JP7050527B2 - Production method of useful substances - Google Patents

Description

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

Technology for producing useful substances such as amino acids and proteins by microorganisms is widely used in industrial fields such as medical treatment.
As a microorganism that produces useful substances such as amino acids and proteins, a method of producing by Escherichia coli is known. However, there are problems that useful substances to be produced are limited and secretory production is difficult. Therefore, by using fungi such as Gram-positive bacteria, yeast and basidiomycete, it is possible to secrete and produce a wide range of useful substances (for example, Non-Patent Documents 1 and 2).

However, such fungi such as Gram-positive bacteria, yeasts and basidiomycetes have a cell wall made of thick polysaccharide in order to withstand stress from the outside world. This cell wall made of polysaccharide causes adsorption of useful substances and suppression of permeation of useful substances, and the secretory efficiency deteriorates, which is a big problem in secretory production.

Therefore, in yeast, for the purpose of thinning the cell wall due to polysaccharides, S. There is a technique (Non-Patent Document 3) for increasing the amount of protein secretion by using a sugar synthesis-related gene-disrupted strain of S. cerevisiae or Yarrowia lipolytica. However, it has been pointed out that the glycoprotein obtained by this method is limited to proteins that do not have glycosylated chains, and no useful solution has been found yet.

Bioindustry Association, Fermentation Handbook, Kyoritsu Shuppan, July 2001 New Biochemical Engineering (2nd Edition), Sankyo Publishing, March 2013, p17-21 J. Bacteriol, 1998, vol. 180, no. 24, p6736-6742

An object of the present invention is to provide a method for producing a useful substance capable of efficiently secreting a useful substance produced by yeast into a culture solution.

The present inventors have arrived at the present invention as a result of diligent studies to solve these problems.
That is, the present invention is a method for producing a useful substance by secreting and producing a useful substance in the culture solution by the yeast contained in the culture solution, wherein the yeast is composed of the genus Kluyveromyces, the genus Hansenula, the genus Pichia, the genus Yarrowia and the genus Candida. At least one selected from the group, the culture solution contains an ionic surfactant (A) and a nonionic surfactant (B), and the ionic surfactant (A) is an anionic surfactant (A1) and a cation. One or more surfactants selected from the group consisting of a surfactant (A2) and an amphoteric surfactant (A3), and the nonionic surfactant (B) is a nonionic surfactant having an HLB value of 3 to 20. A method of producing a useful substance.

By using the method for producing a useful substance of the present invention, the useful substance produced by yeast can be secreted into the culture solution with high efficiency.

The method for producing a useful substance of the present invention is a method for producing a useful substance by secreting and producing a useful substance in a culture solution by yeast contained in the culture solution, wherein the yeast belongs to the genus Kluyveromyces, the genus Hansenula, the genus Pichia, and the genus Yarrowia. And at least one selected from the group consisting of the genus Pichia, characterized in that the culture solution contains an ionic surfactant (A) and a nonionic yeast (B). The ionic surfactant (A) is one or more surfactants selected from the group consisting of an anionic surfactant (A1), a cationic surfactant (A2) and an amphoteric surfactant (A3), and is a nonion. The surfactant (B) is a nonionic surfactant having an HLB value of 3 to 20.

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

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

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

Examples of other essential nutrients include inorganic salts, and examples of the inorganic salts include primary potassium phosphate, ferric potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, and copper sulfate. , Calcium carbonate and the like are used.

The yeasts in the present invention belong to the genera Kluyveromyces, Hansenula, Pichia, Yarrowia and Candida from the viewpoints of gene manipulation, ease of industrial use, and production of useful substances.

The useful substance in the present invention is not particularly limited, and includes proteins (enzymes, hormone proteins, antibodies, peptides, etc.), oligosaccharides, nucleic acids, and the like.

Examples of proteins include enzymes {oxidation-reducing enzymes (cholesterol oxidase, glucose oxidase, ascorbic acid oxidase, peroxidase, etc.), hydrolyzing enzymes (lysoteam, protease, serine protease, amylases, lipase, cellulase, glucoamylase, etc.), isomerizing enzymes (such as isomerase). Glucose isomerase, etc.), transferases (acyltransferase, sulfotransferase, etc.), synthases (fatty acid synthase, phosphate synthase, citrate synthase, etc.) and desorption enzymes (peptidrianase, etc.)}, hormonal proteins {bone-forming factors (bone-forming factors, etc.) BMP), interferon α, interferon β, interleukins 1-12, growth hormone, erythropoetin, insulin, granular colony stimulator (G-CSF), tissue plasminogen activator (TPA), sodium diuretic peptide, blood coagulation Factor VIII, somatomedin, glucagon, growth hormone releasing factor, serum albumin and calcitonin, etc.}, antibody {single-stranded antibody, IgG large subunit, IgG small subunit, etc.}, antigen protein {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 in amino acid composition, oligopeptides, dipeptides, etc.) Tripeptide, etc.) and the like.

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

Examples of the nucleic acid include DNA, RNA, inosinic acid, adenosine monophosphate, guanosine monophosphate and the like.

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

When the useful substance is a protein, it is preferable that the protein has a property that a part or all of the protein is transferred to periplasm after being expressed in the microorganism. More preferably, it is a protein that encodes the signal sequence required for the transition to periplasm in the ORF.
Periplasm is the space outside the cytoplasmic membrane of microorganisms and the cell wall layer containing polysaccharides.
Examples of the signal sequence required for the transition to periplasm include a Sec secretory signal sequence, a TAT secretory signal, an α-signal sequence, a signal sequence of the PHO1 gene, and the like.

As the surfactant used in the method for producing a useful substance of the present invention, it is necessary to use an ionic surfactant (A) and a nonionic surfactant (B) in combination.
The ionic surfactant (A) is at least one ionic surfactant selected from the group consisting of anionic surfactant (A1), cationic surfactant (A2) and amphoteric surfactant (A3). ..

Examples of the anionic surfactant (A1) include ether carboxylic acid (A11) and its salt, sulfate ester (A12) or its salt, ether sulfate ester (A13) and its salt, sulfonate (A14), and sulfosuccinate (A14). A15), phosphate ester (A16) and its salt, ether phosphate ester (A17) and its salt, fatty acid salt (A18), acylated amino acid salt and naturally occurring carboxylic acid and its salt (kenodeoxycholic acid and coli acid and Deoxycholic acid, etc.) and the like.

Examples of the ether carboxylic acid (A11) or a salt thereof include an ether carboxylic acid having a hydrocarbon group (8 to 24 carbon atoms) and a salt thereof.
The ether is preferably an alkylene oxide adduct, more preferably one or two adducts of ethylene oxide and propylene oxide, and particularly preferably an ethylene oxide adduct, from the viewpoint of secretory efficiency of useful substances and cytotoxicity. Is.
The degree of polymerization of the alkylene oxide is preferably 1 to 10 from the viewpoint of secretory efficiency of useful substances and cytotoxicity.
Specifically, (A11) or a salt thereof is polyoxyethylene lauryl ether acetic acid, polyoxyethylene lauryl ether acetate sodium salt, polyoxyethylene tridecyl ether acetate sodium salt, polyoxyethylene octyl ether acetate sodium salt and lauryl glycol acetate. Examples include sodium salts.

Examples of the sulfate ester (A12) and a salt thereof include a sulfate ester having a hydrocarbon group (8 to 24 carbon atoms) and a salt thereof. Specific examples of (A12) and its salts include sodium lauryl sulfate and triethanolamine lauryl sulfate.

Examples of the ether sulfate ester (A13) and a salt thereof include an ether sulfate ester having a hydrocarbon group (8 to 24 carbon atoms) and a salt thereof.
The ether is preferably an alkylene oxide adduct, more preferably one or two adducts of ethylene oxide and propylene oxide, and particularly preferably an ethylene oxide adduct, from the viewpoint of secretory efficiency of useful substances and cytotoxicity. Is.
The degree of polymerization of the alkylene oxide is preferably 1 to 10 from the viewpoint of secretory efficiency of useful substances and cytotoxicity.
Specific examples of (A1-3) and its salts include polyoxyethylene lauryl ether sulfate sodium salt and polyoxyethylene lauryl ether sulfate triethanolamine salt.

Examples of the sulfonate (A14) include dodecyldiphenyl ether disulfonic acid sodium salt, dodecylbenzene sulfonic acid sodium salt, naphthalene sulfonic acid sodium salt and the like.

Examples of the sulfosuccinate (A15) include polyoxyethylene lauryl sulfosuccinate disodium salt, sulfosuccinate lauryl disodium salt, sulfosuccinate polyoxyethylene lauroylethanolamide disodium salt and the like.

Examples of the phosphoric acid ester (A16) include disodium octyl phosphate and disodium lauryl phosphate.

Examples of the ether phosphate ester (A17) include polyoxyethylene octyl ether phosphate disodium salt and polyoxyethylene lauryl ether phosphate disodium salt.

Examples of the fatty acid salt (A18) include sodium octylate salt, sodium lauryl acid salt, sodium stearate salt and the like.

As the anionic surfactant (A1), a compound represented by the following general formula (1) is preferable.

In the general formula (1), R ¹ represents a monovalent hydrocarbon group having 1 to 30 carbon atoms, (OA) represents an oxyalkylene group (for example, oxyethylene, oxypropylene, oxybutylene, etc.), and s is. It is an integer of 1 or more, and Q represents a sulfonic acid (salt) group, a carboxylic acid (salt) group, or a phosphoric acid (salt) group.
In addition, sulfonic acid (salt) means sulfonic acid and / or sulfonate, carboxylic acid (salt) means carboxylic acid and / or carboxylate, and phosphoric acid (salt) means phosphoric acid and / Or means phosphate. Examples of the salt include alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.) and onium cation salts (ammonium cations, quaternary ammonium cations, tertiary sulfonium cations, quaternary sulfonium cations, etc.). Quaternary phosphonium cations, tertiary oxonium cations, etc.) are included.

In the general formula (1), R ¹ is preferably a monovalent hydrocarbon group having 8 to 22 carbon atoms, and more preferably 1 having 10 to 18 carbon atoms, from the viewpoint of secretory efficiency of useful substances and cytotoxicity. It is a valent hydrocarbon group.
As the hydrocarbon group, an aliphatic hydrocarbon group is preferable from the viewpoint of secretory efficiency of useful substances and cytotoxicity, and more preferably, a linear and / or branched aliphatic hydrocarbon having 0 to 3 unsaturated bonds. It is a hydrogen group, particularly preferably an alkyl group and an alkenyl group having 1 to 3 unsaturated bonds.
As the oxyalkylene group, an oxyethylene group is preferable from the viewpoint of secretory efficiency of useful substances and cytotoxicity.
From the viewpoint of secretory efficiency of useful substances and cytotoxicity, s is preferably an integer of 1 to 10, and more preferably an integer of 1 to 5.

Examples of the product represented by the general formula (1) include polyoxyethylene (average 2.5 mol adduct) sodium lauryl ether sulfate, polyoxyethylene (3 mol adduct) sodium lauryl ether acetate and the like.

Examples of the cationic surfactant (A2) include an amine salt-type cationic surfactant (A21) and a quaternary ammonium salt-type cationic surfactant (A22).

As the amine salt type cationic surfactant (A21), the primary to tertiary amines are inorganic acids (hydrochloric acid, nitric acid, sulfuric acid, hydroiodic acid, etc.) or organic acids (acetic acid, formic acid, oxalic acid, lactic acid, gluconic acid, adipine). Includes acid, alkyl phosphate, etc.) neutralized. For example, primary amine salt types include inorganic or organic acid salts of aliphatic higher amines (higher amines such as laurylamine, stearylamine, cetylamine, hardened beef amine, rosinamine); higher grades of lower amines. 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 the tertiary amine salt type include aliphatic amines (triethylamine, ethyldimethylamine, N, N, N', N'-tetramethylethylenediamine, etc.) and aliphatic amine ethylene oxide (2 mol). Additives, alicyclic amines (N-methylpyrrolidin, N-methylpiperidin, N-methylhexamethyleneimine, N-methylmorpholin, 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 diethylmethylethanol Examples thereof include inorganic acid salts and organic acid salts of tertiary amines such as amines.

Examples of the quaternary ammonium salt-type cationic surfactant (A22) include tertiary amines and quaternary agents (alkylating agents such as methyl chloride, methyl bromide, ethyl chloride, benzyl chloride and dimethyl sulfate; ethylene oxide and the like). Includes those obtained by reaction with. For example, lauryltrimethylammonium chloride, didecyldimethylammonium chloride, dioctyldimethylammonium bromide, stearyltrimethylammonium bromide, lauryldimethylbenzylammonium chloride (benzalkonium chloride), cetylpyridinium chloride, polyoxyethylenetrimethylammonium chloride, stealamide ethyldiethyl. Examples thereof include methylammonium metosulfate.

As (A22), a compound represented by the following general formula (2) is preferable from the viewpoint of secretory efficiency of useful substances and cytotoxicity.

一般式（２）中、Ｒ²、Ｒ³、Ｒ⁴及びＲ⁵はそれぞれ炭素数１～３０の一価の炭化水素基を表し、Ｚ^-はカウンターアニオンを表す。
一般式（２）において、有用物質の分泌効率及び細胞毒性の観点から、Ｒ²、Ｒ³、Ｒ⁴及びＲ⁵のうち少なくとも１つは炭素数８～２２の一価の炭化水素基であることが好ましく、さらに好ましくはＲ²、Ｒ³、Ｒ⁴及びＲ⁵のうち少なくとも１つは炭素数１０～１８の一価の炭化水素基であることである。
Ｒ²、Ｒ³、Ｒ⁴及びＲ⁵において、炭化水素基としては、有用物質の分泌効率及び細胞毒性の観点から、脂肪族炭化水素基が好ましく、さらに好ましくは０～３個の不飽和結合を有する直鎖及び／又は分岐鎖の脂肪族炭化水素基であり、特に好ましくはアルキル基及び不飽和結合を１～３個有するアルケニル基である。
また、Ｚ^-としては、有用物質の分泌効率及び細胞毒性の観点から、ハロゲンイオンが好ましく、さらに好ましくは塩化物イオンである。
また、一般式（２）において、炭化水素基は、炭化水素基のいずれかの位置に、水酸基、エーテル基、カルボニル基及びエステル基からなる群より選ばれる少なくとも１種の置換基を有していてもよい。

In the general formula (2), R ² , R ³ , R ⁴ and R ⁵ each represent a monovalent hydrocarbon group having 1 to 30 carbon atoms, and Z ^- represents a counter anion.
In the general formula (2), at least one of R ² , R ³ , R ⁴ and R ⁵ is a monovalent hydrocarbon group having 8 to 22 carbon atoms from the viewpoint of secretory efficiency of useful substances and cytotoxicity. It is preferable, and more preferably, at least one of R ² , R ³ , R ⁴ and R ⁵ is a monovalent hydrocarbon group having 10 to 18 carbon atoms.
In R ² , R ³ , R ⁴ and R ⁵ , the hydrocarbon group is preferably an aliphatic hydrocarbon group, more preferably 0 to 3 unsaturated bonds, from the viewpoint of secretory efficiency of useful substances and cytotoxicity. It is a straight chain and / or a branched chain aliphatic hydrocarbon group having, particularly preferably an alkyl group and an alkenyl group having 1 to 3 unsaturated bonds.
Further, as Z ⁻ , halogen ions are preferable, and chloride ions are more preferable, from the viewpoint of secretion efficiency of useful substances and cytotoxicity.
Further, in the general formula (2), the hydrocarbon group has at least one substituent selected from the group consisting of a hydroxyl group, an ether group, a carbonyl group and an ester group at any position of the hydrocarbon group. May be.

Examples of the amphoteric surfactant (A3) include a carboxylate-type amphoteric surfactant (A31), a sulfate ester-type amphoteric surfactant (A32), a sulfonate-type amphoteric surfactant (A33), and a phosphate ester salt. Type amphoteric surfactant (A34) and the like are included.

Examples of the carboxylate-type amphoteric tenside agent (A31) include an amino acid-type amphoteric tenside agent (A311), a betaine-type amphoteric tenside agent (A312), and an imidazoline-type amphoteric tenside agent (A313).

Examples of the amino acid type amphoteric tenside agent (A311) include amphoteric surfactants having an amino group and a carboxyl group in the molecule, and a compound represented by the following general formula (3).

一般式（３）中、Ｒ⁶は炭素数１～３０の一価の炭化水素基である。ｎは１以上の整数である。ｍは１以上の整数である。Ｍはプロトン；又はアルカリ金属、アルカリ土類金属、アンモニウム（アミン及びアルカノールアミン等由来のカチオンを含む）及び第４級アンモニウム等の１価又は２価のカチオンである。
Ｒ⁶において、炭化水素基の炭素数は、有用物質の分泌効率及び細胞毒性の観点から、８～２２が好ましく、さらに好ましくは１０～１８である。
Ｒ⁶において、炭化水素基としては、有用物質の分泌効率及び細胞毒性の観点から、脂肪族炭化水素基が好ましく、さらに好ましくは０～３個の不飽和結合を有する直鎖又は分岐鎖の脂肪族炭化水素基であり、特に好ましくはアルキル基及び不飽和結合を１～３個有するアルケニル基である。
また、（Ａ３１１）として具体的には、アルキルアミノプロピオン酸型両性界面活性剤（ドデシル－β－アミノプロピオン酸ナトリウム、コカミノプロピオン酸ナトリウム、ステアリルアミノプロピオン酸ナトリウム及びラウリルアミノプロピオン酸ナトリウム等）；アルキルアミノ酢酸型両性界面活性剤（ラウリルアミノ酢酸ナトリウム等）及びＮ－ラウロイル－Ｎ’－カルボキシメチル－Ｎ’－ヒドロキシエチルエチレンジアミンナトリウム等が挙げられる。

In the general formula (3), R ⁶ is a monovalent hydrocarbon group having 1 to 30 carbon atoms. 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 cations derived from amines and alkanolamines) and quaternary ammonium.
^In R6, the number of carbon atoms of the hydrocarbon group is preferably 8 to 22, more preferably 10 to 18, from the viewpoint of secretory efficiency of useful substances and cytotoxicity.
^In R6, as the hydrocarbon group, an aliphatic hydrocarbon group is preferable from the viewpoint of secretory efficiency of useful substances and cytotoxicity, and more preferably, a linear or branched fat having 0 to 3 unsaturated bonds. It is a group hydrocarbon group, and particularly preferably an alkyl group and an alkenyl group having 1 to 3 unsaturated bonds.
Specifically, as (A311), an alkylaminopropionic acid type amphoteric surfactant (sodium dodecyl-β-aminopropionate, sodium cocaminopropionate, sodium stearylaminopropionate, sodium laurylaminopropionate, etc.); Examples thereof include alkylaminoacetic acid type amphoteric surfactants (sodium laurylaminoacetate, etc.) and N-lauroyl-N'-carboxymethyl-N'-hydroxyethylethylenediamine sodium.

The betaine-type amphoteric tenside agent (A312) is an amphoteric tenside agent having a quaternary ammonium salt-type cation moiety and a carboxylic acid-type anion moiety in the molecule. Examples of (A312) include compounds represented by the following general formula (4).

In the general formula (4), R ⁷ is a monovalent hydrocarbon group having 1 to 30 carbon atoms.
In R ⁷ , the number of carbon atoms of the hydrocarbon group is preferably 8 to 22, more preferably 10 to 18, from the viewpoint of secretory efficiency of useful substances and cytotoxicity.
In R ⁷ , the hydrocarbon group is preferably an aliphatic hydrocarbon group, more preferably a straight chain and / or a branched chain having 0 to 3 unsaturated bonds, from the viewpoint of secretory efficiency of useful substances and cytotoxicity. It is an aliphatic hydrocarbon group of the above, and particularly preferably an alkyl group and an alkenyl group having 1 to 3 unsaturated bonds.

Specifically, as (A312), alkyldimethylbetaine (stearyldimethylaminoacetic acid betaine, lauryldimethylaminoacetic acid betaine, etc.), amide betaine (palm oil fatty acid amide propyl betaine, etc. (palm oil fatty acid amide propyldimethylaminoacetic acid betaine, etc.)) and Examples thereof include lauric acid amide propyl betaine), alkyldihydroxyalkyl betaine (lauryl dihydroxyethyl betaine, etc.), and cured coconut oil fatty acid amide propyldimethylaminoacetic acid betaine.

Examples of the imidazoline-type amphoteric tenside agent (A313) include 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine.

Other amphoteric surfactants include glycine-type amphoteric surfactants such as sodium lauroyl glycine, sodium lauryl diaminoethyl glycine, lauryl diaminoethyl glycine hydrochloride and dioctyl diaminoethyl glycine hydrochloride; Amphoteric surfactant; Coleamidepropyldimethylammoniopropanesulfonic acid (CHASP), Coleamidepropyldimethylammonio2-hydroxypropanesulfonic acid (CHASPO); Alkylamineoxide type amphoteric surfactant such as lauryldimethylamineoxide included.

As the amphoteric tenside agent (A3), compounds represented by the following general formulas (5) to (7) are preferable from the viewpoint of secretory efficiency of useful substances and cytotoxicity.

In the general formula (5), R ⁸ , R ⁹ and R ¹⁰ each represent a monovalent hydrocarbon group having 1 to 30 carbon atoms, p is an integer of 1 or more, and X is a sulfonate anion, a carboxylate anion or a carboxylate anion. Represents a phosphonate anion.
In the general formula (6), R ¹¹ and R ¹² each represent a monovalent hydrocarbon group having 1 to 30 carbon atoms, q is an integer of 1 or more, Y is a sulfonic acid (salt) group, and a carboxylic acid (salt). Represents a (salt) group or a phosphate (salt) group.
In the general formula (7), R ¹³ , R ¹⁴ , R ¹⁵ and R ¹⁶ each represent a monovalent hydrocarbon group having 1 to 30 carbon atoms, and r is an integer of 1 or more.

In addition, sulfonic acid (salt) means sulfonic acid and / or sulfonate, carboxylic acid (salt) means carboxylic acid and / or carboxylate, and phosphoric acid (salt) means phosphoric acid and / Or means phosphate. Examples of the salt include alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.) and onium cation salts (ammonium cations, quaternary ammonium cations, tertiary sulfonium cations, quaternary sulfonium cations, etc.). Quaternary phosphonium cations, tertiary oxonium cations, etc.) are included.

Further, in the general formulas (5) to (7), the hydrocarbon group is at least one substituent selected from the group consisting of a hydroxyl group, an ether group, a carbonyl group and an ester group at any position of the hydrocarbon group. May have.

In the general formula (5), at least one of R ⁸ , R ⁹ and R ¹⁰ is preferably a monovalent hydrocarbon group having 8 to 22 carbon atoms from the viewpoint of secretory efficiency of useful substances and cytotoxicity. More preferably, at least one of R ⁸ , R ⁹ and R ¹⁰ is a monovalent hydrocarbon group having 10 to 18 carbon atoms.
^In R8, ^R9 and ^R10 , as the hydrocarbon group, an aliphatic hydrocarbon group is preferable from the viewpoint of secretory efficiency of useful substances and cytotoxicity, and more preferably a direct having 0 to 3 unsaturated bonds. It is an aliphatic hydrocarbon group having a chain and / or a branched chain, and is particularly preferably an alkenyl group having 1 to 3 alkyl groups and unsaturated bonds.
Further, p is preferably an integer of 1 to 10, and more preferably an integer of 1 to 5, from the viewpoint of secretory efficiency of useful substances and cytotoxicity.

In the general formula (6), at least one of R ¹¹ and R ¹² is preferably a monovalent hydrocarbon group having 8 to 22 carbon atoms, more preferably, from the viewpoint of secretory efficiency of useful substances and cytotoxicity. Is that at least one of R ⁶¹¹ and R ⁷¹² is a monovalent hydrocarbon group having 10 to 18 carbon atoms.
In R ¹¹ and R ¹² , as the hydrocarbon group, an aliphatic hydrocarbon group is preferable from the viewpoint of secretory efficiency of useful substances and cytotoxicity, and more preferably, a straight chain having 0 to 3 unsaturated bonds and / /. Alternatively, it is an aliphatic hydrocarbon group having a branched chain, and particularly preferably an alkyl group and an alkenyl group having 1 to 3 unsaturated bonds.
From the viewpoint of secretory efficiency of useful substances and cytotoxicity, q is preferably an integer of 1 to 10, and more preferably an integer of 1 to 5.
As the salt, an alkali metal salt is preferable, and a sodium salt is more preferable, from the viewpoint of secretion efficiency of useful substances and cytotoxicity.

In the general formula (7), at least one of R ¹³ , R ¹⁴ , R ¹⁵ and R ¹⁶ is a monovalent hydrocarbon group having 8 to 20 carbon atoms from the viewpoint of secretory efficiency of useful substances and cytotoxicity. It is preferable, and more preferably, at least one of R ¹³ , R ¹⁴ , R ¹⁵ and R ¹⁶ is a monovalent hydrocarbon group having 10 to 18 carbon atoms.
In R ¹³ , R ¹⁴ , R ¹⁵ and R ¹⁶ , the hydrocarbon group is preferably an aliphatic hydrocarbon group, more preferably 0 to 3 unsaturated bonds, from the viewpoint of secretory efficiency of useful substances and cytotoxicity. It is a straight chain and / or a branched chain aliphatic hydrocarbon group having, particularly preferably an alkyl group and an alkenyl group having 1 to 3 unsaturated bonds.
Further, r is preferably an integer of 1 to 10, and more preferably an integer of 1 to 5, from the viewpoint of secretory efficiency of useful substances and cytotoxicity.

Specific examples of the compound represented by the general formula (5) include those in which X is a sulfonate anion {dodecyldimethyl (3-sulfopropyl) ammonium hydroxide, 3- [tetradecyldimethylammonio] propan-1-sulfonate. And arachidyldimethyl (3-sulfopropyl) ammonium hydroxide etc.}, X is a carboxylate anion {lauryldimethylaminoacetic acid betaine and stearyldimethylaminoacetic acid betaine etc.}, X is a phosphonate anion {dodecyldimethyl (dodecyldimethyl) 3-phosphopropyl) ammonium hydroxide} and the like.

Specific examples of the compound represented by the general formula (6) include those in which Y is a sulfonic acid (salt) {(2-dodecylamino) sodium ethanesulfonate, 2-[(1-oxododecyl) amino] ethane. Sodium sulfonate and 2- (N-methyl-N-palmitoylamino) sodium ethanesulfonate, etc.}, Y is a carboxylic acid (salt) {3- (dodecylamino) sodium propanoate and dodecyl-β-aminopropion Sodium acid such as}, those in which Y is a phosphoric acid (salt) {(2-dodecylamino) sodium ethane phosphate} and the like can be mentioned.

Specific examples of the compound represented by the general formula (7) include miltefosine, dodecylphosphorylcholine, hexadecylphosphorylcholine, 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine and the like.

In the present invention, the ionic surfactant (A) may be used as it is, or may be mixed with water if necessary and used as an aqueous diluted solution (aqueous solution or aqueous dispersion liquid).

As the ionic surfactant (A), an amphoteric tenside agent (A3) and a cationic surfactant (A2) are preferable, and carbonization having 8 to 20 carbon atoms is more preferable, from the viewpoint of secretory efficiency of useful substances and cytotoxicity. An amphoteric surfactant (A3) having a hydrogen group and a cationic surfactant (A2) having a hydrocarbon group having 8 to 20 carbon atoms, particularly preferably an amphoteric surfactant having a hydrocarbon group having 10 to 18 carbon atoms (A). A3) and a cationic surfactant (A2) having a hydrocarbon group having 10 to 18 carbon atoms.

The amount of the ionic 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 the useful substance to be produced, and the type of the extraction method. Based on the weight of the culture solution at the time, 0.0001 to 10% by weight is preferable, and more preferably 0, from the viewpoint of cytotoxicity, the amount of useful substance obtained, the secretory efficiency of useful substances, and the difficulty of protein denaturation. It is 0005 to 5% by weight, more preferably 0.001 to 1% by weight, and particularly preferably 0.005 to 0.1% by weight.

The nonionic surfactant (B) used together with the ionic surfactant (A) in the method for producing a useful substance of the present invention is a nonionic surfactant having an HLB value of 3 to 20 from the viewpoint of producing a useful substance.

HLB is known as a measure for showing the hydrophilicity and hydrophobicity of the nonionic surfactant (B). The higher the HLB value, the higher the hydrophilicity. The HLB in the present invention is a numerical value calculated by the following formula (1) (Takehiko Fujimoto, Introduction to Surfactants, p. 212, published by Sanyo Chemical Industries, Ltd.).
HLB = 10 × (inorganic / organic) (1)

As the nonionic surfactant (B) used in the method for producing a useful substance of the present invention, a nonionic surfactant having an HLB value of 3 to 20 may be used, but the amount of useful substance obtained and the difficulty of protein modification are used. From the viewpoint of the efficiency of secreting useful substances, it is preferable to use a nonionic surfactant (B1) having a HLB of 13 or more and less than 13 and a nonionic surfactant (B2) having an HLB of 13 or more and 20 or less in combination.

As the nonionic surfactant (B), the weight ratio of the ionic surfactant (A) and the nonionic surfactant (B1) in the culture solution is the amount of useful substance obtained, the difficulty of protein denaturation, and the useful substance. From the viewpoint of secretion efficiency, it is preferably 1: 0.5 to 1: 5, and more preferably 1: 1 to 1: 5.
The weight ratio of the ionic surfactant (A) and the nonionic surfactant (B2) is 1: 1 to 1: 100 from the viewpoints of the amount of useful substance obtained, the difficulty of protein denaturation, and the efficiency of secretion of useful substances. , More preferably 1: 1 to 1:20, and particularly preferably 1: 1 to 1:10.
The weight ratio of the nonionic surfactant (B1) to the nonionic surfactant (B2) is 1: 0.001 to 1 from the viewpoint of the amount of useful substance obtained, the difficulty of protein denaturation, and the efficiency of secreting useful substances. : 100 is preferable, and more preferably 1: 0.01 to 1:10.

The nonionic surfactant (B) is an esterified product of a fatty acid and a polyether alcohol; a monoalcohol alkylene oxide (hereinafter, the alkylene oxide may be abbreviated as AO) adduct; an alkylphenol AO adduct; a fatty acid AO adduct. Products; Examples thereof include polyhydric alcohol-type nonionic surfactants and pluronic-type nonionic surfactants.

Regarding the esterified product of fatty acid and polyether alcohol, the fatty acid contains a fatty acid having 10 to 22 carbon atoms, and specifically, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and coconut. Examples include oil fatty acids.
The polyether alcohol includes an alkylene oxide adduct having 2 to 4 carbon atoms to a compound having an active hydrogen group.
Examples of the compound having an active hydrogen group include a hydroxyl group-containing compound (water, polyvalent (2 to 8-valent or higher) alcohol, polyvalent (2 to 3-valent) phenol, bisphenol compound, etc.), and an amino group-containing compound (ammonia, Monoamines, poly (2 to 3 or more) amines, alkanolamines and other polyamines), carboxyl group-containing compounds [aliphatic polycarboxylic acids (succinic acid, adipic acid, etc.), aromatic polycarboxylic acids (phthalic acid, tri) Merit acid etc.) and poly (2-100) carboxylic acid polymer {(meth) acrylic acid (co) polymer} etc.], thiol group-containing compounds (ethylenedithiol and 1,6-hexanedithiol etc.), phosphoric acid Included are system compounds (phosphoric acid, phobic acid, phosphonic acid, etc.) and mixtures of two or more of these.
Examples of the alkylene oxide having 2 to 4 carbon atoms include ethylene oxide (hereinafter abbreviated as EO), 1,2-propylene oxide (hereinafter abbreviated as PO) and 1,2-butylene oxide (hereinafter abbreviated as BO). It is abbreviated.) And the like.

Specific examples of the esterified product of the fatty acid and the polyether alcohol include lauric acid ester EO15 mol PO30 mol addition (HLB = 7.6), oleic acid ester EO15 mol PO30 mol addition (HLB = 7.0), and lauryl. Acid ester EO 30 mol PO30 mol adduct (HLB = 9.9), oleic acid ester EO 30 mol PO30 mol adduct (HLB = 9.4), lauric acid ester EO290 mol adduct (HLB = 18.4) and the like. Be done.

Alcohol AO adducts include polyoxyethylene alkyl ethers and polyoxyalkylene alkyl ethers.
Specifically, 0 to 20 mol of ethylene oxide (hereinafter, ethylene oxide is abbreviated as EO) of higher alcohols having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, palm oil alkyl alcohol, octadecyl alcohol, oleyl alcohol, etc.) and / Or propylene oxide (hereinafter, propylene oxide is abbreviated as PO) 1 to 20 mol adduct (including block adduct and / or random adduct; the same applies hereinafter) [For example, EO 8 mol / PO 7 mol block adduct of decyl alcohol. ] Is included.

More specifically, as the alcohol AO adduct, a lauryl alcohol EO 7 mol adduct (HLB = 12.4), an oleyl alcohol EO 5 mol adduct (HLB = 9.0), and an oleyl alcohol EO 6 mol adduct (HLB = 10. 2), oleyl alcohol EO 7 mol adduct (HLB = 11.0), oleyl alcohol EO 10 mol adduct (HLB = 12.4), 1,2-dodecanediol monooxyethylene adduct and the like can be mentioned.

The alkylphenol AO adduct includes an alkylphenol AO adduct having an alkyl group having 6 to 24 carbon atoms.
Specific examples thereof include EO 1 to 20 mol and / or PO 1 to 20 mol adduct of octylphenol and EO 1 to 20 mol and / or PO 1 to 20 mol adduct of nonylphenol. Further, TRITONTMX-114 (HLB = 12.4), igepalTMCA-520 (HLB = 10.0), igepalTMCA-630 (HLB = 13.0) and the like are easily available from the market.

Fatty acid AO additions include EO-1 to 20 mol and / or PO1 to 20 mol of fatty acids having 8 to 24 carbon atoms (decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, coconut oil fatty acid, etc.). Includes additives.
Specifically, as the fatty acid AO adduct, an oleic acid EO 9 mol adduct (HLB = 11.8), a dioleic acid EO 12 mol adduct (HLB = 10.4), and a dioleic acid EO 20 mol adduct (HLB = 12.9). ) And EO9 mol adduct of stearate (HLB = 11.9) and the like.

Examples of the polyhydric alcohol-type nonionic surfactant include EO and / or PO adducts of 2- to 8-valent polyhydric alcohols (glycerin, trimethylolpropane, pentaerythritol, sorbit, sorbitan, etc.) having 3 to 36 carbon atoms; Includes fatty acid esters of polyhydric alcohols and their EO adducts, as well as fatty acid esters of sucrose, fatty acid alkanolamides (such as coconut oil fatty acid diethanolamide) and their AO adducts.

Specific examples of the polyhydric alcohol-type nonionic surfactant include sorbitan tetraoleic acid ester EO adduct (HLB = 11.4), sorbitan hexaoleic acid ester EO adduct (HLB = 10.2), and glycerin PO 50 mol. An object (HLB = 4.2) and the like can be mentioned.

Pluronic nonionic surfactants include block copolymers of EO and PO.
From the viewpoint of difficulty in denaturing useful substances, cytotoxicity, and secretory efficiency of useful substances, the number average molecular weight (hereinafter abbreviated as Mn) is preferably 1000 to 50,000, and more preferably 3000 to 30,000.
The molar ratio of EO to PO (EO / PO) is preferably 2/1 to 3500/1 from the viewpoint of difficulty in denaturing useful substances, cytotoxicity, and secretory efficiency of useful substances.

Specifically, as the pluronic nonionic surfactant, EO60PO30 molblock copolymer (HLB = 13.1), EO142PO30 molblock copolymer (HLB = 15.6), EO168PO34 molblock copolymer (HLB = 15.). 8), EO290PO56 mol block copolymer (HLB = 15.9) can be mentioned.

Among these nonionic surfactants (B), esterified products of fatty acids and polyether alcohols and pluronic nonionic surfactants are preferable from the viewpoint of cytotoxicity and secretory efficiency of useful substances.

The amount of the nonionic surfactant (B) used in the method for producing a useful substance of the present invention is appropriately selected depending on the target microorganism, the type of the useful substance produced, and the type of the extraction method. From the viewpoint of the amount of useful substance obtained, the difficulty of protein denaturation, and the efficiency of secretion of useful substances based on the weight of the culture solution at the time, 0.0001 to 20% by weight is preferable, and 0.005 to more preferably. It is 10% by weight, more preferably 0.007 to 7% by weight, and particularly preferably 0.01 to 5% by weight.
The amount of the nonionic surfactant (B1) having an HLB of 3 or more and less than 13 is the amount of the useful substance obtained, the difficulty of protein denaturation, the secretory efficiency of the useful substance, and the cytotoxicity at the start of the culture. Based on the weight of the culture broth, it is preferably 0.005 to 10% by weight, more preferably 0.007 to 7% by weight, and particularly preferably 0.01 to 1% by weight.
The amount of the nonionic surfactant (B2) having an HLB of 13 to 20 is the amount of the useful substance obtained, the difficulty of protein denaturation, the secretory efficiency of the useful substance, and the cell toxicity. Based on the weight of the liquid, it is preferably 0.005 to 10% by weight, more preferably 0.007 to 7% by weight, and particularly preferably 0.01 to 5% by weight.

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

The secretory efficiency indicates that the useful substance in the microorganism is secreted to the outside of the microorganism (in the culture solution) by the surfactant.
In the present invention, it is defined by the following formula.
Secretion efficiency (%) = 100 × (X / OD) / {(X / OD) + (Y / OD)}
X (culture supernatant): Concentration of useful substances in the culture solution remaining after removal of the cells by centrifugation Y (cell surface): Wash the cells collected by centrifugation and use the same amount as the culture solution at the time of recovery. Concentration of useful substances in cell suspension resuspended in physiological saline OD: Cell concentration during centrifugation

As for the secretory efficiency, for example, if the protein produced in the microorganism is more periplasmically transferred, the secretory efficiency is increased, and if the protein produced in the microorganism is not transferred to the periplasm, the secretory efficiency is decreased. In addition, the secretory efficiency can be increased by selecting a surfactant having a high secretory efficiency by screening.

The ionic surfactant (A) and the nonionic surfactant (B) may be added to the culture solution in which the microorganism is suspended later, in addition to being used by mixing with the culture solution in advance. Mixing with the culture solution can be performed by adding a surfactant to the culture solution and stirring with a stirring blade, a stirrer or the like. When mixing later, it can be added while stirring with a stirring blade or the like.

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

The turbidity of the culture solution in the method for producing a useful substance of the present invention is preferably 1 to 300 OD, more preferably 10 to 300 OD, and particularly preferably 20 to 300 OD.
When the turbidity of the culture solution is 1 OD or more, the amount of useful substances produced increases, and when the turbidity is 300 OD or less, the production of useful substances is easy, which is preferable.
In the method for producing a useful substance of the present invention, within the above range, the greater the turbidity of the culture solution, the greater the amount of the useful substance produced.

In the method for producing a useful substance of the present invention, from the viewpoint of the production amount of the useful substance, the time during which the turbidity of the culture solution is within the above range is 10% or more of the time required for the step of secreting the useful substance. It is preferable, more preferably 50% or more.

The turbidity of the culture can be increased, for example, by feeding at an appropriate rate using a semi-batch culture method under sufficient aeration conditions and reduced by performing batch cultures under restricted aeration conditions. be able to. Further, it can be increased by lengthening the time from the start of culturing to the addition of the surfactant, and can be decreased by shortening the time from the start of culturing to the addition of the surfactant. Further, it can be increased by slowing the charging rate of the surfactant and decreasing by increasing the charging rate.

In the method for producing a useful substance of the present invention, the production method for secretory production of a useful substance includes a microbial exocrine production method including the following steps (a) and (b). In the following steps, the step of secreting and producing useful substances is step (a).
Step (a): A step of simultaneously presenting a culture solution for culturing a microorganism (yeast or the like) that produces a useful substance and a surfactant to secrete the useful substance to the outside of the microorganism (in the culture solution).
Step (b): After step (a), a step of separating useful substances from the culture broth.

The following is an example of a method for producing a useful substance using the surfactant of the present invention.
(I) Genetic recombination (i-1) A messenger RNA (mRNA) is isolated from cells expressing the target protein, a single-stranded cDNA is synthesized from the mRNA, and then a double-stranded DNA is synthesized, and the two strands are synthesized. Incorporate strand DNA into phage DNA or plasmid. The obtained recombinant phage or plasmid is transformed into a host microorganism to prepare a cDNA library.
(I-2) As a method for screening a phage DNA or a plasmid containing the target DNA, a hybridization method between the phage DNA or the plasmid and a DNA probe encoding a part of the target protein gene or complementary sequence can be mentioned. ..
(I-3) The target gene by cutting out the target cloned DNA or a part thereof from the phage or plasmid after screening and ligating the cloned DNA or a part thereof downstream of the promoter in the expression vector. Expression vector can be prepared. DNA encoding a signal sequence that translocates the intima (a signal sequence that expresses the target substance in the periplasm) can also be ligated at the same time.
(Ii) Culture (ii-1) The host microorganism is transformed with an expression vector to prepare a recombinant microorganism, and the recombinant microorganism is precultured. Preculture is usually carried out on an agar medium at 15-43 ° C. for 3-72 hours.
(Ii-2) The culture medium used for the production of useful substances is autoclaved at 121 ° C. for 20 minutes, and the recombinant microorganisms precultured on an agar medium are cultured here. Culturing is usually carried out at 15 to 43 ° C. for 12 to 72 hours. When a surfactant is used at the same time as the start of culturing, the same operation is performed by using a homogenized mixture of the surfactant and the culture broth as the culture broth. When the surfactant is added 6 to 72 hours after the culture, the culture is continued for 1 to 1000 hours after the surfactant is added.
(Iii) Purification (iii-1) The protein secreted in the culture medium is 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 treatments such as an ion exchange column, a gel filtration column, a hydrophobic column, an affinity column and an extraordinary column, and is subjected to a precipitation treatment such as ethanol precipitation, ammonium sulfate precipitation and polyethylene glycol precipitation. It is separated and purified by performing it as needed.

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

When column treatment is performed in the above purification step (iii), examples of the filler used for column chromatography include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer and the like, and commercially available products include the Capto series. Examples thereof include Sephadex series, Sephadex series, Sepharose series (hereinafter referred to as 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 yeast can be secreted into the culture medium with high efficiency, and a high yield can be obtained in a short time. Can be achieved. Further, in the method for producing a useful substance of the present invention, since the useful substance is secreted into the culture broth, the purification of the useful substance is easy.

Since the useful substance obtained by the production method of the present invention is obtained by the above method, it has a higher specific activity than the conventional one.

The method for producing a useful substance of the present invention comprises a step of simultaneously presenting a surfactant and yeast to secrete the useful substance into the culture broth.

In this step, as long as the yeast is alive, it is presumed that the yeast can produce useful substances and secrete them into the culture medium. Further, if yeast has the ability to produce a useful substance, it is presumed that the production method of the present invention can be used regardless of the type of the useful substance to be produced.
The method for producing a useful substance of the present invention is particularly effective when the useful substance produced in yeast is transferred to the periplasm of yeast. The transition of useful substances to periplasm facilitates the secretion of useful substances into the culture medium.

The present invention will be further described with reference to the following Examples and Comparative Examples, but the present invention is not limited thereto. Unless otherwise specified, parts mean parts by weight.
Further, in the following, Examples 25 and 50 mean Reference Examples 1 and 2.

<Examples 1 to 25>
Luciferase-expressing yeast (Pichia pastoris) was transformed using Pichia pastoris distributed by (Biotechnology Center, Product Evaluation Technology Infrastructure Organization). The pPICZα vector (manufactured by Thermo) was cleaved with XhoI and XbaI, and an artificially synthesized (consigned synthesis by Thermo) luciferase gene having XhoI and XbaI cleavage sites was incorporated into both ends. Transformation of the luciferase gene-incorporated pPICZα vector into yeast was carried out by the method described in the pPICZα vector instruction manual. A 125 ml BMGY culture was prepared by inoculating luciferase-expressing yeast into a BMGY culture solution using a loop loop and shaking it at 30 ° C. for 15 hours at 200 rpm so that the final OD was 0.1 OD / ml. Liquid (1 wt% Yeast extract manufactured by Difco, 2 wt% Bacto peptone manufactured by Difco, 1.34 wt% Yeast nitrogen base w / o amino acid 1.34 wt% manufactured by Difco, re-potassium hydrogen phosphate, pH 6.0). Suspended. Further, the resuspension was continuously stirred and cultured at 30 ° C. for about 17 hours at 1100 rpm until the OD reached 20 to 30. When the OD reached 20 to 30, the culture broth was centrifuged (1500 g, 10 minutes), resuspended in 125 ml BMMY culture broth, and cultured at 30 ° C. and 1100 rpm for 2 hours. After 2 hours, the surfactants shown in Tables 1 and 2 were added to the weight% shown in Tables 1 and 2, and the cells were cultured at 30 ° C. and 1100 rpm for 8 hours. Then, sampling was performed for measuring turbidity (turbidity at the end of culture), and the cells and the culture supernatant were separated from the culture solution by a centrifuge, and the culture supernatant and the cells were collected.

<Comparative Example 1>
In Example 1, the same procedure was carried out except that pure water was added instead of the surfactant, and the culture supernatant and the cells were collected.

<Examples 26 to 50>
Acid phosphatase-expressing yeast (Candida boidinii known literature (Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii, H. Yurimoto et al., Biochimica et Biophysica Acta, 1493, 2000, the method described in 56-63) Inoculated into BMGY medium using platinum ears and shake-cultured at 30 ° C. for 15 hours at 200 rpm to prepare a culture solution having a final OD of 0.1 OD / ml, 125 ml MeOH. It was resuspended in the medium. Further, the resuspension was continuously stirred and cultured at 30 ° C. for about 17 hours at 1100 rpm until the OD became 2 to 5. When the OD reached 2 to 5, methanol was added so that the OD was 2 v / v%, and the cells were cultured at 30 ° C. and 1100 rpm for 2 hours. After 2 hours, the compounds (1) to (13) shown in Tables 3 and 4 were added by the weights shown in Tables 3 and 4, and cultured at 30 ° C. and 1100 rpm for 8 hours. After 8 hours, sampling was performed for measuring turbidity (turbidity at the end of culture), cells and culture supernatant were separated from the culture solution by a centrifuge, and the culture supernatant and cells were collected.
The cells were washed by adding 50 mM NaBf acetate (pH 4.0) to the cells and centrifuging. The washed cells were resuspended with 50 mM NaBf acetate (pH 4.0) to prepare a sample for measuring acid phosphatase activity on the surface of the cells.

<Comparative Example 2>
In Example 26, the same procedure was carried out except that pure water was added instead of compound (4), and the culture supernatant and bacterial cells were collected.

The following compounds (1) to (14) were used in Tables 1 to 4. As the compounds (1) to (8), compounds having an alkyl group having 12 carbon atoms were used.
Compound (1): Polyoxyethylene (added 2.5 mol of EO) Sodium lauryl ether sulfate Compound (2): Polyoxyethylene (EO3 mol) Sodium lauryl ether acetate compound (3): 2- (Dodecylamino) Sodium ethanesulfonate Compound (4): Dodecyl-β-aminoethylsulfonate sodium compound (5): Dodecyldimethyl (3-sulfopropyl) ammonium hydroxyd compound (6): Dodecylphosphorylcholine compound (7): Lauryldimethylaminoacetic acid betaine compound (8) ): Lauryltrimethylammonium chloride compound (9): Glycerin PO 50 mol adduct, HLB = 4.2
Compound (10): Oleyl acid ester EO 15 mol PO 30 mol adduct, HLB = 7.0
Compound (11): Oleyl acid ester EO 30 mol PO 30 mol adduct, HLB = 9.4
Compound (12): EO60PO30 molblock copolymer, HLB = 13.1
Compound (13): EO142PO30 molblock copolymer, HLB = 15.6
Compound (14): Lauric acid ester EO290 mol adduct, HLB = 18.4

With respect to the culture supernatants obtained in Examples 1 to 50 and Comparative Examples 1 and 2, the turbidity of the culture solution, the amount of secreted production of useful substances, the secretory efficiency and the like were evaluated as described below.
The results are shown in Tables 1-4.

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

<Evaluation of secretory production of useful substances: Measurement of luciferase activity of secreted and produced useful substances>
20 μl of each culture supernatant obtained in Examples 1 to 25 and Comparative Example 1 diluted 10 to 1000 times with 0.2 M Tris-HCl buffer (pH 7.4) was mixed with 60 μl of the following luciferin solution added. Using the solution as a sample, the luminescence intensity measured under the following conditions using a luminometer was used as the luciferase activity of the useful substance secreted and produced by applying the value obtained by applying the following formula.
Luminometer: "Glomax Navigator" manufactured by Promega Co., Ltd.
Luciferin solution: New England Biolab Japan Co., Ltd. Product measurement temperature: 25 ° C
Detection device: Luminescent detector Detection wavelength: 350-700nm
[Luciferase activity of secreted and produced useful substances] = [Emission intensity] x Dilution ratio / [Turbidity at the end of culture]
In Tables 1 and 2, the luciferase activity was represented by a relative value when Comparative Example 1 was set to 1.
The higher the luciferase activity of the secreted and produced useful substance, the larger the amount of secreted and produced useful substance.

<Measurement of luciferase activity of useful substances remaining on the cell surface>
To the cells obtained in Examples 1 to 25 and Comparative Example 1, 200 μl of 0.2 M Tris-HCl buffer (pH 7.4) in the same amount as the removed supernatant was added, and the cells were washed by centrifugation. bottom. The washed cells were resuspended in 200 μl of 0.2 M Tris-HCl buffer (pH 7.4) in the same amount as the washed supernatant, and 10 in 0.2 M Tris-HCl buffer (pH 7.4). A sample diluted ~ 1000 times was used as a sample for measuring the luciferase activity on the cell surface.
Using a mixed solution prepared by adding 60 μl of the above luciferin solution to 20 μl of the sample for measuring luciferase activity on the surface of the cells as a sample, the luminescence intensity was measured under the same conditions as in "Measurement of luciferase activity of secreted and produced useful substances", and the measured luminescence was measured. The intensity was applied to the following formula, and the value obtained was taken as the luciferase activity of the useful substance remaining on the cell surface.
[Luciferase activity of useful substances remaining on the cell surface] = [ Luminescent value] x Dilution ratio / [Turbidity at the end of culture]
In Tables 1 and 2, the luciferase activity was represented by a relative value when Comparative Example 1 was set to 1.

<Evaluation of secretory efficiency>
Using the culture supernatants and cells obtained in Examples 1 to 25 and Comparative Example 1, the secretory efficiency in each example was calculated by the following formula.
Secretory efficiency (%) = 100 x "Luciferase activity of secreted and produced useful substances" / "Total luciferase activity"
The total luciferase activity means "luciferase activity of secreted and produced useful substance" + "luciferase activity of useful substance remaining on the cell surface".

<Measurement of protein amount: Acid phosphatase activity measurement>
To each of the culture supernatants obtained in Examples 26 to 50 and Comparative Example 2, 1/40 amount of 2M sodium acetate buffer (pH 4.0) was added to 5 μl. A substrate solution (p-nitrophenylphosphate 0.64 mg / ml-containing 50 mM sodium acetate buffer pH 4.0) was added to the culture supernatant to which the buffer was added or the cell surface measurement samples obtained in Examples 17 to 32 by volume ratio = 1. The mixture was mixed at 1: 1 and reacted at 35 ° C. for 10 minutes.
After the reaction, an equal amount of 10% trichloroacetic acid solution as the reaction solution was added to terminate the reaction. To the solution in which the reaction was stopped, an equal amount of a saturated solution of sodium carbonate was added to the whole reaction solution to develop a color. The insoluble fraction was removed from the developed liquid at 1500 × g in 5 minutes, and the absorbance at 420 nm was measured using (Biotek microplate reader PowerWave). The reference wavelength was 630 nm.
The acid phosphatase activity was defined as the value calculated from the following formula for each of the culture supernatant and the cell surface measurement sample. In the result of the acid phosphatase activity of the obtained culture supernatant, the relative value when the value of Comparative Example 2 was set to 1 was defined as the "acid phosphatase activity of the secreted and produced useful substance", and the acid phosphatase activity of the cell surface measurement sample. The relative values when the value of Comparative Example 2 was set to 1 are shown in Tables 3 and 4 as "acid phosphatase activity of useful substances remaining on the cell surface".
Acid phosphatase activity = {(Abs420nm)-(Abs630nm)} / (turbidity at the end of culture)

<Evaluation of secretory efficiency>
Using the values of "acid phosphatase activity of the useful substance secreted and produced" and "acid phosphatase activity of the useful substance remaining on the cell surface" obtained in Examples 26 to 50 and Comparative Example 2, the secretory efficiency was calculated from the following formula. Calculated. The results are shown in Tables 3 and 4.
Secretory 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 the useful substance secreted and produced" + "acid phosphatase activity of the useful substance remaining on the cell surface".

From Tables 1 to 4, it can be seen that the inclusion of the ionic surfactant (A) and the nonionic surfactant (B) in the culture solution increases the secretory efficiency of useful substances to the outside of the cells.
From the comparison with Examples 24 and 25 and Comparative Example 1 in Tables 2 and 4, and the comparison with Examples 49 and 50 and Comparative Example 2, the nonionic surfactant (B1) or ( It can be seen that the inclusion of either one of B2) increases the secretory efficiency, although the degree of increase in the secretory efficiency varies depending on the strain used.
Further, from Tables 1 to 4, it can be seen that the secretion efficiency is extremely high by combining the three types of the ionic surfactant (A), the nonionic surfactant (B1) and the nonionic surfactant (B2).
Further, from Examples 1 to 4 and 26 to 29, when the ionic surfactant (A) is excessively added, the secretory efficiency increases, but the turbidity decreases due to cytotoxicity, and the susceptibility to cytotoxicity depends on the strain. It can be seen that the difference is about 10 times.
Further, from Examples 2, 13 to 16, 27 and 38 to 41, the HLB of the nonionic surfactant (B1) is about 7 for the highest secretory efficiency, and the nonionic surfactant (B2) has an HLB of about 15 for the nonionic surfactant (B2). It can be seen that the secretory efficiency is high.
From Examples 2, 17 to 23 and 27, 42 to 48, the effect on the secretory efficiency when the ionic surfactant (A) having different ionicity was used was found, and the effects of the amphoteric surfactant and the cationic surfactant were found. It turns out that is high.
Further, in the examples, since the total luciferase activity is large, it can be seen that a large amount can be obtained without denaturing useful substances.

The method for producing a useful substance of the present invention can be used when extracting a useful substance such as a protein from eukaryotes. Examples of proteins include enzymes, hormonal proteins, antibodies and peptides. When the produced protein is an enzyme (protease, cellulase, lipase, amylase, etc.), it can be suitably used for food processing, cleaning agent, fiber processing, papermaking, enzyme conversion, etc., especially biopharmaceuticals. It is useful for use in the production of.

Claims (5)

A method for producing a useful substance by secreting and producing a useful substance in the culture solution by yeast contained in the culture solution. Yeast is at least one selected from the group consisting of the genus Pichia and the genus Candida , and is contained in the culture solution. Containing an ionic surfactant (A) and a nonionic surfactant (B),
The ionic surfactant (A) is one or more surfactants selected from the group consisting of an anionic surfactant (A1), a cationic surfactant (A2) and an amphoteric surfactant (A3), and is a nonionic surfactant. The agent (B) is a nonionic surfactant having an HLB value of 3 to 20, and the nonionic surfactant (B) is an esterified product of a fatty acid and a polyether alcohol, a polyhydric alcohol type nonionic surfactant and a pluronic type nonionic. One or more surfactants selected from the group consisting of surfactants, and the content of the nonionic surfactant (B) is 0. A method for producing a useful substance containing a nonionic surfactant (B1) having an HLB value of 3 or more and less than 13 as a nonionic surfactant (B) having an HLB value of 01 to 5% by weight . The method for producing a useful substance according to claim 1 , wherein the nonionic surfactant (B) contains a nonionic surfactant (B2) having an HLB value of 13 or more and 20 or less. The nonionic surfactant (B) contains a nonionic surfactant (B1) having an HLB of 3 or more and less than 13 and a nonionic surfactant (B2) having an HLB of 13 or more and 20 or less, and has ionic surfactant activity in a culture solution. The weight ratio of the agent (A) to the nonionic surfactant (B1) is 1: 0.5 to 1: 5, and the weight ratio of the ionic surfactant (A) to the nonionic surfactant (B2) is 1. The method for producing a useful substance according to claim 1 or 2 , which is 1 to 1: 100. The method for producing a useful substance according to any one of claims 1 to 3 , wherein the nonionic surfactant (B) contains an esterified product of a fatty acid and a polyether alcohol and / or a pluronic nonionic surfactant. The method for producing a useful substance according to any one of claims 1 to 4 , wherein the useful substance is a protein.