JP2022140734A - Production method of useful substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: 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

COPYRIGHT: (C)2022,JPO&INPIT

Description

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

Microorganisms are widely used to produce useful substances such as amino acids and proteins. Gram-negative bacteria (eg, Escherichia coli), Gram-positive bacteria, and yeast are widely used as microorganisms for producing useful substances.

Techniques for efficiently producing useful proteins by introducing genes of pharmaceutically and industrially useful proteins into Escherichia coli have been known with the progress of genetic engineering techniques.

When the protein is expressed using E. coli, the desired useful protein is produced within the microorganism. Physical disruption methods such as ultrasonic waves, high-pressure homogenizers, and French presses are required to extract the produced target protein from the E. coli. Since a large amount of substances other than the target protein present in the protein are also mixed in, there is a problem that the purity is lowered.

On the other hand, when Gram-positive bacteria or yeast are used to produce useful substances, the produced useful substances are secreted outside the cells and are less contaminated with substances other than the intended useful substances (for example, Non-Patent Documents 1 and 2). However, Gram-positive bacteria and yeast have a problem that their protein-producing ability is lower than that of E. coli.

As a protein production method that solves these problems, a production method that uses a surfactant to secrete a useful substance is known (for example, Patent Document 1). When secretory production is performed, high productivity can be maintained, and lengthening the production time leads to an improvement in productivity.

However, in the method of Patent Document 1, since the secretory productivity per cell is low, the production of useful substances such as proteins obtained by extracellular secretion is small, and it is difficult to improve high productivity. I have a problem.

International Publication No. 2010/137624

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

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

The present inventors arrived at the present invention as a result of conducting studies to achieve the above object.

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

The method for producing a useful substance of the present invention has the effect of enabling efficient secretion and production of a useful substance by a microorganism by increasing the secretion productivity per cell.

1 is a drawing showing a growth curve for explaining the timing of addition of a surfactant (A) and its OD, the stationary phase of culture and its ODS in the method for producing a useful substance of the present invention.

The method for producing a useful substance of the present invention is a method for producing a useful substance by secretion production of a useful substance into a culture solution by microorganisms contained in the culture solution, wherein the turbidity OD at 600 nm of the culture solution during cultivation, A surfactant (A) is added to the culture medium when the ratio OD/ODS to the turbidity ODS at 600 nm of the culture medium in the stationary phase of the culture is in the range of 0.009 to 0.90. .

The turbidity OD of the culture solution is calculated by the following measuring method and formula.
<Method for measuring turbidity OD of culture solution>
Using a culture solution containing the sampled microorganisms, turbidity is measured using a turbidity meter [eg UV-1700 manufactured by Shimadzu Corporation] using a quartz cell with an optical path length of 1 cm.

The culture is centrifuged at 1500 rpm for 5 minutes at 4° C. and the supernatant is discarded. Add the same volume of physiological saline as the volume of the supernatant discarded from the precipitate, stir to resuspend, and dilute with physiological saline to obtain an appropriate absorbance (0.1 to 0.8). Absorbance at 600 nm is measured.

The turbidity OD of the culture solution is calculated by the following formula.
Turbidity (OD) of the culture solution = (absorbance at 600 nm of the diluted culture solution) x dilution ratio of the culture solution In the present invention, the turbidity OD of the culture solution relatively represents the number of microorganisms in the culture solution. I believe.

The ratio OD/ODS of the turbidity OD at 600 nm of the culture solution during culture when the surfactant (A) is added to the culture solution and the turbidity ODS at 600 nm of the culture solution during the stationary phase of the culture is It is 0.009 to 0.90, preferably 0.009 to 0.80 from the viewpoint of the secretion productivity of useful substances per one. 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 secrete and produce useful substances without killing the cells. .

The stationary phase of culture in the present invention means that, in culture without addition of a surfactant, as shown in FIG. This is the period when the turbidity of the culture solution does not increase or decrease because some of them die while they proliferate.

The cultured stationary phase ODS in the present invention can be determined by carrying out a test culture once before the main examination, using the same composition and amount of medium and bacterial cells as in the main examination and culturing under the same culture conditions. . In the test culture, by measuring the turbidity OD of the culture solution at 600 nm successively from the start of culture and drawing a growth curve, the turbidity ODS of the culture solution at 600 nm in the stationary phase of the culture can be obtained. .

Surfactant (A) is added when OD/ODS is in the range of 0.009 to 0.90 in turbidity ODS and turbidity OD in the stationary phase of culture shown in FIG. As for this period, it is preferable to add the surfactant (A) in the logarithmic growth phase, which is the period when microorganisms start dividing and grow logarithmically. On the other hand, if it is added during the culture induction period (the preparation period for microorganisms to adapt to the medium environment and start growing, and the microorganisms are hardly growing), the growth of microorganisms is inhibited, and the secretion productivity of useful substances is reduced. decreases.
Surfactants (A) used in the method for producing useful substances of the present invention include nonionic surfactants (A1), anionic surfactants (A2), amphoteric surfactants (A3) and cationic surfactants (A4). At least one surfactant selected from the group consisting of (A1) preferably has an HLB of 0.1 to 16.

Examples of nonionic surfactants (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 surfactants (A1d) and the like are included.

HLB is known as a measure of the hydrophilicity and hydrophobicity of the nonionic surfactant (A1). A higher HLB value means a higher hydrophilicity. The HLB in the present invention is a numerical value calculated by the following formula (Written by Takehiko Fujimoto, Introduction to Surfactants, page 212, published by Sanyo Chemical Industries, Ltd.).
HLB = 10 x (inorganic/organic)
The HLB of the nonionic surfactant (A1) is preferably 0.1-16, more preferably 1-16, from the viewpoint of secretion efficiency.

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.

Alcohol AO adducts (A1a) include polyoxyethylene alkyl ethers, polyoxyalkylene alkyl ethers and polyalkylene glycols.
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, ethylene oxide is abbreviated as EO) ) 0 to 20 mol and/or propylene oxide (hereinafter, propylene oxide may be abbreviated as PO) 1 to 20 mol adduct (including block adduct and/or random adduct; hereinafter the same) [eg 8 mol EO/7 mol PO block adduct of decyl alcohol].
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) ), 7 mol oleyl alcohol EO adduct (HLB=11.0), 10 mol oleyl alcohol EO adduct (HLB=12.4), 1,2-dodecanediol monooxyethylene adduct, and the like.

Alkylphenol AO adducts (A1b) include alkylphenol AO adducts having an alkyl group having 6 to 24 carbon atoms.

Specific examples of (A1b) include adducts of 1 to 20 mol of EO and/or 1 to 20 mol of PO of octylphenol and adducts of 1 to 20 mol of EO and/or 1 to 20 mol of PO of nonylphenol.

TRITONTMX-114 (HLB=12.4), igepalTMCA-520 (HLB=10.0) and igepalTMCA-630 (HLB=13.0) are readily available on the market.
As the fatty acid AO adduct (A1c), 1 to 20 mol of EO and/or PO1 of a fatty acid having 8 to 24 carbon atoms (decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, coconut oil fatty acid, etc.) ~20 molar adducts are included.
Specific examples of (A1c) include 9 mol EO oleic adduct (HLB=11.8), 12 mol EO dioleate (HLB=10.4), and 20 mol EO dioleate (HLB=12.9). and stearic acid EO9 mol adduct (HLB=11.9).
Polyhydric alcohol-type nonionic surfactants (A1d) include EO and/or PO additions of dihydric to octahydric polyhydric alcohols having 3 to 36 carbon atoms (glycerin, trimethylolpropane, pentaerythritol, sorbitol, sorbitan, etc.) products; fatty acid esters of the above polyhydric alcohols and their EO adducts, sucrose fatty acid esters, fatty acid alkanolamides (coconut oil fatty acid diethanolamide, etc.) and their AO adducts.
Specific examples of (A1d) include sorbitan tetraoleate EO adducts (HLB=11.4) and sorbitan hexaoleate EO adducts (HLB=10.2).

Among these nonionic surfactants (A1), alcohol AO adducts (A1a) and polyhydric alcohol type nonionic surfactants (A1d) are preferred. More preferred are the decyl alcohol EO adduct of (A1a) and coconut oil fatty acid diethanolamide of polypropylene glycol EO block adduct (A1d).
Examples of anionic surfactants (A2) include ether carboxylic acids (A2a) and salts thereof, sulfate esters (A2b) or salts thereof, ether sulfate esters (A2c) and salts thereof, sulfonates (A2d), sulfosuccinates ( A2e), phosphate esters (A2f) and salts thereof, ether phosphate esters (A2g) and salts thereof, fatty acid salts (A2h), acylated amino acid salts and naturally occurring carboxylic acids and salts thereof (chenodeoxycholic acid, cholic acid and deoxycholic acid, etc.).

Ethercarboxylic acids (A2a) or salts thereof include ethercarboxylic acids having a hydrocarbon group (8 to 24 carbon atoms) and salts 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. sodium salts and the like.

Sulfuric acid esters (A2b) and salts thereof include sulfuric acid 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.

Ether sulfates (A2c) and salts thereof include ether sulfates having a hydrocarbon group (8 to 24 carbon atoms) and salts thereof.

Specific examples of (A2c) and salts thereof include sodium polyoxyethylene lauryl ether sulfate and triethanolamine polyoxyethylene lauryl ether sulfate.

The sulfonate (A2d) includes sodium dodecyldiphenyletherdisulfonate, sodium dodecylbenzenesulfonate and sodium naphthalenesulfonate.

Sulfosuccinates (A2e) include disodium polyoxyethylene lauryl sulfosuccinate, disodium lauryl sulfosuccinate and disodium polyoxyethylene lauroylethanolamide sulfosuccinate.

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

The ether phosphate (A2g) includes disodium polyoxyethylene octyl ether phosphate and disodium polyoxyethylene lauryl ether phosphate.

Fatty acid salts (A2h) include sodium octylate, sodium laurate and sodium stearate.

Among these anionic surfactants (A2), ether carboxylic acids (A2a) and sulfonates (A2d) are preferred.

More preferred are polyoxyethylene lauryl ether sodium salt of (A2a) and sodium dodecyldiphenyl ether disulfonic acid of (A2d).

From the viewpoint of 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. is 6.

Specific examples of anionic groups having a pKa of 1 to 5 include carboxyl group (--COOH), sulfate group (--OSO ₃ H), sulfo group (--SO ₃ H), sulfino group (--SO ₂ H), sulfeno Acidic groups such as a group (--SOH), a phosphate group {--OP(=O)(--OH) ₂ }, and groups of salts thereof can be mentioned.

In addition, when the anionic surfactant (A2) has two or more anionic groups, any one pKa may be within the above range, and the pKa of all anionic groups is further within the above range. preferable.

Examples of amphoteric surfactants (A3) include carboxylate type amphoteric surfactants (A3a), sulfate type amphoteric surfactants (A3b), sulfonate type amphoteric surfactants (A3c) and phosphate ester salts. amphoteric surfactants (A3d) and the like.

Carboxylate type amphoteric surfactants (A3a) include amino acid type amphoteric surfactants (A3a1), betaine type amphoteric surfactants (A3a2) and imidazoline type amphoteric surfactants (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 monovalent hydrocarbon group having 1 to 20 carbon atoms. 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 protons, alkali metals, alkaline earth metals, ammonium (including cations derived from amines and alkanolamines, etc.), and quaternary ammonium.
Specific examples of (A3a1) include alkylaminopropionic acid-type amphoteric surfactants (sodium cocaminopropionate, sodium stearylaminopropionate and sodium laurylaminopropionate, etc.); alkylaminoacetic acid-type amphoteric surfactants (laurylamino sodium acetate, etc.) and N-lauroyl-N'-carboxymethyl-N'-hydroxyethylethylenediamine sodium.

The betaine-type amphoteric surfactant (A3a2) is an amphoteric surfactant having a quaternary ammonium salt-type cationic moiety and a carboxylic acid-type anionic moiety in the molecule.

(A3a2) includes compounds represented by the following general formula (2).
RN ⁺ (CH ₃ ) ₂ -CH2COO ^- (2)
In general formula (2), R is a monovalent hydrocarbon group having 1 to 20 carbon atoms.

Specific examples of (A3a2) include alkyldimethylbetaine (betaine stearyldimethylaminoacetate and betaine lauryldimethylaminoacetate, etc.), amidobetaine (coconut fatty acid amidopropylbetaine, etc. (coconut fatty acid amidopropyldimethylaminoacetic acid betaine, etc.), and lauramidopropyl betaine, etc.), alkyldihydroxyalkylbetaines (lauryldihydroxyethyl betaine, etc.), hardened coconut oil fatty acid amidopropyldimethylaminoacetate betaine, and the like.

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

Other amphoteric surfactants include glycine-type amphoteric surfactants such as sodium lauroylglycine, sodium lauryldiaminoethylglycine, lauryldiaminoethylglycine hydrochloride and dioctyldiaminoethylglycine hydrochloride; sulfobetaine-type surfactants such as pentadecylsulfotaurine. amphoteric surfactants; cholamidopropyldimethylammoniopropanesulfonic acid (CHAPS), cholamidopropyldimethylammonio-2-hydroxypropanesulfonic acid (CHAPSO); alkylamine oxide type amphoteric surfactants such as lauryldimethylamine oxide; included.

Among these amphoteric surfactants (A3), carboxylate type amphoteric surfactants (A3a) and phosphate ester salt type amphoteric surfactants (A3d) are preferred.

More preferred are (A3a) betaine lauryldimethylaminoacetate and sodium cocaminopropionate.

From the viewpoint of 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. is 6. The pKa of the cationic group of the amphoteric surfactant (A3) is preferably 8-14, more preferably 9-14, particularly preferably 9.2-14.

Examples of the anionic group having a pKa of 1 to 5 are the same as those exemplified for the amphoteric surfactant (A3).

Cationic groups with a pKa of 8 to 14 include quaternary ammonio groups, primary to tertiary amino groups, and salts thereof.

In addition, when the amphoteric surfactant (A3) has two or more anionic groups, any one pKa may be within the above range, and the pKa of all anionic groups may be within the above range. Preferably, when it has two or more cationic groups, the pKa of any one of them should be within the above range, and it is more preferable that the pKa of all cationic groups be within the above range.

Cationic surfactants (A4) include amine salt-type cationic surfactants (A4a) and quaternary ammonium salt-type cationic surfactants (A4b).

As the amine salt type cationic surfactant (A4a), primary to tertiary amines are mixed with 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, primary amine salts include inorganic or organic acid salts of higher aliphatic amines (higher amines such as laurylamine, stearylamine, cetylamine, hardened beef tallow amine, and rosin amine); Examples include fatty acid (stearic acid, oleic acid, etc.) salts.

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 tertiary amine salt types include aliphatic amines (triethylamine, ethyldimethylamine, N,N,N',N'-tetramethylethylenediamine, etc.), aliphatic amine ethylene oxide (2 mol Above) adducts, alicyclic amines (N-methylpyrrolidine, N-methylpiperidine, N-methylhexamethyleneimine, N-methylmorpholine, 1,8-diazabicyclo(5,4,0)-7-undecene, etc.) , inorganic or organic acid salts of nitrogen-containing heterocyclic aromatic amines (4-dimethylaminopyridine, N-methylimidazole, 4,4'-dipyridyl, etc.); triethanolamine monostearate, stearamidoethyldiethylmethylethanol Examples include inorganic acid salts or organic acid salts of tertiary amines such as amines.

The quaternary ammonium salt-type cationic surfactant (A4b) includes tertiary amines and quaternizing agents (alkylating agents such as methyl chloride, methyl bromide, ethyl chloride, benzyl chloride and dimethyl sulfate; ethylene oxide and the like). and those obtained by the reaction with

For example, lauryltrimethylammonium chloride, didecyldimethylammonium chloride, dioctyldimethylammonium bromide, stearyltrimethylammonium bromide, lauryldimethylbenzylammonium chloride (benzalkonium chloride), cetylpyridinium chloride, polyoxyethylenetrimethylammonium chloride, stearamidoethyldiethyl and methylammonium methosulfate.

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

From the viewpoint of secretion efficiency of useful substances, the cationic surfactant (A4) preferably has a pKa of 8 to 14, more preferably 9 to 14, and particularly preferably 9.2 to 14, due to the cationic group.

Cationic groups with a pKa of 8 to 14 include quaternary ammonio groups, primary to tertiary amino groups, and salts thereof.

In addition, when the cationic surfactant (A4) has two or more cationic groups, any one pKa may be within the above range, and the pKa of all cationic groups is further within the above range. preferable.

As the surfactant (A) in the present invention, the surfactant (A) may be used as it is, or if necessary, it may be mixed with water and used as an aqueous dilution (aqueous solution or water dispersion). .

The total concentration of the surfactant (A) in the aqueous dilution is appropriately selected depending on the target microorganism, the type of physiologically active substance, and the type of extraction method. 0.1 to 99% by weight, more preferably 1 to 50% by weight, based on the weight of the aqueous dilution.

The amount (% by weight) of the surfactant (A) used in the method for producing a useful substance of the present invention is appropriately selected depending on the target microorganism, the type of useful substance to be produced, and the type of extraction method. , based on the weight of the culture medium, from the viewpoint of cytotoxicity, secretion efficiency of useful substances and resistance to protein denaturation, 0.0001 to 10% by weight is preferable, more preferably 0.001 to 8% by weight. 0.005 to 5% by weight, and particularly preferably 0.01 to 2.5% by weight.

As the culture solution used in the production method of the present invention, any cell culture medium generally used in the art can be used without particular limitation, including carbon sources, nitrogen sources and other essential nutrients, natural media, synthetic Any medium may be used.

Carbon sources 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.

Nitrogen sources include ammonium salts of inorganic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, ammonium salts of organic acids, and other nitrogen-containing compounds, as well as 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, and copper sulfate. , calcium carbonate and the like are used.

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

Examples of proteins include enzymes {oxidoreductases (cholesterol oxidase, glucose oxidase, ascorbic acid oxidase, peroxidase, etc.), hydrolases (lysozyme, protease, serine protease, amylase, lipase, cellulase, glucoamylase, etc.), isomerases ( glucose isomerase, etc.), transferases (acyltransferase, sulfotransferase, etc.), synthetic enzymes (fatty acid synthase, phosphate synthase, citrate synthase, etc.) and releasing enzymes (pectin lyase, etc.), hormone proteins {bone morphogenetic factors ( 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, etc.}, antibodies {single-chain antibodies, IgG large subunit, IgG small subunit, etc.}, antigen proteins {hepatitis B surface antigen, etc.}, functional proteins {Pronectin (registered trademark), antifreeze peptides, antibacterial peptides, etc.}, fluorescent proteins (GFP, etc.), luminescent proteins (lucerase, etc.) and peptides (not particularly limited to amino acid compositions, oligopeptides, dipeptides, tripeptides, etc.) ) and the like.

Oligosaccharides include sucrose, lactose, trehalose, maltose, raffinose, panose, cyclodextrins, galactooligosaccharides and fructooligosaccharides.

Nucleic acids include inosine monophosphate, adenosine monophosphate, guanosine monophosphate, and the like.

Among these useful substances, proteins are preferable, and antibodies, enzymes and hormone proteins are more preferable, from the viewpoint of easiness of production of useful substances.

When the useful substance is a protein, it is preferable that the protein has a property that part or all of the protein is translocated to the periplasm or outside the cell after being expressed in the microorganism.

More preferably, it is a protein encoding a signal sequence required for translocation into the periplasm in the ORF.

The periplasm is the space outside the cytoplasmic membrane of microorganisms to the outermost surface of microorganisms.

Signal sequences necessary for translocation to the periplasm include Sec secretory signal sequences, TAT secretory signals, α-signal sequences and the like.

Microorganisms in the present invention are exemplified below, but are not limited thereto.

Examples of microorganisms include bacteria and eukaryotes having a cell wall containing polysaccharides in the outermost layer. The outermost layer is a layer located outside the cell membrane and is in direct contact with the external environment on the outermost surface of the cell. Polysaccharides are compounds in which monosaccharides form multimers through glycosidic bonds, and do not include lipopolysaccharides.

Bacteria include eubacteria and archaea. Eubacteria include Gram-negative and Gram-positive bacteria. Eukaryotes having a cell wall containing polysaccharides in the outermost layer include fungi, algae, and the like. Gram-negative bacteria include Escherichia, Thermus, Rhizobium, Pseudomonas, Shewanella, Vibrio, Salmonella, Acetobacter, Synechocystis and the like. Gram-positive bacteria include Bacillus, Streptmyces, Corynebacterium, Brevibacillus, Bifidobacterium, Lactococcus, Enterococcus, Pediococcus and Leuconostoc. Fungi include the phylum Basidiomycota, Ascomycota, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Microsporidia, Glomeromycota, Mucoromycotina, Entomophthoromycotina, Zoopagomycotina and Kickxellomycotina.子嚢菌門（Ａｓｃｏｍｙｃｏｔａ）としては、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、Ｓａｉｔｏｅｌｌａ、Ｓａｐｒｏｃｈａｅｔｅ、Ｓａｔｕｒｎｉｓｐｏｒａ、Ｓｃｈｉｚｏｂｌａｓｔｏｓｐｏｒｉｏｎ、Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ、Ｓｐｏｒｏｐａｃｈｙｄｅｒｍｉａ、Ｓｔａｒｍｅｒｅｌｌａ、Ｓｔｅｐｈａｎｏａｓｃｕｓ、Ｓｙｍｂｉｏｔａｐｈｒｉｎａ、Ｓｙｍｐｏｄｉｏｍｙｃｅｓ、Ｔｅｔｒａｐｉｓｉｓｐｏｒａ、Ｔｏｒｕｌａｓｐｏｒａ、Ｔｒｉｇｏｎｏｐｓｉｓ、Ｗｉｃｋｅｒｈａｍｉａ、Ｗｉｃｋｅｒｈａｍｉｅｌｌａ、Ｗｉｌｌｉｏｐｓｉｓ、Ｙａｒｒｏｗｉａ、Ｚｙｇｏａｓｃｕｓ 、Ｚｙｇｏｓａｃｃｈａｒｏｍｙｃｅｓ、Ｚｙｇｏｚｙｍａ、Ｂａｎｎｏａ、Ｂｅｎｓｉｎｇｔｏｎｉａ、Ｂｕｌｌｅｒａ、Ｂｕｌｌｅｒｏｍｙｃｅｓ、Ｃｒｙｐｔｏｃｏｃｃｕｓ、Ｃｕｒｖｉｂａｓｉｄｉｕｍ、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, Sterigmatomy Strains such as S. ces, Sterigmatosporidium, Sympodiomycopsis, Tausonia, Tilletiopsis, Trichosporiella, Trichosporon, Tsuchiyaea, Udeniomyces, and Xanthophyllomyces. Examples of algae include eubacterial cyanobacteria (blue-green algae), eukaryotic 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 Gram-negative bacteria, Gram-positive bacteria, and yeast belonging to the phylum Mycosidomycota.

More preferably, it is at least one selected from the group consisting of Escherichia, Corynebacterium, Saccharomyces, Pichia, Candida, Schizosaccharomyces, Yarrowia, Hansenula, Ogataea, Kluyveromyces and Pseudozyma. 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 (%) of the surfactant in the culture medium is preferably 55 to 100, more preferably 60 to 100, and even more preferably 65, from the viewpoint of productivity. ~100, particularly preferably 70-100.

The secretion efficiency indicates that the surfactant secretes useful substances in the microorganism to the outside of the microorganism (into the culture medium).

In the present invention, it is defined by the following formula (5).
Secretion efficiency (%) = 100 x (X)/{(X) + (Y)} (5)
X (culture supernatant): Weight of useful substances in the culture medium remaining after removal of the cells by centrifugation Y (cell surface): Cell suspension obtained by washing and resuspending the cells collected by centrifugation Weight of useful substance inside The secretion efficiency is increased by, for example, allowing the protein produced in the microorganism to migrate to the periplasm more, and decreasing the secretion efficiency to prevent it from translocating to the periplasm. In addition, the secretion efficiency can be increased by selecting a surfactant with high secretion efficiency by screening.

The surfactant (A) and the culture solution can be mixed by adding the surfactant to the culture solution at 4° C. to 99° C. and stirring with a stirring blade, stirrer, or the like. When mixing, it can be performed by adding while stirring with a stirring blade or the like.

In the method for producing a useful substance of the present invention, the production method for secretion production of a useful substance includes a microbial exocrine production method including the following steps (a) and (b). In the following steps, step (a) is the step of secreting and producing a useful substance.
Step (a): A step of coexisting a culture solution for culturing a microorganism (such as yeast) that produces a useful substance and a surfactant to secrete the useful substance outside the microorganism (into the culture solution).
Step (b): After step (a), a step of separating useful substances from the culture medium.

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) Separating messenger RNA (mRNA) from cells expressing the target protein, synthesizing single-stranded cDNA from the mRNA, then synthesizing double-stranded DNA, Stranded DNA is incorporated into phage DNA or plasmids. The resulting recombinant phage or plasmid is transformed into a microorganism to create a cDNA library.
(i-2) Methods for screening phage DNAs or plasmids containing the DNA of interest include hybridization of phage DNAs or plasmids with DNA probes encoding part of the target protein gene or complementary sequence. .
(i-3) excision of the cloned DNA of interest or part thereof from the phage or plasmid after screening, and ligation of the cloned DNA or part thereof downstream of the promoter in the expression vector, resulting in expression vector can be created. A DNA encoding a signal sequence that translocates the inner membrane (a signal sequence that expresses the target substance in the periplasm) can also be ligated at the same time.
(ii) Culturing (ii-1) Transforming a host microorganism with an expression vector to prepare a recombinant microorganism, and pre-cultivating the recombinant microorganism. Preculture is usually carried out on an agar medium at 15 to 43° C. for 3 to 72 hours.
(ii-2) The culture medium used for the production of useful substances is autoclaved at 121° C. for 20 minutes, and the recombinant microorganism precultured on the agar medium is cultured therein. Cultivation is usually carried out at 15-43° C. for 12-72 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, or the like.
(iii-2) The protein-containing culture medium is subjected to repeated column treatments such as ion exchange column, gel filtration column, hydrophobic column, affinity column and ultra column, and precipitation treatment such as ethanol precipitation, ammonium sulfate precipitation and polyethylene glycol precipitation. Separation and purification can be carried out as necessary.

The host microorganism isolated in (iii-1) can then be further cultured by supplying a new culture medium. Continuous production of useful substances can be carried out by further subjecting the culture solution to the step (iii) and repeating purification and cultivation.

When column treatment is performed in the purification step (iii) above, fillers used in column chromatography include silica, dextran, agarose, cellulose, acrylamide and vinyl polymers. Examples include Sephadex series, Sephacryl series, Sepharose series (all of which are GE Healthcare), Bio-Gel series (Bio-Rad), and the like.

By using the method for producing a useful substance of the present invention, the useful substance produced by the microorganism can be secreted into the culture medium with high efficiency, and a high yield can be obtained in a short time. can be achieved. In addition, in the method for producing a useful substance of the present invention, the useful substance is easily purified because the useful substance is secreted into the culture medium.

The method for producing a useful substance of the present invention includes a step of making a surfactant and a microorganism exist at the same time to secrete a useful substance into a culture medium. In this process, it is presumed that as long as the microorganisms are alive, they can produce useful substances and secrete them into the culture medium. Furthermore, it is presumed that the production method of the present invention can be used regardless of the type of useful substance to be produced, as long as the microorganism has the ability to produce the useful substance.

The useful substance production method of the present invention is particularly effective when useful substances produced in microorganisms migrate to the periplasm of the microorganism. Transfer of useful substances to the periplasm facilitates their secretion into the culture medium.

EXAMPLES The present invention will be further described below with reference to Examples and Comparative Examples, but the present invention is not limited to these. Hereinafter, unless otherwise specified, % means % by weight, and part means part by weight.
Production Example 1 <Production of Surfactant (A1-1)>
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 are charged into a stainless steel pressurized reaction apparatus, and after nitrogen substitution, the temperature is 110 to After 50 parts of ethylene oxide was injected at 130° C. over 4 hours, reaction was continued at the same temperature for 10 hours to obtain a surfactant (A1-1). (A1-1) had HLB=1.8 and Mn=2,200.
Production Example 2 <Production of surfactant (A1-2)>
In Production Example 1, instead of 450 parts of Sannics PP-2000, 101 parts of Sannics PP-3000 [manufactured by Sanyo Chemical Co., Ltd.; polyoxypropylene glycol (number average molecular weight: 3200)] was used, and ethylene oxide was press-fitted. A surfactant (A1-2) was obtained in the same manner except that the amount was changed from 50 parts to 399 parts. HLB of (A1-2) was 15.9 and Mn was 16,000.

The number average molecular weights Mn of surfactants (A1-1) and (A1-2) used in Examples and Comparative Examples were measured by the following method.

The number average molecular weight was measured under the following conditions using GPC (Gel Permeation Chromatography) on the soluble matter in tetrahydrofuran (hereinafter abbreviated as THF).

Measuring device: "HLC-8120" manufactured by Tosoh Corporation
Column: "TSKgelGMHXL" (2 pieces) manufactured by Tosoh Corporation and "TSKgelMultiporeHXL-M" (1 piece) manufactured by Tosoh Corporation
Sample solution: 0.25 wt% THF solution Injection volume of sample solution into column: 100 μL
Flow rate: 1 mL/min Measurement temperature: 40°C
Detection device: Refractive index detector Reference material: 12 standard polystyrene (TSK standard POLYSTYRENE) 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 cellulase secretion productivity and secretion efficiency using E. coli>
The pET-22b plasmid (Novagen) was cleaved at the NcoI restriction enzyme site and the BamHI restriction enzyme site, and artificially synthesized (commissioned synthesis by Thermo). .

Then, the BL21(DE3) E. coli strain (Novagen) was transformed with this plasmid to prepare a cellulase expression strain. Localization of the expressed cellulase in the periplasmic fraction was analyzed and confirmed based on the method of METHODS IN ENZYMOLOGY vol.353, p.121, 2002. The prepared cellulase-expressing E. coli was inoculated into 2 mL of LB medium [LB Broth, Miller, manufactured by Difco Laboratories] using a platinum loop, and cultured with shaking at 30° C. for 15 hours at 150 rpm to prepare a preculture solution. 1 mL of medium [yeast extract [manufactured by Nihon Pharmaceutical Co., Ltd.] 24 mg, polypeptone [manufactured by Nihon Pharmaceutical Co., Ltd.] 12 mg, phosphorus dipotassium acid 9.4 mg, monopotassium phosphate 2.2 mg, ammonium sulfate 7 mg, disodium phosphate dodecahydrate 13.2 mg, sodium citrate dihydrate 0.4 mg, glycerol 4 mg, lactalbumin hydrolyzate 3 mg , 1 mM magnesium sulfate, trace metal solution [0.38 µg calcium chloride, 10 µg iron(III) chloride, 0.18 µg zinc sulfate heptahydrate, 0.1 µg copper sulfate, 0.13 µg manganese chloride tetrahydrate, cobalt chloride 0.1 μg, tetrasodium ethylenediaminetetraacetate 4 μg], 100 mg/L ampicillin], and stirring culture was started at 30° C. and 150 rpm.

After 2 hours from the initiation of the culture, Isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 1 mM, and stirring culture was continued.

When the culture time described in Table 1 was reached, the surfactant (A) described in Table 1 (pure water in Comparative Example 1) was added to the weight of the culture solution [medium, pre-culture solution, Isopropyl β-D-1- Based on the total weight of thiogalactopyranoside and the surfactant (A) to be added], the contents were added so as to achieve the weight % described in Table 1, and the agitation culture was continued.

After 30 hours from the start of the culture, the culture solution was sampled, the cells and the culture supernatant were separated from the culture solution using a centrifuge, and the culture supernatant and the cells were recovered. 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 performing test culture in the same manner as in Comparative Example 3 to determine the culture stationary phase ODS.

なお、表１～３中、界面活性剤（Ａ１－３）～（Ａ４－１）は下記を使用した。
（Ａ１－３）：ソルビタンヤシ油脂肪酸エステル、ＨＬＢ＝８．５、Ｍｎ＝３４６
（Ａ２－１）：ポリオキシエチレン（ＥＯ２．５モル）ラウリルエーテル硫酸ナトリウム、ｐＫａ＝１．９９
（Ａ２－２）：ポリオキシエチレン（ＥＯ３モル）ラウリルエーテル酢酸ナトリウム、ｐＫａ＝３．５５
（Ａ３－１）：２－(ドデシルアミノ)エタンスルホン酸ナトリウム、ｐＫａ＝１．９９と１０．７７
（Ａ３－２）：ドデシル－β－アミノプロピオン酸ナトリウム、ｐＫａ＝３．５５と１０．７７
（Ａ３－３）：ミルテホシン、ｐＫａ＝２．１５と１４．０
（Ａ３－４）：ラウリルジメチルアミノ酢酸ベタイン、ｐＫａ＝３．５５と１４．０
（Ａ４－１）：塩化ラウリルトリメチルアンモニウム、ｐＫａ＝１４．０
なお、大腸菌体１個あたりのセルラーゼの分泌生産性と分泌効率の測定方法と計算方法は以下の方法で行う。
＜培養液の濁度の測定方法＞
サンプリングで回収した微生物を含む培養液を用いて、濁度計［（株）島津製作所社製、ＵＶ－１７００］を用いて、光路長１ｃｍの石英セルを用いて濁度の測定を行った。

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 (3 mol of EO) 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): betaine lauryldimethylaminoacetate, pKa = 3.55 and 14.0
(A4-1): Lauryltrimethylammonium chloride, pKa = 14.0
The cellulase secretion productivity and secretion efficiency per E. coli organism are measured and calculated by the following methods.
<Method for measuring turbidity of culture solution>
Turbidity was measured using a turbidity meter [UV-1700, manufactured by Shimadzu Corporation] using a quartz cell with an optical path length of 1 cm using a culture solution containing microorganisms collected by sampling.

The culture was centrifuged at 1500 rpm for 5 minutes at 4° C. and the supernatant was discarded. The precipitate was resuspended in the same volume of saline as the sample solution, diluted with saline to an appropriate absorbance (0.1-0.8), and measured for absorbance at 600 nm. The turbidity of the culture solution was calculated by the following 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: activity measurement of secreted and produced cellulase>
Endo-1,4-β-glucanase activity was measured as cellulase activity.

Carboxymethyl cellulose (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in a 100 mM phosphate buffer aqueous solution of pH 7.5 to a concentration of 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 are mixed, and the mixture is measured by a viscometer. Transfer to a (TVE-22L viscometer, 2 rpm) thermostated cup 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 time (minutes) on the X axis and viscosity (mPa s) on the Y axis. The cellulase activity (endo-1,4-β-glucanase activity) was defined as the absolute value of the slope when a straight line was drawn by the method of least squares.
[Activity of secreted-produced cellulase]=[slope of straight line]×dilution ratio [secretion productivity per cell]=[activity of secreted-produced cellulase]/[turbidity in stationary phase of culture]
The "secretion productivity per cell (relative value)" in Table 1 is the same as the above "secretion productivity per cell" of Comparative Example 1 (blank without surfactant). It is expressed as a relative ratio with 1.00.
<Measurement of activity of cellulase remaining on the surface of bacterial cells>
To the cells obtained in Examples 1 to 18 and Comparative Examples 1 to 3, 200 μL of 0.2M Tris-HCl buffer (pH 7.4), the same amount as the supernatant removed, was added and centrifuged to remove the cells. was washed. The washed cells were resuspended in 200 μL of 0.2 M Tris-HCl buffer (pH 7.4), which was the same amount as the washed supernatant, to prepare a sample for cellulase activity measurement on the cell surface.

Using a mixture 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 fungus, the viscosity was measured under the same conditions as in "Measurement of activity of secreted and produced cellulase". The value obtained by fitting was taken as the activity of cellulase, which is a useful substance remaining on the surface of the cells.
[Activity of cellulase remaining on bacterial cell surface]=[slope of straight line]×dilution ratio <evaluation of cellulase secretion efficiency>
Using the culture supernatants 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 x "activity of secreted cellulase"/"total cellulase activity"
The total cellulase activity means "activity of secreted and produced cellulase" + "activity of cellulase remaining on the surface of the fungus".

The "secretion efficiency (%)" in Table 1 corresponds to the above "secretion-produced cellulase activity" and "cellulase activity remaining on the cell surface" of Comparative Example 1 (blank without surfactant). is expressed as a relative ratio when the is 1.00.
Examples 19 to 39 and Comparative Examples 4 to 6 <Evaluation of luciferase secretion productivity and secretion efficiency using luciferase-expressing yeast>
The luciferase-expressing yeast (Pichia pastoris) was transformed using Pichia pastoris provided by (National Institute of Technology and Evaluation, Biotechnology Center).

A pPICZα vector (manufactured by Thermo) was cleaved with XhoI and XbaI, and a luciferase gene having XhoI and XbaI cleavage sites at both ends was artificially synthesized (commissioned by Thermo).

Transformation of the pPICZα vector containing the luciferase gene into yeast was performed by the method described in the instruction manual for the pPICZα2 vector. The luciferase-expressing yeast was inoculated into the 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 Yeastextract 1 wt%, Difco Bactopeptone 2 wt%, Difco Yeastnitrogen base w/o amino acid 1.34 wt) so that the final OD is 0.05 OD / mL. %, glucose 2 wt %, 100 mM potassium hydrogen phosphate, pH 6.0), and stirred culture was started at 30° C. and 1100 rpm.

At the culture time shown in Table 2, the culture medium was centrifuged (1500 x g, 10 minutes), resuspended in 125 mL of BMMY culture medium, and the surfactant (A) or pure water shown in Table 2 was added. was added so as to obtain the weight % shown in Table 2, and the agitation culture was continued.

After 30 hours from the start of the culture, the culture solution was sampled, the cells and the culture supernatant were separated from the culture solution using 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 which the surfactant (A) was added was determined in advance by performing test culture in the same manner as in Comparative Example 4 to determine the culture stationary phase ODS.

＜菌体１個あたりの分泌生産性の評価：分泌生産した有用物質のルシフェラーゼ活性測定＞
実施例１９～３９及び比較例４～６で得た各培養上清を０．２Ｍ Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ ７．４）で１０～１０００倍希釈したもの２０μＬに、下記のルシフェリン溶液６０μＬ添加した混合液を試料として、ルミノメーターを用いて以下の条件で測定した発光強度を、下記式に当てはめて得られた値を分泌生産した有用物質のルシフェラーゼ活性とした。
ルミノメーター：プロメガ株式会社製の「Ｇｌｏｍａｘ Ｎａｖｉｇａｔｏｒ」
ルシフェリン溶液：ニュー・イングランド・バイオラボ・ジャパン株式会社製品測定温度：２５℃
検出装置：発光検出器
検出波長：３５０～７００ｎｍ
〔分泌生産した有用物質のルシフェラーゼ活性〕＝〔発光強度〕×希釈倍率
〔菌体１個あたりの分泌生産性〕＝〔分泌生産した有用物質のルシフェラーゼ活性〕／〔培養定常期の濁度〕
なお、表２中の「菌体１個あたりの分泌生産性（相対値）」は、比較例４（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。
＜菌体表面に残った有用物質のルシフェラーゼ活性測定＞
実施例１９～３９及び比較例４～６で得た菌体に、除去した上清と同量の０．２Ｍ Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ ７．４）２００μＬを加え、遠心することで菌体を洗浄した。洗浄した菌体に、洗浄した上清と同量の０．２Ｍ Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ ７．４）２００μＬで再懸濁し、０．２Ｍ Ｔｒｉｓ－ＨＣｌ緩衝液（ｐＨ ７．４）で１０～１０００倍希釈したものを菌体表面のルシフェラーゼ活性測定用サンプルとした。

<Evaluation of secretion productivity per cell: luciferase activity measurement of secreted and produced useful substances>
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 resulting mixture as a sample, the luminescence intensity was measured using a luminometer under the following conditions, and the value obtained by applying the following formula was defined as the luciferase activity of the secreted and produced useful substance.
Luminometer: "Glomax Navigator" manufactured by Promega Corporation
Luciferin solution: New England Biolab Japan Co., Ltd. Product measurement temperature: 25°C
Detection device: luminescence detector Detection wavelength: 350-700 nm
[Luciferase activity of secreted and produced useful substance]=[luminescence intensity]×dilution ratio [secretory productivity per cell]=[luciferase activity of secreted and produced useful substance]/[turbidity in stationary phase of culture]
The "secretion productivity per cell (relative value)" in Table 2 is the same as the above "secretion productivity per cell" of Comparative Example 4 (blank without surfactant). It is expressed as a relative ratio with 1.00.
<Measurement of luciferase activity of useful substances remaining on the surface of bacterial cells>
To the cells obtained in Examples 19-39 and Comparative Examples 4-6, 200 μL of 0.2M Tris-HCl buffer (pH 7.4), which is the same amount as the removed supernatant, is added and centrifuged to remove the cells. was washed. The washed cells were resuspended in 200 μL of the same amount of 0.2 M Tris-HCl buffer (pH 7.4) as the washed supernatant, and then resuspended in 0.2 M Tris-HCl buffer (pH 7.4) for 10 minutes. A sample diluted to 1000-fold was used as a sample for luciferase activity measurement on the cell surface.

Using a mixture obtained by adding 60 μL of the above luciferin solution to 20 μL of this luciferase activity measurement sample on the surface of the fungus as a sample, the luminescence intensity was measured under the same conditions as in “luciferase activity measurement of secreted and produced useful substances”, and the measured luminescence The value obtained by applying the intensity to the following formula was taken as the luciferase activity of the useful substance remaining on the cell surface.
[Luciferase activity of useful substance remaining on bacterial cell surface]=[luminescence intensity]×dilution ratio <evaluation of secretion efficiency>
Using the culture supernatants and cells obtained in Examples 19-39 and Comparative Examples 4-6, the secretion efficiency in each Example was calculated by the following formula.

Secretion efficiency (%) = 100 x "luciferase activity of secreted and produced useful substance"/"total luciferase activity"
The total luciferase activity means "luciferase activity of the secreted and produced useful substance" + "luciferase activity of the useful substance remaining on the cell surface".

The "secretion efficiency (%)" in Table 2 corresponds to the above "luciferase activity of secreted and produced useful substances" and "useful substances remaining on the surface of the cells" of Comparative Example 4 (blank without surfactant). It was expressed as a relative ratio when the "luciferase activity of" was set to 1.00.
Examples 40 to 60 and Comparative Examples 7 to 9 <Evaluation of acid phosphatase secretion productivity and secretion efficiency using acid phosphatase-expressing yeast>
酸性ホスファターゼ発現酵母（Ｃａｎｄｉｄａ ｂｏｉｄｉｎｉｉ 公知文献（Ｒｅｇｕｌａｔｉｏｎ ａｎｄ ｅｖａｌｕａｔｉｏｎ ｏｆ ｆｉｖｅ ｍｅｔｈａｎｏｌ－ｉｎｄｕｃｉｂｌｅ ｐｒｏｍｏｔｅｒｓ ｉｎ ｔｈｅ ｍｅｔｈｙｌｏｔｒｏｐｈｉｃ ｙｅａｓｔ Ｃａｎｄｉｄａ ｂｏｉｄｉｎｉｉ， Ｈ． Ｙｕｒｉｍｏｔｏ ｅｔ ａｌ．， Ｂｉｏｃｈｉｍｉｃａ ｅｔ Ｂｉｏｐｈｙｓｉｃａ Ａｃｔａ， １４９３， ２０００， ５６－６３）に記載の方法) was inoculated into the BMGY culture solution using a platinum loop and cultured with shaking at 200 rpm at 30° C. for 15 hours to prepare a preculture solution. The prepared preculture solution was inoculated into 125 mL of BMGY culture solution so that the final OD was 0.05 OD/mL, and stirring culture was started at 30° C. and 1100 rpm.

At the culture time shown in Table 3, the culture solution was centrifuged (1500 x g, 10 minutes), resuspended in 125 mL of BMMY culture solution, and the surfactant (A) or pure water shown in Table 3 was added. was added so as to obtain the weight % shown in Table 4, and the agitation culture was continued.

After 30 hours from the start of the culture, the culture solution was sampled, the cells and the culture supernatant were separated from the culture solution using 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 in 50 mM NaBf acetate (pH 4.0) to obtain a sample for measuring acid phosphatase activity on the cell surface.

The secretion productivity (relative ratio) and secretion efficiency (%) per cell were evaluated, and the results are shown in Table 3.

The culture time for which the surfactant (A) was added was determined in advance by performing test culture in the same manner as in Comparative Example 7 to determine the culture stationary phase ODS.

＜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。

<Evaluation of secretion productivity per cell: measurement of acid phosphatase activity>
To each culture supernatant obtained in Examples 40-60 and Comparative Examples 7-9, 5 μL of 1/40 volume of 2M sodium acetate buffer (pH 4.0) was added. A substrate solution (p-nitrophenylphosphate containing 0.64 mg/mL, 50 mM sodium acetate buffer pH 4.0 ) were mixed at a volume ratio of 1:1 and reacted at 35° C. for 10 minutes.

After the reaction, a 10% trichloroacetic acid solution equivalent to the reaction solution was added to terminate the reaction. Saturated sodium carbonate solution was added to the quenched solution in an amount equal to the total reaction solution to develop color. Insoluble fractions were removed from the colored solution at 1500×g for 5 minutes, and the absorbance at 420 nm was measured using a microplate reader PowerWave manufactured by Biotek. A reference wavelength of 630 nm was measured.

The acid phosphatase activity of each of the culture supernatant and the cell surface measurement sample was calculated from the following formula.
Acid phosphatase activity = {(Abs420nm) - (Abs630nm)}
[Secretion productivity per cell]=[Acid phosphatase activity of secreted and produced useful substance]/[Turbidity in stationary phase of culture]
The "secretion productivity per cell (relative value)" in Table 3 is the same as the above "secretion productivity per cell" of Comparative Example 7 (blank without surfactant). It is expressed as a relative ratio with 1.00.
<Evaluation of secretion efficiency>
Using the values of "acid phosphatase activity of useful substances secreted and produced" and "acid phosphatase activity of useful substances remaining on the cell surface" obtained in Examples 40 to 60 and Comparative Examples 7 to 9, the secretion was obtained from the following formula. Efficiency was calculated. Table 4 shows the results.

Secretion efficiency (%) = 100 x "acid phosphatase activity of secreted and produced useful substances"/"total acid phosphatase activity"
The total acid phosphatase activity means "acid phosphatase activity of secreted and produced useful substances" + "acid phosphatase activity of useful substances remaining on the surface of the cells".
The "secretion efficiency (%)" in Table 3 corresponds to the above "acid phosphatase activity of secreted and produced useful substance" and "useful substance remaining on the surface of the bacteria" in Comparative Example 4 (blank without surfactant). It was expressed as a relative ratio when the "acid phosphatase activity" of the substance was set to 1.00.

Regarding the culture supernatants obtained in Examples 1 to 60 and Comparative Examples 1 to 9, the turbidity of the culture solution, secretion productivity (relative ratio) per cell, and secretion efficiency (%) were evaluated as described above. gone. The results are listed 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 during culture to the turbidity ODS at 600 nm of the culture solution in the stationary phase is 0.009 to 0.009. By adding the surfactant (A) when it is in the range of 90, the relative value when the secretory productivity per cell in Comparative Examples 1, 4, and 7 is set to 1 remarkably increases. It can be seen that

On the other hand, in Comparative Examples 2, 5, and 8 with an OD/ODS of less than 0.009, and Comparative Examples 3, 6, and 9 with an OD/ODS of more than 0.90, secretion per cell compared to the Examples Low relative productivity.

Therefore, it can be seen that the production method of the present invention can obtain 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%, useful substances produced by microorganisms are easily secreted outside the cells, and treatment such as crushing of the microorganisms is not required. It can be seen that the production efficiency is also high because it is possible to obtain a large amount of useful substances. Therefore, it can be seen that the amount of useful substances produced using microorganisms is excellent, and the production efficiency is also high.

The method for producing useful substances of the present invention can be used to produce useful substances such as proteins from microorganisms. Proteins include enzymes, hormone proteins, antibodies, peptides, and the like. When the protein to be produced is an enzyme (protease, cellulase, lipase, amylase, etc.), it can be suitably used for food processing, cleaning agent, fiber treatment, papermaking, enzymatic conversion, etc., especially biopharmaceuticals. It is useful for use in the production of

Claims (4)

A method for producing a useful substance by secreting and producing a useful substance into a culture solution by a microorganism contained in the culture solution, wherein the microorganism is Escherichia coli, the useful substance is cellulase, and In the culture solution when the ratio OD/ODS of the turbidity OD and the turbidity ODS at 600 nm of the culture solution in the stationary phase of the culture when no surfactant is added is in the range of 0.009 to 0.90, A method for producing a useful substance, comprising adding 0.03 to 0.3% by weight of the surfactant (A) based on the weight of the culture solution. Surfactant (A) is a group consisting of nonionic surfactant (A1) with HLB of 0.1 to 16, anionic surfactant (A2), amphoteric surfactant (A3) and cationic surfactant (A4). 2. The method for producing a useful substance according to claim 1, wherein at least one selected from 3. The method for producing useful substances according to claim 2, wherein the nonionic surfactant (A1) has an HLB of 1 to 16 and a number average molecular weight of 100 to 20,000. 4. The method for producing a useful substance according to claim 3, wherein the cellulase is a cellulase that migrates into the periplasm or out of the cell.

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