KR20060020287A - Method of cracking the cell wall of microorganisms - Google Patents

Abstract

The present invention provides a method for crushing a cell wall of a microorganism comprising irradiating microwaves to the microorganism.

Description

Method of cracking the cell wall of microorganisms

Figure 1 shows an embodiment of a microwave irradiation apparatus capable of performing the cell disruption method of the microorganism of the present invention.

1: magnetron

2: container for cell disruption

3: microwave blocking wall

Figure 2a shows the SCOD (Soluble chemical oxygen demand) concentration and calcium concentration over time upon microwave irradiation to aerobic bacteria.

Figure 2b shows the concentration of protein, carbohydrate, and fat over time upon microwave irradiation to aerobic bacteria.

Figure 3a is a photograph of the results observed with an electron microscope before and after irradiating the microwave to aerobic bacteria.

Figure 3b is a photograph of the results observed with an electron microscope before and after irradiation with E. coli microwave.

Figure 3c is a photograph of the results observed with an electron microscope before and after irradiating the microwave to the bird.

The present invention relates to a method for cell disruption of microorganisms, and more particularly, to a method for disrupting cells of microorganisms using microwaves.

In recent years, the study of the biological environment has received great attention. In these studies, the discovery and discovery of new materials that exist in the natural world, but not yet known, are considered to be important research areas in terms of promoting human welfare and securing new resources. For the discovery and discovery of new substances in nature, it is essential to extract and isolate the components of various microorganisms, including tissue cells of animals and plants.

The cell, the basic unit of life, is semipermeable and surrounded by a solid cell wall in its cytoplasm and membrane. The cytoplasm consists of nucleic acids, proteins, carbohydrates, lipids, enzymes, inorganic ions and 80% water, and breaking down cell membranes and / or cell walls is essential for obtaining these substances from cells. Several methods of cell disruption have been developed for cell disruption.

According to Korean Patent Laid-Open Publication No. 2000-34280, a cell crushing apparatus for crushing cells by vertical vibration of the beads using beads is disclosed. Such cell crushing device using beads has a disadvantage of severe power wastage and cannot be applied when crushing a large amount of samples. Since most cell extraction experiments require a technique for simultaneously processing a sample in a short time, the disadvantages described above are problematic.

In addition, among the technologies used in the field of biotechnology, cells such as enzymes (eg, lysis buffer, lysozyme, SDS solution (20% sodium dodecyl sulfate) containing enzyme) are used to disrupt cells. The method suffers from cell disruption due to excessive reagent costs (Sambrook J. and Russell DW (2001). Molecular cloning A Laboratory manual . COLD SPRILNG HARBOR LABORATORY PRESS, NEW YORK. 1.31-1.55).

In addition to the above method, a method of breaking a cell membrane by rotating the ball in a cylinder at high speed (Muller JA (2001) .Prospects and problems of sludge pre-treatment processes.Wat . Sci. Tech. 44 (10), 121-128.) Res. 34 : Mechanical pretreatment of waste activated sludge for anaerobic digestion process.Wah . Res. 34 (8), 2362-2368), Ultrasonic method (Korea Utility Model 20-0193475) (Tiehm A., Nickel K., Zellhorn M. and Neis U. (2001) .Ultrasonic waste activated sludge disintegration for improving anaerobic Wat. Res. 35 (8), 2003-2009), and methods of breaking cell membranes using high temperatures (Wang Q., Noguchi C., Hara Y., Sharon C., Kakimoto K.). and Kato Y. (1997) .Study on anaerobic digestion mechanism: Influence of pretreatment temperature on biodegradation of waste ac tivated sludge. Environ. Technol 18, 999-1008) (Sambrook J. and Russell DW (2001). Molecular cloning A Laboratory manual. COLD SPRILNG HARBOR LABORATORY PRESS, NEW YORK. 1.31-1.55).

As described above, in order to extract nucleic acids, proteins, sugars, lipids, and the like from a sample of cell tissues, a cell disruption process is necessary. However, the cell disruption method described above has problems in terms of function, efficiency, and cost, such as the inability to simultaneously crush a large amount of samples, the generation of noise or vibration during cell disruption, and the high cost. There is. Therefore, there is an urgent need to develop new and improved cell disruption apparatus.

Accordingly, an object of the present invention is to provide a method for breaking up a cell wall of a microorganism, which is capable of simultaneously crushing a large amount of samples, is free of noise and vibration generated during cell crushing, and is inexpensive.

In order to achieve the above object, the present invention provides a method for crushing the cell wall of the microorganism comprising irradiating the microwave to the microorganism.

Most preferably, the wavelength of the microwave may be 2450 MHz, but the present invention is not limited thereto. The microwave irradiation time is preferably 7 minutes to 15 minutes. In addition, the depth of the microbial sample is preferably 0.5 cm to 3 cm.

Hereinafter, the present invention will be described in more detail.

The method for breaking down the cell wall of a microorganism provided by the present invention includes irradiating microwaves to the microorganism.

As used herein, microorganism is a very comprehensive concept that includes fungi, bacteria, viruses, protozoa, cell tissues of animals and plants, and cultures thereof, as defined in the Korean Biotechnology Review Standard.

Microwave is a kind of electromagnetic wave having a frequency in the range of 300 MHz-300 GHz and has a wavelength in the range of 1 cm to 1 m. Because microwaves are a kind of light, reflection, refraction, diffraction, and transmission occur, and the electric field E and the magnetic field H exist vertically. Such microwaves are widely used as heating means because they cause thermal energy by vibrating and rotating polar molecules such as water contained in a sample.

Frequently used in the food industry or home use are 915 MHz and 2450 MHz, mainly 2450 MHz. When microwaves are irradiated to cells, ions and polar substances (water, pigments, fats, proteins, etc.) contained in the cells are initially disorderly arranged in an electric field (2.45x10 ⁹ cycles / second) that changes direction. The rotational energy transitions are repeated at very high speeds. Through this process, the electric field energy is converted into kinetic energy and the kinetic energy is converted into thermal energy by friction. In this process, with the molecular vibration, the cell wall or cell membrane of the microorganism is crushed, and the extraction phenomenon in which the water-soluble organic matter (solute) of the cytoplasm is transferred to the solvent is performed.

In order to irradiate such a microwave to a microbial sample, the general microwave irradiation apparatus as shown in FIG. 1 can be used, for example. Hereinafter, the present invention has been described using the microwave irradiation apparatus shown in FIG. 1, but FIG. 1 shows a state in which a cell disruption vessel containing a sample containing a microorganism is disposed in a general microwave irradiation apparatus. The microwave irradiation method is not limited to the method using the apparatus shown in FIG.

The general microwave irradiation apparatus shown in FIG. 1 is largely composed of a magnetron 1 and a microwave barrier wall 3 for blocking microwaves generated from the magnetron, and contains a microbial sample for carrying out the cell disruption method of the present invention. The state which arrange | positioned the cell crushing container 2 to the microwave irradiation apparatus is shown together in FIG.

In the microwave irradiation apparatus shown in FIG. 1, the magnetron 1 which generates a microwave is located in the upper part. However, the position of the microwave generating magnetron for irradiating the microorganism is not limited thereto, and may be located anywhere as long as it is a position capable of irradiating the microwave to the microorganism.

The frequency of microwaves generated in the magnetron of FIG. 1 is most preferably, but not limited to, 2,450 MHz, which is an international common frequency, and any frequency capable of breaking a cell membrane or cell wall of a microorganism is possible.

In order to carry out the cell disruption method of the present invention, a cell disruption vessel 2 containing a sample containing a microorganism can be placed in a microirradiation apparatus as shown in FIG. According to FIG. 1, the cell crushing container 2 is arranged in a direction in which microwaves are incident vertically, but may be located anywhere as long as the microwaves are reached.

The sample containing the microorganism, which is contained in the cell crushing container, is not limited to the depth of the sample, but may preferably be contained in the range of 0.5 to 3 cm, more preferably 1.1 to 2 cm, most Preferably 1.5 cm deep.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, these Examples and Experimental Examples are only for better understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Example 1

Determination of Cell Breakage and Cytoplasmic Components of Aerobic Bacteria

A. Experimental Method

Aerobic bacteria were collected from aeration tanks at the Pohang Construction Environment Office (Pohang Sewage Treatment Plant). The microorganisms collected in the aeration tank were used as they were in the next experiment. Bacteria that make up the aeration tank is Pseudomonas, Arthrobacter, Comamonas, Lophomonas, Zooglea, Sphaerotilus, Azotobacter, Chromobacterium, Achromobacter, Flavobacterium, it showed that there is such as Bacillus and Nocardia, which the microbes taken in Fig. 3a are Nocardia of bars and It was considered to be Zooglea in the form of cocci .

500 ml of the sample collected from the aeration tank (solid = 25,670 mg / L, volatile solid = 19,650 mg / L, volatile suspended solids = 17,750 mg / L) cell rupture container of Figure 1 having a length of 25 cm, a width of 13.3 cm After placing at a height of 1.5cm in (2), the microwave irradiation time was irradiated with 3, 5, 7, 11, and 15 minutes to quantitatively analyze the soluble proteins, carbohydrates and lipids as components of each cell. Soluble chemical oxygen demand) and calcium ions were measured.

B. Experimental Results

The results of the measurement of SCOD (Soluble Chemical Oxygen Demand) and calcium ions and the results of quantitative analysis of soluble proteins, carbohydrates and lipids are shown in FIGS. 2A and 2B, respectively.

① Increase of SCOD concentration

According to FIG. 2a, the concentration of SCOD increased with microwave irradiation time. This increase in SCOD concentration is a data indicating that microwaves break down cells and break down mutual binding between cellular components such as DNA, RNA, proteins, sugars, and lipids in cells. Therefore, these results indicate that aerobic bacteria are disrupted by microwave irradiation, and intracellular substances are eluted.

However, the concentration of SCOD did not continue to increase proportionally with time, but appeared to saturate after 7 minutes of microwave irradiation. This is because the cell number is limited because of the limited number of cells, and the cell wall is ruptured during microwave irradiation. After some progress, almost all cells are crushed and the newly eluted SCOD decreases.

② increase in calcium ion concentration

According to Figure 2a, the concentration of calcium ions also increases with the microwave irradiation time.

In general, the surface of cells is negatively charged. Between these cells, cations, typically calcium ions, act as bridges (interbridges), which causes the cells to clump together. Therefore, when the microwave crushes the aerobic bacteria, calcium ions, which had a role of crosslinking, are also eluted and their concentration is increased. Therefore, this increase in calcium ion concentration is indirect evidence of cell disruption.

③ increased protein, carbohydrate, and fat concentrations

According to Figure 2b, proteins, carbohydrates, and fats also appear to increase in concentration with increasing microwave irradiation time. This result of increased concentrations of proteins, carbohydrates, and fats, the major constituents of the cytoplasm, indicates that microwave irradiation played a role in cell disruption.

Example 2

A. Experimental Method

In this experiment, electron micrographs were taken before and after microwave irradiation of eukaryotic algae, prokaryotic aerobic bacteria, and E. coli to observe the microbial cell wall disruption effect.

In the case of aerobic bacteria, aerobic bacterial masses collected in an aeration tank of Pohang Construction Environment Office (Pohang Sewage Treatment Plant) were used as in Example 1. For algae, 150 μE in algae ( Chlamydomonas reinhardtii c137 (+)) from UTEX, The Culture Collection of Algae at the University of Texas (www.utex.org), USA, was used in Tri-acetate-phosphate (TAP) medium. / m ² s, 25 ℃, was used by incubating for 7 days at 135rpm. Lastly, in case of E. coli, an experiment was performed on cells cultured for 1 day at 35 ° C. and 140 rpm in Luria-Bertani medium. It was. The components of the TAP medium used above are shown in Table 1 and Table 2, and the components of the LB medium are shown in Table 3.

Table 1. Composition of TAP Medium

Ingredient  g / L NH ₄ Cl 0.4 MgSO ₄ ㆍ 7H ₂ O 0.1 CaCl ₂ ㆍ 2H ₂ O 0.05 K ₂ HPO ₄ 0.108 KH ₂ PO ₄ 0.056 Tris 2.42 Glacial acetic acid (ml) One Trace Ingredients (ml) One Final pH 7

Table 2. Composition of Trace Components

Ingredient g / 1L stock solution Na ₂ EDTA 50 ZnSO ₄ ㆍ 7H ₂ O 22 H ₃ BO ₃ 11.4 MnCl ₂ 4H ₂ O 5.06 FeSO ₄ 7H ₂ O 4.99 CoCl ₂ ㆍ 6H ₂ O 1.61 CuSO ₄ ㆍ 5H ₂ O 1.57 Mo ₇ O ₂₄ (NH ₄ ) ₆ ㆍ 4H ₂ O 1.1 KOH To 16

Table 3. Composition of LB Medium

Ingredient g / L Trypton 10 Yeast extract 5 NaCl 10 Agar 15 Final pH 7

500 ml of aerobic bacteria, E. coli, and algae samples prepared as described above were placed in the cell disruption vessel 2 of FIG. 1 having a length of 25 cm and a length of 13.3 cm, respectively, with a height of 1.5 cm. Irradiation for 7 minutes.

Before and after irradiation, each sample was centrifuged at 10,000 g for 1 minute and then washed in 0.1M sodium phosphate buffer (pH = 7.3). Then, for aerobic bacteria and Escherichia coli, fix the sample in 3% glutaraldehyde diluted with 0.1 M sodium phosphate buffer at 4 ° C. for 8 hours, wash in 0.1 M sodium phosphate buffer, and dilute with 0.1 M sodium phosphate buffer. The sample was fixed again for 4 hours in one 1% osmium tetraoxide. For algae, samples were fixed for one day in FAA solution (10 ml of 37% formaldehyde, 5 ml of acetic acid, 50 ml of ethanol, 35 ml of distilled water).

Each fixed sample was dehydrated with 20, 40, 60, 80, 100% ethanol. The ethanol dehydrated sample was replaced with carbon dioxide by using a critical point dryer. Then, gold-coated samples were observed by electron microscopy at 10,000 times for aerobic bacteria and algae, and 20,000 times for E. coli, and photographed.

B. Experimental Results

The results of the photographing are shown in Figs. 3A, 3B and 3C, respectively.

3a shows that after microwave irradiation the cell wall of aerobic bacteria was destroyed and bacterial masses were scattered.

3B shows that the E. coli cells show the results, and similarly, the cell wall of the rod-shaped cells was destroyed after the microwave irradiation.

3C shows electron micrographs before and after cell disruption of algae. It can be seen that the cells of the spherical form were broken after microwave irradiation.

As described above, according to the cell crushing method of the microorganism of the present invention, cell crushing of various microorganisms can be performed quickly in a short time with a large amount of samples at low cost, and thus, the discovery and discovery of new materials are made easier. You can lose.

Claims (4)

A method of disrupting cells of a microorganism comprising irradiating the microorganisms with microwaves. The method of claim 1, wherein the wavelength of the microwave is 2450 MHz. The method of claim 1 or 2, wherein the microwave irradiation time is 7 minutes to 15 minutes. The method of claim 1 or 2, wherein the depth of the cell sample to which the microwave is irradiated is 0.5 cm to 3 cm.

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