniline as sole source of carbon and nitrogen. It was identified as Pseudomonas species. Optimal temperature and pH for the biodegradation of aniline was 30℃ and pH6~8, respectively. The microorganism grew well and degraded aniline completely in the range of 10 to 35mM of aniline, but substrate inhibition was observed at the concentration of 40mM aniline. The enzyme system involved in aniline biodegradation was repressed by the readily utilizable carbohydrates and nitrite. Ammonia was released from aniline with resting cells and accumulated in the reaction mixtue. And aromatic compounds besides aniline such as phenol, catechol and resorcinol were assimilated by the microorganism.