KR20020020601A - A specific protein induced by aromatic surfactants - Google Patents

A specific protein induced by aromatic surfactants Download PDF

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KR20020020601A
KR20020020601A KR1020000053866A KR20000053866A KR20020020601A KR 20020020601 A KR20020020601 A KR 20020020601A KR 1020000053866 A KR1020000053866 A KR 1020000053866A KR 20000053866 A KR20000053866 A KR 20000053866A KR 20020020601 A KR20020020601 A KR 20020020601A
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protein
aromatic
surfactants
surfactant
specifically induced
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정대원
이강덕
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정대원
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components

Abstract

PURPOSE: Provided is an inducible protein which is specifically expressed by aromatic surfactants and used as a probe for the detection of aromatic surfactants in a waste water disposal site or the like. CONSTITUTION: The protein probe is prepared by massively inducing specific proteins by aromatic surfactants from Candida tropiclisHJ101 then followed by detecting and purifying them. A protein chip for the detection of surfactants is manufactured by using the probe.

Description

방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질{A specific protein induced by aromatic surfactants}A specific protein induced by aromatic surfactants

본 발명은 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"에 관한 것으로서, 단백질이 유도되는 조건 및 이 조건으로 유도된 단백질에 관한 것이다.FIELD OF THE INVENTION The present invention relates to "proteins specifically induced by aromatic containing surfactants" and to conditions under which proteins are derived and proteins derived from these conditions.

지용성 물질을 수용화시키기 위해 다양한 계면활성제가 개발되어 왔으며, 지금도 산업현장에서는 물론 일반가정에서도 계면활성제의 사용이 상용화되어 있다. 계면활성제는 일반적으로 이온성과 비이온성으로 구분되며 종류에 관계없이 일반적으로 방향족을 포함하고 있는 물질들이 매우 많다. 이러한 물질들은 생분해성이 낮으며, 분해과정에서도 독성이 강한 중간체를 생성하는 경향이 있다. 따라서 계면활성제를 다량으로 사용하는 염색, 피혁 가공공장 등에서는 계면활성제가 함유된 폐수의 효율적인 처리를 위한 방법개발에 많은 연구비를 투자하고 있다. 또한 미처리된 계면활성제는 호소로 유입되어 부영양화의 주범이 되고 있으며, 난분해성이므로 생태계에 축적되어 환경오염을 가속시키고 있다. 한편, 정수과정에서도 제거가 어려워 음용수에도 혼입될 가능성을 내포하고 있다. 이러한 계면활성제의 신속하고 정확한 검출을 위한 방법으로 기기분석이 주로 이용되고 있으며, 각 시료에 따라 분석조건을 달리하여야 하므로 샘플분석에는 시간과 경비가 많이 든다. 따라서 간단하고 신속하게 방향족 함유 계면활성제의 확인 내지는 정량을 할 수 있는 in suit type kit의 개발이 요구되고 있다.Various surfactants have been developed to accommodate fat-soluble substances, and the use of surfactants is still commercialized not only in the industrial field but also in homes. Surfactants are generally divided into ionic and nonionic, and there are many substances that generally contain aromatics regardless of their type. These substances are less biodegradable and tend to produce highly toxic intermediates during degradation. Therefore, dyeing and leather processing plants using a large amount of surfactants are investing a lot of research funds to develop a method for efficient treatment of wastewater containing surfactants. In addition, untreated surfactants are introduced into the lake to become the main culprit of eutrophication, and because they are difficult to decompose, they accumulate in the ecosystem and accelerate environmental pollution. On the other hand, it is difficult to remove even in the water purification process, which may be incorporated into drinking water. Instrumental analysis is mainly used for the rapid and accurate detection of such surfactants, and the analysis of samples requires a lot of time and expense because the analysis conditions must be different for each sample. Therefore, there is a need for the development of an in suit type kit that can easily and quickly identify or quantify an aromatic-containing surfactant.

본 발명은 상기한 바와 같은 사정을 고려하여 안출된 것으로서, 본 발명의 목적은 폐수처리장 등에서 방향족 함유 계면활성제의 유입 및 처리정도를 모니터링하거나, 수계에 존재하는 방향족 함유 계면활성제를 신속하고 간단하게 검출할 수 있는 kit 제조를 위한 단백질 프로브를 제공하는 것이다.The present invention has been made in view of the above circumstances, and an object of the present invention is to monitor the inflow and treatment degree of an aromatic-containing surfactant in a wastewater treatment plant or the like, or to quickly and simply detect an aromatic-containing surfactant in an aqueous system. To provide a protein probe for the kit can be made.

도 1은 본 발명에서 유도된 단백질의 크기를 나타낸 전기영동 사진이고1 is an electrophoresis picture showing the size of the protein induced in the present invention

도 2는 본 발명에서 유도된 단백질을 확인한 전기영동 사진이다.Figure 2 is an electrophoresis picture confirming the protein induced in the present invention.

상기한 목적을 달성하기 위하여, 본 발명에 따른 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"은 진핵 미생물군인Candida tropicalisHJ101로 부터 방향족 함유 계면활성제에 의해 특이적으로 유도되는 단백질을 대량으로 유도시킨 후, 유도된 단백질을 검출 및 정제하여 얻어지는 단백질 프로브이다.In order to achieve the above object, the "protein specifically induced by the aromatic containing surfactant" according to the present invention is a large amount of protein specifically induced by the aromatic containing surfactant from the eukaryotic microbial group Candida tropicalis HJ101 After induction, it is a protein probe obtained by detecting and purifying the induced protein.

본 발명의 실시예를 단백질의 유도조건 및 정제방법으로 나누어 상세히 설명한다.The embodiment of the present invention will be described in detail by dividing the protein induction conditions and purification methods.

1. 유도조건1. Induction condition

미생물 배양용 배지에 방향족 함유 계면활성제를 50mg/L이상 첨가시켜 특정 진핵 미생물을 접종시킨 후 약 24시간 이상(대수기 말기) 배양하거나, 500mg/L이상 농도의 계면활성제를 함유한 배양액에 전배양을 통하여 균체수를 늘린(109이상함유) 미생물을 약 2시간이상 반응시켜 단백질을 유도시킨다. 원활하게 대량으로 유도 단백질을 생산하기 위한 미생물 및 배양조건을 상세하게 설명한다.At least 50mg / L of aromatic-containing surfactant is added to the culture medium for incubating microorganisms to inoculate specific eukaryotic microorganisms, followed by incubation for at least 24 hours (end of log phase), or pre-cultivation in culture medium containing at least 500mg / L of surfactant. Through increasing the number of cells (containing more than 10 9 ) microorganisms are reacted for about 2 hours or more to induce proteins. Detailed description of microorganisms and culture conditions for smoothly producing inducible proteins in large quantities.

1) 사용 미생물1) use microorganism

"방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"을 생산하는 미생물은 염색폐수 처리장에서 채취한 시료에서 분리된 진핵미생물군인 효모로서Candida tropicalisHJ101로 동정되었으며, 비병원성균으로 판명되었다. 이 미생물은 일반적으로 염색폐수처리장에 존재하는 효모균의 우점종으로 판명되었다.Microorganisms producing "proteins specifically induced by aromatic-containing surfactants" were identified as Candida tropicalis HJ101 as a yeast, a eukaryotic microorganism isolated from samples taken from dyeing wastewater treatment plants, and identified as non-pathogenic bacteria. This microorganism has generally been found to be a dominant species of yeast in dyeing wastewater treatment plants.

2) 미생물 배양조건2) Microbial Culture Condition

Candida tropicalisHJ101의 원활한 배양은 미생물을 활성화시키기 위한 전배양(1차 배양)과 단백질 대량 유도를 위한 본배양(2차 배양)으로 구분한다.Smooth culture of Candida tropicalis HJ101 is divided into pre-culture (primary culture) for activating microorganisms and main culture (secondary culture) for protein induction.

(1) 전배양(1) Preculture

전배양을 위한 배지로서는 YM배지(조성 : 효모 추출물 3g/L, 맥아 추출물 3g/L, 펩톤 5g/L, 포도당 5g/L, pH 6.2±0.2)를 사용한다. 배지는 121℃에서 15분간 멸균하여 사용한다. 멸균이 끝난 배지 약 300ml를 250℃에서 약 6시간 멸균한 500ml 삼각플라스크에 넣고, 4℃에 보관중인 보존용Candida tropicalisHJ101을 멸균 백금이로 무균적으로 취하여 삼각플라스크에 접종한다. 접종이 끝난 삼각플라스크는 37℃ 진탕배양기에서 150rpm으로 약 12시간정도 배양한다.As a medium for preculture, YM medium (composition: yeast extract 3g / L, malt extract 3g / L, peptone 5g / L, glucose 5g / L, pH 6.2 ± 0.2) is used. The medium is used after sterilization for 15 minutes at 121 ℃. About 300 ml of the sterilized medium is placed in a 500 ml Erlenmeyer flask sterilized at 250 ° C. for about 6 hours, and preservative Candida tropicalis HJ101 stored at 4 캜 is sterilely taken with sterile platinum and inoculated into Erlenmeyer flask. After the inoculation, the Erlenmeyer flask is incubated at 37 rpm shaking incubator for about 12 hours at 150 rpm.

(2) 본배양(2) main culture

전배양이 끝난 배양액을 무균적으로 원심분리하여 상등액을 제거하고 균체만을 수거한 후, 무균적으로 500mg/L이상 농도의 계면활성제가 들어 있는 YM배지에 현탁시켜 멸균 YM배지 500ml가 들어 있는 멸균된 1L 삼각플라스크에 넣는다. 이 플라스크를 37℃에서 150rpm으로 약 2시간정도 진탕배양하여 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"을 유도시킨다.Aseptic centrifugation of the pre-culture was aseptic centrifuged to remove the supernatant, and only the cells were collected, and then sterilely suspended in YM medium containing a surfactant of more than 500mg / L concentration sterilized containing 500ml of sterile YM medium Insert into 1L Erlenmeyer flask. The flask was shaken at 150 rpm at 37 ° C. for about 2 hours to induce “protein specifically induced by aromatic containing surfactant”.

3) 배양 미생물 회수3) Recovery of cultured microorganisms

본 배양이 끝난 배양액을 12000rpm에서 약 30분간 원심분리하여 균체를 회수한다. 회수한 균체는 계면활성제를 제거하기 위하여1차 원심분리한 균체를 증류수에 현탁하여 세척 후, 상기의 균체 회수조건을 3회 이상 반복한다. 회수한 균체는 즉시 파쇄하여 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"의 정제에 사용하며, 여분의 균체는 초저온 냉동고(- 80℃)에 보관한다.The cultured medium is recovered by centrifugation at 12000 rpm for about 30 minutes. The recovered cells are suspended in distilled water by washing the cells centrifuged in the first centrifugation to remove the surfactant, and then the above cell recovery conditions are repeated three or more times. The recovered cells are immediately crushed and used for the purification of "proteins specifically induced by aromatic-containing surfactants" and the extra cells are stored in cryogenic freezers (-80 ℃).

4) 회수한 균체의 파쇄4) Crushing of Recovered Cells

균체의 파쇄는 초음파 파쇄기를 이용한다. 파쇄효율을 높이기 위하여 파쇄 보조용 물질을 첨가하기도 한다. 파쇄조건은 배양미생물의 농도와 양에 따라 조절한다. 경우에 따라서는 고압파쇄기를 이용하여 균체를 파쇄할 수 있다. 균체파쇄의 정도는 광학현미경으로 균체의 상태를 관찰하여 미파쇄된 균체가 거의 보이지 않을때까지 파쇄를 계속한다.Crushing the cells using an ultrasonic crusher. In order to increase crushing efficiency, crushing aids may be added. Crushing conditions are adjusted according to the concentration and amount of cultured microorganisms. In some cases, the cells may be crushed using a high pressure crusher. The degree of cell crushing was observed by observing the state of the cells with an optical microscope, and the crushing was continued until almost crushed cells were hardly seen.

5) 균체 파쇄액의 회수5) Recovery of cell lysate

파쇄한 균체액은 원심분리기를 이용하여 4℃에서 15,000rpm으로 30분간 원심분리하여 파쇄 잔존물과 파쇄액을 분리한다. 분리한 파쇄액은 다시 초원심분리기를 이용하여 4℃에서 50,000rpm으로 3시간 원심분리하여 상등액과 침전물을 분리한다. 이때 분리된 상등액이 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"의 정제를 위한 최종 균체파쇄액이다.The crushed cell solution was centrifuged at 15,000 rpm for 30 minutes at 4 ° C using a centrifuge to separate crushed residues and crushed liquid. The separated crushed liquid was again centrifuged at 50,000 rpm for 3 hours at 4 ° C. using an ultracentrifuge to separate the supernatant and the precipitate. The separated supernatant is the final cell lysate for purification of the "protein specifically derived by the aromatic containing surfactant".

2. 단백질 확인2. Protein Identification

(1) 전기영동장치에 의한 확인(1) Confirmation by electrophoresis device

"방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"은 균체파쇄액을 전기영동장치를 이용하여 SDS PAGE(Sodium dodecyl sulfate polyacrylamide gel eletrophoresis)와 Native PAGE로 확인한다."Protein specifically induced by aromatic-containing surfactants" is confirmed by the cell lysate solution SDS PAGE (Sodium dodecyl sulfate polyacrylamide gel eletrophoresis) and Native PAGE using an electrophoresis device.

전기영동 장치로 확인된 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"의 크기는 SDS page에서 47.9kD, Native PAGE에서 약 105kD으로 확인되었다. 이 결과로부터 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"은 이량체로 판명되었다.(도 1)The size of the "specifically induced protein by the aromatic containing surfactant" confirmed by the electrophoresis device was 47.9kD on the SDS page and about 105kD on the Native PAGE. From this result, "the protein specifically induced by the aromatic containing surfactant" turned out to be a dimer (FIG. 1).

(2) 생리활성에 의한 확인(2) Confirmation by physiological activity

"방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"이 존재하면 발색하는 특수한 물질을 이용하면 Native PAGE상에서 서로 반응하여 특정한 부위에 발색 band를 형성한다. 이 발색 band가 방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질이다.(도 2)In the presence of "proteins specifically induced by aromatic-containing surfactants", the use of special materials that develop color reacts with each other on the native PAGE to form color bands at specific sites. This coloring band is a protein specifically induced by an aromatic containing surfactant (FIG. 2).

3. 정제3. Tablet

"방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"을 다량으로유도할 수 있는 조건으로 배양한 균체를 회수한 후, 파쇄하여 원심분리, 초원심분리를 거처 균체파쇄액을 제조한 후, 겔 여과법, ion교환수지법 등을 이용하여 최종적으로 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"을 얻는다. 이렇게 하여 얻어진 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"은 용도에 따라 in suit 검색 kit의 프로브, 모니터링 시스템의 protein chip 프로브로서 사용할 수 있다.After recovering the cells cultured in a condition that can induce a large amount of "protein specifically induced by the aromatic-containing surfactant", and then crushed by centrifugation, ultracentrifugation to prepare a cell crushing solution, the gel Filtration, ion exchange resin, and the like are finally used to obtain "proteins specifically derived by aromatic-containing surfactants". The "protein specifically derived by the aromatic containing surfactant" obtained in this way can be used as a probe of an in suit search kit and a protein chip probe of a monitoring system depending on a use.

상기한 바와 같은 공정에 의해 얻어지는 본 발명의 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"의 특성 및 작용효과를 설명한다.The properties and effects of the "proteins specifically induced by aromatic-containing surfactants" of the present invention obtained by the process as described above will be described.

전기영동 장치로 확인된 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"은 크기가 SDS page에서 47.9kD, Native PAGE에서 약 105kD으로 된 이량체 단백질로서, 이온성과는 관계없이 방향족을 함유한 계면활성제에 의해 특이적으로 유도되는 특성을 가지고 있다. 따라서 계면활성제 존재유무 및 농도를 파악할 필요성이 있는 폐수처리장 등에서 사용하여 오던 기존의 기기분석을 통한 검출방법과 비교하여 손쉽고 빨리 검출할 수 있어, 시간과 경비를 절감할 수 있는 방향족 함유 계면활성제 검출용 kit개발을 위한 단백질 프로브로서 제공할 수 있다."Protein specifically derived by aromatic-containing surfactants" identified by electrophoresis device is a dimer protein of 47.9 kD in SDS page and about 105 kD in Native PAGE, containing aromatics regardless of ionicity. It has the characteristic specifically induced by surfactant. Therefore, it is easier and faster to detect compared to the existing method of analyzing the existing equipment used in wastewater treatment plants that need to know the presence and concentration of surfactants. It can be provided as a protein probe for kit development.

Claims (6)

방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질Proteins specifically derived by aromatic containing surfactants 제 1항의 제조에 사용되는 균주,Candida tropicalisHJ101 Candida tropicalis HJ101, strain used for the preparation of claim 1 "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"을 항원으로 하여 만들어지는 1차 항체 물질Primary antibody material made from an antigen of "a protein specifically induced by an aromatic containing surfactant" "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"을 항원으로 하여 만들어지는 2차 항체 물질Secondary antibody material made from an antigen of "a protein specifically induced by an aromatic containing surfactant" "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"을 프로브로 하여 만들어지는 계면활성제 검출용 단백질 칩Protein chip for detecting a surfactant made by using a "protein specifically induced by an aromatic containing surfactant" as a probe "방향족 함유 계면활성제에 의해서 특이적으로 유도되는 단백질"을 프로브로 하여 만들어지는 계면활성제 모니터링 시스템Surfactant monitoring system made with probes "proteins specifically induced by aromatic containing surfactants"
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US6059561A (en) * 1995-06-07 2000-05-09 Gen-Probe Incorporated Compositions and methods for detecting and quantifying biological samples
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