KR20060018783A - Glycolipid bioemulsifier produced by marine bacteria Pseudomonas aeruginosa BYK-2 and its purification method - Google Patents
Glycolipid bioemulsifier produced by marine bacteria Pseudomonas aeruginosa BYK-2 and its purification method Download PDFInfo
- Publication number
- KR20060018783A KR20060018783A KR1020040068193A KR20040068193A KR20060018783A KR 20060018783 A KR20060018783 A KR 20060018783A KR 1020040068193 A KR1020040068193 A KR 1020040068193A KR 20040068193 A KR20040068193 A KR 20040068193A KR 20060018783 A KR20060018783 A KR 20060018783A
- Authority
- KR
- South Korea
- Prior art keywords
- bioemulsifier
- glycolipid
- pseudomonas aeruginosa
- byk
- produced
- Prior art date
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- 229930186217 Glycolipid Natural products 0.000 title claims abstract description 66
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Abstract
본 발명은 해양세균 슈도모나스 아에루기노사(Pseudomonas aeruginosa) BYK-2에 의해 생산되는 당지질 생물유화제 및 그 정제방법에 관한 것으로, 이를 보다 상세하게 서술하면, 육상과 해양의 유출유 정화에 동시에 사용할 수 있고 산업적으로 다양하게 사용되어질 수 있는 생물유화제 생산 신규 균주인 해양 세균 슈도모나스 아에루기노사 비와이케이-2(Pseudomonas aeruginosa BYK-2 : 수탁번호 KCTC 18012P)(생명공학연구소 기탁일자 2000년 4월27일)로부터 생산되는 람노리피드와 메칠람노리피드를 동시에 생산하는 당지질 생물유화제로, 상기 균주는 토양유래 슈도모나스 아에루기노사 균주와는 다른 당지질 생물유화제인 람노리피드와 메칠람노리피드를 동시에 생산하는 독특한 생육 특성을 지니고 있는 당지질 생물유화제 및 그 정제방법에 관한 것이다.
슈도모나스 아에루기노사, 당지질 생물유화제, 람노리피드, 메칠람노리피드
The present invention relates to a glycolipid bioemulsifier produced by the marine bacterium Pseudomonas aeruginosa BYK-2 and a purification method thereof, which can be used simultaneously for the purification of effluent oil on land and in the ocean. Pseudomonas aeruginosa BYK-2 ( Accession No. KCTC 18012P) , a new strain producing bioemulsifiers that can be used in a variety of industrial and industrial applications. Glycolipid bioemulsifier to produce both lamnolipid and methyllamnolipid produced at the same time, the strain simultaneously produces the lipophilic bioemulsifiers different from the soil-derived Pseudomonas aeruginosa strain It relates to a glycolipid bioemulsifier having a unique growth characteristics and a purification method thereof.
Pseudomonas aeruginosa, glycolipid bioemulsifier, rhamnolipid, mechilamnolipide
Description
도 1은 본 발명에 따른 해양유류오염지역으로부터 유류분해세균의 분리 방법을 나타낸 블록도.1 is a block diagram showing a method for separating oil decomposition bacteria from the marine oil pollution zone according to the present invention.
도 2는 본 발명의 균주의 동정 결과를 나타낸 도표.Figure 2 is a table showing the identification results of the strain of the present invention.
도 3은 염분 농도의 변화에 따른 슈도모나스 아에루기노사 비와이케이-2의 생육 특성을 나타낸 그래프.Figure 3 is a graph showing the growth characteristics of Pseudomonas aeruginosa biwaikei-2 with a change in salt concentration.
도 4는 슈도모나스 아에루기노사 비와이케이-2를 이용한 난분해성 아라비안 라이트 원유의 생분해능을 나타낸 그래프.Figure 4 is a graph showing the biodegradation of refractory Arabian light crude oil using Pseudomonas aeruginosa biwaikei-2.
도 5는 슈도모나스 아에루기노사 비와이케이-2 이용한 난분해성 벙커씨유의 생분해능을 나타낸 그래프.5 is a graph showing the biodegradability of the hardly decomposable bunker seed oil using Pseudomonas aeruginosa biwaikei-2.
도 6은 본 발명의 균주가 생산하는 생물유화제의 분리정제 방법을 나타낸 블록도.Figure 6 is a block diagram showing a separation and purification method of the bioemulsifier produced by the strain of the present invention.
도 7은 탄소원의 종류에 따른 생산된 생물유화제의 박층크로마토그래피 결과를 나타낸 그래프.7 is a graph showing the thin layer chromatography results of the bioemulsifier produced according to the type of carbon source.
도 8은 정제된 두 종류(BS-1, BS-2)의 생물유화제의 박층크로마토그래피의 결과를 도시한 그래프.8 is a graph showing the results of thin layer chromatography of two purified bioemulsifiers (BS-1 and BS-2).
도 9는 당지질 생물유화제 BS-1의 변환적외선 분광장치 분석 결과를 나타낸 그래프.Figure 9 is a graph showing the results of the conversion infrared spectroscopy apparatus of glycolipid bioemulsifier BS-1.
도 10은 당지질 생물유화제 BS-1의 고속원자충격 질량분석장치 분석결과를 나타낸 그래프.Figure 10 is a graph showing the analysis results of high-speed atomic shock mass spectrometry of glycolipid bioemulsifier BS-1.
도 11은 당지질 생물유화제 BS-1의 수소핵자기공명장치 분석 결과를 나타낸 그래프.11 is a graph showing a hydrogen nuclear magnetic resonance device analysis results of the glycolipid bioemulsifier BS-1.
도 12는 당지질 생물유화제 BS-1의 탄소핵자기공명장치 분석결과를 나타낸 그래프.12 is a graph showing the results of analysis of carbon nuclear magnetic resonance device of glycolipid bioemulsifier BS-1.
도 13은 당지질 생물유화제 BS-1의 뎁트(DEPT) 분광 분석결과를 나타낸 그래프.FIG. 13 is a graph showing DEPT spectroscopic analysis results of glycolipid bioemulsifier BS-1. FIG.
도 14는 당지질 생물유화제 BS-1의 노이지(NOESY) 분광 분석결과를 나타낸 본 발명의 그래프.14 is a graph of the present invention showing the results of NOESY spectroscopy analysis of glycolipid bioemulsifier BS-1.
도 15는 당지질 생물유화제 BS-1의 토코지(TOCSY) 분광 분석결과를 나타낸 본 발명의 그래프.15 is a graph of the present invention showing the results of TOCSY spectroscopy analysis of glycolipid bioemulsifier BS-1.
도 16은 당지질 생물유화제 BS-1의 릴레이(RELAY) 분광 분석결과를 나타낸 본 발명의 그래프.Figure 16 is a graph of the present invention showing the relay (RELAY) spectroscopic analysis of the glycolipid bioemulsifier BS-1.
도 17은 당지질 생물유화제 BS-1의 에치에스큐씨(HSQC) 분광 분석결과를 나타낸 그래프.Fig. 17 is a graph showing the results of HSQC spectroscopy analysis of glycolipid bioemulsifier BS-1.
도 18은 당지질 생물유화제 BS-1의 에치엠비씨(HMBC) 분광 분석결과를 나타낸 본 발명의 그래프.18 is a graph of the present invention showing the results of etch MBC (HMBC) spectroscopic analysis of the glycolipid bioemulsifier BS-1.
도 19는 당지질 생물유화제 BS-1의 구조 분석결과를 나타낸 본 발명의 구조식.19 is a structural formula of the present invention showing the structural analysis of the glycolipid bioemulsifier BS-1.
도 20은 당지질 생물유화제 BS-2의 변환적외선 분광장치 분석 결과를 나타낸 본 발명의 그래프.20 is a graph of the present invention showing the results of the conversion infrared spectroscopy analysis of the glycolipid bioemulsifier BS-2.
도 21은 당지질 생물유화제 BS-2의 수소핵자기공명장치 분석 결과를 나타낸 그래프.Figure 21 is a graph showing the hydrogen nuclear magnetic resonance device analysis results of glycolipid bioemulsifier BS-2.
도 22는 당지질 생물유화제 BS-2의 탄소핵자지공명장치 분석결과를 나타낸 그래프.Figure 22 is a graph showing the results of carbon nuclear magnetic resonance device analysis of glycolipid bioemulsifier BS-2.
도 23은 당지질 생물유화제 BS-2의 뎁트(DEPT)분광 분석결과를 나타낸 그래프.FIG. 23 is a graph showing DEPT spectroscopic analysis results of glycolipid bioemulsifier BS-2. FIG.
도 24는 당지질 생물유화제 BS-2의 릴레이(RELAY)분광 분석결과를 나타낸 그래프.Figure 24 is a graph showing the results of the relay (RELAY) spectroscopy of glycolipid bioemulsifier BS-2.
도 25는 당지질 생물유화제 BS-2의 에치에스큐씨(HSQC)분광 분석결과를 나타낸 그래프.Fig. 25 is a graph showing the results of HSQC spectroscopy of glycolipid bioemulsifier BS-2.
도 26은 당지질 생물유화제 BS-2의 에치엠비씨(HMBC) 분광 분석결과를 나타낸 그래프.26 is a graph showing the results of etch MBC (HMBC) spectroscopy of the glycolipid bioemulsifier BS-2.
도 27은 당지질 생물유화제 BS-2의 구조 분석결과를 나타낸 구조식.Figure 27 is a structural formula showing the structure analysis result of the glycolipid bioemulsifier BS-2.
도 28은 슈도모나스 아에루기노사 BYK-2 균주로부터 분리한 생물유화제의 임계마이셀 농도 결과를 나타낸 그래프.FIG. 28 is a graph showing the critical micelle concentration results of bioemulsifiers isolated from Pseudomonas aeruginosa BYK-2 strains.
도 29는 슈도모나스 아에루기노사 BYK-2 균주로부터 분리한 생물유화제의 친 수-친유 균형(HLB)을 측정한 결과를 나타낸 그래프.29 is a graph showing the results of measuring the hydrophilic-lipophilic balance (HLB) of bioemulsifiers isolated from Pseudomonas aeruginosa BYK-2 strain.
도 30은 슈도모나스 아에루기노사 BYK-2 균주로부터 분리한 생물유화제와 시판용 계면활성제의 담수에서의 유화안정성 분석 결과를 나타낸 그래프.30 is a graph showing the results of emulsion stability analysis in fresh water of bioemulsifier and commercial surfactant isolated from Pseudomonas aeruginosa BYK-2 strain.
도 31은 슈도모나스 아에루기노사 BYK-2 균주로부터 분리한 생물유화제와 시판용 계면활성제의 해수에서의 유화안정성 분석 결과를 나타낸 그래프.FIG. 31 is a graph showing the results of emulsification stability analysis in seawater of a bioemulsifier and a commercial surfactant isolated from Pseudomonas aeruginosa BYK-2 strain.
도 32는 슈도모나스 아에루기노사 BYK-2 균주로부터 분리한 생물유화제의 온도안정성 분석 결과를 나타낸 그래프.32 is a graph showing the results of temperature stability analysis of bioemulsifiers isolated from Pseudomonas aeruginosa BYK-2 strain.
도 33은 슈도모나스 아에루기노사 BYK-2 균주로부터 분리한 생물유화제의 121℃에서 10분간 열처리한 후의 유화안정성 분석 결과를 나타낸 그래프.33 is a graph showing the results of emulsion stability analysis after heat treatment at 121 ° C. for 10 minutes of bioemulsifiers isolated from Pseudomonas aeruginosa BYK-2 strains.
도 34는 현재까지 보고되고있는 람노리피드의 종류를 나타낸 구조식.Fig. 34 is a structural formula showing the kind of rhamno lipid reported to date;
도 35는 현재까지 보고되고있는 메칠람노리피드의 종류를 나타낸 구조식.Fig. 35 is a structural formula showing the kind of methylamnolipid reported to date.
도 36은 현재까지 보고되고있는 변형된 람노리피드의 종류를 나타낸 구조식.36 is a structural formula showing the kind of modified rhamno lipid reported to date.
본 발명은 해양세균 슈도모나스 아에루기노사(Pseudomonas aeruginosa) BYK-2에 의해 생산되는 당지질 생물유화제 및 그 정제방법에 관한 것으로, 이를 보다 상세하게 서술하면, 육상과 해양의 유출유 정화에 동시에 사용할 수 있고 산업적으로 다양하게 사용되어질 수 있는 생물유화제 생산 신규 균주인 해양 세균 슈도모나스 아에루기노사 비와이케이-2(Pseudomonas aeruginosa BYK-2 : 수탁번호 KCTC 18012P)(생명공학연구소 기탁일자 2000년 4월27일)로부터 생산되는 람노리피드와 메칠람노리피드를 동시에 생산하는 당지질 생물유화제로, 상기 균주는 토양유래 슈도모나스 아에루기노사 균주와는 다른 당지질 생물유화제인 람노리피드와 메칠람노리피드를 동시에 생산하는 독특한 생육 특성을 지니고 있는 당지질 생물유화제 및 그 정제방법에 관한 것이다.The present invention relates to a glycolipid bioemulsifier produced by the marine bacterium Pseudomonas aeruginosa BYK-2 and a purification method thereof, which can be used simultaneously for the purification of effluent oil on land and in the ocean. Pseudomonas aeruginosa BYK-2 (Accession No. KCTC 18012P) , a new strain producing bioemulsifiers that can be used in a variety of industrial and industrial applications. Glycolipid bioemulsifier to produce both lamnolipid and methyllamnolipid produced at the same time, the strain simultaneously produces the lipophilic bioemulsifiers different from the soil-derived Pseudomonas aeruginosa strain It relates to a glycolipid bioemulsifier having a unique growth characteristics and a purification method thereof.
일반적으로, 해양 관련 오염중에서 국부적으로 단시간 내에 심각한 피해를 주는 것으로는 역시 유조선을 비롯한 각종 선박들의 해난사고에 의해 해양으로 유출되는 기름일 것이며, 이러한 기름의 유출시에는 해수면에 비교적 두터운 막을 형성하여 해수표면은 완전히 대기와 차단되어 햇빛과 산소의 유입을 중단시키므로, 조류의 다량 폐사와 산소 부족으로 인한 해양환경을 염기성 환경으로 만들어 산소를 필요로 하는 해양생물들이 살아가기가 힘들게 되는 등 문제가 야기되어 왔다.In general, serious damage in a short time within the marine-related pollution is also oil that is spilled into the ocean by the marine accidents of various tankers and other vessels, when the oil spills a relatively thick film on the sea surface to form seawater Since the surface is completely cut off from the atmosphere and stops the inflow of sunlight and oxygen, it causes problems such as marine life that requires oxygen by making the marine environment due to the large amount of algae death and oxygen deficiency into a basic environment. come.
우리 나라의 경우, 해양에서 기름의 유출시 기름의 확산을 막기 위한 일차적인 방법으로 오일팬스를 설치하거나, 그 외에 직접 기름을 빨아들이는 도구를 이용하며 기계적으로 수거하는 방법, 또 직접기름을 태워 없애는 방법 등이 있으나, 이와 같은 방법들은 많은 어려움을 가지고 있으며, 친환경적이지는 못한 것이었다.In our country, oil pans are installed as a primary way to prevent oil from spreading in the event of oil spills from the ocean, or they can be collected mechanically using tools that directly suck oil, or burned directly. There are ways to get rid of them, but these methods have a lot of difficulties and are not environmentally friendly.
따라서, 최근에는 미생물을 이용한 방법이 가장 효과적인 것으로, 이는 기름을 분해하는 미생물을 대량으로 오염지역에 뿌려서 기름을 제거 할 수 있게 함으로서, 미생물의 증식시키는 과정이나 분해시키는 과정에서 전혀 오염물질을 배출하지 않기 때문에 환경오염차원에서 각광받은 획기적인 방법으로 이용되고 있으며, 특히, 자연정화에 관계하고 있는 생물들, 즉 자연에 존재하는 생물을 이용하여 기름 에 대한 오염을 제거하는 방법이 시도되었으며, 그 중 미생물이 생산하는 생물유화제(Bio-surfactant)를 이용하는 것이 크게 주목을 받고 있다.Therefore, in recent years, the method using microorganisms is the most effective, so that the oil can be removed by spraying a large amount of microorganisms that break down the oil in a contaminated area, so that no pollutants are emitted during the growth or decomposition of the microorganisms. As a result, it is being used as a groundbreaking method in terms of environmental pollution, and in particular, a method of removing oil pollution using organisms related to natural purification, that is, organisms existing in nature, has been attempted. The use of this bio-surfactant is attracting much attention.
생물유화제(Bio-surfactant, BS)란 생물에 의해 세포표면에 생성되거나 세포 외로 분비되는 화합물로서 친수성인부분과 소수성인 부분을 동시에 가지는 양친매성 물질로, 유화안정성이 매우 우수하다(Gerson and Zajic, 1979).Bio-surfactant (BS) is a compound produced on the cell surface by the organism or secreted to the outside of the cell and is an amphiphilic substance having both hydrophilic and hydrophobic portions at the same time (Gerson and Zajic, 1979).
생물유화제에는 그들이 생산하는 미생물 종과 생화학적 성질에 기초하여 분류되며, 생물유화제의 종류에는 크게 당지질(glycolipids), 인지질과 지방산(phospholipids and fatty acids), 지단백질(lipopeptides and lipoproteins), 고분자 생물유화제(polymeric surfactants)로 분류할 수 있다(Desai and Desai, 1993).Bioemulsifiers are classified based on the microbial species and their biochemical properties they produce, and bioemulsifiers are largely divided into glycolipids, phospholipids and fatty acids, lipoproteins and lipoproteins, and polymeric bioemulsifiers. polymeric surfactants) (Desai and Desai, 1993).
통상적으로, 슈도모나스 아에루기노사(Pseudomonas aeruginosa) 균주에 관한 연구는 토양으로부터 균을 분리하여, 항균활성에 관한 연구가 최초로 보고되었으며, 1949년 F. J. Jarvis등에 의하여 당지질을 생산함이 확인된 이래로 많은 연구자들에 의해, 슈도모나스 아에로기노사와 그 변종 그리고 이들이 생산하는 당지질 생물유화제(람노리피드)의 종류에 대한 많은 연구가 이루어졌다. 또한 슈도모나스 아에로기노사는 녹농균으로 알려져 있고, 주로 폐수처리와 토양 유류오염지역의 생물학적 정화에 주로 이용되고 있으며, 생물유화제의 생산에 관하여 많은 연구가 이루어 지고있다.In general, studies on Pseudomonas aeruginosa strains have been reported for the first time to isolate the bacteria from the soil, antimicrobial activity, since many researchers have been confirmed to produce glycolipids by FJ Jarvis et al. In 1949 Much research has been done on Pseudomonas aeroginosa, its variants, and the types of glycolipid bioemulsifiers (lamnolipids) they produce. In addition, Pseudomonas aeruginosa is known as Pseudomonas aeruginosa, and is mainly used for wastewater treatment and biological purification of soil oil pollution areas, and much research has been conducted on the production of bioemulsifiers.
슈도모나스 아에로기노사(Pseudomonas aeruginosa)가 생산하는 생물유화제는 많은 연구자들에 의해 주성분이 당지질이며, 당은 람노즈(rhamnose)로 지질은 베타-하이드록시데카노익에시드 (β-hydroxydecanoic acid)로 구성되어 있음이 밝혀졌으며, 환경 등의 조건에 따라 일부 변이종이 나타나 생물유화제의 형태도 달라지는 것으로 보고되고 있다.The bioemulsifier produced by Pseudomonas aeruginosa is the major component of glycolysis by many researchers, the sugar is rhamnose, and the lipid is β-hydroxydecanoic acid. It has been found that consists of, and some variants appear depending on the conditions of the environment, it is reported that the form of the bioemulsifier also changes.
이러한 당 지질 생물유화제인 람노리피드는 람노리피드-1(RL-1), 람노리피드-2(RL-2), 람노리피드-3(RL-3), 람노리피드-4(RL-4)의 4종류의 주요형태가 알려져 있다(도33). 람노리피드의 4가지 주요형태 가운데에서 가장 많이 발견되는 형태는 RL-1과 RL-3으로서 Hisatsuka 등(1971)과 Itoh 등(1972)에 의하여 각각 발견되었으며, RL-2와 RL-4는 오직 휴지기 세포에서만 합성되는 것으로 Syldatk(1985) 등이 보고하고 있다.Rhamnolipids, such as sugar lipid bioemulsifiers, are either rhamnolipid-1 (RL-1), rhamnolipid-2 (RL-2), rhamnolipid-3 (RL-3), or rhamnolipid-4 (RL). Four main types of -4) are known (Fig. 33). Of the four major forms of rhamnolipid, the most common are RL-1 and RL-3, which were found by Hisatsuka et al. (1971) and Itoh et al. (1972), respectively. Syldatk (1985) reported that it is synthesized only in resting cells.
그리고, 메칠 람노리피드는 Hirayama와 Kato(1982) 등이 슈도모나스 아에로기노사 158(Pseudomonas aeruginosa 158)이 RL-1과 RL-3의 구조부위에 베타-하이드록시데카노익에시드의 1번 탄소위치에 메칠기가 결합된 유도체를 보고하였으며(도36), Yamaguchi 등(1976)은 RL-1과 RL-3의 람노즈 구조부위 2번 탄소 위치에 각각 알파-데카노익에시드(a-decanoic acid)가 결합된 람노리피드를 보고하고 있다.Methyl rhamnolipids were prepared by Hirayama and Kato (1982), Pseudomonas aeruginosa 158, and the
그리고, 생산되는 람노리피드의 형태는 미생물 종과 배양조건에 영향을 받는 것으로 보고되고 있으며, RL의 구성 당은 예외 없이 람노즈(rhamnose)이다. 지방산은 탄화수소를 탄소원으로 배양할 때 중간물질로 생성되는 것으로, 이 과정에서 람노즈의 변종을 이루는 여러 종류의 지방산이 유래되는 것으로 알려져 있다.In addition, the form of the produced rhamnolipids is reported to be affected by the microbial species and culture conditions, the constituent sugar of RL is rhamnose without exception. Fatty acids are produced as intermediates when cultivating hydrocarbons as a carbon source, and it is known that various fatty acids that form mutants of lamnose are derived from this process.
람노리피드의 대량생산에 관한 연구는 슈도모나스 종 디에스엠 2874(Pseudomonas sp. DSM 2874)균주를 칼슘 알지네이트 지지체를 이용하여 고정화한 후 유동층 반응기에서 연속 배양하여 생산하는 방법(Siemann and Wagner, 1993)과 발효기를 이용하여 연속 배양한 후 한외 여과법으로 생물유화제를 회수하여 동결 농축하는 방법 등으로 생물유화제를 생산하고 있다(Mulligan and Gibbs, 1993).Studies on the mass production of rhamnolipids were carried out by the method of immobilizing Pseudomonas sp. DSM 2874 strain using calcium alginate support followed by continuous culture in a fluidized bed reactor (Siemann and Wagner, 1993) and Bio-emulsifiers are produced by continuous cultivation using a fermenter, followed by recovery and freeze-concentration of bioemulsifiers by ultrafiltration (Mulligan and Gibbs, 1993).
그러나, 상기 종래 기술의 경우 슈도모나스 아에루기노사가 주로 토양에서 분리되어 연구되었을 뿐 해양에서 분리된 예가 없으며, 토양세균의 경우 대부분이 염분 등에 대해 생육이 저해되는 단점으로 해양 유류오염시 적용이 어렵기 때문에 토양 유출유 처리 및 폐수처리 등에만 주로 이용되어왔으며, 생산되는 생물유화제 또한 물성이 다른 람노리피드와 메칠람노리피드를 이들을 생산하는 각각의 균주에서 따로 생산되고 있으며, 이들을 동시에 생산하는 예가 없으며, 대량분리공정의 경우 기존의 방법에서는 대량 배양후 생물유화제 분리 공정시 한외여과법과 동결건조 등의 방법을 활용하기 때문에 유지관리 비용이 많이 들고, 고순도의 생물 유화제의 회수가 어려운 단점이 있다.However, in the case of the prior art, Pseudomonas aeruginosa was mainly separated from the soil and studied, and there is no example separated from the ocean, and in the case of soil bacteria, growth is inhibited due to salinity, which is difficult to apply in marine oil pollution. Therefore, it has been mainly used only for soil runoff treatment and wastewater treatment, and the bioemulsifiers produced are also produced separately from each strain producing rhamnolipid and methyllamnolipid with different physical properties. In the case of the bulk separation process, the conventional method uses a method such as ultrafiltration and lyophilization during the bioemulsion separation process after mass cultivation, which leads to a high maintenance cost and difficult recovery of high purity bioemulsifier.
본 발명은 상기 종래기술의 문제점을 해결하기 위해서 발명한 것으로, 토양과 해양의 유류 오염지역에 유출유의 효율적인 제거와 각 산업분야에 다양하게 사용되고있는 화학합성 계면활성제를 대신할 환경친화적인 대체 물질의 개발을 위한 것으로, 현재까지 보고되지 않은 해양세균의 슈도모나스 아에로기노사 비와이케이-2(Pseudomonas aeruginosa BYK-2 : 수탁번호 KCTC 18012P) 균주를 통하여, 람노피드와 메칠람노리피드를 동시에 생산할 수 있는 생물유화제 및 그 정제 방법의 특징 을 그 목적으로 한다.The present invention has been invented to solve the problems of the prior art, the efficient removal of effluent oil in the oil and contaminated areas of the soil and ocean and of the environmentally friendly alternative material to replace the chemical synthetic surfactants that are used in various industries For development, through the Pseudomonas aeruginosa BYK-2 (Accession No. KCTC 18012P ) strain of marine bacteria, which have not been reported to date, it is possible to simultaneously produce rhamnofeed and mechilamnolipide . Bioemulsifiers and their purification methods are intended for this purpose.
이하, 상기 목적을 달성하기 위하여, 본 발명에서는 육상과 해양의 유출유 정화에 동시에 사용할 수 있고 산업적으로 다양하게 사용되어질 수 있는 생물유화제 생산 균주인 해양 세균 슈도모나스 아에루기노사 비와이케이-2 (Pseudomonas aeruginosa BYK-2 : 수탁번호 KCTC 18012P) 를 해양에서 분리하여, 상기 균주는 토양유래 슈도모나스 아에루기노사 균주와는 다른 당지질 생물유화제인 람노리피드와 메칠람노리피드를 동시에 생산하는 독특한 생육 특성을 지니고 있는 생물유화제 제공하는 것으로 첨부된 도면을 참조하면서 상세히 설명하기로 한다.Hereinafter, in order to achieve the above object, in the present invention, marine bacteria Pseudomonas aeruginosa biwakei-2 ( Pseudomonas) , which is a bioemulsifier producing strain that can be used simultaneously for purification of effluent oil of land and sea and can be used in various industries. aeruginosa BYK-2: Accession No. KCTC 18012P ) is isolated from the ocean, and the strain has the unique growth characteristics of simultaneously producing a lipid lipid bioemulsifier , Rhamnolipid and Methyllamnolipid , different from Pseudomonas aeruginosa strain derived from soil. It will be described in detail with reference to the accompanying drawings to provide a bioemulsifier having.
1] 슈도모나스 아에루기노사 BTK-2(1] Pseudomonas aeruginosa BTK-2 ( Pseudomonas aeruginosa BYK-2 : 수탁번호 KCTC 18012PPseudomonas aeruginosa BYK-2: Accession No. KCTC 18012P ) 균주Strain
도 1은 해양 유류 오염지역으로부터 유류 분해세균의 분리하는 과정에 대한 개략도이다. 이와 같은 방법으로 균주를 분리할 경우, 유류 분해와 관련된 균주들을 쉽게 분리할 수 있으며, 본 발명의 경우에도 본 방법으로 80여 균주의 유류분해 세균을 확보할 수 있었으며, 그 중 유류 분해능과 생물유화제생산 능이 가장 우수한 균주인 비와이케이-2(BYK-2)를 본 발명에 이용하였으며, 사용된 배지와 분리 방법은 다음과 같다.Figure 1 is a schematic diagram of the process of separating the oil decomposition bacteria from marine oil contaminated area. When the strains are separated in this way, strains related to oil degradation can be easily separated, and in the case of the present invention, over 80 strains of oil-degrading bacteria can be obtained by the present method, among which oil resolution and bioemulsifier BYK-2, the strain having the highest production capacity, was used in the present invention, and the medium and separation method used were as follows.
1) 슈도모나스 아에루기노사 비와이케이-2(Pseudomonas aeruginosa BYK-2 : 수탁번호 KCTC 18012P)의 분리방법1) Pseudomonas aeruginosa BYK-2 ( Pseudomonas aeruginosa BYK-2: Accession No. KCTC 18012P )
우리나라 남해안 유류 오염지역 등 남해안 30여 곳의 지역 해수시료 50㎖를 채취하여, 균주분리용 유류배지[비멸균 아라비안 라이트원유, 0.5g; yeast extract, 5.0㎎; K2HPO4, 0.5㎎; (NH4)2SO4, 50㎎]가 첨가된 삼각플라스크에 넣고, 25℃ 에서 180rpm으로 진탕배양기를 이용하여 7일간 배양하였다.50 ml of seawater samples from 30 areas of the south coast, including the oil pollution area of the south coast of Korea, were collected and strained oil medium [non-sterile Arabian light crude oil, 0.5g; yeast extract, 5.0 mg; K 2 HPO 4 , 0.5 mg; (NH 4 ) 2 SO 4 , 50 mg] was added to an Erlenmeyer flask, to which the mixture was incubated at 180 ° C. at 25 ° C. for 7 days using a shake incubator.
7일간의 배양 기간 중 아라비안 라이트 원유가 완전히 분해된 지역의 해수를 선별하여, 다시 살균된 해수가 들어있는 균주분리용 유류배지에 선별된 유류분산 배지를 각각 2%씩 넣고, 다시 3일간 진탕 배양(25℃에서 180rpm)한 후, 아라비안 라이트 원유의 분해능이 가장 좋은 배지를 선별하여 변형된 해수배지(peptone, 5.0g; yeast extract, 1.0g; ferric citrate, 0.1g; NH4NO3, 1.6mg; K2HPO 4, 8.0㎎; agar, 0.13g; sea water, 1.0ℓ; pH 7.0)에 도말하여 단일 개체들을 분리한 후, 분리용 유류배지에 접종하여 7일간 같은 조건에서 진탕 배양(25℃에서 180rpm)한 후, 원유분해능이 우수한 배지를 재차 선별하여 변형된 해수배지에 도말하여 최종적으로 단일 균주를 분리하였으며, 이를 -75℃ 초저온냉장고에 보관하여 실험에 사용하였다.During the 7 days of culture, seawater from the area where the Arabian Light crude oil was completely decomposed was selected, and 2% of the selected oil dispersion medium was added to the strain-separated oil medium containing the sterilized seawater, followed by shaking culture for 3 days. 180 rpm at 25 ° C.), and then selected the best medium for the resolution of Arabian Light Crude Oil (peptone, 5.0 g; yeast extract, 1.0 g; ferric citrate, 0.1 g; NH 4 NO 3 , 1.6 mg); K 2 HPO 4 , 8.0 mg; agar, 0.13 g; sea water, 1.0 L; pH 7.0), single isolates were isolated, inoculated in a separate oil medium and shaken for 7 days under the same conditions (at 25 ° C). After 180rpm), the medium having excellent crude oil resolution was again selected and spread on a modified seawater medium to finally isolate a single strain, which was stored in a -75 ° C cryogenic refrigerator and used for the experiment.
상기 방법에 의해 분리되어진 균주를 비와이케이-2(BYK-2)라 명명하였으며, 도 2는 본 균주의 동정 결과를 나타낸 도표로서, 먼저 본 균주에 관한 동정은 그램 염색을 통한 현미경 상에서의 형태학적 관찰과 각종 생리, 생화학적 특성을 조사하며 그램 음성의 운동성을 가진 슈도모나스 아에루기노사로 동정되었고 슈도모나스 아에루기노사 비와이케이-2(Pseudomonas aeruginosa BYK-2 : 수탁번호 KCTC 18012P)로 명명하였다.The strain isolated by the above method was named BYK-2, and FIG. 2 is a diagram showing the identification result of the present strain. First, the identification of the present strain was confirmed by morphological microscopy under gram staining. Pseudomonas aeruginosa BYK-2 (accession number KCTC 18012P ) was identified as Pseudomonas aeruginosa with Gram-negative motility.
2) 슈도모나스 아에루기노사 비와이케이-2(Pseudomonas aeruginosa BYK-2 : 수탁버호 KCTC 18012P)의 특성2) Characteristics of Pseudomonas aeruginosa BYK-2 ( Trusted Lake KCTC 18012P )
① 염분농도에 따른 생육특성① Growth characteristics according to salinity concentration
도 3은 슈도모나스 아에루기노사 비와이케이-2의 염분농도에 따른 생육특성을 나타낸 것으로, 변형된 엘비배지에 염분농도를 0~9%까지 넣고 48시간 배양한 후 분광 광도계를 이용하여 배양액의 농도를 660nm에서 측정한 것으로, 일반적으로 토양에서 분리한 슈도모나스 아에루기노사 균주는 6.5% 이상의 염분농도에서는 전혀 균이 성장하지 않는 것으로 보고되고 있으나, 해양에서 분리한 본 균주의 경우에는 0~9% 범위 내에서도 균의 생육이 가능한 것으로 확인되었으며, 해수의 염분농도는 3%이나, 본 균주의 경우에는 염분농도가 4%까지는 전혀 생육에 영향을 받지 않는 것으로 보아 토양이나 해양에서 모두 이용이 가능한 것으로 밝혀졌다.Figure 3 shows the growth characteristics according to the salinity concentration of Pseudomonas aeruginosa biwaikei-2, incubated for 48 hours with a salt concentration of 0 ~ 9% in modified Elbe medium medium using a spectrophotometer It is reported that the Pseudomonas aeruginosa strain isolated from the soil does not grow at all at a salt concentration of 6.5% or higher, but in the case of this strain isolated from the sea, it is 0-9%. It was found that the growth of bacteria was possible within the range, and the salt concentration of seawater was 3%, but the salt concentration of this strain was not affected by growth at all up to 4%. lost.
② 해수와 담수에서 유류의 생분해능② Biodegradability of oil in seawater and fresh water
도 4와 도 5는 본 균주가 해수와 담수 등에서 유류분해능이 우수한지의 여부와 난분해성 유류인 아라비안 라이트 원유와 벙커씨유에 대하여 어느 정도의 유류분해능을 가지는지를 7일간 담수와 해수가 첨가된 변형된 엘비 배지에서 배양하여 확인한 결과를 나타낸 것으로, 난 분해성 유류의 생분해능을 조사하기 위하여 중량법(Mulkins-Phillips 등, 1974)을 사용하였으며, 중량법은 250㎖ 삼각플라스크 각각에 아라비안 라이트 원유 1%와 벙커씨유 1%를 넣고, 변형된 멸균 엘비 배지(tryptone, 10.0g; yeast extract, 5.0g; 해수 또는 담수, 1ℓ) 50㎖를 넣은 후, 14시간 전 배양된 배양액을 1% 접종하고, 25℃에서 180rpm으로 진탕 배양하였 으며, 유류의 생분해능을 조사하기 위하여 배양시간별(24, 48, 72시간)로 50㎖배양액이 든 삼각플라스크를 회수한 다음, 회수된 삼각플라스크에 50㎖벤젠을 가한 후, 잔존 유류를 추출하고, 무수 황산나트륨으로 수분을 제거하여 정량분석에 이용하였고, 측정에 사용된 용기는 먼저 100℃ 건조기에서 1시간 건조한 후, 0.1㎎까지 정량이 가능한 저울로서 무게를 측정하고, 수분이 제거된 벤젠 추출물을 용기에 넣고 80℃ 건조기에서 6시간 동안 건조하고 식힌 후 저울에서 정량하여 생분해능을 측정하였다.4 and 5 are strains added with fresh water and seawater for 7 days to determine whether the strain has excellent oil resolution in seawater and fresh water, and how much oil resolution is achieved for Arabian Light crude oil and bunker seed oil, which are hardly degradable oils. The results obtained by culturing in the cultured Elbe medium were used, and gravimetric method (Mulkins-Phillips et al., 1974) was used to investigate the biodegradability of refractory oils, and the gravimetric method was used for 1% Arabian Light crude oil and bunker in a 250 ml Erlenmeyer flask. 1% of seed oil was added, 50 ml of modified sterile Elbi medium (tryptone, 10.0 g; yeast extract, 5.0 g; seawater or fresh water, 1 L) was added, and the cultured culture was inoculated 1% at 14 hours before, at 25 ° C. The cultured shaker was shaken at 180rpm at, and the Erlenmeyer flask containing 50ml culture solution was recovered by incubation time (24, 48, 72 hours) to investigate the biodegradability of oil. After adding 50 ml benzene, the remaining oil was extracted, water was removed with anhydrous sodium sulfate, and used for quantitative analysis. The vessel used for the measurement was first dried in a 100 ° C. dryer for 1 hour and then weighed to 0.1 mg. The weight was measured, and the benzene extract from which moisture was removed was placed in a container, dried in an 80 ° C. dryer for 6 hours, cooled, and quantified in a balance to measure biodegradability.
그 결과, 아라비안 라이트 원유의 경우(도4)에는 해수와 담수에서 각각 92.1%와 96%가 생분해되었으며, 벙커씨유의 경우(도5)에도 각각 76.2%와 80%가 생분해되어 유류 분해능이 아주 우수한 균주로 확인되었다.As a result, 92.1% and 96% were biodegraded in seawater and fresh water, respectively, in the case of Arabian Light Crude Oil (Fig. 4), and 76.2% and 80% were biodegradable in Bunker Seed Oil (Fig. 5), respectively. It was identified as a strain.
2] 슈도모나스 아에루기노사 BYK-2 균주로부터 분리한 당지질 생물유화제의 분리정제2] Isolation and Purification of Glycolipid Bioemulsifier from Pseudomonas aeruginosa BYK-2 Strain
도 6은 본 균주로부터 당지질 생물유화제를 추출농축한 후 분리정제하는 방법으로, 각종 탄소원을 기질로 하여 균주를 배양한 후, 그 배양액을 4℃에서 8,000g로 10분간 원심분리 한 후, 균주배양 상층액을 회수하였다. 회수한 균주배양 상층액은 5노르말 염산(HCl)으로 수소이온농도(pH)를 2.0으로 맞춘 후, 동량의 클로로포름-메탄올 혼합용매(CM, chloroform : methanol = 1:1, v/v)로 10분간 교반, 추출하고 용매 층을 분리하여 회전진공증발기로 농축하였다. 농축하여 얻어진 시료는 실리카겔이 충진되어 있는 분리관(2.5×100cm)을 이용하여 클로로포름으로 먼저 잔존하는 기질을 제거한 후, 클로로포름-메탄올 혼합용매(CM, chloroform : methanol = 1:1, v/v)를 사용하여 당지질 생물유화제를 1차로 분리한 후 농축하였다. 이렇게 얻어진 생물유화제는 재차 실리카겔이 충진된 분리관으로 클로로포름-메탄올-물 혼합용매(CMW, chloroform : methanol : water = 60:25:4, v/v/v) 혼합용매를 이용하여 용출 시켰다. 용출하여 분별 수집한 시료는 농축하여 박층크로마토그래피로(TLC)로써 순도를 확인하였고, 명확히 분리되지 않은 시료는 재차 회수하여 농축한 후, Sep-pak silica catridge(Waters Co., USA)를 이용하여 분리하였으며, 실리카 카트리지를 이용한 생물유화제 분리방법으로는 먼저, 실리카 카트리지(silica catridge)를 클로로포름 20㎖로 세척한 후, 클로로포름으로 녹인 시료를 실리카 카트리지에 넣고 시료를 흡착시킨다. 흡착시킨 시료는 클로로포름과 메탄올을 이용하여, 농도 비로 용출시켜 정제하였으며, 최종 순도확인은 고속 액체 크로마토그래피 법(HPLC, pharmacia LKB Co, Sweden)으로 하였다.6 is a method of extracting and concentrating a glycolipid bioemulsifier from the present strain and then separating and purifying the strain. After culturing the strain using various carbon sources as substrates, the culture medium is centrifuged at 8,000 g for 10 minutes at 4 ° C., followed by strain culture. The supernatant was recovered. The recovered strain culture supernatant was adjusted to pH 2.0 with 5 normal hydrochloric acid (HCl) and then to 10 with the same amount of chloroform-methanol mixed solvent (CM, chloroform: methanol = 1: 1, v / v). Stirred for minutes, extracted and the solvent layer was separated and concentrated by rotary evaporator. The sample obtained by concentration was first removed with chloroform by using a separation tube (2.5 × 100 cm) filled with silica gel, and then mixed with chloroform-methanol (CM, chloroform: methanol = 1: 1, v / v). Glycolipid bioemulsifier was isolated first and then concentrated. The bioemulsifier thus obtained was again eluted with a silica gel-filled separator using a mixed solvent of chloroform-methanol-water (CMW, chloroform: methanol: water = 60: 25: 4, v / v / v). The eluted fractions were collected and concentrated to confirm purity by thin layer chromatography (TLC). Samples that were not clearly separated were recovered and concentrated again using Sep-pak silica catridge (Waters Co., USA). As a bioemulsifier separation method using a silica cartridge, first, a silica cartridge is washed with 20 ml of chloroform, and a sample dissolved in chloroform is put into a silica cartridge and the sample is adsorbed. The adsorbed sample was purified by eluting with chloroform and methanol in a concentration ratio, and final purity was confirmed by high performance liquid chromatography (HPLC, pharmacia LKB Co, Sweden).
이러한 방법으로 회수할 경우, 추출한 생물유화제 원액 속에 잔류해 있는 탄화수소화합물이나 지방산 등의 제거가 용이하고 고속액체 크로마토그래피(HPLC)등을 이용하지 않고도 빠른 시간 내에 95% 이상의 순도를 가진 당 지질을 회수할 수 있는 장점을 지니고 있다.When recovered in this way, it is easy to remove hydrocarbon compounds and fatty acids remaining in the extracted bioemulsion stock solution and recover sugar lipids with purity of 95% or more in a short time without using high performance liquid chromatography (HPLC). It has the advantages to do it.
도 7은 탄소원의 종류에 따른 당 지질 생물유화제의 종류를 확인한 것으로서, 배지의 성분 중 기질로 사용되는 탄소원을 변화시켜 배양한 후 유기용매로 추출, 농축하여 박층 크로마토그래피(TLC)로 몇 종류의 당 지질 형태의 생물유화제를 생산하는지를 확인한 것으로, 박층크로마토그래피 분석에는 실리카겔 60이 도포된 유리판(silica gel 60 F 254 glass plate)을 사용하고, 전개용매는 클로로포름: 메 탄올: 물이 60 : 25 : 4(부피/부피/부피)로 혼합된 것을 사용하였다.7 shows the types of sugar lipid bioemulsifiers according to the types of carbon sources, which are cultured by changing the carbon source used as a substrate among the components of the medium, and then extracted and concentrated with an organic solvent to obtain several kinds of thin layer chromatography (TLC). It was confirmed that the bioemulsion in the form of sugar lipid was produced. For thin layer chromatography analysis, a silica gel 60 F 254 glass plate was used, and the developing solvent was chloroform: methanol: water 60: 25: A mixture of 4 (volume / volume / volume) was used.
검출시약으로는 에탄올에 20% 황산이 들어 있는 시약과 알파-나프토레소시놀(α-naphtoresorcinol)시약을 사용하였다.For detection reagents, a reagent containing 20% sulfuric acid in ethanol and an alpha-naphtoresorcinol reagent were used.
탄소원의 종류별로 생산된 당지질체의 종류를 살펴보면, 탄화수소화합물을 기질로 사용했을 경우 쿠웨이트 원유, 벙커씨유, 헥사데칸 그리고 테트라데칸에서는 4종류(이동률 0.48, 0.52, 0.65, 0,82)의 당지질이 생산되었고, 유동 파라핀에서는 5종류 (이동률 0.48, 0.52, 0.65, 0.70, 0.82)가 생산되었다.According to the types of glycolipids produced by carbon source, four types of glycolipids (moving rate 0.48, 0.52, 0.65, 0,82) were used in crude oil, bunker seed oil, hexadecane and tetradecane when hydrocarbon compounds were used as substrates. Five kinds (moving rate 0.48, 0.52, 0.65, 0.70, 0.82) were produced in the liquid paraffin.
그리고 지방산을 기질로 한 올레익산과 올리브 유, 그리고 레시틴에서는 3종류(이동률 0.48, 0.65, 0.82)가 생산되었다. 당을 기질로 한 아라비노즈, 락토즈 및 프락토즈는 4종류(이동률 0.48, 0.65, 0.75, 0.82), 트리할로즈, 덱스트로즈, 말토즈의 경우는 5종류 (이동률 0.48, 0.52, 0.65, 0.75, 0.82), 갈락토즈에서는 5종류(이동률 0.48, 0.65, 0.70, 0.75, 0.82), 슈크로즈에서 5종류 (이동률 0.48, 0.65, 0.75, 0.82, 0.91)가 각각 생산되었다. 이러한 결과를 바탕으로 본 발명의 균주가 생산하는 생물유화제는 탄소원의 종류에 따라 다양한 종류의 당 지질체를 생산하는 것을 확인하였으며, 아울러 기질의 종류에 상관없이 3종류(이동률 0.48, 0.65, 0.82)의 당지질체는 공통적으로 생산하는 것으로 확인하였다.Three kinds (migration rate 0.48, 0.65, 0.82) were produced from fatty acid-based oleic acid, olive oil and lecithin. Four kinds of sugar-based arabinose, lactose, and fructose (moving rate 0.48, 0.65, 0.75, 0.82), and five kinds of trihalose, dextrose, maltose (moving rate 0.48, 0.52, 0.65, 0.75, 0.82), and 5 types (moving rate 0.48, 0.65, 0.70, 0.75, 0.82) were produced in galactose, and 5 types (moving rate 0.48, 0.65, 0.75, 0.82, 0.91) were produced in sucrose, respectively. Based on these results, the bioemulsifier produced by the strain of the present invention was confirmed to produce various kinds of sugar lipids according to the type of carbon source, and also three kinds regardless of the type of substrate (migration rate 0.48, 0.65, 0.82). Glycolipids were confirmed to be commonly produced.
따라서 본 균주의 경우 기질의 종류에 따라 생산할 수 있는 당지질체는 총 7종류 (이동률 0.48, 0.52, 0.65, 0.70, 0.75, 0.82, 0.91)로 현재까지 보고된 슈도모나스 아에루기노사 균주들 중 한 균주가 생산할 수 있는 최대 당 지질의 종류는 4가지(RL 1, RL 2, RL 3, RL 4)에 비하여 7종류의 당 지질체 생산 능력은 해양에서 분리한 본 균주가 슈도모나스 아에루기노사 변종으로 해석되어진다.Therefore, in this strain, one of seven strains of Pseudomonas aeruginosa, which has been reported so far, can be produced according to the type of substrate in total (7 transfer rates: 0.48, 0.52, 0.65, 0.70, 0.75, 0.82, 0.91) The maximum amount of sugar lipids that can be produced is 4 strains (
도 8은 정제된 두종류(BS-1, BS-2)의 생물유화제의 박층크로마토그래피의 결과를 도시한 것으로, 그 내용을 살펴보면, 배양액 중 생물유화제의 생산량이 가장 많은 종류인 이동률이 0.48(BS-1), 0.65(BS-2)인 두 종류에 대하여 분리정제한 것으로서 순도는 95%와 96.1% 이었으며, 양은 각각 500㎖과 420㎖을 얻었으며, 이 시료를 이용하여 당지질 생물유화제 BS-1과 BS-2의 구조를 분석하여 보았다.FIG. 8 shows the results of thin layer chromatography of two purified bioemulsifiers (BS-1 and BS-2). Referring to the contents, the migration rate of 0.48 ( BS-1) and 0.65 (BS-2) were separated and purified. The purity was 95% and 96.1%, and the amount was 500ml and 420ml, respectively. The glycolipid bioemulsion agent BS- We analyzed the structure of 1 and BS-2.
1) 당지질 생물유화제 BS-1 시료의 구조 분석1) Structural Analysis of Glycolipid Bioemulsifier BS-1 Sample
:람노리피드(2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid)Rhamno lipid (2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid)
도 9는 당지질 생물유화제 BS-1 시료의 관능기를 확인하기 위하여 변환적외선 분광장치를 이용 분석 결과를 나타낸 것으로, 그 결과를 보면, 흡수파장이 3450cm-1, 2920cm-1, 1750cm-1, 1450cm-1, 1050∼ 1120cm-1 을 가지며, 파장별로 각각 수산화기(hydroxyl group,-OH), 긴 사슬의 탄화수소(long chain hydrocarbon,-CH2-), 에스테르(esters,-CH2-CO-O-R), 메칠기(-methyl,-CH3 ), -진동부위(vibration,-CH2 -)를 가진다.Figure 9 shows an analysis result using the transform infrared spectrometer to determine the glycolipid emulsifier biological functionality of the BS-1 samples, looking at the result, the absorption wavelength of 3450cm -1, 2920cm -1, 1750cm -1 , 1450cm - 1 , 1050-1120cm -1 , and each hydroxyl group (hydroxyl group, -OH), long chain hydrocarbon (-CH 2- ), esters (esters, -CH 2 -CO-OR), It has a methyl group (-methyl, -CH3), -vibration (vibration, -CH 2- ).
도 10은 당지질 생물유화제 BS-1의 고속원자충격 질량분석장치를 이용한 분석결과로서 분자량은 650.37로 화학식은 C32 H58 O13 으로 밝혀졌다.FIG. 10 is a molecular weight of 650.37 and a chemical formula of C 32 H 58 O 13 as a result of analysis using a fast atom impact mass spectrometer of the glycolipid bioemulsifier BS-1.
도 11 내지 도 17은 당지질 생물유화제 BS-1의 구조분석을 위하여 실시한 핵 자기 공명분석법으로서 1차원 분석과 2차원 분석을 동시에 수행하였으며, 도 11은 수소핵자기공명 분석, 도 12는 탄소핵자기공명 분석, 도 13은 뎁트(DEPT)분광 분석, 도 14는 노이지(NOESY) 분광 분석, 도 15는 토코지(TOCSY) 분광 분석, 도 16은 릴레이(RELAY) 분광분석, 도17은 에치에스큐씨(HSQC) 분광 분석, 도 18은 에치엠비씨(HMBC) 분광 분석 결과로서,11 to 17 are nuclear magnetic resonance analysis carried out for structural analysis of the glycolipid bioemulsifier BS-1 was performed simultaneously with the one-dimensional analysis and two-dimensional analysis, Figure 11 is hydrogen nuclear magnetic resonance analysis, Figure 12 is the carbon nuclear magnetic Resonance analysis, FIG. 13 is a DEPT spectroscopy analysis, FIG. 14 is a NOESY spectroscopy analysis, FIG. 15 is a TOCSY spectroscopy analysis, FIG. 16 is a relay spectroscopy analysis, and FIG. (HSQC) spectroscopic analysis, Figure 18 is the HMBC spectroscopic analysis results,
이들의 자료를 종합하여 당지질 생물유화제 BS-1 시료의 구조를 해석하면, 도 19의 구조가 완성되며, 당지질 생물유화제 BS-1의 구조는 2개의 알파 엘람노즈(α-L-rhamnose)와 2개의 베타하이드록시데카노익 에시드(β-hydroxydecanoic acid)가 결합되어 있는 2-오-알파-엘-람노페라노실-알파-엘-람노피라노실-배타-하이드록시데카노일-베타하이드록시데카노익에시드(2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid)로 밝혀졌다. 이 구조는 토양유래 슈도모나스 아에루기노사 디에스엠 2659(Pseudomonas aeruginosaDSM 2659)가 생산하는 4종류의 람노리피드(RL1, RL2, RL3, RL4)(Gruber 등, 1993)중 람노리피드 RL3와 구조가 일치한다.By combining these data, the structure of the glycolipid bioemulsion agent BS-1 sample is analyzed, and the structure of FIG. 19 is completed, and the structures of the glycolipid bioemulsion agent BS-1 are composed of two alpha-L-rhamnose and two. 2-O-Alpha-L-Rhamnoferanosyl-alpha-L-Ranmopyranosyl-Exclusive-Hydroxydecanoyl-Betahydroxydecanoo to which β-hydroxydecanoic acid is bound Ixide (2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid). This structure is based on four different types of lamnolipids (RL1, RL2, RL3, RL4) produced by Pseudomonas aeruginosa DSM 2659 (Gruber et al., 1993). Matches.
2) 당지질 생물유화제 BS-2 시료의 구조 분석2) Structural Analysis of Glycolipid Bioemulsifier BS-2 Sample
:메칠람노리피드(2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxyl decanoyl-β-hydroxydecanoic acid methyl ester)Methyllamnolipid (2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxyl decanoyl-β-hydroxydecanoic acid methyl ester)
도 20은 당지질 생물유화제 BS-2 시료의 관능기를 확인하기 위하여 변환적외선분광장치를 이용 분석 결과를 나타낸 것으로, BS-1과 같은 위치의 흡수파장 3450cm-1, 2920cm-1, 1750cm-1, 1450cm-1, 1050∼1120cm-1 을 가지는 것으로 확인되었으며, 당지질 생물유화제 BS-2의 고속원자충격 질량분석장치를 이용한 분석 결과 분자량은 664로 화학식은 C33 H60 O13으로 밝혀졌다.Figure 20 shows an analysis result using the transform infrared spectrometer to determine the glycolipid emulsifier biological functionality of the BS-2 sample, the absorption wavelength in the same location as the BS-1 3450cm -1, 2920cm -1 , 1750cm -1, 1450cm -1 , 1050 ~ 1120cm -1 was confirmed, the molecular weight was 664 and the chemical formula was C 33 H 60 O 13 as a result of analysis using a fast atom impact mass spectrometer of glycolipid bioemulsifier BS-2.
도 21 내지 도 27은 당지질 생물유화제 BS-2의 구조분석을 위하여 실시한 핵자기 공명분석법으로서 1차원 분석과 2차원 분석을 동시에 수행하였으며, 도 21은 수소핵자기공명 분석, 도 22 탄소핵자기공명 분석, 도 23은 뎁트(DEPT) 분광 분석, 도 24는 릴레이(RELAY) 분광 분석, 도 25는 에치에스큐씨(HSQC) 분광 분석, 도 26은 에치엠비씨(HMBC)분광 분석 결과로서,21 to 27 are nuclear magnetic resonance analysis performed for the structural analysis of glycolipid bioemulsifier BS-2 was performed simultaneously with the one-dimensional analysis and two-dimensional analysis, Figure 21 is hydrogen nuclear magnetic resonance analysis, Figure 22 carbon nuclear magnetic resonance Analysis, FIG. 23 is a DEPT spectroscopic analysis, FIG. 24 is a relay (RELAY) spectroscopy analysis, FIG. 25 is an HSQC spectroscopy analysis, FIG. 26 is an HMBC spectroscopic analysis result.
이들의 자료를 종합하여 당지질 생물유화제 BS-2 시료의 구조를 해석하면, 도 27에 도시된 것과 같은 구조가 완성되며, 당지질 생물유화제 BS-2의 구조는 2개의 알파 엘 람노즈(α-L-rhamnose)와 1개의 베타-하이드록시데카노익에시드(β-hydroxydecanoic acid), 1개의 베타-하이드록시데카노익에시드 메칠 에스테르(β-hydroxydecanoic acid methyl ester)가 결합된 2-오-알파-엘-람노피라노실-알파-엘-람노피라노실-베타-하이드록시데카노일-베타하이드록시데카노익에시드메칠에스테르(2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxy-decanoyl-β-hydroxydecanoic acid methyl ester로 밝혀졌다. 이 구조는 일본의 히라야마 등 (Hirayama et al, 1982)이 토양유래 슈도모나스 아에루기노사 158(Peudomonas aeruginosa158)로부터 최초로 분리하여 보고한 메칠 람노리피드(GL1, GL2), 2개의 구조 중 GL2와 일치하는 것으로 나타난다. 그러 나 그루버 등(Gruber et al, 1193)이 보고한 람노리피드 RL3과 히라야마 등(Hirayama et al, 1982)이 보고한 GL2는 각각 한 균주에서 람노리피드나 메칠 람노리피드를 생산하였지만, 본 발명의 균주의 경우는 한 균주에서 람노리피드와 메칠람노리피드를 동시에 생산한다는 점이 슈도모나스 아에루기노사(Peudomonas aeruginosa)연구에 있어서 현재까지 보고된 적이 없는 새로운 균학적 특성을 지닌 변이 종으로 사료된다.By combining these data, the structure of the glycolipid bioemulsion agent BS-2 sample is analyzed, and the structure as shown in FIG. 27 is completed, and the structure of the glycolipid bioemulsion agent BS-2 is composed of two alpha el lamnoses (α-L). 2-o-alpha combined with rhamnose, 1 beta-hydroxydecanoic acid and 1 beta-hydroxydecanoic acid methyl ester L-Ranmopyranosyl-alpha-L-Ranmopyranosyl-beta-hydroxydecanoyl-betahydroxydecanoic acid methyl ester (2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxy It was identified as -decanoyl-β-hydroxydecanoic acid methyl ester This structure was first reported by Hirayama et al. (1982) isolated from soil-derived Pseudomonas aeruginosa 158 (Peudomonas aeruginosa158). (GL1, GL2), which matches GL2 of the two structures However, Rhamnolipid RL3 reported by Gruber et al (1193) and GL2 reported by Hirayama et al (1982) produced either rhamnolipid or methyl ramnolipid in one strain. In the case of the strain of the present invention, the production of rhamnolipid and methyllamnolipid at the same time in one strain is a mutant species with new bacteriological characteristics that have not been reported so far in the study of Pseudomonas aeruginosa. Is fed.
3] 슈도모나스 아에루기노사 BYK-2(3] Pseudomonas aeruginosa BYK-2 ( Pseudomonas aeruginosa BYK-2 : 수탁번호 KCTC 18012PPseudomonas aeruginosa BYK-2: Accession No. KCTC 18012P ) 균주로부터 분리한 생물유화제의 특성Characteristics of Bioemulsifier Isolated from Strains
1) 임계 마이셀농도를 측정1) measure critical micelle concentration
도 28은 슈도모나스 아에루기노사 비와이케이-2가 생물유화제에 대한 임계마이셀 농도를 측정하였다. 일반적으로 계면활성제들은 낮은 농도에서는 흡착의 형태로 표면장력과 계면장력의 저하에 관여하다가 농도가 진해지면 계면활성제의 흡착이 포화상태에 도달하여 집합체를 형성하는데 이것이 임계 마이셀 농도(CMC)라고 한다. 이러한 아이셀의 중요한 성질은 입자콜로이드와는 달리 수중에서 매우 안정된 형태를 취하고 있어 유화제의 본래 목적인 유화안정성을 유지시키는데 중요한 역할을 하게 된다.FIG. 28 shows the critical micelle concentration for Pseudomonas aeruginosa biwaikei-2 bioemulsifier. In general, surfactants are involved in lowering the surface tension and the interfacial tension in the form of adsorption at low concentrations, and when the concentration increases, the adsorption of the surfactant reaches saturation to form an aggregate, which is called a critical micelle concentration (CMC). Unlike the particle colloid, the important property of the icell is very stable in water, so it plays an important role in maintaining the emulsion stability, which is the original purpose of the emulsifier.
임계 마이셀 농도는 표면장력계를 사용하여, 배양 상층액으로부터 분리된 생물유화제를 각기 다른 농도별(0, 1, 2, 3, 4, 5, 7.5, 10, 25, 50, 100㎎/ℓ)로 10㎖씩 희석하여 제조한 후, 표면장력계(TD-1, Lauda, German)로 표면장력을 측정하여 임계 마이셀 농도(critical micelle concentration)값을 측정하였으며, 본 균주 가 생산하는 당지질 생물유화제의 임계 마이셀 농도는 10㎎/ℓ이였으며, 표면장력은 29.5mN/m 이였다.The critical micelle concentration was determined by using a surface tension meter, and the bioemulsifier separated from the culture supernatant at different concentrations (0, 1, 2, 3, 4, 5, 7.5, 10, 25, 50, 100 mg / l). After diluting with 10 ml each, the surface tension was measured with a surface tension meter (TD-1, Lauda, German) to measure the critical micelle concentration. The critical micelle concentration was 10 mg / l and the surface tension was 29.5 mN / m.
이 농도는 토양에서 분리된 슈도모나스 아에루기노사가 생산하는 람노리피드의 임계 마이셀 농도 15㎎/ℓ(Mulligan et al., 1993)와 슈도모나스 아에루기노사 에이티씨씨 9024(Peudomanas aeruginosaATCC9027)가 생산하는 람노리피드의 임계 마이셀 농도 50㎎/ℓ(Zhang et al., 1992) 보다도 적은 량으로도 임계 마이셀이 형성되며, 유화안정성 또한 아주 우수한 것으로, 이것은 본 균주가 람노리피드와 메칠람노리피드를 동시에 생산하는 특성에 따른 결과를 나타내는 것이다.This concentration is 15 mg / l (Mulligan et al., 1993) and 15 mg / L of critical micelles produced by Pseudomonas aeruginosa isolated from the soil, and Pseudomonas aeruginosa ATCC9027 produced by Pseudomonas aeruginosa ATCC9027. The critical micelles are formed even at a concentration less than 50 mg / l (Zhang et al., 1992) of the rhamnolipids (Zhang et al., 1992), and the emulsion stability is also excellent. At the same time, results are shown according to the characteristics produced.
2) 친수친유 균형값(HLB, hydrophilic lipophilic balanced biosurfactant)2) hydrophilic lipophilic balanced biosurfactant (HLB)
도 29에서 나타낸 친수친유균형값(HLB, hydrophilic lipophilic balanced biosurfactant)은 본 균주가 생산하는 당지질 생물유화제가 친수성이 강한지, 소수성이 강한지를 확인하는 수치로 유화형태나 특성을 나타내는 지표가 되는 것으로, 친수친유 균형값이 1 ~ 7인 것은 물/기름(W/O, water in oil)형이고, 10∼13은 기름/물(O/W, oil in water) 형으로 보고되고 있다(Poter et al., 1994). 그 특징으로는 친수친유 균형값이 1 ~ 4는 불용성이며, 4 ~ 7은 안정성과 분산능이 떨어지며, 7 ~ 9는 안정적이지만 불투명하며, 10 ~ 13은 약간 흐리며, 13 이상은 투명한 용액으로 존재하는 것으로 알려져 있다. 따라서, 친수친유 균형값의 측정은 본 균주가 생산하는 당지질 생물유화제(C. BS)와 정제된 BS-1 그리고 시판용 계면활성제(Triton X-100, SDS, Tween 80, Tween 40, Tween 20, Emulsan)를 사용하여 Porter(1994)등에 의한 적정법으로 비교 측정하였으며, 측정은 0.01g의 각종 계 면활성제를 10㎖의 증류수에 녹인후, 4% 벤젠과 96% 1,4-다이옥산(1,4-dioxane)이 혼합된 유기용제로 적정하였다. 각종 계면활성제가 녹아있는 10㎖의 용액은 교반기로 혼합하여 적정하였으며, 친수친유 균형값은 맑은 종말점이 나올때까지 계속 유기 용제를 적하시켜 적하량으로 구하였다.The hydrophilic lipophilic balanced biosurfactant (HLB) shown in FIG. 29 is a value indicating whether the glycolipid bioemulsifier produced by the strain is hydrophilic or strong hydrophobic, and is an index showing the emulsification form and characteristics. Hydrophilic lipophilic balance values of 1 to 7 are reported as water / oil (W / O), and 10 to 13 are reported as oil / water (Oter). , 1994). Its characteristic is that the hydrophilic lipophilic balance value of 1 to 4 is insoluble, 4 to 7 is poor in stability and dispersibility, 7 to 9 is stable but opaque, 10 to 13 is slightly cloudy, and 13 or more is present as a transparent solution. It is known. Thus, the measurement of the hydrophilic lipophilic balance value is based on the glycolipid bioemulsifier (C. BS) produced by the strain, purified BS-1 and commercially available surfactants (Triton X-100, SDS,
그 결과, 당지질 생물유화제(C. BS)의 친수친유 균형값은 11.1, 정제된 BS-1은 10.6, 에멀산(emulsan)은 11.4, 트리톤 엑스-100(Triton X-100)은 10.0, 에스디에스(SDS)는 10.6, 트윈 80(Tween 80)은 15.0, 트윈 40(Tween 40)은 13.4, 트윈 20(Tween 20)은 12.1로 측정되어 실험에 사용된 각종 계면활성제는 모두 기름/물(O/W) 형으로 확인되었다.As a result, the hydrophilic lipophilic balance value of the glycolipid bioemulsifier (C. BS) is 11.1, the purified BS-1 is 10.6, the emulsan is 11.4, the Triton X-100 is 10.0, and the SD (SDS) was measured as 10.6,
3) 담수와 해수에 대한 유화안정성3) Emulsification Stability for Freshwater and Seawater
도 30과 도 31은 본 균주가 생산하는 생물유화제의 물성 중 담수와 해수에 대한 유화안정성을 분석한 것으로 유화활성 측정법에 의해 유화시킨 후 시간의 변화에 따른 기울기의 변화량으로 시판중인 계면활성제와 비교한 것으로, 각종 시판용 계면활성제(Triton X-100, SDS, Tween 80, Tween 40, Tween 20, Emulsan)를 각각 0.01%씩 넣고 로젠버그 등(Rosenberg E. et al 1979)의 방법으로 유화 활성도를 측정하였으며, 유화안정성은 유화활성도 측정법과 동일한 방법으로 실험을 행한 후 10분 간격으로 620nm에서 흡광도를 측정하며 변화된 값의 기울기로 부터 붕괴상수(Kd 값)을 산출하였다.FIG. 30 and FIG. 31 analyze emulsification stability of freshwater and seawater among physical properties of bioemulsifiers produced by the strain, and compare them with commercially available surfactants in terms of the change in slope with time after emulsification by emulsification activity measurement. In each case, various commercially available surfactants (Triton X-100, SDS,
붕괴상수(Kd) = ( logX2- logX1 ) / 10Decay Constant (Kd) = (logX 2 -logX 1 ) / 10
(X1 : 진탕 후 10분 정치시킨 후의 흡광도 값, X2 : 매 10분 간격마다 측정한 흡광도 값)(X 1 : absorbance value after standing for 10 minutes after shaking, X 2 : absorbance value measured every 10 minutes)
그 결과 담수에서는 본 균주가 생산하는 생물유화제가 붕괴상수(Kd) = -26으로, 해수에서는 에멀산과 트윈 40, 그리고 본 균주가 생산하는 생물유화제가 붕괴상수(Kd) = -36.6, -40.3, -43.8로 다소 에멀산이 높기는 하지만 거의 유사한 안정성을 나타냈었으며, 이러한 결과는 본 균주가 생산하는 생물유화제가 해수나 담수에 대한 유류유 처리에 모두 사용이 가능한 것임을 나타내는 것이다.As a result, in fresh water, the bioemulsifier produced by this strain is the decay constant (Kd) = -26, in seawater the emulsion and
4) 생물유화제의 온도 안정성과 열 안정성4) Temperature stability and thermal stability of bioemulsifier
도 32와 도 33은 본 균주가 생산하는 생물유화제의 온도 안정성과 열안정성을 검토한 것으로, 생물유화제의 온도 및 열 안정성은 유화안정성 측정법괴 동일한 방법으로 행하였으며,32 and 33 illustrate the temperature stability and thermal stability of the bioemulsifier produced by the strain, the temperature and thermal stability of the bioemulsifier was performed by the same method as the emulsification stability measurement method,
먼저, 온도 안정성은 20℃ ~ 100℃까지 각 온도별로(20℃, 40℃, 50℃, 60℃, 70℃, 80℃, 100℃)범위에서 60분간 10분 간격으로 유화안정성을 측정하였으며, 온도 안정성은 30℃에서 붕괴상수(Kd)가 -6.07로 가장 안정하였으며, 그 외 각온도별(20℃, 40℃, 50℃, 60℃, 70℃, 80℃, 100℃), 붕괴상수(Kd) 값이 -6.82, -6.79, -8.73, -9.04, -9.45, -10.7로 20℃ ~ 100℃ 전체 온도 범위에서 고른 유화안정성을 나타내었다(도32).First, the temperature stability was measured at 10 minutes intervals for 60 minutes in each temperature range (20 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 100 ℃) from 20 ℃ to 100 ℃, The temperature stability was the most stable at 30 ℃ as the decay constant (Kd) was -6.07, and for each temperature (20 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 100 ℃), the decay constant ( Kd) values of -6.82, -6.79, -8.73, -9.04, -9.45, and -10.7 showed uniform emulsion stability over the entire temperature range of 20 ° C to 100 ° C (FIG. 32).
또한 열 안정성 실험을 위하여, 생물유화제를 121℃에서 10분간 고압 멸균한 것과, 멸균처리를 하지 않은 시료와 열 안정성을 비교하였으며, 실험은 20mM 트리 스-염산 완충용액(Tris-HCl buffer, pH 7.0)에 0.01%(중량/부피)의 생물유화제를 첨가하여 사용하였다. 그 결과 열처리하지 않은 것은 60분 후, 붕괴 상수(Kd)값은 -6.13이었고, 열처리 한 것의 경우 -6.53으로 열 안정성이 아주 우수한 것으로 확인되었다(도33).In addition, for thermal stability experiments, the bioemulsifier was autoclaved at 121 ° C. for 10 minutes, compared with the non-sterile sample and thermal stability, the experiment was 20mM tris- hydrochloric acid buffer (Tris-HCl buffer, pH 7.0 0.01% (weight / volume) of bioemulsifier was used. As a result, after 60 minutes of heat treatment, the disintegration constant (Kd) value was -6.13, and when heat-treated, it was confirmed that the thermal stability was very good at -6.53 (Fig. 33).
결론적으로,In conclusion,
본 슈도모나스 아에루기노사 BYK-2(Pseudomonas aeruginosa BYK-2 : 수탁번호 KCTC 18012P)의 신규 균주는 토양과 해양의 유류 오염지역에 유출유의 효율적인 제거와 각 산업분야에 다양하게 사용되고있는 화학합성 계면활성제를 대신할 환경친화적인 대체 물질의 개발을 위한 것으로, 본 균주의 생육 특성은 9%의 염분에서도 생육이 가능한 호염성 균주이며, 난분해성 유류인 원유와 벙커씨유의 유류분해능이 우수한 균주로 판명되었으며(도4 ,5),This new strain of Pseudomonas aeruginosa BYK-2 (Accession No. KCTC 18012P ) is an efficient removal of effluent oils in soil and marine oil contaminated areas and chemically synthesized surfactants used in various industries. In order to develop an environmentally friendly substitute material, the growth characteristics of this strain is a basophilic strain capable of growing even at 9% salinity, and it has been proved to be a strain capable of excellent oil resolution of crude oil and bunker seed oil, which are hardly decomposable oils. (Figs. 4 and 5),
탄소원의 종류(탄화수소화합물, 탄수화물, 당류)에 따라 배양하여 배양 상층액으로 부터 추출한 후, 박층 크로마토그래피(TLC)로 각 성분을 분석한 결과 이동률(Rf)이 0.48, 0.52, 0.65, 0.70, 0.82, 0.91인 총 7종류의 당지질 생물유화제를 생산하는 것을 확인하였으며, 이러한 많은 종류의 당지질 생물유화제를 슈도모나스 아에루기노사(Pseudomonas aeruginosa)가 생산한다는 보고는 현재까지 없으며, 7종류의 당지질 생물유화제중 그 생산량이 가장 많은 이동률(Rf)을 보인 0.48, 0.65인 두 종류의 당지질 생물유화제(BS-1, BS-2)를 분리하는 방법을 개발하여 각종 분석 장비를 이용하여 구조분석을 행한 결과, 분자량이 650과 664인 람노리피드(2-O-α-L-rhamno-pyranosyl-α-L-rhamnopyranosyl-β-hydroxyl decanoyl-β- hydroxydecanoic acid)와 메칠람노리피드(2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxyldecanoyl-β-hydroxydecanoic acid methyl ester)로 밝혀졌으며(도 10, 도 20), 본 균주가 생산하는 당지질 생물유화제의 물성을 살펴보면, 임계마이셀농도는 10㎎/L로, 기존에 보고되고있는 슈도모나스 아에루기노사 유래 당지질 생물유화제의 임계마이셀농도(l5㎎/L, 50㎎/L)보다 훨씬 적은 량으로 임계마이셀 농도가 형성되어, 유화분산능이 우수하고 그 사용량이 적기 때문에 생태계에 안정한 환경 친화적인 물질이다. 그리고 표면장력은 29.5mN/m, 계면장력은 0.98mN/m로 확인되었다.After culturing according to the type of carbon source (hydrocarbon compounds, carbohydrates, sugars), extracting them from the culture supernatant, and analyzing each component by thin layer chromatography (TLC), the transfer rate (Rf) was 0.48, 0.52, 0.65, 0.70, 0.82. In addition, it has been confirmed that a total of seven types of glycolipid bioemulsifiers of 0.91 have been produced, and there are no reports that Pseudomonas aeruginosa produces many kinds of glycolipid bioemulsifiers . As a result of developing a method of separating two types of glycolipid bioemulsifiers (BS-1, BS-2), which are 0.48 and 0.65, which exhibited the highest transfer rate (Rf), structural analysis was performed using various analytical equipment. Rhamnolipids (2-O-α-L-rhamno-pyranosyl-α-L-rhamnopyranosyl-β-hydroxyl decanoyl-β-hydroxydecanoic acid) and methylamnolipids (2-O-α-L) -rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxyldec anoyl-β-hydroxydecanoic acid methyl ester) (FIG. 10, 20), and the physical properties of the glycolipid bioemulsifier produced by this strain, the critical micelle concentration is 10 mg / L, Pseudomonas ace The critical micelle concentration is formed in much lower concentration than the critical micelle concentration (l5mg / L, 50mg / L) of the Eruginosa-derived glycolipid bioemulsifier, so it is excellent in emulsifying and dispersing, and its usage is low. It is a substance. The surface tension was 29.5 mN / m and the interface tension was 0.98 mN / m.
한편, 본 발명은 생물유화제의 물성 중 유화안정성을 담수와 해수에서 시판용 계면활성제와 비교한 시판용 계면활성제보다 담수나 해수에서 모두 우수한 유화안정성을 유지하여 담수나 해수의 유출유 처리에 매우 효율적인 것으로 밝혀졌으며, 온도안정성과 121℃에서 10분간 고온 가압멸균 처리한 열 안정성도 매우 우수하여, 열처리 공정을 가지는 식품산업 등과 같은 산업 전반에 안정적으로 활용할 수 있을 것으로 보여 진다.On the other hand, the present invention revealed that the emulsion stability in the physical properties of the bioemulsifier is very efficient in treating freshwater or seawater effluent oil by maintaining excellent emulsion stability in both freshwater and seawater than commercially available surfactants compared to commercially available surfactants in freshwater and seawater. It is also excellent in temperature stability and thermal stability after autoclaving at 121 ° C. for 10 minutes, and it can be used stably throughout the industry such as food industry having heat treatment process.
본 발명에서는 이러한 특성을 바탕으로 각종 배양조건을 검토한 후 생물유화제를 대량배양 후 실리카겔과 유기용매인 클로로포름과 메탄올을 이용한 대량분리공정방법을 개발하였다.In the present invention, after examining various culture conditions based on these characteristics, a large volume separation process using silica gel and organic solvents, chloroform and methanol, was developed after mass culture of bioemulsifier.
이상에서와 같이 본 발명은 비록 상기의 실시예에 한하여 설명하였지만 반드시 여기에만 한정되는 것은 아니며 본 발명의 범주와 사상을 벗어나지 않는 범위내에서 다양한 변형실시가 가능함은 물론이다.As described above, although the present invention has been described with reference to the above embodiments, it is not necessarily limited thereto, and various modifications may be made without departing from the scope and spirit of the present invention.
이상으로 살펴본 바와 같이, 본 발명은 해양세균 슈도모나스 이에루기노사(Pseudomonas aeruginosa) BYK-2에 의해 생산되는 생물유화제 관한 것으로, 육상과 해양의 유출유 처리에 모두 이용할 수 있고, 국내 우점종인 토착세균을 개발하여 유출유에 처리함으로써 외국에서 수입하는 유출유처리 균주보다 훨씬 효율적인 처리가 가능한 장점을 가지고 있다.As described above, the present invention relates to a bioemulsion produced by the marine bacterium Pseudomonas aeruginosa BYK-2, which can be used for both effluent oil treatment on land and the ocean, and uses indigenous bacteria that are domestic dominant species. By developing and treating the spilled oil, it has the advantage of being much more efficient than the spilled oil strains imported from foreign countries.
또한, 본 발명은 육상과 해양의 유출유 처리에 동시에 이용함으로써, 유출유 처리뿐만 아니라 폐수나 오수속에 포함된 난용성, 난분해성 탄화수소들의 제거에도 효율적으로 이용할 수 있다.In addition, the present invention can be efficiently used for the removal of poorly soluble and hardly decomposable hydrocarbons contained in wastewater or sewage, as well as the outflow oil treatment, by simultaneously using the land and ocean for effluent treatment.
그리고, 고 순도의 시료를 얻기 위해서는 주로 고가의 고속능 액체크로마토 그래피법 등에 의하여 기존에는 분리함으로써 많은 비용과 시료 량의 분리능의 한계로 많은 량의 시료를 회수하는 데는 많은 시간이 소비되었으나 본 발명자가 고안한 분리 방법을 사용할 경우 당 지질에 대해서는 많은 양의 고순도(95% 이상)의 시료를 빠른 시간 내에 저 비용으로 손쉽게 분리할 수 있는 장점이 있다.In order to obtain a high-purity sample, the inventors of the present invention mainly used a high-speed, high-performance liquid chromatography method to separate a large amount of time due to the limitation of the cost and the resolution of the sample. In case of using the proposed separation method, a large amount of high purity (more than 95%) of sugar lipids can be easily separated at low cost in a short time.
또한, 본 균주의 경우는 해수에서 분리한 점과 한 균주에서 람노리피드와 메칠람노리피드를 동시에 생산하는 점이 슈도모나스 아에루기노사연구에 있어서 현재까지 보고된 적이 없는 새로운 균 학적 특성을 지니고 있다. 즉, 현재까지 보고된 슈도모나스 아에루기노사가 생산하는 각각의 람노리피드나 메칠 람노리피드 비해, 두 가지 형태의 물질이 혼합된 본 발명의 균주가 생산 생물유화제의 임계마이셀 농도가 이들보다 낮은 농도에서 형성됨으로써 적은 량으로도 훨씬 유화안정성을 유지 할 수 있고, 사용량을 줄일 수 있어 훨씬 환경 친화적인 효과가 있다.In addition, this strain has a new bacteriological characteristic that has not been reported so far in the Pseudomonas aeruginosa study in that it isolates from seawater and simultaneously produces rhamnolipid and mechilamnolipid in one strain. . That is, compared to each of the rhamnolipids and methyl rhamnolipids produced by Pseudomonas aeruginosa so far reported, the strain of the present invention in which the two types of substances are mixed has a lower critical micelle concentration of the production bioemulsifier than these. It can be formed in a concentration to maintain the emulsion stability even in a small amount, and the amount can be reduced, there is a much environmentally friendly effect.
또한, 본 발명은 생물유화제의 유화 안정성도 시판용 계면활성제에 비해 담수나 해수에서 모두 안정하였으며, 온도 및 열 안정성이 있기 때문에 화학합성계면활성제를 대신하여 산업적으로 다양하게 사용할 수 있다.In addition, the emulsion stability of the bioemulsifier is also stable in both fresh water and sea water compared to commercially available surfactants, and because of the temperature and thermal stability can be used in a variety of industries in place of chemical synthetic surfactants.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20090009623A (en) * | 2007-07-20 | 2009-01-23 | 경북대학교 산학협력단 | Usage of natural vegetable oils in the preparation of hydroxy fatty acids by Pseudomonas aeruginosa |
| KR101339907B1 (en) * | 2011-09-14 | 2013-12-10 | 보은군 | Pseudomonas aeruginosa Ab7 to produce surfactant used in cultivation of jujube and method for production of surfactant therefrom |
| JP2014111595A (en) * | 2012-11-26 | 2014-06-19 | Evonik Industries Ag | Process for isolation of rhamnolipids |
| WO2016115048A1 (en) | 2015-01-12 | 2016-07-21 | Logos Technologies, Llc | Production of rhamnolipid compositions |
| CN107406867A (en) * | 2015-01-12 | 2017-11-28 | 罗格斯技术有限责任公司 | The preparation of rhamnolipid composition |
| CN111892254A (en) * | 2020-09-02 | 2020-11-06 | 浙江一清环保工程有限公司 | Method for producing rhamnolipid by fermenting kitchen wastewater and fish meal wastewater in resource utilization mode |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20090009623A (en) * | 2007-07-20 | 2009-01-23 | 경북대학교 산학협력단 | Usage of natural vegetable oils in the preparation of hydroxy fatty acids by Pseudomonas aeruginosa |
| KR101339907B1 (en) * | 2011-09-14 | 2013-12-10 | 보은군 | Pseudomonas aeruginosa Ab7 to produce surfactant used in cultivation of jujube and method for production of surfactant therefrom |
| JP2014111595A (en) * | 2012-11-26 | 2014-06-19 | Evonik Industries Ag | Process for isolation of rhamnolipids |
| WO2016115048A1 (en) | 2015-01-12 | 2016-07-21 | Logos Technologies, Llc | Production of rhamnolipid compositions |
| CN107406867A (en) * | 2015-01-12 | 2017-11-28 | 罗格斯技术有限责任公司 | The preparation of rhamnolipid composition |
| US9884883B2 (en) | 2015-01-12 | 2018-02-06 | Logos Technologies, Llc | Production of rhamnolipid compositions |
| CN107406867B (en) * | 2015-01-12 | 2021-06-01 | 斯泰潘公司 | Preparation of rhamnolipid composition |
| US10829507B2 (en) | 2017-02-06 | 2020-11-10 | Stepan Company | Decolorization of concentrated rhamnolipid composition |
| CN111892254A (en) * | 2020-09-02 | 2020-11-06 | 浙江一清环保工程有限公司 | Method for producing rhamnolipid by fermenting kitchen wastewater and fish meal wastewater in resource utilization mode |
| CN111892254B (en) * | 2020-09-02 | 2022-08-16 | 浙江一清环保工程有限公司 | Method for producing rhamnolipid by fermenting kitchen wastewater and fish meal wastewater in resource utilization mode |
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