균형 생육조건하에서 Actinobacillus sp. El-9에 의한 Poly-β-Hydroxybutyrate의 생산

손홍주,이상준 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.3

토양으로부터 PHB를 생산하는 세균들을 농화배양에 의하여 순수분리하였다. 이들중 주정중류 폐액을 기질로 하여 PHB를 생산하는 균주를 공시균으로 선정하여 형태학적, 배양적, 생화학적 제반 특성을 검토한 결과 Actinobacillus sp.로 동정되었다. 균체생육을 위한 최적 배양온도 및 pH는 각각 30℃, 7.0이었다. 최적 생륙배지의 조성은 포도당 2%, NH_4NO_3 0.15%, Na_2HPO_4·12H_2O 0.25%, KH_2PO_4 0.25%, MgSo_4·7H_2O 0.15%, FeSO_4·7H_2O 0.005%, CaCl_2·2H_2O 0.003%, 미량원소 용액 5 ml/l이었다. Actinobacillus sp. EL-9의 PHB 생산 최적조건은 균형 생육조건(balanced-growth conditions) 즉, 생육 최적조건과 일치하였으며, 보조기질로 propionic acid을 첨가시 PHB/HV copolymer가 합성되었다. Microorganisms growing alcoholic distillery wastes were isolated from soil. Of them, the selected strain EL-9 had a capability of accumulating poly-β-hydroxybutyrate (PHB) when grown in alcoholic distillery wastes. The strain EL-9 was identified as a genus Actinoba-cillus based on the morphological, cultural, and biochemical characteristics. The strain EL-9 was named temporarily as Actinobacillus sp. EL-9. The optimal temperature and pH for cell growth of Actinobacillus sp. EL-9 were 30℃ and 7.0, respectively. The optimal medium compositions for cell growth comprised glucose 2%, NH_4NO_3 0.15%, Na_2HPO_4·12H_2O 0.25%, KH_2PO_4 0.25%, MgSo_4·7H_2O 0.15%, FeSO_4·7H_2O 0.005%, CaCl_2·2H_2O 0.003% and trace element solution 5 ml/l. To investigate the optimal conditions for PHB production, two-stage culture technique was used. The result showed that the conditions for PHB production are identical those for cell growth. When propionic acid was added as a cosubstrate, PHB/HV copolymer was produced.

주정중류 폐액을 이용한 Actinobacillus sp. EL-9로부터 Poly-β-Hyroxybutyrate의 생산 및 폐액의 처리

손홍주,이상준 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.3

본 연구는 국내 주정공장에서 배출되는 주정증류 폐액을 생물전환 공정을 통하여 PHB의 생산에 이용함과 동시에 COD 제거 효율에 따른 폐수처리 효과를 얻는데 그 목적을 두었다. 주정증류 폐액에서 PHB를 생산하는 공시균 Actinobacillus sp. EL-9의 주정증류 폐액에서의 PHB 생산과 주정증류 폐액에서의 COD제거효과에 대해서 시험한 결과는 다음과 같다. 주정증류 페액 원액에 포도당 0.5%, NH_4NO_3 0.05% 첨가시 PHB 축적율이 향상되었으며, 효소 가수분해한 주정증류 폐액에서 가장 높은 PHB 축적율을 보였다. 또한 주정증류 폐액에서의 PHB 생산량은 1.9 g/l이었다. 균체 생육과 PHB 생산에 따른 주정증류 폐액의 COD 제거효율은 배양 24시간만에 약 54%를 나타내었다. Alcoholic distillery wastes are utilized as dual purposes to produce PHB in lower production cost and to reduce the amount of waste to be treated. In this study, various attempts were made to increase PHB production under various conditions by Actinobacillus sp. EL-9 in a shaker culture. The addition of glucose, NH_4NO_3 to alcoholic distillery wastes slightly promoted cell mass and PHB production. Enzyme hydrolysis of alcoholic distillery wastes increased the production of PHB than that of untreated waste and acid hydrolysis treatment. The PHB weight in alcoholic distillery wastes was 1.91 g/l. Fermentation process of PHB production reduced the amount of COD value p to 54%, which reduced organic loading rate and capacity of activated sludge system.

Pseudomonas sp. HJ에 의한 Poly(Hydroxybutyric-Co-Hydroxyvaleric) Acid의 생산

손홍주,민관필,이상준 부산대학교 유전공학연구소 1995 분자생물학 연구보 Vol.11 No.-

하수처리장의 활성오니를 분리원으로 하여 수십종의 PHA 생산균을 분리하였다. 일반적으로 hydroxyvalerate monomer unit의 전구물질로 알려져 있지않은 glucose로부터 비교적 많은 PHA를 생산하는 균주를 공시균으로 선정하여 형태학적, 배양적, 생리학적 제 특성을 검토한 결과 Pseudomonas 속으로 동정되었다. 균체 생육을 위한 최적 배양온도 및 배양 pH는 각각 37℃와 7.0이었으며, 최적 탄소원으로서 glucose 1%, 최적 질소원으로서 (NH_4)_2SO_4 0.2%, K_2HPO_4 0.3%, KH_2PO_4 0.45%였다. 최적 PHA 생산조건을 조사하기 위하여 2단계 배양법을 이용하였다. PHA 생산은 배지성분중 NH_4, SO_4, Mg가 결핍되었을 때 향상되었고, 그중 NH_4의 결핍시 PHA 축적률과 HV monomer의 함량이 가장 높았다. C/N molar ratio 95.2에서 PHA 축적률이 가장 높았다. 공시균주 Pseucomonas sp. HJ는 alkane, alkanoic acid, alcohol을 탄소원으로 하여 PHB/HV를 생산하였다. PHA의 생산량과 HV monomer의 함량은 이용된 기질에 따라 다양하였으며, 특히 hexadecane와 propionate를 탄소원으로 하였을 때 PHA중의 HV monomer의 함량이 49∼74mol%로 매우 높았다. ^1H-NMR로서 공시균으로부터 분리정제된 PHA의 조성을 분석한 결과 PHB/HV copolymer임을 알 수 있었다. To produce PHA(polyhydroxyalkanoic acid) from microbe, dozens of microorganism have been screened from sewage sludge. Selected a strain HJ out of 50 strains of PHA producing bacteria has a capability of accumulating large amounts of PHB/HV copolymer when grown in batch culture with a single carbon source(glucose) that was not generally considered as precusor of hydroxyvalerate monomer unit. The strain HJ was identified as the genus Pseudomonas with respect to morphological, cultural, and biochemical characteristics. The optimal temperature and pH for cell growth were 37℃ and 7.0. The optimal medium compositions for cell growth were glucose 1% as a carbon source, (NH_4)_2SO_4 0.2%, as a nitrogen source, K_2HPO_4 0.3%, and KH_2PO_4 0.45%. To investigate the optimal condition for PHA production two-step cultivation method was employed. PHA production was induced by deficiency of NH^+_4, SO^2_4, Mg^+2. Besides carbon source, deficiency of all nutrients stimulated PHA productivity but deficiency of NH^+_4 stimulated the most HV monomer content. The highest PHA production was C/N molar ratio 95.2. Pseudomonas sp. HJ was also able to produc PHB/HV copolymer when cultivated on alkane, alkanoate, alcohol as carbon sources. Especially Pseudomonas sp. HJ was able to incorporate hydroxyvalerate into PHB/HV to level as high as from 49 to 74 mol% when grown in a medium containing hexadecane and propionate. The purified PHA was identified PHB/HV copolymer by ^1H-NMR analysis.

Actinobacillus sp. EL-9로부터 생산된 생분해성 폴리에스터의 구조 및 특성

손홍주,이건,김근기,김한수,김용균,이상준 부산대학교 유전공학연구소 1998 분자생물학 연구보 Vol.14 No.-

Actinobacillus sp. EL-9로 부터 추출, 정제된 PHB를 GC, IR 및 NMR로 분석한 후, 그 구조를 확인하였으며, 열적 성질, 점도 및 분자량 등을 검토하였다. Actinobacillus sp. EL-9로부터 추출, 정제된 PHB를 IR,500㎒ ^1H-NMR 및 125㎒ ^13C-NMR 를 이용하여 조성분석을 실시한 결과, 본 공시균이 생산한PHB는3-hydroxybutyric acid 의 homopolymer임을 알 수 있었다. 또한 PHB의melting temperature(T_m)은 169.7℃임을 알 수 있었으며, crystallization trmperature(T_c)는 69.13℃이었다. PHB의 점도는 1.08×10^6(±3,000)이었다. In this study, the composition and characteristic of poly-β-hydroxybutyrate(PHB) biosynthesized by Actinobacillus sp. EL-9 are investigated. PHB produced by Actinobacillus sp. EL-9 was identified as the homopolymer of 3-hydroxybutyric acid(PHB)by infrared spectroscopy and nuclear magnetic resonance spectroscopy analysis. The melting temperature(T_m), and crystallization trmperature(T_c)of PHB was 169.7℃ and 69.13℃, respectively. the viscosity on the basis of Brookfield viscometer was 6.01㎗/g. The viscosity-average molecular weight estimated by Mark-Ho-wink-Sakurada equation was 1.08×10^6(±3,000).

Alcaligenes sp. GB-77에 의한 Polyhydroxyalkanoic Acid의 생산

김근배,손홍주,이상준 부산대학교 유전공학연구소 1995 분자생물학 연구보 Vol.11 No.-

Polyhydroxyalkanoic acid(PHA)를 생산하는 세균들을 하수처리장의 활성오니로부터 존화배양에 의하여 분리하였다. 이들 중 단일기질로부터 PHA를 생산하는 균주의 생리학적 성질을 검토한 결과 Alcaligenes 속으로 동정되었다. 균체 생육에 최적인 배양온도, pH는 각각 36℃, pH6.8로 나타났으며, 탄소원으로 과당 10g/l, 질소원으로 polypeptone 5g/l, Na_2HPO_4 1×10^-2 M, KH_2PO_4 1.3×10^2 M이었다. PHA 합성을 위한 최적조건을 조사하기 위하여 2단계 배양법을 이용한 결과, 배양온도 36℃, pH6.8, C/N 비 50이 PHA 합성에 최적인 것으로 나타났다. 탄소원을 간헐적으로 첨가하는 유가배양에 의하여 배양 60시간만에 건조균체량 17.4g/l, PHA 9.1g/l를 얻을 수 있었다. 추출, 정제된 PHA는 NMR에 의하여 분석한 결과 PHA/HV copolymer 임을 확인할 수 있었다. For polyhydroxyalkanoci acid (PHA) production, several microorganisms were isolated from sewage sludge. One of them, GB-77 strain, was chosen from its PHA/HV copolymer production on only fructose without cosubstrate. The isolated strain GB-77 was identified as the genus Alcaligenes. Optimal temperature and pH for cell growth were 36℃ and 6.8. Optimal medium composition was 10 g/l of fructose and 5 g/l of polypeptone, 1×10^-2 M Na_2HPO_4, 1.3×10^-2 M KH_2PO_4. To investigate the optimal condition for polyhydroxyalkanoic acid production two-stage culture technique was used; first stage for cell grown and second stage for PHA production on unbalanced growth conditions. Optimal conditions for high PHA production were C/N ratio 50, temperature 36℃ and pH 6.8. To overcome frutose inhibition on cell growth, intermittent feeding fed-batch culture technique was used. Total cell concentration was 17.4 g/l with 9.1 g/l of PHA. The purified PHA was identified PHA/HV copolymer by NMR analysis.

원유의 미생물적 유화

차미선,손홍주,이건,고명선,이상준,이종근 부산대학교 환경문제연구소 1996 環境硏究報 Vol.14 No.1

Microorganisms producing bioemulsifier were isolated from the sea water in Pusan costal aera. The isolated strain which had the highest emulsification activity and stability was identified as the genus Acinetobacter from the results of morphological, cultural and biochemical tests and named Acinetobacter sp. EL-C6 for convinience. The compositions of optimum medium for emulsification of crude oil by Acinetobacter sp. EL-C6 were crude oil 2%, NH_4NO_3 0.2%, K_2HPO_4 0.01%, MgSO_4·7H_2O 1%, CaCl_2·2H_2O 0.1%, NaCl 3% and the initial pH 7.5. The cultivation for emulsification of crude oil was carried out in 500m1 shaking flask containing 100m1 of the optimum medium at 30℃. The highest emulsification was observed after 5 days. The utilization on the various hydrocarbon of the Acinetobacter sp. EL-C6 was showed that the utilization of n-alkane compounds were better than that of aromatic compounds. Among the petroleum compounds, by Acinetobacter sp. EL-06.

Monascus anka로부터 유기배양에 의한 적색소의 대량생산

김희구,박근태,손홍주 한국식품영양학회 1998 韓國食品營養學會誌 Vol.11 No.6

Monascus anka KFCC 4478의 적색소 생산력을 증가시키기 위하여 발효조 배양조건을 검토한 후, 탄소원을 연속적 또는 간헐적으로 공급하는 유기배양을 하였다. 5l 용량의 발효조를 이용하여 배양시 교반속도 200rpm, 통기량 1vvm 및 종균 접종량 10%(v/v)일 때 색소 생성량이 최대였다. 또한 벽면에 부착된 균사체를 재접종함으로써 색소 생성력이 향상되었다. Intermittent feeding fed-batch culture에 의하여 배양 9일만에 30g/l의 건조균체와 350(A_495)의 색소가 생성되었으며, continuous feeding fed-batch culture에 의하여 22g/l의 건조균체와 190(A_495)의 색소가 얻어졌다. 따라서 본 균주에 의하여 적색소를 생산코자 할 경우는 회분배양보다 유기배양이 더 우수한 것으로 나타났다. The production of red pigment from glucose by fed-batch culture of Monascus anka was investigated. In batch culture using fermentor, 200 rpm of agitation speed, 1vvm of aeration volume, and 10%(v/v) of inoculum size were optimal, respectively. The red pigment production was increased by removal of wall-attached mycelium. In an intermittent feeding fed-batch culture, dry cell weight increased to 30g/l, and the red pigment content reached 350 of absorbance at 495nm. In a continuous feeding fed-batch culture, dry cell weight increased to 22g/l, and the red pigment content reached 190 of absorbance at 495nm.

Monasucs anka의 적색조 생산 특성

김희구,박근태,손홍주 한국식품영양학회 1998 韓國食品營養學會誌 Vol.11 No.6

Monascus anka KFCC 4478의 적색소 생산력을 증가시키기 위하여 배지조성 및 배양조건에 대하여 검토하였다. 공시균주의 색소 생성력은 탄소원으로 glucose 4.0%, 유기질소원으로 peptone 3.0%, 무기질소원으로 NaNO_3 0.15%, 비타민 성분으로 thiamine-HCI 및 nicotinic acid 각 10㎍/l였으며, 배양온도 30℃, 초기 pH 7.0이었다. 특히 유기질소원과 무기질소원을 복합 처리함으로서 색소 생성력이 향상되었다. 결정된 색소생성 최적조건하에서 본 공시균주는 배양 5일만에 최대 균체 증식과 색소 생성력을 나타내었다. Optimal media and cultural conditions for the production of red pigment were established using Monascus anka KFCC 4478. The optimal temperature and initial pH for the production of red pigment were 30℃ and 7.0, respectively. Glucose turned out to be most suitable carbon source for red pigment production. Optimal glucose concentration was 3.0%. Addition combined of nitrogen sources of peptone and NaNO_3 induced good red pigment production. Thiamine-HCL and nicotinic acid were increased the production of red pigment. Under optimal conditions, maximum red pigment production and cell growth were observed after 5 days of incubation.

Burkhoderia sp. AK-17에 의한 잎들깨 재배의 생물학적 조절

김근기,김용균,손홍주,최영환,강규영 Plant molecular biology and biotechnology research 2005 Plant molecular biology and biotechnology research Vol.2005 No.

There are various crop diseases in green houses that are caused by the cultural environments, especially high temperature and moisture. To solve the forementioned problems, farmers are overusing agricultural chemicals, causing other damages by the chemical residue. In this study, antagonistic bacteria as biological control agents were isolated to produce the environmentally-friendly crops for use in green houses. Eighteen species of antagonistic bacteria were totally isolated from the soil and plants in the Perilla fields, and AK-17 showed the highest activity among the isolates. According to the results of anti-fungal spectrum against several pathogens by AK-17, the antagonism effect of the isolates was remarkable against grey mold rot by Botrytis cinerea, sclerotinia rot by Sclerotnia sclerotiorum, and stem rot by Rhizoctonia solini. To evaluate the biological control effects of the isolates against the major diseases of Perilla, studies were carried out to evaluate the preventive and the curative effects of the diseases throughout the pot experiments. According to the forementioned experiments, the preventive and the curative effects by the isolates against sclerotinia rot were respectively showed as 55% and 92%. For the grey mold rot, those were 40% and 78%, respectively. As to the evaluation of the growth-promoting effect by AK-17, the length and the biomass of the tested plants were increased to 120% and to 164%, respectively. For the leaf numbers and area were respectively increased to 120% and 220%. Furthermore, AK-17 was identified as Burkhoderia sp. according to the results of physiological properties and genetic methods. 시설원예단지의 재배환경은 고온다습으로 많은 병해가 발생하며, 이를 방제하기위해 과다한 농약을 사용하므로 농약 잔류성에 대한 피해가 심각한 우려를 낳고 있다. 미생물을 이용한 생물학적 방제법 개발로 청정 시설원예작물을 생산할 목적으로 길항균을 분리하였다. 잎들깨 재배토양과 식물체로부터 길항세균 18종을 분리하였으며 분리한 길항세균 중에 AK-17이 가장 활성이 뛰어나 이를 이용하여 주요 식물병원균에 대한 항균스펙트럼을 조사하였다. 그 결과 잿빛곰팡이병원균의 Botrytis cinerea와 균핵병원균의 Sclerotinia sclerotiorum 및 줄기썩음병원균인 Rhizoctonia solant에 대한 항균효과가 뛰어났다. 잎들깨의 주요 병에 대한 생물학적 방제실험은 병발생 억제효과와 병방제효과를 pot 실험으로 실시했다. 그 결과 균핵병은 55%의 병발생 억제효과와 92%의 방제효과가 있었고, 잿빛곰팡이병은 40%의 병발생 억제효과와 78%의 방제효과를 확인할 수 있었다. AK-17의 식물 생육촉진효과는 신장이 120%, 생체중이 164% 증가되었으며, 엽수와 엽면적은 각각 120%와 220%의 증가효과를 보였다. 그리고 AK-17을 생리·생화학적방법과 유전학적 방법으로 동정한 결과, Burkhoderia sp.로 확인되었다.

65세 이상 노인 급성 심근경색증 환자의 사망에 영향을 미치는 인자들

정봉기,양금열,손준혁,주시내,남홍우,전용덕,이홍순 대한노인병학회 2001 대한노인병학회지 Vol.5 No.2