JP2007037438A - Method for separating and purifying spirosome - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply and efficiently separating and purifying spirosome. <P>SOLUTION: The method for separating and purifying spirosome comprises forming spheroplast by treating a bacterium with a cell-wall degrading enzyme, stirring the obtained spheroplast in a salt solution to separate spirosome in the solution and subjecting the obtained solution to a solid-liquid separation means. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for separating and purifying spirosomes present in, for example, bacterial periplasm.

  Conventionally, when a bacterium is mechanically disrupted, helical microproteins called spirosomes have been found in various bacteria (Non-patent Documents 1 and 2).

  E. coli-derived spirosomes have been found to be multienzymes having both alcohol dehydrogenase activity, pyruvate-formate-lyase-deactivase activity, and acetyl-CoA reductase activity. It is also known that spirosomes derived from other bacteria are related to anaerobic sugar metabolism (Non-Patent Documents 3 to 6).

  Spirosomes (alcohol dehydrogenase E in E. coli) are known to be commonly retained in eukaryotes ranging from dysentery, fungi, plants and humans in addition to bacteria (Non-patent Document 7). -10). Thus, spirosomes (alcohol dehydrogenase E) are conserved in a wide variety of organisms.

  On the other hand, spirosomes were thought to exist in the cytoplasm in bacteria (Non-patent Documents 1 and 11 to 13). Therefore, conventionally, as a method for separating spirosomes, methods have been used in which cells are disrupted and subjected to various fractional centrifugations. For example, in Non-Patent Documents 1, 11 and 13, suspensions obtained by preparing protoplasts or spheroplasts and subjecting them to sonication or osmotic shock or the like, It is disclosed for use as a starting material for separation and purification. Subsequently, these suspensions are subjected to fractional centrifugation with an ultracentrifuge, and further subjected to sucrose density gradient centrifugation or potassium tartrate density gradient centrifugation to purify the pyrosomes. Alternatively, the suspension is precipitated using ammonium sulfate, and then subjected to column chromatography to purify the spirosome. However, in this conventional method for separating spirosomes, the suspension obtained by disrupting the cells that are the starting material contains membrane fragments, ribosomes, nucleic acids, and intracellular proteins, so only the spirosomes are purified. However, there are problems such as requiring many steps, complicated, time consuming, and low final yield.

  Therefore, a method for separating and purifying spirosomes efficiently has not been known so far.

  By the way, as a method for separating an enzyme localized in the periplasm, for example, a method of separating an enzyme protein in a solution without damaging the microbial cells using osmotic shock (or chloroform shock) (non-patent document). 14). However, this method has a problem that the amount of impurities contained in the solution is small but the yield is small.

Kawata, T., "Electron Microscope", 1984, Vol. 19, p. 3-9 Matayoshi, S. and Oda, H., `` Microbiology and Immunology '', 1985, 29, p.13-20 Matayoshi, S. et al., "Journal of General Microbiology", 1989, 135, p.525-529 Matayoshi, S., “The Japanese Journal of Bacteriology”, 1993, 48, p.417-427 Kessler, D. et al., `` FEBS Letters '', 1991, 281, p.59-63 Kessler, D. et al., "The Journal of Biological Chemistry", 1992, Vol. 267, p.18073-18079 Eva E. Avila et al., "Journal of Parasitolgy", 2002, Vol. 88, p.217-222 Brigitte boxma et al., `` Molecular Microbiology '', 2004, 51, p.1389-1399 Ariane Atteia et al., "Plant Molecular Biology", 2003, 53, p.175-188 William G. Gutheil et al., “Biochemistry”, 1992, 31, p.475-481 Imamura, R. “Research in Microbiology”, 1992, 143, 743-753 Yamato, M. et al., `` Microbiology and Immunology '', 1994, 38, p.177-182 Kessel, M. et al., "Journal of Bacteriology", 1981, Vol. 147, p.653-659 Lee, J. E. and Ahn, T. I., `` Research in Microbiology '', 2000, 151, 605-618

  As described above, conventionally, it is considered that spirosomes are present in the cytoplasm in bacteria, and separation of spirosomes from bacteria has been complicated.

  In view of the above-described circumstances, an object of the present invention is to provide a simple and efficient method for separating and purifying spirosomes.

  The present inventors have revealed that in bacteria, spirosomes are localized in large amounts in the periplasm rather than in the cytoplasm. Therefore, as a result of diligent research to solve the above problems, spheroplasts were formed by treating bacteria with cell wall degrading enzymes, and 1.75% to 3% so that the obtained spheroplasts were not disrupted. Spirosomes are liberated in the solution by stirring in the salt solution of, and the resulting solution is subjected to solid-liquid separation means, and it is found that the spirosomes can be separated and purified efficiently, and the present invention is completed. It came to.

The present invention includes the following.
(1) The first step of forming spheroplasts by treating bacteria with cell wall degrading enzymes, and 1.75% to 3% salt so that the spheroplasts obtained in the first step are not disrupted. A second step of releasing the spirosomes in the solution by stirring in the solution, and a step of separating the spirosomes in the supernatant by applying the solution obtained in the second step to the solid-liquid separation means. A method for separating and purifying a spirosome, comprising three steps, wherein the spirosome is separated from the periplasm.

  (2) The method according to (1), comprising a step of subjecting the supernatant to a protein purification means after the third step.

(3) The method according to (1), wherein the bacterium is Escherichia coli.
(4) The method according to (1), wherein the cell wall degrading enzyme is lysozyme.

  (5) The spirosome has one or more enzyme activities selected from the group consisting of alcohol dehydrogenase activity, pyruvate-formate-lyase-deactivase activity, and acetyl-CoA reductase activity. (1) The method described in the above.

(6) The method according to (1), wherein the salt solution is a sodium chloride solution.
(7) The method according to (1), wherein the solid-liquid separation means is selected from the group consisting of centrifugation, filtration and adsorption.

  According to the separation and purification method of the present invention, spirosomes can be efficiently separated and purified from bacteria. In particular, the spirosome obtained by the separation and purification method according to the present invention is non-denatured and can be used for the development of diagnostic agents, therapeutic agents, and therapeutic methods for diseases.

Hereinafter, the present invention will be described in detail.
The spirosome can be separated from the bacterial periplasm by the method for separating and purifying the spirosome according to the present invention (hereinafter simply referred to as “the method for separating and purifying the present invention”).

  In the present invention, the spirosome is one or more enzymes selected from the group consisting of alcohol dehydrogenase activity, pyruvate-formate-lyase-deactivase activity, and acetyl-CoA reductase activity. It means an enzyme having activity. The alcohol dehydrogenase activity is an enzyme activity that catalyzes a redox reaction between an alcohol and an aldehyde. In addition, pyruvate formate lyase diactivation activity refers to the conversion of pyruvate formate lyase, which catalyzes the reaction to produce pyruvate from acetyl-CoA and formic acid, from active to inactive, and vice versa. Is also converting enzyme activity. Furthermore, the acetyl-CoA reductase activity is an activity that reduces acetyl-coenzyme A and is involved in the reaction that generates acetaldehyde from acetyl-CoA.

  In the separation and purification method according to the present invention, bacteria are first treated with a cell wall degrading enzyme. That is, spheroplasts are formed from bacteria. Here, the bacterium is not particularly limited as long as the spirosome is present in the periplasm. For example, Escherichia genus such as E. coli, Clostridium Botulinum (Clostridium) botulinum and Clostridium hiranonis and other Clostridium genus, Spirochaeta stenostrepta and other Spirochaeta genus, Salmonella typhi and Salmonella Salmonella genus Salmonella such as Salmonella enteritidis and Salmonella typhimurium, Shigella genus Shigella fexneri and Enterobacter aerogenes Enterobacter, Pluteus vulgaris (Porteus v ulgaris and Porteus morganii, and other genus Porteus, Yersinia enterocolitica, Yersinia genus Acholeplasma such as granularum and Acholeplasma axanthum, Peptostreptococcus productus such as Peptostreptococcus productus, Eubacterium lentum and Eubacterium lentum Eubacterium aerofaciens and other genus Eubacterium, Treponema phagedenis and other treponema and Listeria monocytogenes and other squirrels such as Listeria monocytogenes Rear (Listeria) bacteria belonging to the genus, and the like. In particular, Escherichia coli is preferable. Examples of the cell wall degrading enzyme to be used include lysozyme, endo-N-acetylmuramidase, and achromopeptidase. In particular, lysozyme is preferable.

For example, the bacteria are incubated in a buffer containing lysozyme, sucrose (0.5 M) and EDTA (2.5 mM) at 30-37 ° C. for 30 minutes to 2 hours. The amount of lysozyme for bacteria is, for example, 100 to 300 μg / ml, particularly 200 to 250 μg / ml for 10 ⁸ bacteria / ml. Spheroplasts can be obtained by removing the buffer solution by decantation after lysozyme treatment.

  Subsequently, the obtained spheroplast is suspended in a salt solution having a concentration that does not disrupt the spheroplast, and the suspension is stirred for 10 to 60 seconds, particularly 20 to 30 seconds using, for example, THERMO-MIXER. . By this step, spirosomes localized in the periplasm of spheroplasts are released into the solution. Examples of the salt solution to be used include sodium chloride solution (hereinafter referred to as “NaCl solution”), phosphate buffer solution or Tris buffer solution in which NaCl is adjusted to 1.75% to 3%. Particularly preferred is a NaCl solution. The salt concentration at which spheroplasts are not crushed can be appropriately determined depending on the potency of the cell wall degrading enzyme such as lysozyme and the bacterial species, and is, for example, 1.75% to 3%.

  Next, the solution containing the released spirosomes is subjected to solid-liquid separation means. After being subjected to solid-liquid separation means, spirosomes are separated in the supernatant. Here, any solid-liquid separation means may be used as long as it can be separated into a supernatant containing spirosomes and other components contained in spheroplasts. Examples of the solid-liquid separation means include centrifugation, salting out, filtration, adsorption, electrophoresis, chromatography and the like, and one or more of these means can be used. For example, the solution containing the released spirosome can be subjected to centrifugation at 3000 to 10000 rpm for 15 to 60 minutes to allow the spheroplast cells to settle, while separating the supernatant containing the spirosome.

  In the present invention, the supernatant containing the spirosome is further subjected to a protein purification means, so that impurities can be removed and the purity of the spirosome can be increased. The protein purification means may be a method generally used for protein purification, and includes, for example, centrifugation, filtration, adsorption, electrophoresis, chromatography and the like, and one or more of these means may be used. it can. For example, the supernatant containing spirosomes is subjected to centrifugation at 10,000 to 40000 rpm for 1 to 4 hours to remove precipitates and concentrate the supernatant to increase the purity of spirosomes. Alternatively, depending on the number of rotations of centrifugation, the purity of spirosomes may be increased by removing the supernatant after centrifugation and concentrating the precipitate.

  As described above, according to the separation and purification method of the present invention, spirosomes can be efficiently separated and purified from the bacterial periplasm. The separated and purified spirosome is a native one having the same enzyme activity as that existing in the microbial cells, that is, one that retains a helical form. The enzyme activity and purity of the spirosome obtained by the separation and purification method according to the present invention can be measured by, for example, an absorbance measurement method, a fluorescence photometry method, an enzyme immunoassay method (EIA) or the like.

  As mentioned above, it has been found that spirosomes are commonly retained in eukaryotes ranging from dysentery amoebae, fungi, plants and humans in addition to bacteria. Therefore, the spirosome obtained by the separation and purification method according to the present invention is highly likely to be used in various fields such as the medical industry and the fermentation industry.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
[Example 1] Localization of spirosomes in E. coli In this example, it was confirmed that spirosomes were localized in the periplasm in E. coli.

First, Escherichia coli E. coli B strain was cultured at 37 ° C. for 6 hours (10 ⁸ cells / ml) in 50 ml of PYG medium (peptone 1%, yeast extract 0.5%, glucose 1%, NaCl 0.5%). After incubation, the cells are washed twice with PBS, 20 μl of the solution is placed on an electron microscope observation grid, allowed to stand for 3 minutes, and then the solution is blotted with filter paper and then 0.6 M sucrose. -A 0.1M phosphate buffer (pH 6.8) containing lysozyme (200 μg / ml) was placed on a grid in an amount of 20 μl and allowed to act for 3 minutes. After 3 minutes of action, the lysozyme solution was blotted with filter paper, then 100 μl of PBS was applied to the grid and left in PBS for 5 minutes. After standing for 5 minutes, PBS was blotted with a filter paper, stained with 1% uranium acetate solution, and then observed with an electron microscope.

  The obtained electron micrographs are shown in FIGS. In FIG. 1A and B, the part shown with the arrow is a spirosome. The scale bar in FIGS. 1A and B is 100 nm. Furthermore, the enlarged photograph of the location shown by the arrow in FIG. 1B is shown in FIG. The scale bar in FIG. 2 is 100 nm.

  As shown in FIGS. 1A and 1B and FIG. 2, it can be seen that spirosomes are found on the surface of spheroplasts and are localized in the periplasm.

[Example 2] Examination of salt concentration at which spheroplasts of Escherichia coli are not destroyed In this example, a salt concentration at which the spheroplasts of Escherichia coli were not destroyed even when vigorously stirred was examined.

  The microbial cells obtained in the same manner as in Example 1 were suspended in the same lysozyme solution and treated at 37 ° C. for 2 hours. As the spheroplasts subsided, the lysozyme solution was discarded by decantation, washed once with 3% NaCl solution, suspended in the same solution, and dispensed 1 ml at a time into test tubes. Next, distilled water was added so that the salt solution concentration of each test tube was 1.0%, 1.25%, 1.5%, 1.75%, and 2.0%, and THERMO-MIXER (MODEL TM-100, TOKYO THERMONICS CO., LTD Japan) for 30 seconds. The spheroplast cells were observed with a differential interference microscope. The observation results showed that 1.25% was almost crushed, 1.5% was slightly crushed, and 1.75% and 2.0% were almost crushed. Therefore, turbidity was measured (wavelength 570 nm) before and after stirring in a 1.75% saline solution to reconfirm that the cells were not crushed. The results are shown in Table 1.

  Further, the spheroplast cells were suspended in 20 ml of a 1.75% NaCl solution, and the suspension before and after stirring was observed with a differential interference microscope. The obtained differential interference micrograph is shown in FIG.

  As shown in Table 1 and FIG. 3, the spheroplast cells of E. coli were not destroyed even when suspended in a 1.75% NaCl solution and stirred. As with 1.75% NaCl solution, E. coli spheroplast cells can be destroyed even when suspended in a NaCl solution with a concentration exceeding 1.75% (for example, 2.0% to 3.0%) and stirred. There wasn't.

[Example 3] Separation and purification of spirosomes from E. coli This example shows a method for separating and purifying spirosomes from spheroplast cells of E. coli.

  First, 1 ml of a 1.75% NaCl solution was gently added to the settled spheroplast cells obtained in the same manner as in Example 2, and the solution was immediately discarded by decantation. The spheroplast cells were suspended by adding 20 ml of a 1.75% NaCl solution and stirred with THERMO-MIXER (MODEL TM-100, TOKYO THERMONICS CO., LTD Japan) for 30 seconds. After stirring, the spheroplast cell suspension was subjected to centrifugation at 3000 rpm for 15 minutes to allow the spheroplast cell suspension to settle. The supernatant was taken out and further centrifuged at 10,000 rpm for 1 hour to remove the precipitate, and the supernatant was further concentrated. To concentrate the supernatant, put 15 ml of the supernatant into a dialysis membrane tube, dialyze against distilled water overnight, and then fill with Spectra / Gel Absorbent (SPECTRUM LABORATORIES INC. USA & CANADA) for dehydration. And concentrated.

  Laemmli method (Laemmli, UK 1970. Cleavage of structural proteins during the assembly of the head of bacteriophase T4. Nature, 227: 680-685 ) Using Spear and Roizman's method (Spear, PG & Roizman, B. 1972. Proteins specified by herpes simplex virus. V. Purification and structural proteins of the herpes virion. Journal of virology, 9, 143-159) And subjected to SDS-PAGE.

  FIG. 4 shows electron micrographs of cells before lysozyme treatment and spheroplast cells after lysozyme treatment. FIG. 5 shows photographs of the cell suspension before lysozyme treatment and the spheroplast cell suspension after lysozyme treatment.

  As shown in FIGS. 4 and 5, it can be seen that the normal gonococcal microbial cells become cocciform by lysozyme treatment, and the microbial cells settle without being crushed.

  On the other hand, the result of SDS-PAGE is shown in FIG. In FIG. 6, each lane is as follows. 1 lane: whole spheroplast cell sample before stirring, 2 lane: whole spheroplast cell sample after stirring, 3 lane: concentrated sample of supernatant after stirring. Moreover, the band shown by the arrow in FIG. 6 is a spirosome.

  As shown in FIG. 6, it can be seen that a large amount of spirosome is present in the concentrated sample of the supernatant after stirring.

[Example 4] Comparison between the separation and purification method according to the present invention and the conventional method In this example, the separation and purification method according to the present invention was compared with the conventional method.

  As the spirosome separated and purified by the separation and purification method according to the present invention, the concentrated sample of the supernatant after stirring described in Example 3 was used.

  On the other hand, as a conventional method, a method of disrupting bacterial cells by ultrasonic treatment and a method of disrupting spheroplast cells by osmotic pressure were used.

  In the method of disrupting cells by sonication, the cultured cells (E. coli E. coli B strain) are suspended in distilled water and treated with an ultrasonic disrupter (Bioruptor, UC100-D, OLYMPUS) for 3 minutes. Complete crushing was confirmed with a differential interference microscope.

  Traditionally, the shape of elongated spirosomes is considered to be weak to mechanical disruption. As a gentler method of disrupting bacterial cells, 2% Triton X- is prepared by lysing cell walls with enzymes to form spheroplasts or protoplasts. A method of crushing by osmotic shock in 100 mM 50 mM Tris-HCl buffer was used. Therefore, a sample was prepared by disrupting spheroplasts of E. coli B strain by osmotic shock in the same manner.

  Next, a concentrated sample of a suspension obtained by crushing bacterial cells by sonication, a concentrated sample of a suspension obtained by crushing spheroplast cells with osmotic pressure, and the description in Example 3 The concentrated sample of the supernatant after stirring was subjected to SDS-PAGE in the same manner as described in Example 3.

  The result of SDS-PAGE is shown in FIG. In FIG. 7, each lane is as follows. 1 lane: concentrated sample of suspension obtained by disrupting cells by sonication, 2 lane: concentrated sample of suspension obtained by disrupting spheroplast cells by osmotic pressure, 3 lanes : Concentrated sample of the supernatant after stirring. In addition, a band indicated by an arrow in FIG. 7 is a spirosome.

  As shown in FIG. 7, in the concentrated sample of the suspension obtained by crushing whole cells by ultrasonic treatment, all the proteins of the cells are mixed (lane 1), and spheroplast cells are also present. Even in the concentrated sample of the suspension obtained by crushing with osmotic pressure, all the proteins in the microbial cells are mixed (lane 2). On the other hand, in the concentrated sample of the supernatant after stirring described in Example 3, only the protein in the periplasm, mainly spirosomes, can be separated, and if further purification of spirosomes is performed thereafter, the accuracy increases, Purification is simple (lane 3).

[Example 5] Concentration of spirosomes using an ultracentrifuge After centrifuging the supernatant described in Example 3 at 40000 rpm for 2 hours, the supernatant was discarded and the precipitate was suspended in 200 μl of PBS. The spirosomes were concentrated. An electron micrograph of the spirosome concentrate thus obtained is shown in FIG. The scale bar in FIG. 8 is 100 nm.

  As shown in FIG. 8, it can be seen that many long spirosomes were confirmed in the sedimentation, and their morphology was sufficiently retained. The fragment-like membrane component seen when the cells are crushed and concentrated is hardly seen.

FIG. 1A shows an electron micrograph of Escherichia coli spheroplast cells. FIG. 1B shows an electron micrograph of spheroplast cells of E. coli. FIG. 2 shows an enlarged photograph of a portion indicated by an arrow in FIG. 1B. FIG. 3 shows differential interference micrographs of the spheroplast cell suspension before and after stirring. FIG. 4 shows electron micrographs of cells before lysozyme treatment and spheroplast cells after lysozyme treatment. FIG. 5 shows photographs of the cell suspension before lysozyme treatment and the spheroplast cell suspension after lysozyme treatment. FIG. 6 shows the results of SDS-PAGE of the whole spheroplast cell sample before and after stirring and the concentrated sample of the supernatant after stirring. FIG. 7 shows the results of SDS-PAGE of the sample obtained by the separation and purification method according to the present invention and the sample obtained by the conventional method. FIG. 8 shows an electron micrograph of a spirosome concentrate obtained using an ultracentrifuge.

Claims (7)

A first step of forming spheroplasts by treating bacteria with cell wall degrading enzymes;
A second step of releasing spirosomes in the solution by stirring the spheroplast obtained in the first step in a 1.75% to 3% salt solution so that it is not crushed;
A third step of separating the spirosome in the supernatant by subjecting the solution obtained in the second step to solid-liquid separation means;
A method for separating and purifying a spirosome, comprising separating the spirosome from the periplasm.   The method according to claim 1, further comprising the step of subjecting the supernatant to a protein purification means after the third step.   The method according to claim 1, wherein the bacterium is Escherichia coli.   The method according to claim 1, wherein the cell wall degrading enzyme is lysozyme.   The spirosome has one or more enzyme activities selected from the group consisting of alcohol dehydrogenase activity, pyruvate-formate-lyase-deactivase activity, and acetyl-CoA reductase activity. The method of claim 1.   The method according to claim 1, wherein the salt solution is a sodium chloride solution.   The method according to claim 1, wherein the solid-liquid separation means is selected from the group consisting of centrifugation, filtration and adsorption.

Images