Cell wall degradation promotion method

Abstract

The present invention provides a method for promoting the decomposition of bacterial cell walls. The method for promoting the decomposition of bacterial cell walls is characterized in that N-acetylmuramidase is allowed to act on bacteria in the presence of a sugar, wherein the sugar is selected from one or more of aldopentose, L-form aldohexose or ketohexose as a monosaccharide, a disaccharide, an oligosaccharide and a polysaccharide having one or more of the monosaccharides as constituent sugars, and derivatives thereof.

Description

Technical Field

[0001] The present invention relates to a method for promoting the decomposition of bacterial cell walls. Background Art

[0002] The bacterial cell wall maintains the cell structure and protects the cell from turgor pressure caused by the presence of a large number of molecules such as proteins in the cell. In addition, since the cell wall is located at the outermost layer of the cell, it plays a role in regulating the interaction between the cell and the external environment. Therefore, in order to clarify the properties of bacteria and seek their utilization, it is important to understand the composition, structure, and function of the cell wall.

[0003] The cell wall of Gram-negative bacteria is composed of a peptidoglycan layer and an outer membrane on the outside, while the cell wall of Gram-positive bacteria is composed of a thicker peptidoglycan layer and is modified by teichoic acid or sugar chains. In peptidoglycan, a sugar chain composed of repeated N-acetylmuramic acid and N-acetylglucosamine is used as the basic skeleton, and peptides composed of several amino acids are bonded to the carboxyl groups of the muramic acid, and the peptides are cross-linked to each other, thereby forming a strong network structure.

[0004] As enzymes that decompose bacterial cell walls, N-acetylmuramidase (systematic name: peptidoglycan N-acetylmuramoyl hydrolase) and the like are known. N-acetylmuramidase is an enzyme that hydrolyzes the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine that constitute peptidoglycan, and is used for nucleic acid extraction, research on cell wall structure or cell wall constituents, preparation of protoplasts, and the like. In addition, it has been reported that if lysozyme, which is one type of N-acetylmuramidase, and mutanolysin, which is also one type of N-acetylmuramidase, are allowed to act on lactic acid bacteria, a synergistic effect can be seen in their bactericidal action (Non-Patent Document 1).

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Kobayashi Osamu et al., Science and Industry, 75(3), 117-122, 2001 Summary of the invention

[0008] However, it has been found that even when lysozyme and mutanolysin are used in combination, the peptidoglycan of the bacterial cell wall may not be sufficiently decomposed. Therefore, an object of the present invention is to provide a method for decomposing the bacterial cell wall simply and efficiently.

[0009] Therefore, the present inventors conducted various studies to explore substances that promote the decomposition reaction in the decomposition of bacterial cell walls using N-acetyl muramidase. As a result, they found that by carrying out the cell wall decomposition reaction in the presence of pentose, L-form aldohexose or ketohexose, the cell wall decomposition rate and / or the initial cell wall decomposition rate are improved, thereby completing the present invention.

[0010] That is, the present invention provides the following [1] to

[12] .

[0011] [1] A method for promoting cell wall degradation, characterized in that N-acetylmuramidase is allowed to act on bacteria in the presence of a sugar, wherein the sugar is one or more selected from the group consisting of aldopentose, L-form aldohexose or ketohexose as a monosaccharide, disaccharides, oligosaccharides and polysaccharides having one or more of the monosaccharides as constituent sugars, and derivatives thereof.

[0012] [2] The method for promoting cell wall decomposition according to [1], wherein the sugar is one or more selected from the group consisting of D-arabinose, L-arabinose, D-lyxose, L-lyxose, D-xylose, L-xylose, D-ribose, L-ribose, L-fucose, L-mannose, L-rhamnose and D-fructose.

[0013] [3] The method for promoting cell wall degradation according to [1] or [2], wherein the N-acetylmuramase is derived from Streptomyces globisporus.

[0014] [4] The method for promoting cell wall degradation according to any one of [1] to [3], wherein the N-acetylmuramidase is mutanolysin.

[0015] [5] The method for promoting cell wall degradation according to any one of [1] to [4], wherein the bacterium is a Gram-positive bacterium.

[0016] [6] The method for promoting cell wall degradation according to any one of [1] to [5], wherein the bacterium is a Lactobacillus bacterium.

[0017] [7] A bacterial cell wall decomposition promoter comprising a sugar as an active ingredient, wherein the sugar is one or more selected from the group consisting of aldopentose, L-form aldohexose or ketohexose as monosaccharides, disaccharides, oligosaccharides and polysaccharides having one or more of the monosaccharides as constituent sugars, and their derivatives.

[0018] [8] The cell wall decomposition promoter according to [7], wherein the sugar is one or more selected from the group consisting of D-arabinose, L-arabinose, D-lyxose, L-lyxose, D-xylose, L-xylose, D-ribose, L-ribose, L-fucose, L-mannose, L-rhamnose and D-fructose.

[0019] [9] A kit for promoting bacterial cell wall decomposition, comprising a sugar and N-acetylmuramidase, wherein the sugar is one or more selected from a monosaccharide such as pentose, L-form aldohexose or ketohexose, a disaccharide, an oligosaccharide or a polysaccharide having one or more of the monosaccharides as constituent sugars, and derivatives thereof.

[0020]

[10] The kit for promoting cell wall decomposition according to [9], wherein the sugar is one or more selected from the group consisting of D-arabinose, L-arabinose, D-lyxose, L-lyxose, D-xylose, L-xylose, D-ribose, L-ribose, L-fucose, L-mannose, L-rhamnose and D-fructose.

[0021]

[11] A use of a sugar for producing a cell wall decomposition promoter, wherein the sugar is one or more selected from the group consisting of monosaccharides such as pentose, L-form aldohexose or ketohexose, disaccharides, oligosaccharides and polysaccharides having one or more of the monosaccharides as constituent sugars, and their derivatives.

[0022]

[12] The use according to

[11] , wherein the sugar is one or more selected from the group consisting of D-arabinose, L-arabinose, D-lyxose, L-lyxose, D-xylose, L-xylose, D-ribose, L-ribose, L-fucose, L-mannose, L-rhamnose and D-fructose.

[0023] According to the present invention, in the decomposition of bacterial cell walls using N-acetyl muramidase, the bacterial cell wall decomposition rate and / or the initial cell wall decomposition rate can be increased by a simple method such as decomposition in the presence of a specific sugar. Thus, cell wall decomposition products can be obtained with high yield and high purity, which can help to clarify the composition, structure and function of bacterial cell walls, cell membranes or cytoplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the effect of adding D-glucose or L-rhamnose in the cell wall decomposition reaction by N-acetylmuramidase.

[0025] Figure 2 It is a graph showing the effect of adding L-rhamnose in the cell wall decomposition reaction by N-acetylmuramidase (A: reaction temperature 37°C, B: reaction temperature 50°C).

[0026] Figure 3 It is a graph showing the effect of adding L-rhamnose in the cell wall decomposition reaction by N-acetylmuramidase (A: mutanolysin derived from Streptomyces sphaeroides ATCC 21553, B: mutanolysin (recombinant)).

[0027] Figure 4 This is a graph showing the effects of adding various sugars on the cell wall decomposition reaction by N-acetylmuramidase.

[0028] Figure 5 The graphs show the effects of adding various sugars on the cell wall decomposition reaction by N-acetylmuramidase (A: the effect of D-arabinose on each enzyme, B: the effect of various sugars on mutanolysin alone).

[0029] Figure 6 This is a graph showing the effect of adding D-arabinose, L-arabinose, or L-rhamnose in the cell wall decomposition reaction by N-acetylmuramidase.

[0030] Figure 7 This is a diagram showing the difference in effect caused by the difference in reaction temperature when D-arabinose is added in the cell wall decomposition reaction using N-acetylmuramidase. DETAILED DESCRIPTION

[0031] The method for promoting bacterial cell wall decomposition of the present invention is characterized in that N-acetylmuramidase is allowed to act on bacteria in the presence of a sugar, wherein the sugar is one or more selected from aldopentose, L-form aldohexose or ketohexose as a monosaccharide, a disaccharide, an oligosaccharide and a polysaccharide having one or more of the monosaccharides as constituent sugars, and derivatives thereof. Promoting cell wall decomposition in the present invention refers to an increase in the cell wall decomposition rate and / or an increase in the initial cell wall decomposition rate.

[0032] In the present invention, the target bacteria for decomposing the cell wall are not particularly limited, and for example, Gram-positive bacteria such as lactic acid bacteria, bifidobacteria, and clostridium can be cited; Gram-negative bacteria such as Escherichia coli and Bacteroides can be cited. Among them, Gram-positive bacteria with thick peptidoglycan are preferred, lactic acid bacteria and bifidobacteria are more preferred, and lactic acid bacteria are particularly preferred. As lactic acid bacteria, lactic acid bacteria belonging to the genus Lactobacillus, Streptococcus, Lactococcus, Leuconostoc, and Micrococcus are preferred, and more specifically, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus jugurti, Lactobacillus delbrueckii subspecies bulgaricus, Streptococcus thermophilus, Lactococcus lactis lactic acid subspecies, Lactococcus lactis cream subspecies, Leuconostoc mesenteroides, Pediococcus pentosaceus, etc. can be cited, and Lactobacillus casei is particularly preferred. As Lactobacillus casei, Lactobacillus casei YIT 9029 (FERM BP-1366) is preferred.

[0033] The preparation method of the bacterium is not particularly limited, as long as it is carried out according to a conventional method. For example, the seed bacteria of the bacterium can be inoculated in a culture medium that can proliferate and cultured. After culture, the method of separating and purifying the bacterium using centrifugation or filtration is used to prepare. As a culture medium, synthetic culture media such as MRS mixed with individual components or culture media using milk components such as milk or skim milk powder can be cited, but it is not limited to these. The obtained bacterium can be directly supplied to the cell wall decomposition reaction, or it can be suspended in a buffer having a pH value of about 7.0 and a buffer function such as tris-maleate buffer, tris-HCl buffer, phosphate buffer, HEPES buffer, and the like, and the suspension is used for the cell wall decomposition reaction.

[0034] The enzyme used in the present invention to decompose the cell wall of bacteria is not particularly limited as long as it is N-acetyl muramidase, and is preferably an N-acetyl muramidase from Streptomyces globisporus. Here, "N-acetyl muramidase from Streptomyces globisporus" may be an enzyme obtained by expressing a gene encoding the N-acetyl muramidase in Escherichia coli or the like, or an enzyme obtained by modifying the gene by gene recombination technology and expressing the gene, and retaining the activity of the N-acetyl muramidase. As N-acetyl muramidase, for example, N-acetyl muramidase M1, N-acetyl muramidase M2, a mixture containing at least N-acetyl muramidase M1 and N-acetyl muramidase M2, etc., and as a representative example, mutanolysin may be exemplified. Alternatively, commercially available N-acetyl muramidase may be used, for example, mutanolysin (manufactured by Sigma-Aldrich Co.) from Streptomyces sphaerocephalus ATCC 21553, mutanolysin (recombinant) (manufactured by A&A Biotechnology), etc. The form of N-acetyl muramidase is not particularly limited, and a dried product of enzyme protein, particles containing enzyme protein, and liquid containing enzyme protein, etc. may be used. In the method for promoting cell wall decomposition of the present invention, the above-mentioned N-acetyl muramidase may be used alone or in combination of two or more, and other cell wall decomposing enzymes may also be used in combination.

[0035] The amount of N-acetylmuramidase used can be appropriately set in consideration of the amount of bacteria used or the size of the reaction system, and is preferably 1 to 1000 U, more preferably 10 to 500 U, and even more preferably 50 to 300 U. Here, 1 U refers to the amount of enzyme that produces a ΔA600nm of 0.01 in 1 minute under the reaction conditions of pH 6.0, 37°C, and 1 mL using a cell wall suspension of Streptococcus faecalis as a substrate.

[0036] The sugar used in the present invention is one or more sugars selected from the group consisting of pentose, L-form aldohexose or ketohexose as monosaccharides, disaccharides, oligosaccharides and polysaccharides with one or more of the monosaccharides as constituent sugars, and their derivatives. Here, disaccharides refer to sugars formed by glycosidic bonds between two molecules of monosaccharides, oligosaccharides refer to sugars formed by glycosidic bonds between about 1 to 10 molecules of monosaccharides, and polysaccharides refer to sugars formed by glycosidic bonds between a plurality of monosaccharides. As sugar derivatives, for example, deoxy sugars formed by the replacement of the hydroxyl group of the sugar with a hydrogen atom, uronic acid formed by the replacement of the hydroxymethyl group of the sugar with a carboxyl group, amino sugars formed by the replacement of the hydroxyl group of the sugar with an amine group, and sugar alcohols formed by the reduction of the carbonyl group of the sugar to an alcohol can be cited, but are not limited to these. Sugars can be natural or synthetic.

[0037] Aldopentose is a monosaccharide with 5 carbon atoms having an aldehyde group at position 1. As an aldopentose, it can be any one of the D body and the L body, specifically, D-arabinose, L-arabinose, D-lyxose and L-lyxose, D-xylose, L-xylose, D-ribose and L-ribose can be mentioned. As a derivative of aldopentose, D-deoxyribose, D-arabitol, xylitol, ribitol, etc. can be mentioned. In addition, as disaccharides, oligosaccharides and polysaccharides, xylobiose, primrose, broad bean sugar, xyloglucan, arabinan, arabinoxylan, rhamnogalacturonan-I, etc. can be mentioned. Among them, from the aspect of the effect of promoting cell wall decomposition, preferably D-arabinose, L-arabinose, D-lyxose and L-lyxose, L-xylose, D-ribose or L-ribose.

[0038] L-form aldohexose is an L-form monosaccharide among monosaccharides having 6 carbon atoms and an aldehyde group at position 1. Specifically, L-mannose, L-allose, L-altrose, L-glucose, L-gulose, L-idose, L-galactose and L-talose can be mentioned. As derivatives of L-form aldohexose, L-fucose, L-rhamnose, L-talomethylose, colisose, iduronic acid, L-mannitol and the like can be mentioned. In addition, as disaccharides, oligosaccharides and polysaccharides, scutose, neohesperidose, rutinose, rutinose, fucoidan, rhamnogalacturonan, rhamnogalacturonan-II and the like can be mentioned. Among them, from the aspect of the effect of promoting the decomposition of the cell wall, L-fucose, L-mannose or L-rhamnose is preferred.

[0039] Ketohexose is a monosaccharide having 6 carbon atoms and a keto group at position 2. Ketohexose may be either a D-form or an L-form, and specific examples thereof include D-fructose, L-fructose, D-psicose, L-psicose, D-sorbose, L-sorbose, D-tagatose, and L-tagatose. Among them, D-fructose is preferred from the perspective of the effect of promoting cell wall decomposition.

[0040] Among the above sugars, from the viewpoint of the effect of promoting cell wall decomposition, aldopentose, L-form aldohexose, ketohexose or derivatives thereof are preferred, and aldopentose, L-form aldohexose or derivatives thereof are more preferred. Specifically, D-arabinose, L-arabinose, D-lyxose, L-lyxose, D-xylose, L-xylose, D-ribose, L-ribose, L-fucose, L-mannose, L-rhamnose or D-fructose are preferred, and D-arabinose, L-arabinose, D-lyxose, L-lyxose, L-xylose, D-ribose, L-ribose, L-fucose, L-mannose or L-rhamnose are more preferred, and D-arabinose, L-arabinose, D-lyxose, L-lyxose, L-xylose, D-ribose, L-ribose or L-fucose are further preferred, and D-arabinose, L-arabinose, D-lyxose, L-lyxose, L-xylose, D-ribose, L-ribose or L-fucose are particularly preferred from the perspective of low cost. These sugars may be used alone or in combination of two or more.

[0041] The amount of sugar used may be appropriately set in consideration of the amount of bacteria used or the size of the reaction system, and is preferably 0.01 to 3 M, more preferably 0.05 to 2 M, further preferably 0.1 to 1.5 M, and particularly preferably 0.5 to 1.5 M, from the viewpoint of the effect of promoting cell wall decomposition and the solubility of the sugar.

[0042] The above-mentioned sugar is useful as a bacterial cell wall decomposition promoter. The above-mentioned sugar can be used directly as a bacterial cell wall decomposition promoter, and can also be used as a raw material for manufacturing a bacterial cell wall decomposition promoter. The bacterial cell wall decomposition promoter of the present invention may also contain a solvent in addition to the above-mentioned sugar, and an aqueous medium can be mentioned as the solvent. As an aqueous medium, in addition to water, various buffers such as phosphate buffer, glycine buffer, Good's buffer, tris buffer, ammonia buffer, etc. can be mentioned, and one or more of them can be included. The bacterial cell wall decomposition promoter of the present invention has good solubility in solvents and is easy to operate. In addition, the bacterial cell wall decomposition promoter of the present invention may also contain a pH buffer, a surfactant, etc. in addition to the above-mentioned sugar and solvent.

[0043] In order to implement the method for promoting bacterial cell wall decomposition of the present invention, it is sufficient to allow N-acetyl muramidase to act on bacteria in the presence of the above-mentioned sugar. The order of adding sugar is not particularly limited, for example, sugar can be added to the bacterial suspension, and then N-acetyl muramidase can be added to perform cell wall decomposition reaction, or N-acetyl muramidase can be added to the bacterial suspension, and then sugar can be added to perform cell wall decomposition reaction.

[0044] The reaction temperature of the cell wall decomposition reaction can be any temperature that can maintain the activity of N-acetyl muramidase. From the viewpoint of enzyme activity, it is preferably 30 to 60° C., more preferably 35 to 55° C., and further preferably 37 to 50° C. The reaction time varies depending on the type of bacteria used, but from the viewpoint of decomposition rate, it is preferably 10 minutes to 48 hours, and more preferably 30 minutes to 24 hours. As a pH value, it is preferably 5.5 to 8.0, and more preferably 6.0 to 7.0.

[0045] The degree of cell wall degradation can be measured, for example, by measuring the turbidity (OD) of the bacterial suspension before and after the reaction using a spectrophotometer. 615 ), and the decomposition rate was calculated according to the following formula (1) for evaluation.

[0046] Decomposition rate = 1-(OD after reaction 615 / OD before reaction 615 )(1)

[0047] The improvement of the cell wall decomposition rate in the present invention means, for example, that the cell wall decomposition rate calculated by the above formula is improved compared to the case in the absence of the above sugar. In addition, the improvement of the initial cell wall decomposition rate in the present invention means that the cell wall decomposition rate after 5 hours, preferably 2 hours, and more preferably 1 hour after the action of N-acetylmuramidase is improved compared to the case in the absence of the above sugar.

[0048] In order to implement the bacterial cell wall decomposition promotion method of the present invention, it is preferred to use a bacterial cell wall decomposition promotion kit containing a sugar and N-acetylmuramidase, wherein the sugar is selected from aldopentose, L-form aldohexose or ketohexose as a monosaccharide, a disaccharide, an oligosaccharide and a polysaccharide having one or more of the monosaccharides as constituent sugars, and their derivatives. If necessary, the kit may also contain a buffer, an instruction manual recording the implementation method, etc.

[0049] According to the method for promoting the cell wall decomposition of bacteria of the present invention, as shown in the following examples, by allowing N-acetyl muramidase to act on bacteria in the presence of the above-mentioned sugar, the cell wall decomposition reaction can be promoted. After the cell wall decomposition reaction is completed, as required, the usual methods such as enzyme treatment, dialysis, chromatography, etc. can be appropriately combined to recover the decomposition product from the reaction solution with a high yield. As this method, there is no particular limitation, for example, as long as the reaction solution is treated with a protease, then treated with a nuclease, and then dialyzed.

[0050] Example

[0051] Next, the present invention will be described in further detail with reference to Examples, but the present invention is not limited to these Examples.

[0052] Example 1 Bacterial cell wall decomposition 1

[0053] (1) Test strains

[0054] Lactobacillus casei YIT 9029 (hereinafter referred to as YIT 9029) was used.

[0055] (2) Preparation of culture medium

[0056] Difco Lactobacilli MRS Broth (manufactured by Becton, Dickinson and Company) was prepared by the method specified by the manufacturer, and 30 mL was dispensed into a 50 mL polypropylene centrifuge tube (manufactured by Eppendorf North America, Inc.).

[0057] (3) Preparation of bacterial solution

[0058] About 2 × 10 9 30 μL of the CFU / mL pre-cultured bacterial solution was inoculated into 30 mL of MRS broth prepared in (2), and the mixture was cultured at 37°C for 24 hours.

[0059] (4) Decomposition of cell walls

[0060] Will contain 2~3×10 9 The bacterial solution of YIT 9029 with CFU / mL was centrifuged (5000×g, 15 minutes), and the precipitate was washed three times with the same amount of sterilized water as the original bacterial solution. The bacteria were suspended in the same amount of 5mM tris maleate buffer (manufactured by Sigma-Aldrich Co.) (pH 6.4) as the original bacterial solution, and 100μg of protein lysozyme, i.e. lysozyme from chicken egg white lyophilized powder (manufactured by Sigma-Aldrich Co., hereinafter referred to as Lysozyme or lysozyme) and 100U of mutanolysin (recombinant) (manufactured by A&A Biotechnology) were added per 1mL of the suspension. The reaction solution was treated at 37°C for 24 hours. In order to examine the effect of sugar addition on the cell wall degradation reaction, a buffer prepared by adding D-glucose or L-rhamnose to the Tris maleate buffer to a final concentration of 0.5 M was used instead of the Tris maleate buffer, and the reaction was carried out in the same manner. Each reaction was carried out three times.

[0061] (5) Determination of decomposition rate

[0062] The turbidity (OD) at 615 nm (maximum absorption wavelength; measurement wavelength range 575-660 nm) of the bacterial suspension was measured over time using a spectrophotometer PiCOSCOPE PAS-110 (manufactured by Ushio Electric Co., Ltd.). 615 ), and the decomposition rate was calculated according to the following formula (1). The decomposition rate was expressed as the average value ± standard deviation of 3 times.

[0063] Decomposition rate = 1-(OD after reaction 615 / OD before reaction 615 ) (1)

[0064] (6) Results

[0065] like Figure 1 As shown in the figure, when D-glucose was added, the cell wall degradation rate was confirmed to be reduced compared with the case where no sugar was added. On the other hand, when L-rhamnose was added, the cell wall degradation rate was confirmed to be increased.

[0066] Example 2 Decomposition of bacterial cell walls 2

[0067] The effect of L-rhamnose on the cell wall decomposition reaction was examined by performing the reaction in the same manner as in Example 1, except that the concentration of L-rhamnose added was changed to a final concentration of 0 (no sugar added), 0.5, 1.0 or 1.5 M and the reaction temperature was changed to 37 or 50° C. Each reaction was performed three times.

[0068] The results, such as Figure 2 As shown, regardless of the reaction temperature, the higher the concentration of L-rhamnose, the faster the increase in the decomposition rate. At a reaction temperature of 37°C, the decomposition rate at the 5th hour of the reaction was 0.36 when no L-rhamnose was added, and 0.83 when 1.5M L-rhamnose was added. At a reaction temperature of 50°C, the decomposition rate at the 1st hour of the reaction was 0.33 when no L-rhamnose was added, and 0.69 when 1.5M L-rhamnose was added. The decomposition rate at the 24th hour of the reaction was 0.97 at a reaction temperature of 37°C and 0.87 at a reaction temperature of 50°C when 1.5M L-rhamnose was added.

[0069] Example 3 Decomposition of bacterial cell walls

[0070] The effect of L-rhamnose on the cell wall decomposition reaction was examined by performing the reaction in the same manner as in Example 1, except that the concentration of L-rhamnose added was adjusted to a final concentration of 1.5 M, 100 U of mutanolysin derived from Streptomyces sphaerocephalus ATCC21553 (manufactured by Sigma-Aldrich Co.) or 100 U of mutanolysin (recombinant) (manufactured by A&A Biotechnology) was used as N-acetylmuramidase, and the reaction temperature was changed to 37 or 50° C. Each reaction was performed once.

[0071] The results, such as Figure 3 As shown, regardless of the reaction temperature or the type of N-acetylmuramidase from Streptomyces sphaerocephalosporus used, an increase in the cell wall decomposition rate was confirmed when L-rhamnose was added compared to the case where no sugar was added. Therefore, it is shown that L-rhamnose promotes cell wall decomposition regardless of the type of N-acetylmuramidase from Streptomyces sphaerocephalosporus.

[0072] Example 4 Decomposition of bacterial cell walls

[0073] The cell wall decomposition promoting effect of each sugar was examined by performing the reaction in the same manner as in Example 1 except that the added sugar was changed to D-arabinose, L-arabinose, D-lyxose, L-lyxose, D-xylose, L-xylose, D-ribose, L-ribose, D-fucose, L-fucose, D-mannose, L-mannose, D-galactose or D-fructose. Each reaction was performed three times.

[0074] The results, such as Figure 4 As shown, it was confirmed that D-arabinose, L-arabinose, D-lyxose, L-lyxose, L-xylose, D-ribose, L-ribose, L-fucose, and L-mannose had a cell wall decomposition promoting effect. In addition, it was confirmed that D-xylose and D-fructose had a cell wall decomposition promoting effect in the first hour of the reaction. In particular, when L-fucose was added, a decomposition rate of about 3 times that of the case where no sugar was added was obtained after 1 hour of reaction, and when D-arabinose or L-arabinose was added, a decomposition rate of about 4 times that of the case where no sugar was added was obtained after 1 hour of reaction.

[0075] Example 5 Verification of the Effect of Sugar Addition 1

[0076] From Examples 1 to 4, it is expected that sugars such as D-arabinose increase the activity of mutanolysin and promote the degradation of cell walls. However, since the possibility of increasing the activity of lysozyme added at the same time cannot be ruled out, the degradation rate when YIT 9029 was treated with mutanolysin or lysozyme alone was studied for some sugars. Specifically, the reaction was carried out in the same manner as in Example 1 except that D-arabinose was added as a sugar at a final concentration of 1.5 M, and mutanolysin (recombinant) (manufactured by A&A Biotechnology) was used alone, lysozyme was used alone, or mutanolysin (recombinant) and lysozyme were used in combination. Each reaction was carried out once. In addition, the reaction was carried out in the same manner as in Example 1 except that L-rhamnose, L-mannose, L-fucose, or L-arabinose was added as a sugar at a final concentration of 0.5, 1.0, or 1.5 M, and mutanolysin (recombinant) (manufactured by A&A Biotechnology) was used alone. Each reaction was carried out 3 times.

[0077] The results, such as Figure 5 As shown in A, D-arabinose did not show significant degradation promotion when lysozyme was used alone, but showed degradation promotion when mutanolysin was used alone and when mutanolysin and lysozyme were used in combination. Figure 5 As shown in Figure B, L-rhamnose, L-mannose, L-fucose, and L-arabinose, which were confirmed to have a decomposition promoting effect in Example 4, also showed a decomposition promoting effect when mutanolysin was used alone. Therefore, it was shown that various sugars increase the activity of mutanolysin.

[0078] Example 6 Verification of the Effect of Sugar Addition 2

[0079] The superiority of the promoting effect of the D form and the L form and the optimal conditions for adding arabinose were investigated for D-arabinose and L-arabinose, which had a high rate of increase in mutanolysin activity in Example 4. Specifically, the reaction was carried out in the same manner as in Example 1 except that D-arabinose or L-arabinose was added at a final concentration of 0, 0.1, 0.5, 1.0 or 1.5 M and the reaction temperature was changed to 37 or 50° C. Each reaction was carried out three times.

[0080] The results, such as Figure 6 As shown in the figure, there is no difference in the promoting effect between the D form and the L form in the concentration range of 0.1M to 1.5M. In addition, at 1.0M and 1.5M, the decomposition rate exceeded 80% in the first hour of the reaction, but the decomposition rate at each time was slightly higher at 1.5M than at 1.0M, and concentration dependence was observed between 0.1M and 1.0M. Therefore, it is believed that 1.5M, which is close to the upper limit of solubility in water at room temperature, is the optimal concentration. Furthermore, as Figure 7As shown, in the short-time reaction, the decomposition rate at 50°C was higher than that at 37°C, and when 1.5 M D-arabinose was added, the decomposition rate reached 93% in 1 hour.

Claims

1. A method for promoting cell wall decomposition, characterized in that: N-acetylmuramidase is allowed to act on the bacteria in the presence of a monosaccharide selected from the group consisting of D-lyxose, L-lyxose, D-ribose, L-ribose, L-fucose, L-rhamnose and D-fructose.

2. The method for promoting cell wall decomposition according to claim 1, wherein: The N-acetylmuramidase is from Streptomyces globisporus.

3. The method for promoting cell wall decomposition according to claim 1 or 2, wherein: The N-acetyl muramidase is mutanolysin.

4. The method for promoting cell wall decomposition according to claim 1 or 2, wherein: The bacteria are Gram-positive bacteria.

5. The method for promoting cell wall decomposition according to claim 1 or 2, wherein: The bacteria are of the genus Lactobacillus.

6. A kit for promoting the decomposition of bacterial cell walls, comprising a monosaccharide and N-acetyl muramidase, wherein the monosaccharide is selected from the group consisting of D-lyxose, L-lyxose, D-ribose, L-ribose, L-fucose, L-rhamnose and D-fructose.

7. Use of a monosaccharide for producing a cell wall decomposition promoter, wherein the monosaccharide is selected from the group consisting of D-lyxose, L-lyxose, D-ribose, L-ribose, L-fucose, L-rhamnose and D-fructose.

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