JP6496509B2 - Method of producing useful substance - Google Patents

Description

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

Bacteria are widely used to produce useful substances such as amino acids and proteins. In particular, in recent years, it is known that techniques for efficiently producing useful proteins are known using transformed bacteria in which genes of proteins useful for pharmaceuticals and industry are introduced into bacteria using genetic engineering techniques. It has become.

  As bacteria used for protein production, E. coli, which is a kind of gram negative bacteria, is widely used. Development of technology for utilizing E. coli for protein production is in progress, and this technology is widely used in the production of industrial proteins, food processing proteins and pharmaceutical proteins.

  Examples of methods for extracting proteins produced by E. coli include physical disruption methods such as ultrasonic waves, high-pressure homogenizers, and French press, and these are widely put to practical use. These physical disruption methods kill E. coli cells at the same time as removing proteins.

  However, physical disruption methods that destroy E. coli and extract proteins from E. coli have problems such as the amount of E. coli 1 cell production being limited because proteins accumulate in E. coli.

On the other hand, in recent years, there has been disclosed a technique of secreting a protein outside the bacteria of E. coli, causing E. coli to continuously produce a protein, and accumulating the protein at a high concentration in a culture solution (see, for example, Patent Document 1). Patent Document 1 further describes using, as an antifoamer, a silicone-based antifoamer generally used at the time of culture of bacteria.
WO 2010/137624

However, in the production method of Patent Document 1 using a silicone antifoaming agent, the antifoaming agent suddenly loses its antifoaming ability during bacterial culture in the presence of a surfactant to cause rapid foaming, resulting in various lines. There is a problem that the yield is lowered due to the interruption of the culture by the backflow of the culture solution, the interruption of the culture for the bubbling treatment and the improper adjustment of the culture solution.
An object of the present invention is to provide a method for producing a useful substance which can produce a useful substance in a high yield without causing rapid bubbling during culture in the presence of a surfactant.

The present inventors arrived at the present invention as a result of earnestly examining in order to solve said subject. That is, the present invention relates to a method of producing a protein secretory production in a culture medium a protein with Gram-negative bacteria contained in the culture solution, carboxylate surfactants in culture (A1-1) And an organic polar compound antifoam (B), wherein the weight ratio of the carboxylate type surfactant (A1-1) to the organic polar compound antifoam (B) ((A1-1) / (B) The method for producing a protein , wherein 10) to 100) .

  The method for producing a useful substance of the present invention can produce a useful substance with high yield without causing rapid bubbling during culture in the presence of a surfactant.

FIG. 1 shows a culture apparatus of Example 1.

  The method for producing a useful substance according to the present invention is a method for producing a useful substance in which a useful substance is secreted and produced in a culture solution using bacteria contained in the culture solution, which comprises a surfactant (A) and It is a manufacturing method of the useful substance containing organic polar compound type | system | group antifoamer (B).

  As the antifoaming agents, conventionally, lower alcohol antifoaming agents, organic polar compound antifoaming agents, mineral oil antifoaming agents and silicone resin antifoaming agents are known (Fujimoto Takehiko, introduction to surfactants, Sanyo Chemical Industries, Ltd., 2007, p. In the present invention, it has been found that, by using an organic polar compound-based antifoaming agent as an antifoaming agent, rapid foaming does not occur during culture in the presence of a surfactant.

The organic polar compound antifoaming agent (B) is an antifoaming agent containing an organic polar compound as a defoaming component. Examples of the organic polar compounds include lower alcohols having 1 to 4 carbon atoms (B1), aliphatic alcohols having 5 to 18 carbon atoms (B2), fatty acids (B3), polyalkylene glycols (B4), fatty acid alkanolamides (B5), Examples thereof include polyhydric alcohol or alkylene oxide adduct of polyhydric alcohol and ester of fatty acid (B6), and alkylene oxide adduct of monohydric or trihydric or higher alcohol or phenolic compound (B7).
The organic polar compounds may be used alone or in combination of two or more.

  As a C1-C4 lower alcohol (B1), methanol, ethanol, propanol or butanol is mentioned.

  As a C5-C18 aliphatic alcohol (B2), a C5-C18 monohydric aliphatic alcohol, specifically, pentyl alcohol, 2, 6- dimethyl -4- heptanol, octyl alcohol, decyl Alcohol, lauryl alcohol, stearyl alcohol, oleyl alcohol, 2-ethylhexanol and the like can be mentioned.

  The fatty acid (B3) contains a higher fatty acid having 12 to 30 carbon atoms, and specifically, lauric acid, myristic acid, pentadecyl acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, and vacenic acid , Linoleic acid, ricinoleic acid, linolenic acid, arachidic acid, behenic acid, lignoceric acid, montanic acid and melisic acid etc. and their metals {alkali metals (Li, Na, K etc.), alkaline earth metals (Mg, Ca) , Ba, etc.), Zn, Cu, Ni, Co, Al, etc.} salts and the like can be mentioned.

As the polyalkylene glycol (B4), an alkylene oxide having 2 to 4 carbon atoms of an alkylene group to an alkylene glycol having 2 to 4 carbon atoms (such as ethylene glycol, propylene glycol and butylene glycol) (hereinafter described as AO) Addition polymers are included.
Examples of AO include ethylene oxide, 1,2- or 1,3-propylene oxide, 1,2-, 1,3- or 1,4-butylene oxide.
The polymerization of AO may be homopolymerization or copolymerization. In the case of copolymerization, any of block copolymerization, random copolymerization, combined use of block and random, etc. may be used. The degree of polymerization is preferably 1 to 1000, more preferably 1 to 250, from the viewpoints of suppressing sudden bubbling, reducing the ability to culture bacteria, and obtaining a large amount of useful substance.
As (B4), a copolymer of ethylene oxide and 1,2-propylene oxide is preferable, and a block polymer of ethylene oxide and 1,2-propylene oxide is more preferable.

As fatty acid alkanolamide (B5), the amidated compound of amino alcohol and fatty acid is mentioned.
Examples of the amino alcohol include those having 1 to 10 carbon atoms, and monoethanolamine, diethanolamine, monoisopropanolamine and the like are preferable.
As fatty acid, the above-mentioned fatty acid (B3) is mentioned.
As (B5), coconut oil fatty acid diethanolamide etc. are mentioned.

The polyhydric alcohol or an AO adduct of a polyhydric alcohol and an ester of a fatty acid (B6) is an ester of a polyhydric alcohol or an AO adduct of a polyhydric alcohol and a fatty acid and having an HLB of less than 10.0 and AO adducts of fatty acids having an HLB less than 10.0 may be mentioned.
The ester may be a monoester, a diester, a triester or more.

HLB in the present invention is a numerical value calculated by the following formula (1) (Fujimoto Takehiko, Introduction to surfactants, page 142, published by Sanyo Chemical Industries, Ltd.).
HLB = 20 × {molecular weight of hydrophilic group / molecular weight of surfactant} (1)
HLB is known as a measure of the hydrophilicity and hydrophobicity of surfactants. The higher the value of HLB, the higher the hydrophilicity.

Examples of polyhydric alcohols include C2-C4 alkylene glycols and tri- to octahydric alcohols (glycerin, trimethylolpropane, pentaerythritol, sucrose, sorbit, sorbitan and the like).
As a fatty acid, the above-mentioned fatty acid (B3) etc. are mentioned.
Examples of (B6) include sorbitan monopalmitate (HLB = 6.7), sorbitan monostearate (HLB = 4.7), sorbitan trioleate (HLB = 1.8), polyoxyethylene (Degree of polymerization = 10) polyoxypropylene (degree of polymerization = 30) glycol oleic acid monoester (HLB = 3.6), polyoxyethylene (degree of polymerization = 10) polyoxypropylene (degree of polymerization = 10) glycol oleic acid monoester Ester (HLB = 6.7), polyoxyethylene (degree of polymerization = 5) polyoxypropylene (degree of polymerization = 10) glycol oleic acid monoester (HLB = 4.0), polyethylene glycol (degree of polymerization = 200) monolauric acid Ester (HLB = 9.8), polyethylene glycol (degree of polymerization = 2) 0) Dilaurate (HLB = 6.6), polyethylene glycol (degree of polymerization = 400) dilaurate (HLB = 9.8), polyethylene glycol (degree of polymerization = 200) monostearate (HLB = 8.5) ), Polyethylene glycol (degree of polymerization = 200) distearate (HLB = 5.4), polyethylene glycol (degree of polymerization = 400) distearate (HLB = 5.4), polyethylene glycol (degree of polymerization = 200) oleic acid Ester (HLB = 8.4), polyethylene glycol (degree of polymerization = 200), dioleate (HLB = 5.3), polyethylene glycol (degree of polymerization = 400) dioleate (HLB = 8.4), etc. may be mentioned. .

As the AO adduct of monohydric or trihydric alcohol or phenol compound (B7), AO adduct of monohydric alcohol, AO adduct of trihydric alcohol or more, and AO adduct of monohydric phenol compound Etc.
As the monohydric alcohol, the above-mentioned C1-C4 lower alcohol (B1), the above-mentioned C5-C18 aliphatic alcohol (B2), and the C19-C24 monovalent aliphatic alcohol (nona Decyl alcohol, 1-tetracosanol, etc.) and monovalent aromatic aliphatic alcohol having 6 to 24 carbon atoms (benzyl alcohol, t-butyl benzyl alcohol, etc.), etc. Alcohol (glycerin, trimethylolpropane, pentaerythritol, sorbit and sorbitan etc.) and the like.
Examples of the monovalent or higher phenol compounds include phenol and 1- or 2-naphthol, and alkyl-substituted phenol compounds in which a hydrogen atom on the aromatic ring thereof is substituted with a linear or branched alkyl group having 1 to 24 carbon atoms (octylphenol, nonylphenol Etc.).
As (B7), an AO adduct of a C 8-18 monohydric aliphatic alcohol, an AO adduct of a C 19-24 monohydric aliphatic alcohol among those exemplified in the above (B 2), AO adducts of alcohols having a valence of 3 or more and AO adducts of alkyl-substituted phenol compounds substituted with a linear or branched alkyl group having 1 to 24 carbon atoms are preferable, and carbons among those exemplified in the above (B2) are more preferable. AO adducts of monovalent aliphatic alcohols of 8 to 18 having an HLB of less than 7.0, AO adducts of monovalent aliphatic alcohols having 19 to 24 carbon atoms, having an HLB of 7. AO adducts of less than 0, AO adducts of trihydric to octahydric alcohols having an HLB of less than 8.0 and alkyl substituted phenol compounds substituted with linear or branched alkyl groups having 1 to 24 carbon atoms so It is those having an HLB of less than 8.0 me.
Particularly preferred is an AO adduct of a C 8-18 monovalent aliphatic alcohol having an HLB of less than 7.0 {oleyl alcohol EO 2 molar adduct (HLB = 4.9) etc.}, C 1 AO adducts of alkyl-substituted phenol compounds substituted with a linear or branched alkyl group of 24 to 24 and having an HLB of less than 8.0 {nonylphenol EO 2 molar adduct (HLB = 5.7) etc.} and 3 to 8 AO adducts of polyhydric alcohols having an HLB less than 8.0 (PO50 molar adduct of glycerin (HLB = 0), etc.).

  As a defoaming agent (B), from the viewpoint that a large amount of useful substance can be obtained because the culture ability of bacteria is unlikely to decrease, aliphatic alcohol (B2) having 5 to 18 carbon atoms, fatty acid (B3), polyalkylene glycol ( B4) at least one selected from the group consisting of fatty acid alkanolamides (B5), esters of polyhydric alcohols and fatty acids (B6), and monohydric or trihydric alcohols or AO adducts of phenolic compounds (B7) is preferred More preferably, aliphatic alcohol (B2) having 5 to 18 carbon atoms, polyalkylene glycol (B4), ester of polyhydric alcohol and fatty acid (B6) and AO adduct of monohydric or trihydric alcohol or phenolic compound (B7) at least one member selected from the group consisting of (B7), particularly preferably polyalkylene glycol (B4) at least one selected from the group consisting of esters of polyhydric alcohols and fatty acids (B6) and monohydric or trihydric alcohols or AO adducts of phenolic compounds (B7), most preferably poly Alkylene glycol (B4) and / or ester of polyhydric alcohol and fatty acid (B6).

  The organic polar compound-based antifoaming agent (B) is not limited to the organic polar compound described above, but may contain other components (fatty acids, amides, animal and vegetable oils, hydrocarbon oils, hydrophobic silica, hydrophilic silica, water-soluble polymers, etc.) JP-A 2004-305882) and water and / or a known solvent (solvent handbook, Kodansha, 1982, page 143-881, etc.) may be included.

  The content of the organic polar compound contained in the organic polar compound-based antifoaming agent (B) is preferably 5 to 100% by weight, more preferably 10 to 100% by weight, from the viewpoint of defoaming property. Is 50 to 100% by weight.

  The amount of the antifoaming agent (B) used is preferably 0.00001 to 20% by weight, more preferably 0.0005 to 10% by weight based on the weight of the culture fluid, from the viewpoint of secretion efficiency of useful substances %.

  The antifoaming agent (B) may be added later to the culture solution in which the microorganism is suspended, in addition to being used by mixing with the culture solution in advance. The mixing with the culture solution can be performed by adding an antifoaming agent (B) to the culture solution at 4 ° C. to 99 ° C. and stirring with a stirring blade or a stirrer. When mixing later, it can carry out by adding, stirring with a stirring blade etc.

  Examples of bacteria in the present invention include, but are not limited to, the following. Bacteria include eubacteria and archaebacteria. Eubacteria are divided into outer membrane-bearing bacteria (bacteria with outer membrane) and bacteria without outer membrane. Outer membrane-bearing bacteria include gram negative bacteria and a few gram positive bacteria. Gram-negative bacteria include Escherichia (Escherichia), Thermus (Thermus), Rhizobium (Rhizobium), Pseudomonas (Pseudomonas), Shewanella (Shewanella), Vibrio (Vibrio), and Salmonella. Bacteria (Salmonella), Acetobacter (Acetobacter), Synechocystis (Synechocystis), and the like. Examples of gram-positive bacteria include Bacillus, Streptmyces, Corynebacterium, Brevibacillus, Bifidobacterium, and Lactococcus. And Enterococci (Enterococcus), Pediococcus (Pediococcus), Leuconostoc (Leuconostoc), Streptomyces, and the like. Examples of gram-positive bacteria that are outer membrane-containing bacteria include Deinococcus (Deinococcus radiodurans) and Corynebacterium (Corynebacterium).

  Among these, from the viewpoint of obtaining a large amount of useful substance, outer membrane-containing bacteria are preferable, more preferably gram-negative bacteria, particularly preferably Escherichia genus, and most preferably E. coli.

  Useful substances in the present invention include, but are not particularly limited to, proteins (enzymes, hormone proteins, antibodies, peptides and the like), 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.), isomerase ( Glucose isomerase etc., transferase (acyltransferase and sulfotransferase etc.), synthetase (fatty acid synthase, phosphate synthase and citrate synthase etc.) and eliminase (pectin lyase etc.) etc.}, hormone protein {bone morphogenetic protein BMP), interferon alpha, interferon beta, 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, serum albumin and calcitonin etc}, antibody {single chain antibody, IgG large subunit, IgG small subunit etc., antigen protein {hepatitis B surface antigen etc}, functional protein {pronectin (registered trademark), antifreeze peptide, antibacterial peptide etc}, fluorescent protein (GFP etc), luminescent protein (ruchelase etc) And peptides (in particular, the amino acid composition is not limited, and oligo peptides, dipeptides, tripeptides and the like) and the like can be mentioned.

  Examples of oligosaccharides include sucrose, lactose, trehalose, maltose, raffinose, panose, cyclodextrin, galactooligosaccharides and fructooligosaccharides.

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

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

In the case where the useful substance is a protein, it is preferable that the protein has a property of transferring to the periplasm partially or entirely after the protein is expressed in bacteria. More preferably, they are proteins encoding in the ORF a signal sequence necessary for transition to the periplasm.
Periplasm is the space from the bacterial plasma membrane to the outermost surface of the bacteria.
Signal sequences necessary for transfer to the periplasm include Sec secretion signal sequences, TAT secretion signals and the like.

  The surfactant (A) used in the method of producing a useful substance of the present invention includes an amphoteric surfactant (A1), an anionic surfactant (A2), a nonionic surfactant (A3) and a cationic surfactant It is at least one surfactant selected from the group consisting of (A4).

  As the amphoteric surfactant (A1), carboxylate type amphoteric surfactant (A1-1), sulfate ester type amphoteric surfactant (A1-2), sulfonate type amphoteric surfactant (A1-3) And phosphoric ester salt type amphoteric surfactants (A1-4) and the like.

  As the carboxylate type amphoteric surfactant (A1-1), an amino acid type amphoteric surfactant (A1-1-1), a betaine type amphoteric surfactant (A1-1-2) and an imidazoline type amphoteric surfactant (A1-1-3) etc. are mentioned.

The amino acid type amphoteric surfactant (A1-1-1) is an amphoteric surfactant having an amino group and a carboxyl group in the molecule, and examples thereof include compounds represented by the following general formula (1).
_{[R-NH- (CH 2)} n -COO -] m M (1)
In general formula (1), 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 proton; or a monovalent or divalent cation such as an alkali metal, an alkaline earth metal, ammonium (including a cation derived from an amine and an alkanolamine etc.) and a quaternary ammonium.
Further, specifically as (A1-1-1), alkylaminopropionic acid type amphoteric surfactants (sodium cocaminopropionate, sodium stearylaminopropionate and sodium laurylaminopropionate, etc.); alkylaminoacetic acid type amphoteric Surfactants (such as sodium lauryl aminoacetate) and N-lauroyl-N'-carboxymethyl-N'-hydroxyethyl ethylenediamine sodium and the like can be mentioned.

The betaine type amphoteric surfactant (A1-1-2) is an amphoteric surfactant having a quaternary ammonium salt type cationic moiety and a carboxylic acid type anionic moiety in the molecule. Examples of (A1-1-2) include compounds represented by the following general formula (2). Specifically as (A1-1-2), alkyl dimethyl betaine (stearyl dimethylamino acetic acid betaine and lauryl dimethylamino acetic acid betaine etc), amido betaine (coconut oil fatty acid amido propyl betaine etc (coconut fatty acid amido propyl dimethylamino acetic acid) Betaine etc.) and lauric acid amidopropyl betaine etc.) and alkyl dihydroxy alkyl betaines such as lauryl dihydroxyethyl betaine etc., hardened coconut oil fatty acid amidopropyl dimethylamino acetic acid betaine etc.
^{_{R-N + (CH 3)}} 2 -CH 2 COO - (2)
In general formula (2), R is a C1-C20 monovalent hydrocarbon group.

  Examples of the imidazoline type amphoteric surfactant (A1-1-3) include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine and the like.

  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; cholamidopropyl dimethyl ammoniopropane sulfonic acid (CHAPS), cholamidopropyl dimethyl ammonio 2-hydroxypropane sulfonic acid (CHAPSO); alkylamine oxide type amphoteric surfactants such as lauryldimethylamine oxide It can be mentioned.

  As the anionic surfactant (A2), ether carboxylic acid (A2-1) and its salt, sulfuric acid ester (A2-2) or its salt, ether sulfuric acid ester (A2-3) and its salt, sulfonic acid salt ( A2-4), sulfosuccinate (A2-5), phosphoric acid ester (A2-6) and salts thereof, ether phosphoric acid ester (A2-7) and salts thereof, acylated amino acid salts, and naturally occurring carboxylic acids and Its salts (quenodeoxycholic acid, cholic acid, deoxycholic acid, etc.) and the like can be mentioned.

  The ether carboxylic acid (A2-1) or a salt thereof includes an ether carboxylic acid having a hydrocarbon group (having 8 to 24 carbon atoms) and a salt thereof. Specifically as (A2-1) or a salt thereof, 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 Examples thereof include glycol acetate sodium salt and the like.

  The sulfuric acid ester (A2-2) and the salt thereof include sulfuric acid ester having a hydrocarbon group (having 8 to 24 carbon atoms) and the salt thereof. Specific examples of (A2-2) and salts thereof include sodium lauryl sulfate and triethanolamine lauryl sulfate.

  The ether sulfate (A2-3) and the salt thereof include ether sulfates having a hydrocarbon group (having 8 to 24 carbon atoms) and the salt thereof. Specific examples of (A2-3) and salts thereof include polyoxyethylene lauryl ether sulfate sodium salt and polyoxyethylene lauryl ether sulfate triethanolamine salt.

  Examples of the sulfonate (A2-4) include sodium dodecyl diphenyl ether disulfonate, sodium dodecyl benzene sulfonate and sodium naphthalene sulfonate.

  Examples of the sulfosuccinate salt (A2-5) include polyoxyethylene laurylsulfosuccinate disodium salt, lauric acid disodium laurylsulfosuccinate, sulfosuccinic acid polyoxyethylene lauroyl ethanolamide disodium salt and the like.

  Examples of phosphoric acid esters (A2-6) include octyl phosphate disodium salt and lauryl phosphate disodium salt.

  Examples of the ether phosphate (A2-7) include polyoxyethylene octyl ether disodium salt and polyoxyethylene lauryl ether disodium salt.

As the nonionic surfactant (A3), the aforementioned AO adduct of monohydric or trivalent or higher alcohol or phenol compound (B6), AO adduct of fatty acid (A3-3) and fatty acid of the above polyhydric alcohol Ester (B5) etc. are mentioned.
As surfactant (A), the same thing as the said organic polar compound contained in antifoamer (B) can also be used.

Examples of the nonionic surfactant (A3) include monohydric aliphatic alcohols having 8 to 18 carbon atoms among those exemplified as the above-mentioned monohydric or trivalent or higher alcohols or the AO adducts of phenol compounds (B6) AO adduct having HLB = 7.0 or more (A3-1-1), AO adduct of aliphatic alcohol having 19 to 24 carbon atoms having HLB = 7.0 or more (A3-1) -2) and an AO adduct of an alkyl-substituted phenol compound substituted with a linear or branched alkyl group having 1 to 24 carbon atoms, wherein HLB is at least 8.0 (A3-2), an AO adduct of a fatty acid A3-3) A fatty acid ester of a polyhydric alcohol selected from those having HLB = 10.0 or more (A3-3-1) and fatty acid esters of polyhydric alcohol (B5), HLB = 10.0 or more Also (A3-4-1) is preferable.

  Among the above (A3-1-1) and (A3-1-2), more preferable ones are 10 mol adduct of decyl alcohol EO (HLB = 14.7) and 7 mol adduct of lauryl alcohol ELO (HLB = 12.4). ), 5 mol adduct of oleyl alcohol EO (HLB = 9.0), 6 mol adduct of oleyl alcohol EO (HLB = 10.2), 7 mol adduct of oleyl alcohol EO (HLB = 11.0) and 10 mol adduct of oleyl alcohol EO (HLB = 12.4), 1,2-dodecanediol monooxyethylene adduct and the like.

Among the above (A3-2), more preferable ones include 1 to 20 moles of EO with 1 to 20 moles of EO and / or 1 to 20 moles of PO and 1 to 20 moles of nonylphenol with EO 1 to 20 moles and / or PO 1 to 20 moles. ^{, TRITON TM X-114 (HLB} = 12.4), readily available from ^{igepal TM CA-520 (HLB =} 10.0) and ^{igepal TM CA-630 (HLB =} 13.0) and the like market.

  Among the above (A3-3-1), more preferred are EO 1 to C 8 -C 24 fatty acids (decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, coconut oil fatty acid, etc.) 20 moles and / or PO 1 to 20 moles adduct [EO 9 moles adduct (HLB = 11.8), dioleate EO 12 moles adduct (HLB = 10.4), dioleate EO 20 moles adduct (HLB = 12 .9) and stearic acid EO 9 molar adduct (HLB = 11.9) etc.].

  Among the above (A3-4-1), further preferred are sorbitan monooleate ester EO 5 molar adduct (HLB = 10.0) and the like.

  As the nonionic surfactant (A3), one having an HLB of 7.0 to 13.0 among the above (A3-1-1) from the viewpoint of obtaining a large amount of useful substance and secretion efficiency (A3-1- 1A), among the above (A3-1-2), one having an HLB of 7.0 to 13.0 (A3-1-2A), and one having an HLB of 8.0 to 13.0 among the above (A3-2) The thing (A3-2A), the thing whose HLB is 10.0-13.0 among said (A3-3-1) (A3-3-1A), and HLB is 10. of the above-mentioned (A3-4-1). The thing (A3-4-1A) of 0-13.0 is preferable, More preferably, said (A3-1-1A), said (A3-1-2A), said (A3-2A), said (A3- 2A) Among 3-1A), HLB is 12.0-13.0 (A3-3-1B) and the above (A3-4-1A).

  As a cationic surfactant (A4), an amine salt type cationic surfactant (A4-1), a quaternary ammonium salt type cationic surfactant (A4-2), etc. are mentioned.

  As the amine salt type cationic surfactant (A4-1), an inorganic acid (hydrochloric acid, nitric acid, sulfuric acid, hydroiodic acid, etc.) or an organic acid (acetic acid, formic acid, oxalic acid, lactic acid, gluconic acid) is used. , Adipic acid, alkyl phosphoric acid, etc.). For example, as the primary amine salt type, inorganic or organic acid salts of aliphatic higher amines (higher amines such as laurylamine, stearylamine, cetylamine, hardened beef tallow amine, rosin amine and the like); higher acids of lower amines Examples include fatty acid (stearic acid, oleic acid, etc.) salts and the like. Examples of secondary amine salt types include inorganic acid salts or organic acid salts such as ethylene oxide adducts of aliphatic amines. Moreover, as a tertiary amine salt type, for example, aliphatic amines (triethylamine, ethyldimethylamine, N, N, N ', N'-tetramethylethylenediamine etc.), ethylene oxide (2 moles of aliphatic amines) Or more) adducts, alicyclic amines (N-methyl pyrrolidine, N-methyl piperidine, N-methyl hexamethylene imine, N-methyl morpholine, 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, stearamide ethyl diethyl methyl ethanol Inorganic acid salts or organic acid salts of tertiary amines such as amines may be mentioned.

  Examples of the quaternary ammonium salt type cationic surfactant (A4-2) include tertiary amines and quaternizing agents (alkyl chlorides such as methyl chloride, methyl bromide, ethyl chloride, benzyl chloride and dimethyl sulfate; ethylene oxide Etc.) are included. 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 Methyl ammonium methosulfate etc. are mentioned.

  The surfactant (A) is preferably an amphoteric surfactant (A1), an anionic surfactant (A2) and a nonionic surfactant (A3) from the viewpoint of production amount of useful substance and secretion efficiency, and further, Preferred are the above (A1), the above (A2), the above (A3-1-1), (A3-1-2) and the above (A3-2), and particularly preferred are the above (A1) and the above (A2). And (A3-1-1) and (A3-1-2), and particularly preferably a carboxylate type amphoteric surfactant (A1-1), an ether carboxylic acid (A2-1), and the above (A3- 1). 1-1) and (A3-1-2), and most preferably the above (A1-1).

  As a combination of surfactant (A) and organic polar compound based antifoamer (B), surfactant (A) from the viewpoint that the culture ability of bacteria is not reduced and the defoaming ability is not reduced during the culture. Preferred as organic polar compounds, aliphatic alcohols having 5 to 18 carbon atoms (B2), polyalkylene glycols (B4), esters of polyhydric alcohols and fatty acids (B6) and monohydric or trihydric alcohols or phenolic compounds A combination with an organic polar compound based antifoamer comprising at least one member selected from the group consisting of AO adducts of (B7) is preferred, and one interface selected from the above (A1), (A2) and (A3) An active agent (A) and an organic polar compound based antifoaming agent comprising at least one organic polar compound selected from the group consisting of (B2), (B4), (B6) and (B7) above Combination is more preferable, and one surfactant (A) selected from the above (A1-1), (A2-1), (A3-1) and (A3-3) and the above (B2), (B4) Particularly preferred is the combination with an organic polar compound based antifoamer comprising at least one organic polar compound selected from the group consisting of (B6) and (B7), and the above (A1-1) and (A3-1) And one surfactant selected from (A3-3) and at least one organic polar compound selected from the group consisting of (B2), (B4), (B6) and (B7) above. Most preferred is a combination with an organic polar compound antifoam.

  The weight ratio ((A) / (B)) to the surfactant (A) organic polar compound antifoaming agent (B) in the culture solution does not decrease the culture ability of bacteria, does not cause the backflow of the culture solution, and From a viewpoint of obtaining a useful substance, 0.005-990000 are preferable, More preferably, it is 0.1-100,000, Especially preferably, it is 0.1-100.

In the present invention, the surfactant (A) may use the surfactant (A) as it is, or may be mixed with water if necessary, and used as an aqueous dilution (aqueous solution or aqueous dispersion). .
The total concentration of the surfactant (A) in the aqueous dilution liquid is appropriately selected according to the target microorganism, the type of physiologically active substance and the type of extraction method, but from the viewpoint of the secretion and handling of useful substance, 0.1 to 99% by weight is preferred, preferably 1 to 50% by weight, based on the weight of the aqueous diluent.

  From the viewpoint of productivity, the secretion efficiency (%) when producing a useful substance using the surfactant of the present invention is preferably 1 to 100, more preferably 5 to 100, and particularly preferably 10 to 100. , Most preferably 50-100.

The secretion efficiency of the surfactant indicates that the useful substance in the bacteria is secreted out of the bacteria (in the culture solution) by the surfactant.
In the present invention, it is defined by the following formula.
Secretion efficiency (%) = 100 × {(X / Y) -Z}
X: Useful substance Y in culture solution remaining after removal of cells by centrifugation Y: Total useful substance Z in culture solution: ratio of lysed bacteria, defined by the following equation.
Z = Z1 / Z2
Z1: Intracytoplasmic localization substance Z2 in the culture solution remaining after removal of cells by centrifugation: Whole cytoplasmic localization substance in culture solution The intracytoplasmic localization substance is a substance existing in the cytoplasm and Refer to the substance that is eluted in the culture medium by lysis.

  The secretion efficiency is increased, for example, if the protein produced in bacteria is further transferred to the periplasm, and the secretion efficiency is decreased if it is not transferred to the periplasm. In addition, secretion efficiency can be increased by selecting a surfactant with high secretion efficiency by screening.

  The amount (% by weight) of the surfactant (A) used in the method of producing a useful substance of the present invention is appropriately selected according to the target microorganism, the type of useful substance to be produced and the type of extraction method. From the viewpoint of secretion efficiency and degradation of useful substances (especially proteins), preferably 0.0001 to 10, more preferably 0.005 to 10, particularly preferably 0. 1 to 5.

  The surfactant (A) may be added later to the culture solution in which the microorganism is suspended, in addition to being used by mixing with the culture solution in advance. The mixing with the culture solution can be performed by adding the surfactant (A) to the culture solution at 4 ° C. to 99 ° C. and stirring with a stirring blade or a stirrer. When mixing later, it can carry out by adding, stirring with a stirring blade etc.

  In using surfactant (A), you may mix and use several types other than using the said surfactant alone.

  In the production method of the present invention, in addition to the surfactant (A) and the organic polar compound antifoam (B), the culture solution may contain another antifoam (C) other than (B). .

Other antifoam agents (C) include lower alcohol antifoam agents, mineral oil antifoam agents and silicone antifoam agents.
The lower alcohol-based antifoaming agents include aliphatic alcohols having 1 to 4 carbon atoms, and specific examples include methanol, ethanol, isopropanol, sec-butanol and n-butanol.

  Mineral oil based antifoaming agents include antifoaming agents containing a mineral oil that is liquid at 25 ° C. as a defoaming component, and as mineral oils, vacuum distillation, oil removal, solvent extraction, hydrocracking, solvent dewaxing A product obtained by appropriately combining sulfuric acid washing, clay refining, hydrorefining and the like can be used. Cosmo Pure Spin G, Cosmo Pure Spin E, Cosmo SP-10, Cosmo SP-32 and Cosmo SC22 can be used as trade names. (As mentioned above, Cosmo Oil Co., Ltd., "Cosmo" and "Pure Spin" are registered trademarks of the company.) MC Oil P-22, S-10S (above, Idemitsu Kosan Co., Ltd.), and Stanol 40 ( Exxon Mobil Corporation) and the like.

As a silicone type antifoamer, the antifoamer which uses dimethyl silicone oil and modified silicone oil as an antifoaming component is contained.
As a silicone oil, polydimethylsiloxane etc. of dynamic viscosity 10-10000 (mm < ² > / s, 25 degreeC) etc. are mentioned, A cyclooctamethyl tetrasiloxane etc. are also contained.
As a modified silicone oil, a part of the methyl group of the above polydimethylsiloxane is an alkyl group having 2 to 6 carbon atoms, an alkoxyl group having 2 to 4 carbon atoms, a phenyl group, a hydrogen atom, a halogen (such as chlorine and bromine) atom And / or those substituted with an aminoalkyl group having 2 to 6 carbon atoms, and the like.

The content of the other antifoaming agent (C) in the culture solution is preferably 0.00001 to 20% by weight, more preferably 0 based on the weight of the culture solution, from the viewpoint of antifoaming and foam suppressing properties. It is .0000 1 to 10% by weight.
1 type may be used for another antifoamer (C), and 2 or more types may be used together.

In the present invention, the dry cell density refers to the weight of bacteria contained in 1 L of culture solution at any time from the start of culture to the end of culture in the secretory production of a useful substance. The weight of the bacteria is the weight of the bacteria in a dry state.
The dry cell density is determined by the following procedures (1) to (5).
Procedure (1): Weigh the container (centrifuge tube) in advance.
Procedure (2): Put 100 ml of the culture solution into a container whose weight was measured in procedure (1), centrifuge it (4,000 G, 15 minutes, 4 ° C.), remove the supernatant, and collect bacteria. Step (3): Wash the collected bacteria in the container with 0.9% by weight aqueous NaCl solution [the same volume as the culture solution used in step (2)] and centrifuge again (4,000 G, 15 minutes, 4 Remove the supernatant and collect bacteria.
Procedure (4): With the bacteria obtained in procedure (3) still in the container, dry at 105 ° C. for 10 hours, and measure the total weight of the container and the bacteria.
Procedure (5): After drying at 105 ° C. for 2 hours after procedure (4), the total weight of the container and bacteria is measured to confirm that there is no change in weight. If the weight is further reduced, drying at 105 ° C. is continued until there is no weight change.
The dry cell density is determined by applying the measured values of step (5) and step (1) and the volume (L) of the culture solution used in step (2) to the following equation.
Dry cell density (g / L) = ([measurement value of procedure (5)]-[measurement value of procedure (1)] / 0.1

The dry cell density in the method for producing the useful substance of the present invention is preferably 1.5 to 500 g / L, more preferably 3 g / L to 200 g / L, particularly preferably 4 based on the volume of the culture solution. -100 g / L. The dry cell density of 1.5 g / L or more is preferable because the amount of useful substance produced is increased. Moreover, since a bacteria can produce a useful substance efficiently because it is 500 g / L or less, it is preferable.
The dry cell density when the bacterium is E. coli is preferably 1.5 to 500 g / L, more preferably 3 to 100 g / L, from the viewpoint of being able to produce a useful substance based on the volume of the culture solution. And particularly preferably 10 to 50 g / L, and most preferably 12 to 27 g / L.
In the method for producing the useful substance of the present invention, within the above range, the production amount of the useful substance increases as the dry cell density increases.

  In the method of producing a useful substance of the present invention, from the viewpoint of the amount of production of useful substance, the time during which the dry cell density is within the above range is preferably 10% or more of the time required for the step of secreting the useful substance. More preferably, it is 50% or more.

  The dry cell density can be increased, for example, by feeding at a suitable rate using semi-batch culture under sufficient aeration conditions, and reduced by performing batch cultures under limited aeration conditions Can. In addition, it can be increased by increasing the time from the start of culture to the addition of surfactant, and can be reduced by shortening the time from the start of culture to the addition of surfactant.

In the method for producing the useful substance of the present invention, the production method for secretion production of the useful substance includes an extracellular secretion production method including the following steps (a) and (b). In the following steps, the step of secretory production of the useful substance is step (a).
Step (a): A culture solution for culturing a bacterium producing a useful substance (gram-negative bacteria etc.), a surfactant (A) and an antifoam agent (B) simultaneously and extracellularly a useful substance Secrete).
Step (b): a step of separating the useful substance from the culture solution after the step (a).

Hereinafter, an example of a method of producing a useful substance using the surfactant of the present invention will be shown.
(I) genetically modified (i-1) messenger RNA (mRNA) is isolated from cells expressing the target protein, single-stranded cDNA is then synthesized from the mRNA, and double-stranded DNA is then synthesized; The strand DNA is incorporated into phage DNA or a plasmid. The resulting recombinant phage or plasmid is transformed into host E. coli to prepare a cDNA library.
(I-2) The method for screening phage DNA or plasmid containing the DNA of interest includes hybridization of phage DNA or plasmid with a DNA probe encoding a part of the target protein gene or complementary sequence. .
(I-3) A target gene is obtained by cutting out a target cloned DNA or a part thereof from a phage or plasmid after screening, and ligating the cloned DNA or a part thereof downstream of a promoter in an expression vector Can be constructed. It is also possible to simultaneously link DNA encoding a signal sequence that causes the inner membrane to translocate (a signal sequence that causes the periplasm to express a target substance).
(Ii) Cultivation (ii-1) Transform the host bacteria with an expression vector to produce recombinant bacteria, and preculture the recombinant bacteria. The preculture is usually performed on an agar medium at 15 to 43 ° C. for 3 to 72 hours.
(Ii-2) The culture solution used for the production of the useful substance is autoclaved at 121 ° C. for 20 minutes, and the recombinant bacteria precultured in an agar medium are cultured here. The culture is usually performed at 15 to 43 ° C. for 12 to 72 hours. In addition, when using surfactant (A) and antifoamer (B) simultaneously with culture | cultivation start, what mixed surfactant (A) and antifoamer (B), and a culture solution and was homogenized is used. Perform the same operation using as a culture solution. Moreover, when surfactant (A) and an antifoamer (B) are added 6 to 72 hours after culture | cultivation, after adding a surfactant and an antifoamer, culture | cultivation is continued 1 to 1000 hours.
(Iii) Purification (iii-1) Proteins secreted into the culture solution are separated from microorganisms and microbial residues by centrifugation, hollow fiber separation, filtration and the like.
(Iii-2) The culture solution containing the protein is repeatedly subjected to column treatment 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 are carried out by performing as appropriate.

  The host cells separated in (iii-1) can be further cultured by newly supplying a culture fluid. The culture solution or the like is further subjected to the step of (iii), purification, and culture are repeated, whereby continuous production of useful substances can be performed.

  Examples of the packing agent used for column chromatography in the above-mentioned step (iii) of protein separation / extraction step include silica, dextran, agarose, cellulose, acrylamide and vinyl polymer, etc. In the commercial product, Sephadex series, Sephacryl series Sepharose series (above, Pharmacia), Bio-Gel series (Bio-Rad), and the like.

The method for producing the useful substance of the present invention facilitates purification of the useful substance because the useful substance is secreted into the culture solution. In addition, since a high yield can be obtained in a short time, a high production amount can be achieved.
In addition, by using the method for producing a useful substance of the present invention, the culture solution is not backflowed, and even in a culture vessel equipped with a foam control device equipped with a foam-cutting blade, a foam surface sensor, etc. Demonstrates the effect of suppressing backflow. Furthermore, since the culture tank is not filled with foam, the culture capacity of bacteria is not reduced, and a large amount of useful substance can be obtained.

  The useful substance obtained by the production method of the present invention has a specific activity higher than that of the prior art because it is obtained by the above method.

The method for producing the useful substance of the present invention comprises the step of simultaneously causing the surfactant (A) and the organic polar compound antifoam (B) and the bacteria to secrete the useful substance into the culture solution. In this process, as long as the bacteria survives, the bacteria can make a useful substance and secrete it into the culture solution. Furthermore, the production method of the present invention can be used regardless of the type of useful substance to be produced, as long as the bacteria has the ability to produce a useful substance.
The method for producing useful substance of the present invention is particularly effective when the useful substance produced in bacteria is transferred to the periplasm of bacteria. Transfer of the useful substance to the periplasm facilitates the secretion of the useful substance into the culture solution.

  The present invention is further described by the following examples and comparative examples, but the present invention is not limited thereto. Unless otherwise stated, parts mean parts by weight.

<Production Example 1>
The alkaline phosphatase (phoA) gene of E. coli strain W3110 was amplified by PCR using primers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2). The PCR fragment was treated with restriction enzymes NdeI and BamHI, and then ligated to the NdeI restriction enzyme site and BamHI restriction enzyme site of pET-22b plasmid (Novagen). Thereafter, this plasmid was transformed into an AG1 (DE3) E. coli strain prepared by modifying E. coli strain AG1 (ToYoBo) using λDE3 Lysogenization Kit (Novagen) to prepare an alkaline phosphatase expression strain (α). Localization of the expressed alkaline phosphatase in the periplasmic fraction was analyzed and confirmed based on the method of METHODS IN ENZYMOLOGY 353 2002 p. 121.

Example 1
The expression strain (α) is inoculated into 1 mL of LB culture solution (containing 100 mg / L of ampicillin) and cultured at 30 ° C. for 12 hours to prepare an overnight culture, 0.5 ml of LB culture solution (ampicillin 100 mg / L) Inoculation in 50 ml) and shaking culture at 30 ° C. for 3 hours to prepare a pre-preculture fluid. The pre-preculture solution was inoculated into 500 ml of LB culture solution (containing 100 mg / L of ampicillin) and shake culture was carried out at 30 ° C. for 12 hours to prepare a pre-culture solution.
The content of each component in 2.5 L of the culture broth (2.5 L of the culture broth) was 60 g of yeast extract (manufactured by Nippon Pharmaceutical Co., Ltd.), 30 g of Polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), and potassium potassium phosphate 23.3. 5 g, 5.5 g of monopotassium phosphate, 17.5 g of ammonium sulfate, 33 g of disodium phosphate dihydrate, 1 g of sodium citrate dihydrate, 10 g of glycerol, 75 g of lactalbumin hydrolyzate, surfactant (A) ( 25 g of sodium polyoxyethylene tridecyl ether acetate, 2.5 g of antifoam (B) (pentyl alcohol), 1 mM magnesium sulfate, trace metal solution (calcium chloride 945 μg, iron (III) chloride 25 mg, zinc sulfate heptahydrate 450 μg, copper sulfate 255 μg, manganese chloride tetrahydrate 335 μg, cobalt chloride 245 μg, ethylene diamine 4 Microorganism culture apparatus (product name "BMS-P" by Able, Inc.) equipped with a dropping line for inoculating 4 sodium acetate (10 mg), 100 mg / L ampicillin] and dropping ammonia water and lactic acid aqueous solution respectively and a pH sensor The cultivation was carried out at 30 ° C. while maintaining the pH at 6.8 by dropping ammonia water or an aqueous solution of lactic acid as required.
After the start of culture, 7.5 mL of 1 M IPTG solution was added. From 14 hours after the start of culture, 2 L of glycerol / protein solution (50% by weight glycerol, 50 g / L lactalbumin hydrolyzate, antifoamer (pentyl alcohol, 0.1% by weight), 100 mg / L ampicillin) was added dropwise (60 ml / liter). h) started.
The culture was carried out for 48 hours, and the presence or absence of a sudden reflux during the culture and the height of the foam of the culture solution were measured by the following method, and the results are shown in Table 1. Moreover, the turbidity was measured by the following method, and it described in Table 1.
The culture was terminated at 48 hours, the culture solution was recovered, and the total protein in the culture solution was analyzed by SDS-PAGE to quantify the amount of recombinant protein produced from the quantification of the protein band.

Examples 2 to 71
In Example 1, as the surfactant (A), those described in Tables 1 to 4 are used, and as the antifoaming agent (B), those described in Tables 1 to 4 in place of “pentyl alcohol” and their concentrations E. coli is cultured in the same manner as in Example 1 except for using it, and the presence or absence of sudden backflow, the height of foam in the culture tank and the turbidity of the culture solution are measured in the same manner as in Example 1 It described to 1-4. The amount of recombinant protein produced in the same manner as in Example 1 was quantified for the culture solution after completion of culture, and the results are shown in Tables 1 to 4.

<Comparative Examples 1 to 39>
In Example 1, those in Tables 4 and 5 are used as surfactant (A), and in place of “Pentyl alcohol” as antifoaming agents, those in Tables 4 and 5 and their concentrations are used or no antifoaming agent is used E. coli was cultured in the same manner as in Example 1 except for the following, and the presence or absence of sudden backflow, the height of foam in the culture tank and the turbidity of the culture solution were measured in the same manner as in Example 1; Described in 6. The amount of recombinant protein produced in the same manner as in Example 1 was quantified for the culture solution after completion of culture, and the results are shown in Tables 5 and 6.
In addition, culture products were stopped at the time when backflow occurred when sudden backflow was caused by bubbling, and measurement of turbidity and quantification of the produced recombinant protein were not performed.

<Method for evaluating the presence or absence of sudden backflow and the height of foam in culture>
Throughout the culture time of E. coli, the presence or absence of sudden backflow from the vent line of the device was observed. At 48 hours from the start of the culture, the height of the foam deposited on the top of the culture was measured while the culture was kept stirred.

<Measurement of turbidity of culture solution>
The turbidity was measured using a 1 m quartz cell using a turbidimeter (manufactured by Shimadzu, UV-1700).
The culture solution was diluted with physiological saline to have an appropriate absorbance and then measured. The same culture broth was diluted at the same dilution rate as described above except that it did not contain bacteria, and the absorbance was measured as a blank. The turbidity of the culture solution was calculated by the following equation.
Turbidity of culture solution = [(turbidity measurement value of diluted culture fluid)-(blank turbidity measurement value)] × dilution factor

Each component used the following in Tables 1-5.
Silicone antifoaming agent: Shin-Etsu Chemical Co., Ltd. product, "KM-70"
Mineral oil-based antifoaming agent: San Nopco Co., Ltd. "Dappo H-106"

From the results of Tables 1 to 6, Examples 1 to 71, which are production methods of the present invention using an organic polar compound-based antifoaming agent, are conventional production methods using only the other antifoaming agent (C). As compared with Comparative Examples 1 to 39, it can be seen that foaming of the culture solution is suppressed and sudden backflow of the culture solution does not occur. Further, in the comparative example, as a result of stopping the culture due to the occurrence of a sudden backflow, or the result of bubbling, the pH could not be adjusted, resulting in a decrease in protein production, but the example of the present invention It turns out that all were able to obtain a large amount of protein which is a useful substance.
From this, in the production method of the present invention, by using the surfactant (A) and the antifoaming agent (B), it is possible to produce a useful substance without causing the culture solution to reversely flow back. Furthermore, it turns out that a large amount of useful substances can be obtained.

  The method for producing a useful substance of the present invention can be used in the secretory production of a useful substance such as a protein using bacteria. When the protein to be produced is an enzyme (protease, cellulase, lipase, amylase or the like), it can be suitably used as food processing, detergent, fiber processing, paper making use, enzyme conversion use, etc.

1: Culture tank 2: Stirring blade 3: Thermometer 4: Aeration nozzle 5: Exhaust port 6: Culture fluid 7: Foaming blade 8: Foam surface sensor 9: pH sensor

Claims (4)

A method for producing a protein in which a protein is secreted and produced in a culture solution using a gram negative bacterium contained in the culture solution, which comprises a carboxylate surfactant (A1-1) and an organic polar compound system in the culture solution The weight ratio ((A1-1) / (B)) of the carboxylic acid salt type surfactant (A1-1) to the organic polar compound antifoam agent (B) including the antifoaming agent (B) is 10 to 100. A method of producing a protein that is The method for producing a protein according to claim 1, wherein the protein has the property of transferring to the periplasm. The amount of the antifoaming agent (B) used is 0.00001 to 0.1 % by weight based on the weight of the culture solution
The method for producing a protein according to claim 1 or 2, which is The organic polar compound antifoaming agent (B) is an aliphatic alcohol having 5 to 18 carbon atoms (B1), a fatty acid (B2), a polyalkylene glycol (B3), a fatty acid alkanolamide (B4),
4. The method according to any one of claims 1 to 3, comprising at least one organic polar compound selected from the group consisting of esters of polyhydric alcohols and fatty acids (B5) and alkylene oxide adducts of alcohols (B6).
A method for producing a protein according to any one of the preceding claims.

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