JP4382746B2 - Lysing agent - Google Patents

Description

  The present invention relates to a lysing agent used when extracting useful substances such as proteins from useful substance-producing bacteria and a method for producing useful substances.

Microorganisms are widely used as hosts for producing useful substances such as amino acids and proteins. Particularly in recent years, a technique for efficiently producing a useful substance using a transformed microorganism into which a gene of an industrially useful protein has been introduced using genetic engineering techniques has been known.
Examples of preferred microorganisms that produce useful substances include Gram-negative bacteria such as Escherichia coli and Pseudomonas, Gram-positive bacteria such as Bacillus and Lactobacillus, yeasts such as Saccharomyces and Candida, Aspergillus and Penicillium Examples include filamentous fungi, actinomycetes such as Streptomyces and Rhodococcus.
The first step in the purification of useful substances such as proteins is a step in which cells producing these useful substances are lysed to release cellular components.
Cell lysis methods include physical methods and chemical methods. Among them, the chemical cell lysis method includes a method of destroying the integrity of the cell membrane or cell wall using a surfactant.
Nonionic surfactants that have been proposed include nonionic surfactants having a sugar chain (for example, Patent Document-1), alkylamine ethylene oxide adducts (for example, Patent Document-2), and sorbitan fatty acid esters. There are ethylene oxide adducts (for example, Patent Document 3). As ionic surfactants, anionic surfactants and amphoteric surfactants have been proposed in addition to cationic surfactants such as quaternary ammonium salts (for example, Patent Document 4) (for example, Patent Document- 5, 6).
JP 2004-504330 A JP 2002-119288 A JP-A-6-153947 JP 2002-335969 A JP 2002-199885 A JP-A-5-64584

  However, the conventional method using a nonionic surfactant is not suitable for mass synthesis because the lysis power is insufficient, and the conventional method using an ionic surfactant is useful for extracted proteins and the like. There was a problem that the material was denatured and the three-dimensional conformation was destroyed.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have excellent lysis power by using a cationic surfactant whose counter ion is a carboxylate anion, and without modifying useful substances, The present inventors have found a bacteriolytic agent that can release and extract high-quality useful substances, and have reached the present invention.

That is, the present invention is useful by using a lytic agent characterized in that it comprises a cationic surfactant that is a carboxylate anion composed of a trivalent or tetravalent polyvalent carboxylic acid as a counter ion, and the lytic agent. This is a method for producing useful substances from substance-producing bacteria.

The lysing agent of the present invention has an improved lysing power as compared with the conventional lysing agent for extracting useful substances from useful substance producing bacteria such as Escherichia coli. There is little to do.
Therefore, a high-quality useful substance, for example, an enzyme having a low degree of denaturation and high activity can be obtained. Moreover, productivity of saccharides etc. is also favorable.

An essential component of the lysing agent of the present invention is a cationic surfactant (A) whose counter ion is a carboxylate anion, and (A) is a surfactant having a hydrophobic group in its cation moiety.
Examples of the cation moiety include the following quaternary ammonium cations (q1) and amine salt cations (q2). The quaternary ammonium cation (q1) is preferable from the viewpoint of lytic ability.
(Q1) Quaternary ammonium cation For example, the compound shown by General formula (1) is mentioned.

In the formula, R ¹ , R ² , R ³ and R ⁴ are linear or branched hydrocarbon groups having 1 to 22 carbon atoms, and at least one of R ^{1 to} R ⁴ has 6 or more carbon atoms. And R ^{1 to} R ⁴ may be the same as or different from each other.

Examples of the hydrocarbon group represented by R ^{1 to} R ⁴ include an aliphatic hydrocarbon group and an aromatic hydrocarbon group.
Examples of the aliphatic hydrocarbon group include linear or branched alkyl groups (methyl group, ethyl group, propyl group, n-butyl group, pentyl group, n-hexyl group, heptyl group, n-octyl group, nonyl group, decyl group). Groups, undecyl groups, dodecyl groups, i-, sec-, and t-butyl groups, 2-methylbutyl groups, 2,2-dimethylpropyl groups, 3-methylbutyl groups, 2-ethylhexyl groups, etc.) and alkenyl groups (vinyl groups) , Allyl group, methallyl group, etc.).
Aromatic hydrocarbon groups include phenyl, arylalkyl groups (benzyl, phenylethyl, phenylpropyl, phenylbutyl, etc.) and alkylaryl groups (methylphenyl, ethylphenyl, octylphenyl, nonylphenyl) Group, decylphenyl group, dodecylphenyl group, etc.).

The following (q11) to (q14) may be mentioned as preferred combinations of the hydrocarbon groups of R ^{1 to} R ⁴ .
(Q11) A combination in which only one is an aliphatic hydrocarbon group having 6 to 22 carbon atoms and the other is an alkyl group having 1 to 4 carbon atoms;
Dodecyltrimethylammonium, tetradecyltrimethylammonium, hexadecyltrimethylammonium, octadecyltrimethylammonium, dodecyldimethylethylammonium, tetradecyldimethylethylammonium, hexadecyldimethylethylammonium, octadecyldimethylethylammonium, dodecylmethyldiethylammonium, tetradecylmethyldiethylammonium Hexadecylmethyldiethylammonium and octadecylmethyldiethylammonium.
(Q12) A combination in which only two are aliphatic hydrocarbon groups having 6 to 22 carbon atoms and others are alkyl groups having 1 to 4 carbon atoms;
Octyldecyldimethylammonium, dioctyldimethylammonium, didecyldimethylammonium, decyldodecyldimethylammonium, didodecyldimethylammonium, octyldecylmethylethylammonium, dioctylmethylethylammonium, didecylmethylethylammonium, didodecylmethylethylammonium, didecylmethyl A combination in which only one (q13) such as propylammonium, didodecylethylpropylammonium and distearyldimethylammonium is an aromatic hydrocarbon group having 6 to 22 carbon atoms and the other is a short chain alkyl group having 1 to 4 carbon atoms;
Benzyltrimethylammonium, benzyltriethylammonium, benzyltripropylammonium, benzylethyldimethylammonium and the like.
(Q14) A combination in which only one is an aliphatic hydrocarbon group having 6 to 22 carbon atoms, only one is an aromatic hydrocarbon group having 6 to 22 carbon atoms, and the other is an alkyl group having 1 to 4 carbon atoms;
Decyl dimethyl benzyl ammonium, dodecyl dimethyl benzyl ammonium, tetradecyl dimethyl benzyl ammonium, hexadecyl dimethyl benzyl ammonium and coconut oil alkyl dimethyl benzyl ammonium and the like.

Among (q1), from the viewpoint of lysis power, (q12) is preferable, and more preferably, two of R ^{1 to} R ⁴ are alkyl groups having 8 to 14 carbon atoms.

(Q2) Amine salt type cation Examples of the amine salt type cation include primary to tertiary amine salt type cations.
Examples of the primary amine constituting the primary amine cation include monoalkyl or cycloalkylamine having 6 to 18 carbon atoms (for example, monohexylamine, monocyclohexylamine, monooctylamine and monododecylamine).
Examples of the secondary amine constituting the secondary amine cation include dialkylamines having 6 to 18 carbon atoms in at least one alkyl group (for example, hexylmethylamine, octylethylamine, and methyldodecylamine).
Examples of the tertiary amine constituting the tertiary amine cation include a trialkylamine in which at least one alkyl group has a carbon number (for example, dimethyldodecylamine).

The counter ion of the cationic surfactant in the present invention is a carboxylate anion (a), which is a counter ion having —COO ⁻ ion obtained by removing a hydrogen atom cation from a carboxylic acid (a ₀ ).
Examples of the carboxylic acid (a ₀ ) constituting the carboxylate anion (a) include the following monovalent carboxylic acid (a ₀ 1) and polyvalent carboxylic acid (a ₀ 2).

(A ₀ 1) monovalent carboxylic acid;
Aliphatic saturated monocarboxylic acids (acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, verargonic acid, lauric acid, myristic acid, stearic acid, behenic acid, 2-ethylhexanoic acid, etc. ; Aliphatic unsaturated monocarboxylic acids (such as oleic acid)
And aliphatic oxymonocarboxylic acids (glycolic acid, lactic acid, tartaric acid, gluconic acid, etc.).
(A ₀ 2) a polyvalent carboxylic acid;
Aliphatic saturated dicarboxylic acids (oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, etc.); aliphatic unsaturated dicarboxylic acids (maleic acid, fumaric acid, itaconic acid) Aromatic dicarboxylic acids (phthalic acid, isophthalic acid, terephthalic acid, etc.); tricarboxylic acids (trimellitic acid, citric acid, etc.); tetracarboxylic acids (pyromellitic acid, butanetetracarboxylic acid, cyclopentanetetracarboxylic acid, Ethylenediaminetetraacetic acid and the like); and pentacarboxylic acid (diethylenetriaminepentaacetic acid and the like).
Of these, (a ₀ 2) is preferable from the viewpoint of difficulty of denaturation of proteins and the like, more preferable is a divalent or trivalent to octavalent polyvalent carboxylic acid, and particularly preferable are tricarboxylic acid and tetracarboxylic acid. Acids, most preferred are tetracarboxylic acids.

Examples of the method for producing the cationic surfactant (A) in the present invention include the following three methods.
(1) A method in which a quaternary ammonium cation alkyl carbonate and an equivalent amount of a carboxylic acid (a ₀ ) are added, and the mixture is reacted by stirring at 60 to 100 ° C. for 3 to 20 hours, followed by purification.
Examples of the alkyl group of the alkyl carbonate include an alkyl group having 1 to 4 carbon atoms, such as a methyl group, an ethyl group, a propyl group, and a butyl group.
(2) A method in which a quaternary ammonium halide is subjected to salt exchange with a strong basic salt of carboxylic acid and then purified. Examples of the strongly basic salt of carboxylic acid include the alkali metal salts (sodium salt, potassium salt, etc.), ammonium salts and amine salts of (a ₀ ).
(3) A method in which quaternary ammonium hydroxide is subjected to salt exchange with a strong basic salt of carboxylic acid and then purified. Examples of the strongly basic salt of carboxylic acid include the alkali metal salts (sodium salt, potassium salt, etc.), ammonium salts and amine salts of (a ₀ ).

  The lysis agent used in the present invention may contain only the above-mentioned cationic surfactant (A), but within the range not inhibiting the effect of the present invention, the organic solvent (B) and other surfactants (C ) And / or a stabilizer (D).

Examples of the organic solvent (B) include hydrocarbon solvents (xylene, toluene, etc.), aliphatic alcohol solvents (methanol, ethanol, etc.), ketone solvents (acetone, methyl ethyl ketone, etc.) and carboxylic acid ester solvents (ethyl acetate, Propyl acetate, methyl formate, etc.).
The use ratio of the organic solvent (B) is preferably 10% by weight or less, more preferably 5% or less (hereinafter, unless otherwise specified,% represents% by weight), particularly preferably 3%. It is as follows.

  Other surfactants (C) include the following nonionic surfactants (C1), cationic surfactants other than (A) (C2), anionic surfactants (C3), and amphoteric interfaces. 1 or more types chosen from an activator (C4) are mentioned.

Nonionic surfactant (C1);
(C11) Higher alcohol alkylene oxide (hereinafter abbreviated as AO) adduct:
1 to 20 moles of ethylene oxide (hereinafter abbreviated as EO) and / or propylene oxide (hereinafter referred to as PO) of a higher alcohol having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl alcohol and oleyl alcohol). Abbreviated) 1 to 20 mol adduct (including block adduct and / or random adduct, the same applies hereinafter) [for example, EO8 mol / PO7 mol block adduct of decyl alcohol].
(C12) AO adduct of alkylphenol having 6 to 24 carbon atoms:
EO1-20 mol and / or PO1-20 mol adducts of octyl or nonylphenol (for example, TRITON® X-100 and TRITON® X-114, etc.).
(C13) Polypropylene glycol EO adduct and polyethylene glycol PO adduct:
Examples include pluronic surfactants.
(C13) Fatty acid AO adduct:
Examples include EO 1-20 mol and / or PO 1-20 mol adducts of fatty acids having 8 to 24 carbon atoms (such as decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and coconut oil fatty acid).
(C14) Polyhydric alcohol type nonionic surfactant:
EO and / or PO adducts of 2 to 8 polyhydric alcohols having 3 to 36 carbon atoms (such as glycerin, trimethylolpropane, pentaerythritol, sorbit and sorbitan); fatty acid esters of the polyhydric alcohols and EO adducts thereof (Eg, TWEEN® 20 and TWEEN® 80); alkyl glucosides (eg, N-octyl-β-D-maltoside, n-dodecanoyl sucrose, n-octyl-β-D-glucopyranoside, etc. And fatty acid esters of sugar, fatty acid alkanolamides and their AO adducts (such as polyoxyethylene fatty acid alkanolamides); and fatty acids include those mentioned above.

Examples of the cationic surfactant (C2) other than (A) include cationic surfactants having at least one counter ion selected from a halogen anion, a hydroxy anion, an alkyl sulfate anion and a superacid anion as a counter ion. It is done.
Halogen anion includes fluoride ion, chloride ion, bromide ion, iodide ion, alkyl sulfate anion includes methyl sulfate ion, ethyl sulfate ion, etc., super strong acid anion includes tetrafluoroborate ion, trifluoromethanesulfonic acid And ions.
In addition, the cation part which comprises (C2) can mention the cation part similar to what was mentioned by (A).
Specific examples of (C2) include benzalkonium chloride and cetyltrimethylammonium bromide.

  Examples of the anionic surfactant (C3) include ether carboxylic acids or salts thereof, sulfate esters or ether sulfate esters and salts thereof, sulfonates, sulfosuccinates, phosphorus having a hydrocarbon group having 8 to 24 carbon atoms. Examples include acid esters or ether phosphate esters and salts thereof, fatty acid salts, acylated amino acid salts, and naturally-occurring carboxylic acids and salts thereof (for example, chenodeoxycholic acid, cholic acid, and deoxycholic acid).

Examples of the amphoteric surfactant (C4) include betaine-type amphoteric surfactants and amino acid-type amphoteric surfactants.
Specific examples include amidosulfobetaine, coleamidopropyldimethylammoniopropanesulfonic acid (CHAPS), coleamidopropyldimethylammonio 2-hydroxypropanesulfonic acid (CHAPSO), carboxybetaine, lauroylsarcosine, and methylbetaine.

The proportion of other surfactant (C) used is preferably 60% or less, more preferably 40% or less, particularly preferably 20% or less, based on the weight of the lysing agent, from the viewpoint of the difficulty of denaturing the protein. is there.
In addition, the content of (C) based on the content of (A) is preferably 60% or less, more preferably 40% or less, from the viewpoint of difficulty in denaturing the protein.
From the viewpoint of the difficulty of denaturing proteins, the contents of (C1) to (C4) based on the weight of (A) are as follows.
(C1) is preferably 50% or less, more preferably 30% or less, (C2) is preferably 20% or less, more preferably 10% or less, and (C3) is preferably 20% or less, more preferably 10% or less, (C4) is preferably 20% or less, more preferably 10% or less.

Examples of the stabilizer (D) include chelating agents, organic acids and salts thereof, and polyhydric alcohols.
Examples of the chelating agent include ethylenediaminetetraacetic acid and its salt, polyphosphoric acid and its salt, metaphosphoric acid and its salt.
Examples of the organic acid and its salt include lactic acid and its salt, hyaluronic acid and its salt, and the like.
Examples of the polyhydric alcohol include glycerin, sorbitol, mannitol, maltitol, pentaerythritol, xylitol, polyethylene glycol and propylene glycol.

  The weight ratio (A) / (B) / (C) / (D) of each component in the lysing agent of the present invention is preferably 30 to 100/0 to 10/0 from the viewpoint of difficulty in denaturing the protein. -60/0 to 50, more preferably 50 to 100/0 to 5/0 to 40/0 to 30, particularly preferably 70 to 100/0 to 3/0 to 20/0 to 20.

The lysing agent of the present invention is usually liquid.
In use, the lysing agent of the present invention can be diluted with water as necessary, and used as an aqueous solution or aqueous dispersion lysing solution.
The concentration of components other than water in the aqueous solution of the lysing agent is appropriately selected depending on the cells of interest, the type of useful substance, and the type of extraction method, but is 0.01 to 99.9%, preferably 0.8. 1-50%.

Another embodiment of the present invention is a method for producing a useful substance from a useful substance-producing bacterium using the above lysing agent.
Since the useful substance obtained by the useful substance production method of the present invention is obtained by the lysis method described above, it has a higher purity than before and is excellent in lysis ability, so that a high yield can be obtained.
Examples of the production method of the useful substance of the present invention include a production method according to the following sequence of steps. (One example when the useful substance is a recombinant protein)
(1) Protein culturing step: Recombinant protein is cultured in a protein producer such as E. coli.
(2) Lysis process: The inclusion body in the protein producing body is taken out by using a lysing agent.
(3) Unfolding step: 0.5 mol / L or more unfolding agent and 20 mmol / L or less reducing agent are added to an inclusion body suspension (for example, 10 mg protein / mL), and the mixture is gently stirred and left at room temperature for several hours. .
(4) Refolding step: A refolding agent is added to the unfolded protein suspension so as to have a concentration of 0.2 to 6 mol / L, and the mixture is gently stirred and left at room temperature overnight. Alternatively, refolding is performed by diluting with a refolding buffer.
(5) Separation / removal step: The target normal protein is separated from the suspension by column chromatography or the like.

Examples of the protein producer in the protein culturing step (1) include the following bacterial cells, Escherichia and Bacillus.
Bacterial cells include streptococci, staphylococci, escherichia, streptomyces and bacillus cells, fungal cells such as yeast cells and aspergillus Aspergillus cells, insect cells: eg Drosophila S2, Spodoptera Sf9 (Spodoptera Sf9) cells, animal cells: eg CHO, COS, Hela, C127, 3T3, BHK, 293 and Bowes melanoma cells A plant cell etc. are mentioned.
As Escherichia (Escherichia), Escherichia coli (E. coli) K12DH1 [Proc. Natl. Acad. Sci. USA, Volume 60, 160 (1968)], JM103 (see Nucleic Acids Research, Vol. 9, 309 (1981)), JA221 (Journal of Molecular Biology). ) 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (196) See year)], C600 [Genetics (Genetics) 39 vol, see 440 pp (1954)], MM294 [Nature (Nature) 217 Vol see 1110 pages to (1968)] and the like.
Examples of Bacillus include Bacillus subtilis MI114 (see Gene, 24, 255 (1983)), 207-21 (Journal of Biochemistry 95, 87). Page (1984)].

Specific methods for producing recombinant proteins include the following methods.
(I) A messenger RNA (mRNA) is isolated from the target protein-producing cell, a single-stranded cDNA and then a double-stranded DNA are synthesized from the mRNA, and the complementary DNA is incorporated into a phage or a plasmid.
(Ii) A host is transformed with the obtained recombinant phage or plasmid, and after culture, contains the target DNA by hybridization with a DNA probe encoding a part of the target protein or by immunoassay using an antibody. Isolate phage or plasmid.
(Iii) It can be produced by cutting out the desired cloned DNA from the recombinant DNA and ligating the cloned DNA or a part thereof downstream of the promoter in the expression vector.
Thereafter, the host is transformed with the expression vector and cultured by an appropriate method. Culture is usually performed at 15 to 43 ° C. for 3 to 24 hours, and aeration and agitation can be added as necessary.

  As a protein refolding method in the unfolding step and the refolding step, after using the lysis agent of the present invention, the three-dimensional structure of the protein is disrupted with the unfolding agent (unfolding), and then a dilution method and a dialysis method. Refolding can be carried out by any of the methods described above, surfactant utilization method, artificial chaperone utilization method and the method described in Japanese Patent Application No. 2005-235980. In particular, the method described in Japanese Patent Application No. 2005-235980 is preferable from the viewpoint of productivity and versatility.

Examples of the unfolding agent used in the unfolding step include guanidine hydrochloride, urea and a combination thereof.
In addition, when the protein is a protein containing an S—S bond in the molecule, 2-mercaptoethanol, dithiothreitol, cystine, thiophenol or the like is further added as a reducing agent in addition to guanidine hydrochloride and / or urea. May be.

  Examples of the packing material used for column chromatography in the protein separation / removal process include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer, etc., and commercially available products such as Sephadex series, Sephacryl series, Sepharose series (above, Pharmacia series). And Bio-Gel series (Bio-Rad) are available.

As useful substances in the production method of useful substances of the present invention,
Examples include proteins (P1), amino acids (P2), nucleic acids (P3), antibiotics (P4), saccharides (P5), and vitamins (P6).

Examples of the protein (P1) include an enzyme (P1-1) and a recombinant protein (P1-2). Examples of the enzyme (P1-1) include a hydrolase, an isomerase, an oxidoreductase, a transferase, a synthetic enzyme, and a desorbing enzyme.
Examples of hydrolases include protease, serine protease, amylase, lipase, cellulase, glucoamylase and the like.
An example of the isomerase is glucose isomerase.
Examples of the oxidoreductase include peroxidase.
Examples of the transferase include acyltransferase and sulfotransferase.
Synthetic enzymes include fatty acid synthase, phosphate synthase, citrate synthase and the like.
Examples of the desorption enzyme include pectin lyase.

As a recombinant protein (P1-2),
Examples include protein preparations and vaccines.
Examples of protein preparations include interferon α, interferon β, interleukins 1 to 12, growth hormone, erythropoietin, insulin, granular colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), natriuretic peptide, Examples include blood coagulation factor VIII, somatomedin, glucagon, growth hormone releasing factor, serum albumin, calcitonin and the like.
Examples of the vaccine include hepatitis A vaccine, hepatitis B vaccine, and hepatitis C vaccine.

Examples of the amino acid (P2) include glutamic acid, tryptophan, alanine, and dipeptide.
Examples of the nucleic acid (P3) include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Antibiotics (P4) include streptomycin and vancomycin.
Examples of the saccharide (P5) include hyaluronic acid, albumin, ceramide, erythritol, trehalose, lipopolysaccharide, and cyclodextrin.
Examples of vitamins (P6) include vitamin A and derivatives thereof and salts thereof, vitamin B and derivatives thereof such as vitamin B6 and vitamin B12, and salts thereof, vitamin C and derivatives thereof and salts thereof. .

  Among these, the lysis agent of the present invention is suitable for the production of (P1) and (P2), particularly (P1).

Another embodiment of the present invention is a protein, amino acid, nucleic acid, antibiotic, saccharide or vitamin obtained by the above-mentioned useful substance production method, and examples thereof include those described above.
Of these, (P1) and (P2), particularly (P1) are preferred.

[Example]
The following examples further illustrate the invention, but the invention is not limited thereto.

Production Example 1:
Production of dipentyldimethylammonium cyclopentanetetracarboxylate;
In a 50 ml Erlenmeyer flask, 16.04 g (0.04 equivalents as a cationic group) of methyl carbonate of didecyldimethylammonium was added and 2.46 g (0.04 equivalents as a carboxyl group) of cyclopentanetetracarboxylic acid was added while stirring. Added in small portions. When stirring was continued for 8 hours while heating to 80 ° C. in a thermostat equipped with a stirrer, carbon dioxide and methanol were released out of the system, and 15.49 g of dipentyldimethylammonium cyclopentanetetracarboxylate (yield 99.99 g). 9%).

Production Examples 2 to 4:
The following cationic surfactants were produced in the same manner as in Production Example 1 except that the type of carboxylic acid and the charged weight were changed (the equivalent of the carboxyl group was not changed).
Didecyldimethylammonium citrate,
Didecyldimethylammonium adipate,
And acetate of didecyldimethylammonium.

Production Example 5:
Instead of didecyldimethylammonium methyl carbonate, distearyldimethylammonium methyl carbonate was used, and the charge weight was changed (the equivalent weight as the cationic group was not changed). Thus, cyclopentanetetracarboxylate of distearyldimethylammonium was produced.

  As a comparative lysing agent, a xylene solution of laurylamine EO 2 mol adduct (70% xylene), a mixture of tetradecyl and pentadecyl alcohol (weight ratio 6/4) EO 2 mol adduct, and didecyldimethylammonium chloride were used.

With respect to the above lysing agents, the lysis power, the difficulty of denaturing proteins, and the productivity of sugars were evaluated.
The evaluation method is as follows.

<Bacterial power>
To a 2 ml screw tube, 0.5 ml of the bacterial cell solution and an aqueous solution of a lysate diluted to a concentration of 50% as a surfactant were added with a 50 μl micropipette and mixed by shaking. The number of cells was measured with an ultradeep shape measuring microscope (manufactured by KEYENCE, VK-8500) using a sample that was stored at 20 ° C. for 3 days. The lysis power was calculated by the following formula. The results are shown in Table 1.
In the blank, 50 μl of ion-exchanged water was used instead of the lysing agent aqueous dilution.
Lysis power (%) = (1−number of cells in sample / number of cells in blank) × 100

Judgment criteria:
Lysis power ≧ 80% ・ ・ ・ ・ ・ ・ 5 points 60% ≦ Bacterial power <80% ・ ・ ・ 4 points 40% ≦ Bacterial power <60% ・ ・ ・ 3 points 20% ≦ Bacterial power <40% ・ ・・ 2-point lysis power <20% ・ ・ ・ ・ ・ ・ ・ 1 point

<Difficult to denature proteins (enzymes)>
In a 2 ml centrifuge tube, 0.6 ml of a 1% cetylmethylcellulose aqueous dispersion, 0.6 ml of an aqueous lysate diluted to a concentration of 0.2% as a surfactant, and cellulase (manufactured by Nagase) 10 μl of a 100 ppm aqueous solution of Cellulizer HT) was added and shaken and mixed. The mixture was allowed to stand at 37 ° C. for 5 minutes, and then centrifuged (10,000 rpm × 3 minutes) with a centrifuge (Microfuge 11 manufactured by Beckman), and the upper layer was separated and collected. In a 20 ml test tube, 0.25 ml of the upper layer, 0.25 ml of ion exchanged water and 0.5 ml of 5% aqueous phenol solution were added and mixed.
Further, 2.5 ml of concentrated sulfuric acid was added, allowed to stand at room temperature for 10 minutes, mixed, and then allowed to stand at 20 ° C. for 20 minutes to obtain a sample solution. The absorbance at 490 nm of the sample solution (absorption of the product obtained by decomposing cetylmethylcellulose with an enzyme) was measured with an ultraviolet-visible spectrophotometer (UV-2550, manufactured by Shimadzu Corporation). In the blank, 0.6 ml of ion-exchanged water was used instead of the lysing agent water dilution. When cellulase (enzyme) is not denatured in the lysis step, the activity is maintained and cetylmethylcellulose is efficiently decomposed, the absorbance increases (the absorbance is close to that of a blank).
The difficulty of protein denaturation was calculated by the following formula and evaluated according to the following criteria.
Hardness of protein denaturation (%) = (absorbance of sample solution / absorbance of blank) × 100
The results are shown in Table 1.

Criteria Difficult to denature proteins ≧ 90% ・ ・ ・ ・ ・ ・ 5 points 70% ≦ Difficult to denature proteins <90% ・ ・ ・ 4 points 50% ≦ Difficult to denature proteins <70% ・・ ・ 3 points 30% ≦ Difficult to denature proteins <50% ・ ・ ・ Difficult to denature proteins 2 points <30% ・ ・ ・ ・ 1 point

<Productivity of sugar (hyaluronic acid)>
1 liter of medium consisting of 5% glucose, 0.2% potassium potassium phosphate, 1.0% polypeptone, 0.5% yeast extract is heat sterilized, then inoculated with Streptococcus mutans,
The cells were cultured at 37 ° C. with stirring for 2 days. Each lysing agent was added to the cultured medium so as to have a final concentration of 0.4% by weight, followed by stirring for 1 hour. After removing cell debris by centrifugation, the supernatant was ethanol precipitated twice. The precipitate was vacuum dried at 40 ° C. to obtain purified hyaluronic acid.
The productivity of saccharides was evaluated from the yield of hyaluronic acid (g / L) using the following criteria. The results are shown in Table 1.

Judgment criteria Yield of hyaluronic acid ≧ 5 (g / L) ... 5 points 3 (g / L) ≤ Yield of hyaluronic acid ≥ 5 (g / L) ··· Yield of 3 points hyaluronic acid <3 (g / L) ... 1 point

  The lysis agent of this invention can be used in the process of extracting useful substances, such as protein, from a production microbe. Useful substances include proteins, amino acids, nucleic acids, antibiotics, sugars or vitamins. In addition, the lysis agent of the present invention can be used as a vector for introducing a gene into cells, and can also be used as a cell disruption agent by combining the lysis agent of the present invention with an enzyme or the like.

Claims (5)

Lysing agent characterized by containing the counterion is 3-4 valent polyhydric composed of carboxylic acid carboxylate anion and a cationic surfactant (A).   The lysis agent according to claim 1, wherein the cationic surfactant is a quaternary ammonium salt type surfactant.   The lysing agent according to claim 1 or 2, further comprising an organic solvent, another surfactant and / or a stabilizer.   The method to produce a useful substance from microorganisms using the lysis agent in any one of Claims 1-3.   The method for producing a useful substance according to claim 4, wherein the useful substance is a protein, amino acid, nucleic acid, antibiotic, saccharide or vitamin.