KR20020012627A - A specific protein induced by aromatic surfactants - Google Patents

Abstract

PURPOSE: A protein specifically induced by aromatic surfactant regardless of the kind of surfactant is provided to detect presence of aromatic surfactant contained in wastewater easily, speedily and economically. The protein can be confirmed using electrophoresis to biomass homogenate as SDS PAGE of 47.5 kDa and Native PAGE of about 150 kDa. This protein can be provided as protein probe for developing in suit type kit to detect aromatic surfactant. CONSTITUTION: The protein is induced by following steps: (i) inoculate specific eukaryotic microorganism by adding more than 50 mg per liter of aromatic surfactant to culture medium for culturing microorganism; (ii) or react for more than 2 hours microorganism that is increased in strain number by prepropagation in culture medium containing more than 500 mg per liter of surfactant. The microorganism used to produce protein is deferminated as strain Candida tropicalisHJ101 an eukaryotic microorganism fermentum that is separated from a sample taken from dying wastewater. By using the specifically induced protein as antigens primary antibody and secondary antibody are prepared and probe to detect aromatic surfactant is also prepared.

Description

A specific protein induced by aromatic surfactants

The present invention relates to proteins specifically induced by aromatic containing surfactants, and to conditions under which proteins are induced and proteins derived by these conditions. 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 factories that use a large amount of surfactants have invested a lot of research funds in developing methods for efficient treatment of surfactant-containing wastewater. 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.

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.

1 is a SDS PAGE picture of a protein specifically induced by an aromatic-containing surfactant in the present invention, SDS PAGE picture of a protein derived by the concentration of a specific aromatic-containing surfactant

2 is a photograph showing the SDS PAGE of a protein specifically induced by an aromatic-containing surfactant in the present invention, a photograph confirming the SDS PAGE of a protein specifically induced according to the type and concentration conditions of the aromatic-containing surfactant

Figure 3 is a photograph showing a protein specifically induced by an aromatic-containing surfactant in the present invention in Native PAGE

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 microorganism Candida tropicalis HJ101 The protein probe is then obtained by detecting and purifying the induced protein.

The embodiment of the present invention will be described in detail by dividing the protein induction conditions and purification methods.

1. Induction condition

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) use microorganism

A microorganism producing a protein specifically induced by an aromatic-containing surfactant was identified as Candida tropicalis HJ101 as a yeast, a eukaryotic microorganism isolated from a sample collected from a dye wastewater treatment plant, and identified as a non-pathogenic bacterium. This microorganism has generally been found to be a dominant species of yeast in dyeing wastewater treatment plants.

2) Microbial Culture Condition

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) Preculture

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. 300 ml of the prepared YM medium is put into a 500 ml Erlenmeyer flask and sterilized at 121 ° C. for 15 minutes. Preserved Candida tropicalis HJ101 stored at 4 ° C in sterile medium is aseptically taken with sterile platinum and inoculated into a Erlenmeyer flask. After the inoculation, the Erlenmeyer flask is incubated at 37 rpm shaking incubator for about 12 hours at 150 rpm.

(2) main culture

After centrifugation of the preculture, the supernatant is removed by aseptic centrifugation, and only the cells are collected. The suspension is aseptically suspended in 500 ml of sterile YM medium containing a surfactant of 500 mg / L or more and put into a sterilized lL Erlenmeyer flask. . The flask was shaken at 150 rpm at 37 ° C. for about 2 hours to induce a protein specifically induced by the aromatic containing surfactant.

3) Recovery of cultured microorganisms

The culture is recovered by centrifuging the culture medium at 5,000 rpm for about 30 minutes. The recovered cells were washed with 0.75% physiological saline suspended in primary centrifuged cells to remove the surfactant, and then the above cell recovery conditions were repeated three or more times. The recovered cells are immediately crushed and used for the purification of proteins specifically induced by aromatic-containing surfactants. The extra cells are stored in cryogenic freezers (-80 ℃).

4) Crushing of Recovered Cells

Crushing the cells using an ultrasonic crusher. In order to increase the 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) Recovery of cell lysate

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 lysate is again centrifuged at 50,000 rpm at 4 ° C. for 3 hours using an ultracentrifuge to separate the supernatant and the precipitate. At this time, the purified supernatant is used to purify the protein specifically induced by the aromatic-containing surfactant. Final cell lysate.

2. Protein Identification

(1) Confirmation by electrophoresis device

Proteins specifically induced by aromatic-containing surfactants are identified by SDS PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) and Native PAGE using an electrophoresis device.

1 is a photograph showing the SDS PAGE of a protein specifically induced by an aromatic-containing surfactant in the present invention, showing the SDS PAGE results of the protein induced by concentration for a specific aromatic-containing surfactant (Fig. 1). 2 is a photograph showing the SDS PAGE of the protein specifically induced by the aromatic-containing surfactant in the present invention, the result of confirming the protein specifically induced by the type and concentration conditions of the aromatic-containing surfactant by the SDS PAGE (FIG. 2) Figure 3 is a result of confirming the protein specifically induced by the aromatic-containing surfactant in the present invention by the native PAGE in the purification process, GF is a protein subjected to Gel Filtration process, IEx is purified through Ion Exchange process. Native PAGE results for the protein (Figure 3).

The size of the protein specifically induced by the aromatic containing surfactant confirmed by electrophoresis was 47.5 kDa in SDS PAGE and about 105 kDa in Native PAGE. From this result, the protein specifically induced by the aromatic containing surfactant turned out to be a dimer.

(2) Confirmation by physiological activity

If there is a protein that is specifically induced by an aromatic-containing surfactant, using a special material that develops color reacts with each other on the native PAGE to form a color band on a specific site. At this time, the band of the site | part to develop is a protein specifically induced by an aromatic containing surfactant.

3. Tablet

After recovering the cells cultured in a condition that can induce a large amount of protein specifically induced by the aromatic-containing surfactant, the cells were crushed and centrifuged, ultracentrifuged to prepare a cell crushing solution, gel filtration, Finally, a protein specifically derived by an aromatic-containing surfactant is obtained by using an ion exchange resin method or the like. The protein specifically induced by the aromatic-containing surfactant thus obtained can be used as a probe of an in suit search kit and a protein chip probe of a monitoring system, depending on the application.

The characteristics and effect of the protein specifically induced by the aromatic-containing surfactant of the present invention obtained by the above process will be described.

Proteins specifically induced by aromatic surfactants identified by electrophoretic devices are dimer proteins of size 47.5 kDa in SDS PAGE and about 105 kDa in Native PAGE, which contain aromatics regardless of ionicity It has a characteristic specifically induced by. Therefore, it is possible to save time and expense in a simpler and faster way than the existing method of analysis through the analysis of existing equipment such as wastewater treatment plant where it is necessary to grasp the concentration of the aromatic-containing surfactant for the purpose of confirming the presence of surfactant and environmental impact assessment. It can be provided as a protein probe for the development of a kit for detecting an aromatic-containing surfactant.

Claims (6)

Proteins specifically derived by aromatic containing surfactants Candida tropicalis HJ101, strain used for the preparation of claim 1 Primary antibody substance made from antigen as protein specifically induced by aromatic containing surfactant Secondary antibody material produced by using antigen as a protein specifically induced by aromatic containing surfactant Protein chip for detecting aromatic-containing surfactant, which is made by using a protein specifically induced by aromatic-containing surfactant Aromatic-containing surfactant monitoring system made by using a protein specifically induced by aromatic-containing surfactant