JP2019170202A - Production method of useful substance - Google Patents

Abstract

To provide a production method capable of enhancing secretion productivity for one bacterial cell, for efficiently secreting and producing a useful substance by microorganisms.SOLUTION: There is provided a production method of a useful substance for secreting and producing a useful substance into a culture broth by microorganisms included in the culture broth, in which, when a ratio OD/ODS between turbidity OD in 600 nm of the culture broth in culture time, and turbidity ODS in 600 nm of the culture broth in a culture stationary phase, is in a range of 0.009-0.90, a surfactant (A) is added into the culture broth.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for producing useful substances.

Microorganisms are widely used for producing useful substances such as amino acids and proteins. Gram negative bacteria (for example, Escherichia coli), Gram positive bacteria, and yeast are widely used as microorganisms used for producing useful substances.
With the advancement of genetic engineering technology, a technology for efficiently producing a useful protein by introducing a gene of a protein useful in medicine or industry into Escherichia coli is known.
When the protein is expressed using E. coli, the target useful protein is produced in the microorganism. In order to extract the produced target protein outside the body of E. coli, physical disruption methods such as ultrasonic waves, high-pressure homogenizers, and French presses are required. There is a problem that purity is lowered because substances other than the target protein present in are mixed in a large amount.
On the other hand, when a Gram-positive bacterium or yeast is used for production of useful substances, the produced useful substances are secreted outside the cells, and there is little contamination with substances other than the target useful substances (for example, Non-Patent Documents 1 and 2). However, Gram-positive bacteria and yeast have a problem that their protein production capacity is lower than that of E. coli.

As a protein production method for solving these problems, a production method for secreting useful substances using a surfactant is known (for example, Patent Document 1). When secretory production is performed, high productivity can be maintained, and a longer production time leads to improved productivity.
However, in the method of Patent Document 1, since the secretory productivity per cell is low, the production amount of useful substances such as proteins obtained by being secreted outside the cell is small, and it is difficult to improve high productivity. There are challenges.

International Publication No. 2010/137624

Bioindustry Association, Fermentation Handbook, Kyoritsu Publishing, July 2001 New Biochemical Engineering (2nd edition), Sankyo Publishing, March 2013, p17-21

An object of the present invention is to provide a production method capable of efficiently secreting and producing a useful substance by microorganisms by increasing the secretion productivity per cell.

The inventors of the present invention have reached the present invention as a result of studies to achieve the above object.
That is, the present invention is a method for producing a useful substance that secretes and produces a useful substance in the culture solution by microorganisms contained in the culture solution. The turbidity OD at 600 nm of the culture solution at the time of culture and the culture in the stationary culture phase This is a useful substance production method in which a surfactant (A) is added to a culture solution when the ratio OD / ODS of the solution to the turbidity ODS at 600 nm is in the range of 0.009 to 0.90.

  The production method of the useful substance of the present invention has an effect that the useful substance can be efficiently secreted and produced by the microorganism by increasing the secretion productivity per cell.

In the method for producing a useful substance of the present invention, it is a drawing showing a growth curve explaining the time when a surfactant (A) is added and its OD, the culture stationary phase and its ODS.

The method for producing a useful substance of the present invention is a method for producing a useful substance that secretes and produces a useful substance in a culture solution by microorganisms contained in the culture solution, and includes a turbidity OD at 600 nm of the culture solution during culture, The surfactant (A) is added to the culture solution when the ratio OD / ODS of the culture solution in the stationary culture phase to the turbidity ODS at 600 nm is in the range of 0.009 to 0.90. .

The turbidity OD of the culture solution is calculated by the following measurement method and calculation formula.
<Measurement method of turbidity OD of culture solution>
Using the culture solution containing the sampled microorganisms, turbidity is measured using a turbidimeter [for example, UV-1700, manufactured by Shimadzu Corporation] using a quartz cell having an optical path length of 1 cm.
The culture solution is centrifuged at 1500 rpm for 5 minutes at 4 ° C., and the supernatant is discarded. Add the same amount of physiological saline as the supernatant that discarded the precipitate, stir and resuspend, and dilute with physiological saline to obtain an appropriate absorbance (0.1 to 0.8). The absorbance at 600 nm is measured.
The turbidity OD of the culture solution is calculated by the following formula.
Turbidity of culture broth (OD) = (absorbance at 600 nm of diluted culture broth) × dilution ratio of culture broth In the present invention, the turbidity OD of this culture broth relatively represents the number of microorganisms in the culture broth. I believe.

The ratio OD / ODS of the turbidity OD at 600 nm of the culture solution at the time of culture when the surfactant (A) is added to the culture solution to the turbidity ODS at 600 nm of the culture solution in the stationary culture phase is From the viewpoint of secretion productivity of useful substances per one, it is 0.009-0.90, preferably 0.009-0.80. That is, by adding the surfactant (A) when the OD / ODS is in the range of 0.009 to 0.90, it is possible to continuously produce and secrete useful substances without killing the cells. .

  In the culture stationary phase in the present invention, in the culture without adding a surfactant, as shown in FIG. 1, the logarithmic growth phase ends due to nutrient depletion or accumulation of toxic metabolites, and the microorganisms in the culture solution While it grows, some die, so there is no increase or decrease in the turbidity of the culture.

  The culture stationary phase ODS in the present invention can be obtained by carrying out a test culture once using the same composition and amount of medium and cells used in the present study and cultivating under the same culture conditions before the present study. . In the test culture, the turbidity ODS at 600 nm of the culture solution in the stationary culture phase can be obtained by measuring the turbidity OD at 600 nm of the culture solution at the time of culture from the start of culture and drawing a growth curve. .

  Surfactant (A) is added when OD / ODS is in the range of 0.009 to 0.90 in the turbidity ODS and turbidity OD in the culture stationary phase shown in FIG. As this period, it is preferable to add the surfactant (A) in the logarithmic growth period, which is a period when the microorganism starts to divide and begins to grow logarithmically. On the other hand, when added during the culture induction period (a period during which microorganisms start to adapt to the medium environment and start to grow, and the microorganisms are hardly growing), the growth of microorganisms is inhibited and the secretion productivity of useful substances Will fall.

The surfactant (A) used in the production method of the useful substance of the present invention is a nonionic surfactant (A1), an anionic surfactant (A2), an amphoteric surfactant (A3) and a cationic surfactant (A4). In the case of (A1), those having an HLB of 0.1 to 16 are preferable.

  Examples of the nonionic surfactant (A1) include alcohol alkylene oxide (hereinafter, alkylene oxide may be abbreviated as AO) adduct (A1a), alkylphenol AO adduct (A1b), fatty acid AO adduct (A1c), Polyhydric alcohol type nonionic surfactant (A1d) etc. are mentioned.

HLB is known as a measure of the hydrophilicity and hydrophobicity of the nonionic surfactant (A1). A higher HLB value means higher hydrophilicity.
The HLB in the present invention is a numerical value calculated by the following formula (written by Takehiko Fujimoto, Introduction to Surfactant, page 212, published by Sanyo Chemical Industries, Ltd.).
HLB = 10 × (inorganic / organic)

  From the viewpoint of secretion efficiency, the HLB of the nonionic surfactant (A1) is preferably from 0.1 to 16, and more preferably from 1 to 16.

The number average molecular weight of the nonionic surfactant (A1) is preferably from 100 to 20,000, more preferably from 200 to 20,000, from the viewpoint of secretion efficiency of useful substances.

Examples of the alcohol AO adduct (A1a) include polyoxyethylene alkyl ether, polyoxyalkylene alkyl ether, and polyalkylene glycol.
Specifically, as (A1a), ethylene oxide of a higher alcohol having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl alcohol, oleyl alcohol, etc.) (hereinafter abbreviated as EO) 0-20 mol and / or propylene oxide (hereinafter, propylene oxide may be abbreviated as PO) 1-20 mol adduct (including block adduct and / or random adduct; the same shall apply hereinafter) [For example, decyl alcohol EO8 mol / PO7 mol block adduct].
More specifically as (A1a), lauryl alcohol EO 7 mol adduct (HLB = 12.4), oleyl alcohol EO 5 mol adduct (HLB = 9.0), oleyl alcohol EO 6 mol adduct (HLB = 10.2). ), Oleyl alcohol EO 7 mol adduct (HLB = 11.0), oleyl alcohol EO 10 mol adduct (HLB = 12.4), 1,2-dodecanediol monooxyethylene adduct and the like.

Examples of the alkylphenol AO adduct (A1b) include alkylphenol AO adducts having an alkyl group having 6 to 24 carbon atoms.
Specific examples of (A1b) include EO1-20 mol and / or PO1-20 mol adducts of octylphenol, and EO1-20 mol and / or PO1-20 mol adducts of nonylphenol.
TRITONTMX-114 (HLB = 12.4), igepalTMCA-520 (HLB = 10.0), igepalTMCA-630 (HLB = 13.0), and the like can be easily obtained from the market.

As fatty acid AO adduct (A1c), EO 1-20 mol of fatty acids having 8 to 24 carbon atoms (decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, coconut oil fatty acid, etc.) and / or PO1 ˜20 molar adduct is mentioned.
Specifically as (A1c), oleic acid EO 9 mol adduct (HLB = 11.8), dioleic acid EO 12 mol adduct (HLB = 10.4), dioleic acid EO 20 mol adduct (HLB = 12.9). And stearic acid EO 9 mol adduct (HLB = 11.9).

As polyhydric alcohol type nonionic surfactant (A1d), EO and / or PO addition of 2 to 8 carbon number polyhydric alcohols having 3 to 36 carbon atoms (such as glycerin, trimethylolpropane, pentaerythritol, sorbit and sorbitan) Products; fatty acid esters of polyhydric alcohols and EO adducts thereof, fatty acid esters of sucrose, fatty acid alkanolamides (such as coconut oil fatty acid diethanolamide), and AO adducts thereof.
Specific examples of (A1d) include sorbitan tetraoleate EO adduct (HLB = 11.4) and sorbitan hexaoleate EO adduct (HLB = 10.2).

Of these nonionic surfactants (A1), the alcohol AO adduct (A1a) and the polyhydric alcohol type nonionic surfactant (A1d) are preferable. More preferred are decyl alcohol EO adduct of (A1a) and coconut oil fatty acid diethanolamide of polyprolene glycol EO block adduct (A1d).

  Examples of the anionic surfactant (A2) include ether carboxylic acid (A2a) and a salt thereof, sulfate ester (A2b) or a salt thereof, ether sulfate ester (A2c) and a salt thereof, sulfonate (A2d), sulfosuccinate ( A2e), phosphate ester (A2f) and its salt, ether phosphate ester (A2g) and its salt, fatty acid salt (A2h), acylated amino acid salt and naturally occurring carboxylic acid and its salt (chenodeoxycholic acid, cholic acid and Deoxycholic acid and the like).

Examples of the ether carboxylic acid (A2a) or a salt thereof include an ether carboxylic acid having a hydrocarbon group (8 to 24 carbon atoms) and a salt thereof.
Specific examples of (A2a) or salts thereof include polyoxyethylene lauryl ether acetic acid, polyoxyethylene lauryl ether acetic acid sodium salt, polyoxyethylene tridecyl ether acetic acid sodium salt, polyoxyethylene octyl ether acetic acid sodium salt and lauryl glycol acetic acid A sodium salt etc. are mentioned.

Examples of the sulfate ester (A2b) and salts thereof include sulfate esters having a hydrocarbon group (8 to 24 carbon atoms) and salts thereof.
Specific examples of (A2b) and salts thereof include sodium lauryl sulfate and triethanolamine lauryl sulfate.

Examples of the ether sulfate ester (A2c) and salts thereof include ether sulfate esters having a hydrocarbon group (8 to 24 carbon atoms) and salts thereof.
Specific examples of (A2c) and salts thereof include polyoxyethylene lauryl ether sulfate sodium salt and polyoxyethylene lauryl ether sulfate triethanolamine salt.

  Examples of the sulfonate (A2d) include dodecyl diphenyl ether disulfonate sodium salt, dodecylbenzene sulfonate sodium salt, and naphthalene sulfonate sodium salt.

  Examples of the sulfosuccinate (A2e) include polyoxyethylene lauryl sulfosuccinic acid disodium salt, sulfosuccinic acid lauryl disodium salt, and sulfosuccinic acid polyoxyethylene lauroyl ethanolamide disodium salt.

  Examples of the phosphate ester (A2f) include octyl phosphate disodium salt and lauryl phosphate disodium salt.

  Examples of the ether phosphate (A2g) include polyoxyethylene octyl ether phosphate disodium salt and polyoxyethylene lauryl ether phosphate disodium salt.

  Examples of the fatty acid salt (A2h) include sodium octylate, sodium laurate, and sodium stearate.

Of these anionic surfactants (A2), ether carboxylic acid (A2a) and sulfonate (A2d) are preferable.
More preferred are (A2a) polyoxyethylene lauryl ether acetic acid sodium salt and (A2d) dodecyl diphenyl ether disulfonic acid sodium salt.

From the viewpoint of the secretion efficiency of useful substances, the pKa of the anionic group of the anionic surfactant (A2) in water (25 ° C.) is preferably 1 to 5, more preferably 1 to 4, particularly preferably 1 to 3. 6.
Specific examples of the anionic group having a pKa of 1 to 5 include a carboxyl group (—COOH), a sulfate group (—OSO ₃ H), a sulfo group (—SO ₃ H), a sulfino group (—SO ₂ H), and a sulfeno group. Examples include an acidic group such as a group (—SOH) and a phosphoric acid group {—OP (═O) (— OH) ₂ } and a salt group thereof.
When the anionic surfactant (A2) has two or more anionic groups, any one pKa may be in the above range, and the pKa of all anionic groups is in the above range. preferable.

  As the amphoteric surfactant (A3), a carboxylate type amphoteric surfactant (A3a), a sulfate ester type amphoteric surfactant (A3b), a sulfonate type amphoteric surfactant (A3c) and a phosphate ester salt Type amphoteric surfactant (A3d) and the like are included.

  Examples of the carboxylate type amphoteric surfactant (A3a) include an amino acid type amphoteric surfactant (A3a1), a betaine type amphoteric surfactant (A3a2), and an imidazoline type amphoteric surfactant (A3a3).

  The amino acid type amphoteric surfactant (A3a1) is an amphoteric surfactant having an amino group and a carboxyl group in the molecule, and includes compounds represented by the following general formula.

[R—NH— (CH ₂ ) n —COO ⁻ ] m · Mm ⁺ (1)

 In general formula (2), R is a C1-C20 monovalent hydrocarbon group. n is an integer of 1 or more. m is an integer of 1 or more. M is a monovalent or divalent cation such as proton, alkali metal, alkaline earth metal, ammonium (including cations derived from amines and alkanolamines) and quaternary ammonium.

 Specifically, as (A3a1), an alkylaminopropionic acid type amphoteric surfactant (sodium cocaminopropionate, sodium stearylaminopropionate, sodium laurylaminopropionate, etc.); an alkylaminoacetic acid type amphoteric surfactant (laurylamino) Sodium acetate) and N-lauroyl-N′-carboxymethyl-N′-hydroxyethylethylenediamine sodium.

The betaine type amphoteric surfactant (A3a2) is an amphoteric surfactant having a quaternary ammonium salt type cation moiety and a carboxylic acid type anion moiety in the molecule.
Examples of (A3a2) include compounds represented by the following general formula (2).

RN ⁺ (CH ₃ ) ₂ —CH ₂ COO ⁻ (2)

  In general formula (2), R is a C1-C20 monovalent hydrocarbon group.

Specific examples of (A3a2) include alkyldimethylbetaines (such as stearyldimethylaminoacetic acid betaine and lauryldimethylaminoacetic acid betaine), amide betaines (such as coconut oil fatty acid amidopropyl betaine) (such as coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine), and Lauric acid amidopropyl betaine) and alkyldihydroxyalkyl betaines (lauryl dihydroxyethyl betaine), hydrogenated coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine, and the like.

  Examples of the imidazoline type amphoteric surfactant (A3a3) include 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine.

  Other amphoteric surfactants include glycine-type amphoteric surfactants such as sodium lauroyl glycine, sodium lauryl diaminoethyl glycine, lauryl diaminoethyl glycine hydrochloride and dioctyl diaminoethyl glycine hydrochloride; sulfobetaine types such as pentadecyl sulfotaurine Amphoteric surfactants; Coleamidopropyldimethylammoniopropanesulfonic acid (CHAPS), Coleamidopropyldimethylammonio 2-hydroxypropanesulfonic acid (CHAPSO); Alkylamine oxide amphoteric surfactants such as lauryldimethylamine oxide included.

Of these amphoteric surfactants (A3), preferred are the carboxylate type amphoteric surfactants (A3a) and the phosphate ester type amphoteric surfactants (A3d).
More preferred are (A3a) lauryldimethylaminoacetic acid betaine, cocaminopropionic acid sodium salt.

From the viewpoint of the secretion efficiency of useful substances, the pKa of the anionic group of the amphoteric surfactant (A3) in water (25 ° C.) is preferably 1 to 5, more preferably 1 to 4, particularly preferably 1 to 3. 6. Moreover, 8-14 are preferable, as for pKa by the cationic group of an amphoteric surfactant (A3), More preferably, it is 9-14, Most preferably, it is 9.2-14.
The anionic group having a pKa of 1 to 5 is the same as that exemplified for the amphoteric surfactant (A3).
Examples of the cationic group having a pKa of 8 to 14 include a quaternary ammonio group, a primary to tertiary amino group, and a salt group thereof.
When the amphoteric surfactant (A3) has two or more anionic groups, any one pKa may be in the above range, and the pKa of all anionic groups is in the above range. Preferably, when it has two or more cationic groups, any one pKa should just be the said range, and it is still more preferable that pKa of all the cationic groups is the said range.

  Examples of the cationic surfactant (A4) include an amine salt type cationic surfactant (A4a) and a quaternary ammonium salt type cationic surfactant (A4b).

As the amine salt type cationic surfactant (A4a), primary to tertiary amines are inorganic acids (hydrochloric acid, nitric acid, sulfuric acid, hydroiodic acid, etc.) or organic acids (acetic acid, formic acid, oxalic acid, lactic acid, gluconic acid, adipine) Acid, alkyl phosphoric acid, etc.).
For example, the primary amine salt type includes inorganic or organic acid salts of higher aliphatic amines (higher amines such as laurylamine, stearylamine, cetylamine, hardened tallow amine, and rosinamine); Examples include fatty acid (stearic acid, oleic acid, etc.) salts and the like.
Examples of the secondary amine salt type include inorganic acid salts or organic acid salts such as ethylene oxide adducts of aliphatic amines. Examples of the tertiary amine salt type include aliphatic amines (triethylamine, ethyldimethylamine, N, N, N ′, N′-tetramethylethylenediamine, etc.), ethylene oxides of aliphatic amines (2 moles). Above) Adducts, alicyclic amines (N-methylpyrrolidine, N-methylpiperidine, N-methylhexamethyleneimine, N-methylmorpholine, 1,8-diazabicyclo (5,4,0) -7-undecene, etc.) , Inorganic acid salts or organic acid salts of nitrogen-containing heterocyclic aromatic amines (4-dimethylaminopyridine, N-methylimidazole, 4,4′-dipyridyl, etc.); triethanolamine monostearate, stearamide ethyl diethyl methyl ethanol Examples include inorganic acid salts or organic acid salts of tertiary amines such as amines.

As quaternary ammonium salt type cationic surfactant (A4b), tertiary amines and quaternizing agents (alkylating agents such as methyl chloride, methyl bromide, ethyl chloride, benzyl chloride, dimethyl sulfate; ethylene oxide, etc.) And those obtained by the reaction with.
For example, lauryl trimethyl ammonium chloride, didecyl dimethyl ammonium chloride, dioctyl dimethyl ammonium bromide, stearyl trimethyl ammonium bromide, lauryl dimethyl benzyl ammonium chloride (benzalkonium chloride), cetyl pyridinium chloride, polyoxyethylene trimethyl ammonium chloride, stearamide ethyl diethyl Examples include methylammonium methosulfate.

Of these cationic surfactants (A4), preferred are quaternary ammonium salt type cationic surfactants (A4b), and more preferred is lauryltrimethylammonium chloride.

From the viewpoint of the secretion efficiency of useful substances, the pKa by the cationic group of the cationic surfactant (A4) is preferably 8 to 14, more preferably 9 to 14, particularly preferably 9.2 to 14.
Examples of the cationic group having a pKa of 8 to 14 include a quaternary ammonio group, a primary to tertiary amino group, and a salt group thereof.
When the cationic surfactant (A4) has two or more cationic groups, any one pKa may be in the above range, and the pKa of all the cationic groups may be in the above range. preferable.

In the present invention, as the surfactant (A), the surfactant (A) may be used as it is, or may be mixed with water as necessary and used as an aqueous diluent (aqueous solution or aqueous dispersion). .
The total concentration of the surfactant (A) in the aqueous diluent is appropriately selected depending on the target microorganism, the type of the physiologically active substance and the type of extraction method. From the viewpoint of secretion and handling properties of useful substances, Based on the weight of the aqueous diluent, it is preferably 0.1 to 99% by weight, more preferably 1 to 50% by weight.

The amount (% by weight) of the surfactant (A) used in the production method of the useful substance of the present invention is appropriately selected depending on the target microorganism, the kind of useful substance produced and the kind of extraction method. From the viewpoint of cytotoxicity, secretion efficiency of useful substances, and difficulty in denaturing proteins based on the weight of the culture solution, 0.0001 to 10% by weight is preferable, and 0.001 to 8% by weight is more preferable. Next, it is further preferably 0.005 to 5% by weight, particularly preferably 0.01 to 2.5% by weight.

The culture solution used in the production method of the present invention can be used without particular limitation as long as it is a cell culture medium generally used in the art, and is a natural medium containing a carbon source, a nitrogen source or other essential nutrients, or a synthetic medium. Any of the media may be used.

Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.

Examples of the nitrogen source include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor and the like.

Other essential nutrients include inorganic salts, such as monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate. Calcium carbonate or the like is used.

Useful substances in the present invention are not particularly limited, but include proteins (enzymes, hormone proteins, antibodies, peptides, etc.), oligosaccharides, nucleic acids and the like.

 Proteins include enzymes (oxidoreductases (cholesterol oxidase, glucose oxidase, ascorbate oxidase, peroxidase, etc.), hydrolases (lysozyme, protease, serine protease, amylase, lipase, cellulase, glucoamylase, etc.), isomerase ( Glucose isomerase, etc.), transferases (acyltransferase, sulfotransferase, etc.), synthetic enzymes (fatty acid synthase, phosphate synthase, citrate synthase, etc.) and elimination enzymes (pectin lyase, etc.)}, hormone proteins {bone morphogenetic factor ( BMP), interferon α, interferon β, interleukin 1-12, growth hormone, erythropoietin, insulin, granular colony stimulating factor (G-CSF) , Tissue plasminogen activator (TPA), natriuretic peptide, blood coagulation factor VIII, somatomedin, glucagon, growth hormone releasing factor and calcitonin}, antibody {single chain antibody, IgG large subunit, IgG small subunit Unit, etc.}, antigenic protein {hepatitis B surface antigen, etc.}, functional protein {pronectin (registered trademark), antifreeze peptide, antibacterial peptide, etc.}, fluorescent protein (GFP, etc.), photoprotein (lucerase, etc.) and peptide ( The amino acid composition is not particularly limited, and examples include oligopeptides, dipeptides and tripeptides).

  Examples of the oligosaccharide include sucrose, lactose, trehalose, maltose, raffinose, panose, cyclodextrin, galactooligosaccharide and fructooligosaccharide.

  Examples of nucleic acids include inosine monophosphate, adenosine monophosphate, guanosine monophosphate, and the like.

  Of these useful substances, proteins are preferable from the viewpoint of ease of preparation of useful substances, and antibodies, enzymes, and hormone proteins are more preferable.

When the useful substance is a protein, it is preferable that the protein has a property that part or all of the protein is transferred to the periplasm or extracellular after the protein is expressed in the microorganism.
More preferably, it is a protein encoding a signal sequence necessary for transition to the periplasm in the ORF.
Periplasm is the space outside the cytoplasmic membrane of a microorganism and to the outermost surface of the microorganism.
Examples of the signal sequence necessary for the transition to the periplasm include a Sec secretion signal sequence, a TAT secretion signal, and an α-signal sequence.

Examples of the microorganisms in the present invention are shown below, but are not limited thereto.
Examples of the microorganism include bacteria and eukaryotes having a cell wall containing a polysaccharide in the outermost layer. The outermost layer is a layer located outside the cell membrane and in direct contact with the external environment on the outermost surface of the cell. A polysaccharide is a compound in which a monosaccharide takes a multimer by a glycosidic bond, and does not include lipopolysaccharide.
Bacteria include eubacteria and archaea. Eubacteria include gram negative bacteria and gram positive bacteria. Examples of eukaryotes having a cell wall containing a polysaccharide in the outermost layer include fungi and algae. Examples of gram-negative bacteria include Escherichia, Thermus, Rhizobium, Pseudomonas, Shewanella, Vibrio, Salmonella, Acetobacter, Synechocystis and the like. Examples of Gram-positive bacteria include Bacillus, Streptomyces, Corynebacterium, Brevibacterium, Bifidobacterium, Lactococcus, Enterococcus, Pediococcus and Leuconostoc. The fungi include: Basidiomycota, Ascomycota, Chytridymycota, Neocallimastigomycota, Blastomycophyta, Blastomia Gates (Gromeromycota), Mucormycotina, Entomophthoromycotina, Zoopamycotina, Kickxellomycotina, etc. The Ascomycota (Ascomycota), Ambrosiozyma, Arxula, Babjevia, Blastobotrys, Candida, Citeromyces, Clavispora, Debaryomyces, Dekkera, Dipodascus, Galactomyces, Geotrichum, Hanseniaspora, Hansenula, Kazachstania, Kloeckera, Kluyveromyces, Lipomyces, Lodderomyces, Metschnikowia, Myxozyma , Nadsonia, Ogataea, minuta, Pachysolen, Pichia, Saccharomyces, Saccharomycopsis, Sai oella, Saprochaete, Saturnispora, Schizoblastosporion, Schizosaccharomyces, Sporopachydermia, Starmerella, Stephanoascus, Symbiotaphrina, Sympodiomyces, Tetrapisispora, Torulaspora, Trigonopsis, Wickerhamia, Wickerhamiella, Williopsis, Yarrowia, Zygoascus, Zygosaccharomyces, Zygozyma, Bannoa, Bensingtonia, Bullera, Bulleromyces, Cryptococcus, Curvibasidiu , Cystofilobasidium, Dioszegia, Erythrobasidium, Dioszegia, Erythrobasidium, Fellomyces, Fibulobasidium, Filobasidium, Filobasidiella, Guehomyces, Kockovaella, Kondoa, Kurtzmanomyces, Leucosporidium, Malassezia, Mastigobasidium, Mrakia, Occultifur, Pseudozyma, Rhodosporidium, Rhodotorula, Sakaguchia, Sirobasidium, Sporidiobolus, Sporobolomyces , Sterigmaty strains such as Ces, Sterigmatosporidium, Symposiomycosis, Tausonia, Tillethiopsis, Trichosporiella, Trichosporon, Tsuchiyaea, Udeniomyces, and Xanthophyllomyces. Examples of algae include eubacteria cyanobacteria (cyanobacteria), eukaryotes and unicellular organisms (diatoms, yellow green algae, dinoflagellates, etc.), and the like.

Among these, from the viewpoint of productivity of useful substances, the microorganism of the present invention is preferably at least one selected from the group consisting of yeasts that are Gram-negative bacteria, Gram-positive bacteria, and E.coccus fungi.
More preferably, it is at least one species selected from the group consisting of Escherichia, Corynebacterium, Saccharomyces, Pichia, Candida, Schizosaccharomyces, Yarrowia, Hansenula, Ogataea, Kluyveromyces, and Pseudozyme. One type of microorganism may be used, or two or more types may be used in combination.

In the method for producing a useful substance of the present invention, the secretion efficiency (%) by the surfactant in the culture solution is preferably 55 to 100, more preferably 60 to 100, and still more preferably 65 from the viewpoint of productivity. -100, particularly preferably 70-100.

Secretion efficiency indicates that useful substances in the microorganism are secreted outside the microorganism (in the culture medium) by the surfactant.
In the present invention, it is defined by the following formula (5).
Secretion efficiency (%) = 100 × (X) / {(X) + (Y)} (5)
X (culture supernatant): weight of useful substance in culture remaining after removal of cells by centrifugation Y (surface of cells): cell suspension washed and resuspended by centrifugation Weight of useful substances in

  For example, if the protein produced in the microorganism is transferred to the periplasm more, the secretion efficiency is increased, and if the protein produced in the microorganism is not transferred to the periplasm more, the secretion efficiency is decreased. Moreover, secretion efficiency can be raised by selecting surfactant with high secretion efficiency by screening.

  Mixing of the surfactant (A) and the culture solution can be performed by adding the surfactant to the culture solution at 4 ° C to 99 ° C and stirring with a stirring blade or a stirrer. When mixing, it can carry out by adding, stirring with a stirring blade etc.

In the production method of the useful substance of the present invention, the production method for producing secretory production of the useful substance includes a microbial exocrine production method including the following steps (a) and (b). In the following steps, the step of producing secreted useful substances is step (a).
Step (a): A step in which a culture medium for culturing microorganisms (such as yeast) producing useful substances and a surfactant are simultaneously present to secrete useful substances out of the microorganisms (in the culture liquid).
Step (b): A step of separating useful substances from the culture solution after the step (a).

An example of a method for producing useful substances using the surfactant (A) of the present invention is shown below.
(I) Genetic recombination (i-1) Isolating messenger RNA (mRNA) from cells expressing the target protein, synthesizing single-stranded cDNA and then double-stranded DNA from the mRNA, Strand DNA is incorporated into phage DNA or plasmid. The resulting recombinant phage or plasmid is transformed into a microorganism to prepare a cDNA library.
(I-2) Examples of a method for screening phage DNA or plasmid containing the target DNA include a hybridization method of the phage DNA or plasmid and a DNA probe encoding a part of the target protein gene or complementary sequence. .
(I-3) The target cloned DNA or a part thereof is cut out from the screened phage or plasmid, and the cloned DNA or a part thereof is ligated downstream of the promoter in the expression vector. Expression vectors can be prepared. DNAs encoding a signal sequence for translocating the inner membrane (a signal sequence for expressing the target substance in the periplasm) can be linked simultaneously.
(Ii) Culture (ii-1) A host microorganism is transformed with an expression vector to prepare a recombinant microorganism, and the recombinant microorganism is pre-cultured. Pre-culture is usually performed at 15 to 43 ° C. for 3 to 72 hours on an agar medium.
(Ii-2) The culture solution used for the production of useful substances is autoclaved at 121 ° C. for 20 minutes, and the recombinant microorganism pre-cultured on an agar medium is cultured here. The culture is usually performed at 15 to 43 ° C. for 12 to 72 hours.
(Iii) Purification (iii-1) The protein secreted into the culture solution is separated from microorganisms and microbial residues by centrifugation, hollow fiber separation, filtration and the like.
(Iii-2) The culture solution containing protein is subjected to precipitation treatment such as ethanol precipitation, ammonium sulfate precipitation and polyethylene glycol precipitation by repeating column treatments such as ion exchange column, gel filtration column, hydrophobic column, affinity column and ultra-column. Separation and purification are carried out as necessary.

  Thereafter, the host microorganism isolated in (iii-1) can be further cultured by supplying a new culture solution. By further subjecting the culture solution and the like to the step (iii) and repeating the purification and culture, continuous production of useful substances can be performed.

When performing column treatment in the purification step (iii) above, examples of the filler used for column chromatography include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer, and the like, and commercially available products such as Capto series, Examples include Sephadex series, Sephacryl series, Sepharose series (hereinafter, GE Healthcare), Bio-Gel series (Bio-Rad).

By using the production method of useful substances of the present invention, useful substances produced by microorganisms can be secreted into the culture solution with high efficiency, and a high yield can be obtained in a short time. Can be achieved. In addition, the useful substance production method of the present invention facilitates purification of the useful substance because the useful substance is secreted into the culture solution.

The method for producing a useful substance of the present invention includes a step of causing a surfactant and a microorganism to simultaneously exist and secreting the useful substance into the culture solution. In this step, as long as the microorganism is alive, it is presumed that the microorganism can make a useful substance and secrete it into the culture solution. Furthermore, if the microorganism has the ability to produce a useful substance, it is presumed that the production method of the present invention can be used regardless of the kind of useful substance to be produced.
The useful substance production method of the present invention is particularly effective when the useful substance prepared in the microorganism is transferred to the periplasm of the microorganism. By transferring the useful substance to the periplasm, the useful substance is easily secreted into the culture medium.

  Hereinafter, although an example and a comparative example explain the present invention further, the present invention is not limited to these. Hereinafter, unless otherwise specified, “%” represents “% by weight” and “parts” represents “parts by weight”.

Production Example 1 <Production of Surfactant (A1-1)>
A stainless steel pressure reactor was charged with 450 parts of SANNICS PP-2000 [manufactured by Sanyo Chemical Co., Ltd .; polyoxypropylene glycol (number average molecular weight: 2000)] and 0.05 part of sodium hydroxide. After injecting 50 parts of ethylene oxide over 4 hours at 130 ° C., the mixture was further reacted at the same temperature for 10 hours to obtain a surfactant (A1-1). It was HLB = 1.8 and Mn = 2,200 of (A1-1).

Production Example 2 <Production of Surfactant (A1-2)>
In Production Example 1, 101 parts of Sanix PP-3000 [manufactured by Sanyo Chemical Co., Ltd .; polyoxypropylene glycol (number average molecular weight: 3200)] was used in place of 450 parts of Sanniks PP-2000, and ethylene oxide was injected. A surfactant (A1-2) was obtained by performing the same operation except that the amount was changed from 50 parts to 399 parts. It was HLB = 15.9 of (A1-2) and Mn = 16,000.

The number average molecular weight Mn of the surfactants (A1-1) and (A1-2) used in Examples and Comparative Examples was measured by the following method.
The number average molecular weight was measured under the following conditions using GPC (Gel Permeation Chromatography) with respect to the soluble component in tetrahydrofuran (hereinafter abbreviated as THF).
Measuring device: “HLC-8120” manufactured by Tosoh Corporation
Column: “TSKgelGMHXL” (2) manufactured by Tosoh Corporation and “T SKgelMultiporeHXL-M” (1) manufactured by Tosoh Corporation
Sample solution: 0.25 mass% THF solution Injection amount of sample solution to the column: 100 μL
Flow rate: 1 mL / min Measurement temperature: 40 ° C
Detection device: Refractive index detector Reference material: 12 standard polystyrene (TSK standard POLYSYRENE) manufactured by Tosoh Corporation (number average molecular weight: 500, 1050, 2800, 5970, 9100, 18100, 37900, 96400, 190000, 355000, 1090000) 2890000).

Examples 1 to 18 and Comparative Examples 1 to 3 <Evaluation of secretory productivity and secretory efficiency of cellulase using E. coli>
The pET-22b plasmid (Novagen) was cleaved at the NcoI restriction enzyme site and the BamHI restriction enzyme site, and the bglC gene of Bacillus licheni formis having NcoI and BamHI cleavage sites at both ends was artificially synthesized (consigned synthesis by Thermo). .
Thereafter, this plasmid was transformed into BL21 (DE3) E. coli strain (Novagen) to prepare a cellulase expression strain. Based on the method of METHODS IN ENZYMOLOGY 353 2002 page 121, it was confirmed that the expressed cellulase was localized in the periplasmic fraction. The prepared cellulase-expressing Escherichia coli was inoculated into 2 mL of LB medium (manufactured by LB Broth, Miller, Difco Laboratories) using platinum ears, and cultured with shaking at 150 rpm at 30 ° C. for 15 hours to prepare a preculture solution.
The prepared pre-culture solution is 1 mL of medium [yeast extract [manufactured by Nippon Pharmaceutical Co., Ltd.] 24 mg, polypeptone [manufactured by Nippon Pharmaceutical Co., Ltd.] 12 mg, phosphorus so that the final turbidity (OD) is 0.03 OD / mL. 9.4 mg of dipotassium acid, 2.2 mg of monopotassium phosphate, 7 mg of ammonium sulfate, 13.2 mg of disodium phosphate 12 hydrate, 0.4 mg of sodium citrate dihydrate, 4 mg of glycerol, 3 mg of hydrolyzed lactalbumin 1 mM magnesium sulfate, trace metal solution [calcium chloride 0.38 μg, iron (III) chloride 10 μg, zinc sulfate heptahydrate 0.18 μg, copper sulfate 0.1 μg, manganese chloride tetrahydrate 0.13 μg, cobalt chloride 0.1 μg, ethylenediaminetetraacetic acid 4 sodium 4 μg], 100 mg / L ampicillin] and inoculated at 150 ° C. for 150 r And stirring was started culture by m.
Two hours after the start of the culture, Isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 1 mM, and stirring culture was continued.
When the culturing time shown in Table 1 was reached, the surfactant (A) shown in Table 1 (pure water in Comparative Example 1) was added to the weight of the culture solution [medium, preculture solution, Isopropyl β-D-1- The total weight of thiogalactopyranoside and the surfactant (A) to be added] was added to the weight% shown in Table 1, and the stirring culture was continued.
The culture solution was sampled 30 hours after the start of the culture, and the cells and the culture supernatant were separated from the culture solution by a centrifuge, and the culture supernatant and the cells were collected. The secretion productivity and secretion efficiency (%) per cell were evaluated, and the results are shown in Table 1.
The culture time for adding the surfactant (A) was determined in advance by determining the culture stationary phase ODS by conducting test culture in the same manner as in Comparative Example 3.

In Tables 1 to 3, the following surfactants (A1-3) to (A4-1) were used.
(A1-3): Sorbitan coconut oil fatty acid ester, HLB = 8.5, Mn = 346
(A2-1): Polyoxyethylene (EO 2.5 mol) sodium lauryl ether sulfate, pKa = 1.99
(A2-2): Polyoxyethylene (EO 3 mol) sodium lauryl ether acetate, pKa = 3.55
(A3-1): Sodium 2- (dodecylamino) ethanesulfonate, pKa = 1.99 and 10.77
(A3-2): sodium dodecyl-β-aminopropionate, pKa = 3.55 and 10.77
(A3-3): Miltefosine, pKa = 2.15 and 14.0
(A3-4): lauryldimethylaminoacetic acid betaine, pKa = 3.55 and 14.0
(A4-1): Lauryltrimethylammonium chloride, pKa = 14.0

In addition, the measurement method and calculation method of the secretion productivity and secretion efficiency of cellulase per Escherichia coli are carried out by the following methods.

<Measurement method of turbidity of culture solution>
Using a culture solution containing microorganisms collected by sampling, turbidity was measured using a quartz cell having an optical path length of 1 cm using a turbidimeter [manufactured by Shimadzu Corporation, UV-1700].
The culture solution was centrifuged at 1500 rpm for 5 minutes at 4 ° C., and the supernatant was discarded. The precipitate was resuspended in the same amount of physiological saline as the amount of the sample solution, diluted with physiological saline to obtain an appropriate absorbance (0.1 to 0.8), and the absorbance at 600 nm was measured. The turbidity of the culture solution was calculated by the following mathematical formula (1).
Turbidity (OD) of culture solution = (absorbance at 600 nm of diluted culture solution) × dilution ratio of culture solution (1)

<Evaluation of secretory productivity per cell: measurement of the activity of secreted cellulase>
Endo-1,4-β-glucanase activity was measured as cellulase activity.
Carboxymethylcellulose (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in a 100 mM phosphate buffer aqueous solution of pH 7.5 so as to be 17.5 g / L to prepare a substrate solution.
5 μL of the culture supernatant obtained by centrifuging each culture solution obtained in Examples 1 to 18 and Comparative Examples 1 to 3 (13000 G × 15 min) and 3 mL of the substrate solution were mixed, and this mixture was used as a viscometer. Transfer to a temperature-controlled cup (TVE-22L viscometer, 2 rpm) at 40 ° C.
Read the viscosity immediately after mixing, read the viscosity after 1, 2, 3, 4, 5 and 6 minutes, and plot the XY coordinate diagram with the time (minutes) as the X axis and the viscosity (mPa · s) as the Y axis. The absolute value of the slope when a straight line was drawn by the least square method was defined as cellulase activity (endo-1,4-β-glucanase activity).
[Activity of secreted cellulase] = [Slope of line] × dilution ratio [secretory productivity per cell] = [activity of secreted cellulase] / [turbidity in stationary phase of culture]
The “secretory productivity per bacterial cell (relative value)” in Table 1 is the above-mentioned “secretory productivity per bacterial cell” in Comparative Example 1 (blank without using a surfactant). The relative ratio was expressed as 1.00.

<Measurement of the activity of cellulase remaining on the cell surface>
To the cells obtained in Examples 1 to 18 and Comparative Examples 1 to 3, 200 μL of 0.2 M Tris-HCl buffer (pH 7.4) in the same amount as the removed supernatant was added, and the cells were centrifuged. Was washed. The washed cells were resuspended with 200 μL of 0.2 M Tris-HCl buffer (pH 7.4) in the same amount as the washed supernatant to obtain a cellulase activity measurement sample on the surface of the cells.
Using a mixed solution obtained by adding 3 mL of the above carboxymethylcellulose substrate solution to 5 μL of the cellulase activity measurement sample on the surface of the bacterial cell, the viscosity was measured under the same conditions as in “Measurement of activity of secreted cellulase”. The value obtained by fitting was used as the activity of cellulase, a useful substance remaining on the surface of the cells.
[Activity of cellulase remaining on the cell surface] = [Slope of straight line] × dilution ratio

<Evaluation of cellulase secretion efficiency>
Using each culture supernatant and cells obtained in Examples 1 to 18 and Comparative Examples 1 to 3, the secretion efficiency in each example was calculated by the following formula.
Secretion efficiency (%) = 100 × “activity of cellulase produced by secretion” / “total cellulase activity”

The total cellulase activity means “activity of secreted cellulase” + “activity of cellulase remaining on the cell surface”.
In addition, “secretion efficiency (%)” in Table 1 is the above-mentioned “activity of cellulase produced by secretion” and “activity of cellulase remaining on the cell surface” in Comparative Example 1 (blank not using a surfactant). Is expressed as a relative ratio when.

Examples 19 to 39 and Comparative Examples 4 to 6 <Evaluation of secretion productivity and secretion efficiency of luciferase using luciferase-expressing yeast>
Luciferase-expressing yeast (Pichia pastoris) was transformed using Pichia pastoris distributed by (Biotechnology Center, National Institute of Technology and Evaluation).
A pPICZα vector (manufactured by Thermo) was cleaved with XhoI and XbaI, and luciferase genes having XhoI and XbaI cleavage sites were incorporated at both ends artificially synthesized (combined synthesis by Thermo).
Transformation of the pPICZα vector incorporating the luciferase gene into yeast was performed by the method described in the pPICZα2 vector instruction manual. A luciferase-expressing yeast was inoculated into a BMGY culture solution using a platinum loop, and cultured at 30 ° C. for 15 hours with shaking at 200 rpm to prepare a preculture solution. 125 mL BMGY culture solution (Difco's Yeast extract 1 wt%, Difco's Bactopeptone 2 wt%, Difco's Yeastnitrogen base w / o amino acid 1.34) so that the prepared preculture solution has a final OD of 0.05 OD / mL. %, Glucose 2 wt%, 100 mM potassium hydrogen phosphate, pH 6.0), and stirring culture was started at 30 ° C. and 1100 rpm.
When the culture time shown in Table 2 is reached, the culture solution is centrifuged (1500 × g, 10 minutes), resuspended in 125 mL of BMMY culture solution, and the surfactant (A) or pure water shown in Table 2 is used. Was added so as to achieve the weight% described in Table 2, and stirring culture was continued.
The culture solution was sampled 30 hours after the start of the culture, and the cells and the culture supernatant were separated from the culture solution by a centrifuge, and the culture supernatant and the cells were collected.
The secretion productivity (relative ratio) and secretion efficiency (%) per cell were evaluated, and the results are shown in Table 2.
The culture time for adding the surfactant (A) was determined in advance by determining the culture stationary phase ODS by conducting test culture in the same manner as in Comparative Example 4.

<Evaluation of secretory productivity per cell: measurement of luciferase activity of useful substances secreted and produced>
60 μL of the following luciferin solution was added to 20 μL of each culture supernatant obtained in Examples 19 to 39 and Comparative Examples 4 to 6 diluted 10 to 1000 times with 0.2 M Tris-HCl buffer (pH 7.4). Using the obtained mixed solution as a sample, the luminescence intensity measured under the following conditions using a luminometer was applied to the following formula to obtain the value obtained as secreted luciferase activity of the useful substance.
Luminometer: “Glomax Navigator” manufactured by Promega Corporation
Luciferin solution: New England Biolab Japan Co., Ltd. Product temperature: 25 ° C
Detector: Luminescence detector Detection wavelength: 350-700 nm
[Luciferase activity of useful substance secreted and produced] = [luminescence intensity] × dilution ratio [secretory productivity per bacterial cell] = [luciferase activity of useful substance produced and secreted] / [turbidity in stationary phase of culture]
The “secretory productivity per bacterial cell (relative value)” in Table 2 is the above-mentioned “secretory productivity per bacterial cell” in Comparative Example 4 (blank without using a surfactant). The relative ratio was expressed as 1.00.

<Measurement of luciferase activity of useful substances remaining on the cell surface>
To the bacterial cells obtained in Examples 19 to 39 and Comparative Examples 4 to 6, 200 μL of 0.2 M Tris-HCl buffer (pH 7.4) in the same amount as the removed supernatant was added, and the bacterial cells were centrifuged. Was washed. The washed cells are resuspended in 200 μL of 0.2 M Tris-HCl buffer (pH 7.4) in the same amount as the washed supernatant, and 10 times with 0.2 M Tris-HCl buffer (pH 7.4). A sample diluted ˜1000 times was used as a sample for measuring the luciferase activity on the surface of the cells.
Luminescence intensity was measured under the same conditions as in “Measurement of Luciferase Activity of Useful Substances Secreted and Produced” using a mixed solution obtained by adding 60 μL of the above luciferin solution to 20 μL of this luciferase activity measurement sample on the surface of the cells. The value obtained by applying the intensity to the following formula was defined as the luciferase activity of the useful substance remaining on the surface of the cells.
[Luciferase activity of useful substance remaining on cell surface] = [luminescence intensity] × dilution rate

<Evaluation of secretion efficiency>
The secretion efficiency in each Example was calculated by the following formula using each culture supernatant and cells obtained in Examples 19 to 39 and Comparative Examples 4 to 6.
Secretion efficiency (%) = 100 × “luciferase activity of useful substance secreted and produced” / “total luciferase activity”

The total luciferase activity means “luciferase activity of useful substance secreted and produced” + “luciferase activity of useful substance remaining on the cell surface”.
In addition, “secretion efficiency (%)” in Table 2 indicates the above-mentioned “luciferase activity of a useful substance produced by secretion” and “useful substance remaining on the surface of the fungus” in Comparative Example 4 (blank without using a surfactant). The luciferase activity of ”was expressed as a relative ratio when 1.00.

Examples 40 to 60 and Comparative Examples 7 to 9 <Evaluation of secretory productivity and secretory efficiency of acid phosphatase using acid phosphatase expressing yeast>
Acid phosphatase-expressing yeast (Regandation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida body.
al. , Biochimica et Biophysica Acta, 1493, 2000, 56-63). ) Was inoculated into the BMGY medium using platinum ears and cultured with shaking at 30 ° C. for 15 hours at 200 rpm to prepare a preculture liquid. The prepared preculture was inoculated into 125 mL of BMGY culture so that the final OD was 0.05 OD / mL, and stirring culture was started at 30 ° C. and 1100 rpm.
When the culture time shown in Table 3 is reached, the culture solution is centrifuged (1500 × g, 10 minutes), resuspended in 125 mL of BMMY culture solution, and the surfactant (A) or pure water shown in Table 3 is used. Was added so that it might become the weight% as described in Table 4, and stirring culture was continued.
The culture solution was sampled 30 hours after the start of the culture, and the cells and the culture supernatant were separated from the culture solution by a centrifuge, and the culture supernatant and the cells were collected. 50 mM NaBf acetate (pH 4.0) was added to the cells, and the cells were washed by centrifugation. The washed cells were resuspended with 50 mM NaBf acetate (pH 4.0) to obtain a sample for measuring acid phosphatase activity on the surface of the cells.
The secretion productivity (relative ratio) and secretion efficiency (%) per cell were evaluated, and the results are shown in Table 3.
The culture time for adding the surfactant (A) was determined in advance by determining the culture stationary phase ODS by conducting test culture in the same manner as in Comparative Example 7.

＜菌体１個あたりの分泌生産性の評価：酸性ホスファターゼ活性測定＞
実施例４０〜６０及び比較例７〜９で得た各培養上清に１／４０量の２Ｍ 酢酸ナトリウム緩衝液（ｐＨ４．０）５μＬを添加した。緩衝液を添加した培養上清又は実施例４０〜６０及び比較例７〜９で得た各菌体表面測定サンプルに基質溶液（ｐ−ｎｉｔｒｏｐｈｅｎｙｌｐｈｏｓｐｈａｔｅ ０．６４ｍｇ／ｍＬ含有 ５０ｍＭ酢酸ナトリウム緩衝液ｐＨ４．０）を容量比＝１：１で混合し、３５℃で１０分間反応した。
反応後、反応液と等量の１０％トリクロロ酢酸溶液を添加し反応停止をした。反応停止をした溶液に、炭酸ナトリウム飽和溶液を反応液全体と等量加え、発色させた。発色させた液は、１５００×ｇ、５分で不溶画分を除去し、（Ｂｉｏｔｅｋ社製マイクロプレートリーダーＰｏｗｅｒＷａｖｅ）を用いて４２０ｎｍの吸光度を測定した。参照波長には６３０ｎｍを測定した。
培養上清、菌体表面測定サンプルそれぞれについて、下記の式より算出した値を酸性ホスファターゼ活性とした。
酸性ホスファターゼ活性＝｛（Ａｂｓ４２０ｎｍ）−（Ａｂｓ６３０ｎｍ）｝
〔菌体１個あたりの分泌生産性〕＝〔分泌生産した有用物質の酸性ホスファターゼ活性〕／〔培養定常期の濁度〕
なお、表３中の「菌体１個あたりの分泌生産性（相対値）」は、比較例７（界面活性剤を用いないブランク）の上記の「菌体１個あたりの分泌生産性」を１．００とした場合の相対比で表した。

<Evaluation of secretory productivity per cell: measurement of acid phosphatase activity>
To each culture supernatant obtained in Examples 40 to 60 and Comparative Examples 7 to 9, 1/4 μl of 2 M sodium acetate buffer (pH 4.0) 5 μL was added. A culture solution to which a buffer solution was added or each cell surface measurement sample obtained in Examples 40 to 60 and Comparative Examples 7 to 9 contained a substrate solution (p-nitrophenyl phosphate 0.64 mg / mL, 50 mM sodium acetate buffer pH 4.0). ) Was mixed at a volume ratio of 1: 1 and reacted at 35 ° C. for 10 minutes.
After the reaction, 10% trichloroacetic acid solution equivalent to the reaction solution was added to stop the reaction. To the solution whose reaction was stopped, a saturated sodium carbonate solution was added in an amount equivalent to the whole reaction solution to cause color development. From the colored solution, the insoluble fraction was removed at 1500 × g for 5 minutes, and the absorbance at 420 nm was measured using (Biotek Microplate Reader PowerWave). A reference wavelength of 630 nm was measured.
For each of the culture supernatant and the cell surface measurement sample, the value calculated from the following formula was defined as the acid phosphatase activity.
Acid phosphatase activity = {(Abs 420 nm) − (Abs 630 nm)}
[Secretory productivity per cell] = [acid phosphatase activity of useful substances secreted and produced] / [turbidity in stationary culture phase]
The “secretory productivity per bacterial cell (relative value)” in Table 3 is the above-mentioned “secretory productivity per bacterial cell” in Comparative Example 7 (blank without using a surfactant). The relative ratio was expressed as 1.00.

<Evaluation of secretion efficiency>
Using the values of “acid phosphatase activity of useful substance secreted and produced” and “acid phosphatase activity of useful substance remaining on the surface of the cell” obtained in Examples 40 to 60 and Comparative Examples 7 to 9, secretion was performed according to the following formula. Efficiency was calculated. The results are shown in Table 4.
Secretion efficiency (%) = 100 × “acid phosphatase activity of useful substances secreted and produced” / “total acid phosphatase activity”

The total acid phosphatase activity means “acid phosphatase activity of a useful substance secreted and produced” + “acid phosphatase activity of a useful substance remaining on the cell surface”.
In addition, “secretion efficiency (%)” in Table 3 is the above-mentioned “acid phosphatase activity of useful substance produced by secretion” and “useful remaining on the cell surface” of Comparative Example 4 (blank without using a surfactant). It was expressed as a relative ratio when the acid phosphatase activity of the substance was 1.00.

  For the culture supernatants obtained in Examples 1 to 60 and Comparative Examples 1 to 9, the turbidity of the culture solution, the secretion productivity (relative ratio) per bacterial cell, and the secretion efficiency (%) were evaluated as described above. went. The results are shown in Tables 1-3.

In Examples 1 to 60 in Tables 1 to 3, the ratio OD / ODS of the turbidity OD at 600 nm of the culture solution at the time of culture and the turbidity ODS at 600 nm of the culture solution in the stationary culture phase is 0.009 to 0.00. When it is in the range of 90, by adding the surfactant (A), the relative value when the secretion productivity per cell of Comparative Examples 1, 4, and 7 is set to 1 is significantly increased. You can see that
On the other hand, Comparative Examples 2, 5, and 8 having an OD / ODS of less than 0.009, and Comparative Examples 3, 6, and 9 having an OD / ODS of more than 0.90 are secreted per bacterial cell as compared to the Examples. The relative value of productivity is low.
Therefore, it can be seen that the production method of the present invention provides a large amount of useful substances. Furthermore, in the examples using the production method of the present invention, the secretion efficiency is greater than 50%, the useful substances produced by the microorganisms are easily secreted outside the cells, and treatments such as crushing the bacteria are not required. From the fact that a large amount of useful substances can be obtained, it can be seen that the production efficiency is also high. Therefore, it can be seen that the production amount of useful substances using microorganisms is excellent and the production efficiency is also high.

The method for producing useful substances of the present invention can be used when producing useful substances such as proteins from microorganisms. Examples of proteins include enzymes, hormone proteins, antibodies and peptides. When the protein to be produced is an enzyme (protease, cellulase, lipase, amylase, etc.), it can be suitably used for food processing, detergents, fiber processing, papermaking, enzyme conversion, etc. This is useful in the production of

Claims (7)

A method for producing a useful substance that secretes and produces a useful substance in a culture solution by microorganisms contained in the culture solution, the turbidity OD at 600 nm of the culture solution at the time of culture, and the stationary culture condition when no surfactant is added A method for producing a useful substance in which a surfactant (A) is added to a culture solution when the ratio OD / ODS of the culture solution at 600 nm to the turbidity ODS at 600 nm is in the range of 0.009 to 0.90. The surfactant (A) is a group consisting of a nonionic surfactant (A1), an anionic surfactant (A2), an amphoteric surfactant (A3) and a cationic surfactant (A4) having an HLB of 0.1 to 16. The method for producing a useful substance according to claim 1, wherein the method is at least one selected from the group. The method for producing a useful substance according to claim 1 or 2, wherein the nonionic surfactant (A1) has an HLB of 1 to 16 and a number average molecular weight of 100 to 20,000. The method for producing a useful substance according to any one of claims 1 to 3, wherein the content of the surfactant (A) at the end of the cultivation is 0.0001 to 10% by weight based on the weight of the medium. The method for producing a useful substance according to claim 1, wherein the useful substance is a protein. The method for producing a useful substance according to claim 5, wherein the protein is a protein that moves into the periplasm or out of the cell. The method for producing a useful substance according to any one of claims 1 to 6, wherein the microorganism is a gram-negative bacterium, a gram-positive bacterium, or a yeast.

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