JP5808529B2 - Useful substance production method - Google Patents

Description

  The present invention relates to a method for producing useful substances. Specifically, the present invention relates to a method for producing a useful substance that secretes and produces a useful substance by causing a surfactant and bacteria to exist simultaneously.

Bacteria are widely used for producing useful substances such as amino acids and proteins. Escherichia coli is widely used as a bacterium for the production of useful substances. Along with the advancement of genetic engineering technology, we know the technology for efficiently producing useful proteins by introducing genes of proteins useful in medicine and industry into E. coli. It has been.
Usually, when protein is expressed using Escherichia coli, physical disruption methods such as ultrasonic waves, high-pressure homogenizers, and French presses are used to extract the target protein. However, these physical disruption methods have a problem that purity is lowered because a large amount of substances other than the target protein present in the cells of E. coli are mixed when the protein is extracted.
As a method for producing a useful substance that solves this problem, a method for secreting and producing a useful substance is known. Secretion production of a useful substance is effective both for the purpose of obtaining the target useful substance with high purity and for achieving high productivity.

In secretory production of useful substances, a method using a surfactant is known. For example, in the production of glutamic acid, polyoxyethylene sorbitan monopalmitate, which is a kind of surfactant, is used (Patent Document 1). A surfactant is also used in the production of proteins such as cellulase (Non-patent Document 1).
When secretory production is performed, high productivity can be maintained, and a longer production time leads to improved productivity. However, it is not known about factors necessary to maintain high productivity and prolong production time.

International Publication No. 99/07853 Pamphlet

Bioindustry Association Fermentation and Metabolism Study Group, “Fermentation Handbook”, Kyoritsu Shuppan, July 2001, page 253

  The problem to be solved by the present invention is to provide a culture method for sustaining bacterial secretory production in a secretory production method for useful substances using a surfactant.

The inventor of the present invention has arrived at the present invention as a result of studies to achieve the above object.
That is, the present invention is a method for extracellular secretion production of a useful substance in which the following step (a) is performed and further step (b) is performed, and the amount of Kjeldahl nitrogen in the culture solution at the start of culture is based on the volume of the culture solution As a useful substance production method of 4.5 to 50 g / L.
Step (a): A step of secreting a useful substance out of the cell by simultaneously presenting a culture solution for culturing bacteria producing the useful substance and a surfactant.
Step (b): A step of separating useful substances from the culture solution after the step (a).

The present invention has the following effects.
By using the culturing method of the present invention in the secretory production process of useful substances using surfactants, the yield of useful substances can be improved, higher productivity can be maintained, and the production time can be extended. As a result, the productivity of useful substances can be improved.

  Examples of bacteria in the present invention are given below, but are not limited thereto. Bacteria include eubacteria and archaea. Eubacteria include gram negative bacteria and gram positive bacteria. Gram-negative bacteria include Escherichia, Thermus, Rhizobium, Pseudomonas, Shewanella, Vibromo, Vibromo, Vibromo Examples include Salmonella, Acetobacter genus, Synechocystis genus, and the like. Gram-positive bacteria include the genus Bacillus (genus Bacillus), the genus Streptomyces (genus Streptomyces), the genus Corynebacterium (genus Corynebacterium), the genus Brevibacillus, the genus Bifidobacterium, and the genus Bifidobacterium. Examples include the genus Coccus (genus Lactococcus), the genus Enterococcus (genus Enterococcus), the genus Pediococcus (genus Pediococcus), the genus Leuconostoc (genus Leuconostoc), and the genus Streptomyces. Moreover, the bacterium to be used may have a specific gene deletion or gene overexpression.

  Among these, Escherichia bacteria are preferable from the viewpoint of productivity of useful substances, and Escherichia coli is more preferable.

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

  Recombinant proteins include enzymes (oxidoreductases (cholesterol oxidase, glucose oxidase, ascorbate oxidase, peroxidase, etc.), hydrolases (lysozyme, protease, serine protease, alkaline protease, amylase, lipase, cellulase, glucoamylase, etc.), Isomerase (such as glucose isomerase), transferase (such as acyltransferase and sulfotransferase), synthase (such as fatty acid synthase, phosphate synthase, and citrate synthase) and elimination enzyme (such as pectin lyase)}, hormone protein { Bone morphogenetic factor (BMP), interferon α, interferon β, interleukin 1-12, growth hormone, erythropoietin, insulin, granular Colony stimulating factor (G-CSF), tissue plasminogen activator (TPA), natriuretic peptide, blood coagulation factor VIII, somatomedin, glucagon, growth hormone releasing factor, serum albumin and calcitonin}, antibody {1 Chain antibody, IgG large subunit, IgG small subunit, etc.}, fluorescent protein (GFP, etc.), photoprotein (lucerase, etc.) and the like. The peptide is not particularly limited in amino acid composition, and examples thereof include dipeptides and tripeptides.

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

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

  Among these useful substances, peptides and proteins are preferable from the viewpoint of productivity of useful substances, more preferably recombinant proteins, and further preferably enzymes, single chain antibodies and hormone proteins.

  In the method for producing a useful substance of the present invention, the culture solution is a liquid containing components for growing bacteria.

  The culture solution contains water, a carbon source, a nitrogen source, and the like. As the carbon source, bacteria can synthesize ATP (adenosine triphosphate) and can be assimilated by metabolism, and include sugars such as glucose, lipids and proteins. Nitrogen sources are those that bacteria can use for the biosynthesis of proteins and nucleic acids, and include proteins, amino acids, ammonia, urea, and ammonium sulfate.

  In the production method of the present invention, the amount of Kjeldahl nitrogen in the culture solution at the start of culture is 4.5 to 50 g / L based on the volume of the culture solution, preferably 5 to 25 g / L from the viewpoint of productivity, Preferably it is 6-20 g / L. When the amount of Kjeldahl nitrogen is less than 4.5 g / L, the production efficiency of the recombinant protein is deteriorated, and when it is more than 50 g / L, the growth or survival of bacteria is adversely affected.

  In the present invention, the Kjeldahl nitrogen amount is a nitrogen amount determined by the Kjeldahl measurement method. The Kjeldahl measurement method is a method for measuring the total amount of trivalent nitrogen contained in an organic substance or the like. Nitrogen detected by this measurement method can be used to increase production of useful substances and serves as an index of nutrient sources for cell culture.

  The Kjeldahl measurement method is a method for quantifying ammonia generated in the presence of selenium used as a catalyst in a measurement method defined by JIS (JIS K0400-42-60).

The amount of Kjeldahl nitrogen in the culture solution at the start of the culture can be easily prepared by adding a substance containing nitrogen atoms to the culture solution and adjusting the amount.
Examples of substances containing nitrogen atoms added to the culture solution include proteins and protein degradation products, amino acids, urea, ammonia, and ammonium salts such as ammonium sulfate.
As the substance containing nitrogen atoms added to the culture solution, proteins and protein degradation products, amino acids and urea are preferable from the viewpoint of improving the productivity of useful substances.

  In the present invention, the start of culture refers to the time of inoculating bacteria in the culture solution.

  In the present invention, the surfactant (B) includes an amphoteric surfactant (B1), an anionic surfactant (B2), a nonionic surfactant (B3), and a cationic surfactant (B4).

  Examples of the amphoteric surfactant (B1) include a carboxylate amphoteric surfactant (B1-1), a sulfate ester amphoteric surfactant (B1-2), and a sulfonate amphoteric surfactant (B1-3). ) And phosphate ester type amphoteric surfactant (B1-4).

Carboxylate type amphoteric surfactant (B1-1) includes amino acid type amphoteric surfactant (B1-1-1), betaine type amphoteric surfactant (B1-1-2), and imidazoline type amphoteric surfactant ( B1-1-3) and the like.
The amino acid type amphoteric surfactant (B1-1-1) is an amphoteric surfactant having an amino group and a carboxyl group in the molecule, and examples thereof include a compound 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 2. m is an integer of 1 or 2. M is a monovalent or divalent cation such as a hydrogen atom, an alkali metal, an alkaline earth metal, ammonium (including cations derived from amines and alkanolamines), and quaternary ammonium.
Further, (B1-1-1) is an alkylaminopropionic acid type amphoteric surfactant (such as sodium cocaminopropionate, sodium stearylaminopropionate and sodium laurylaminopropionate); an alkylaminoacetic acid type amphoteric surfactant ( And sodium N-lauroyl-N′-carboxymethyl-N′-hydroxyethylethylenediamine and the like.

The betaine-type amphoteric surfactant (B1-1-2) is an amphoteric surfactant having a quaternary ammonium salt-type cation moiety and a carboxylic acid-type anion moiety in the molecule. ) Alkyldimethylbetaines (stearyl dimethylaminoacetic acid betaine and lauryldimethylaminoacetic acid betaine etc.), amide betaines (coconut oil fatty acid amidopropyl betaine and lauric acid amidopropyl betaine etc.) and alkyldihydroxyalkyl betaines (lauryl dihydroxy) Ethyl betaine, etc.).
RN ⁺ (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 (B1-1-3) include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine.

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

  Examples of the anionic surfactant (B2) include ether carboxylic acid (B2-1) and its salt, sulfate ester (B2-2) or its salt, ether sulfate ester (B2-3) and its salt, sulfonate ( B2-4), sulfosuccinate (B2-5), phosphate ester (B2-6) and salt thereof, ether phosphate ester (B2-7) and salt thereof, fatty acid salt (B2-8), acylated amino acid Examples thereof include salts and naturally-derived carboxylic acids and salts thereof (such as chenodeoxycholic acid, cholic acid, and deoxycholic acid).

  The ether carboxylic acid (B2-1) or a salt thereof includes an ether carboxylic acid having a hydrocarbon group having 8 to 24 carbon atoms and a salt thereof, polyoxyethylene lauryl ether acetic acid, polyoxyethylene lauryl ether acetic acid sodium salt, Examples include sodium polyoxyethylene tridecyl ether acetate, sodium polyoxyethylene octyl ether acetate, and sodium lauryl glycol acetate.

  Examples of the sulfate ester (B2-2) and salts thereof include sulfate esters having a hydrocarbon group having 8 to 24 carbon atoms and salts thereof, such as sodium lauryl sulfate and triethanolamine salt of lauryl sulfate.

  Ether sulfate (B2-3) and salts thereof include ether sulfates having a hydrocarbon group having 8 to 24 carbon atoms and salts thereof, polyoxyethylene lauryl ether sulfate sodium salt and polyoxyethylene lauryl ether sulfate. A triethanolamine salt etc. are mentioned.

  Examples of the sulfonate (B2-4) include sodium dodecyl diphenyl ether disulfonate, sodium dodecylbenzene sulfonate, and sodium naphthalene sulfonate.

  Examples of the sulfosuccinate (B2-5) include polyoxyethylene lauryl sulfosuccinic acid disodium salt, sulfosuccinic acid lauryl disodium salt, and sulfosuccinic acid polyoxyethylene lauroyl ethanolamide disodium salt.

  Examples of the phosphate ester (B2-6) include octyl phosphate disodium salt and lauryl phosphate disodium salt.

  Examples of the ether phosphate (B2-7) include polyoxyethylene octyl ether phosphate disodium salt and polyoxyethylene lauryl ether phosphate disodium salt.

  Examples of the fatty acid salt (B2-8) include sodium octylate, sodium laurate, and sodium stearate.

  Nonionic surfactants (B3) include higher alcohol alkylene oxide (hereinafter abbreviated as AO) adduct (B3-1), alkylphenol AO adduct (B3-2), fatty acid AO adduct (B3-3) and Polyhydric alcohol type nonionic surfactant (B3-4) etc. are contained.

  The higher alcohol AO adduct (B3-1) includes polyoxyalkylene alkyl ethers and higher alcohols having 8 to 24 alkyl ether carbon atoms (decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl alcohol, oleyl alcohol, etc. ) Ethylene oxide (hereinafter abbreviated as EO) 1-20 mol and / or propylene oxide (hereinafter abbreviated as PO) 1-20 mol adduct (including block adducts and / or random adducts, etc.), etc. Is mentioned.

  The alkylphenol AO adduct (B3-2) includes an AO adduct of an alkylphenol having an alkyl group having 6 to 24 carbon atoms, an EO1 to 20 mol and / or PO1 to 20 mol adduct of octylphenol, and an EO1 of nonylphenol. -20 mol and / or PO 1-20 mol adducts and the like. Moreover, TRITON (registered trademark) X-114, igepal (registered trademark) CA-520, igepal (registered trademark) CA-630, and the like can be easily obtained from the market.

  As fatty acid AO adduct (B3-3), EO 1-20 mol of fatty acids having 8 to 24 carbon atoms (decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, coconut oil fatty acid, etc.) and / or Or PO1-20 mol adduct is contained, An oleic acid EO9 mol adduct, a dioleic acid EO12 mol adduct, a dioleic acid EO20 mol adduct, a stearic acid EO9 mol adduct, etc. are mentioned.

  Examples of the polyhydric alcohol type nonionic surfactant (B3-4) include EO and / or 2-8 polyhydric alcohols having 3 to 36 carbon atoms (such as glycerin, trimethylolpropane, pentaerythritol, sorbit and sorbitan). Or PO adducts, fatty acid esters of polyhydric alcohols and EO adducts thereof, sugar fatty acid esters, fatty acid alkanolamides (such as coconut oil fatty acid diethanolamide) and AO adducts thereof, and sorbitan tetraoleic acid Examples include ester EO adducts and sorbitan hexaoleate EO adducts.

  Examples of the cationic surfactant (B4) include an amine salt type cationic surfactant (B4-1) and a quaternary ammonium salt type cationic surfactant (B4-2).

  As amine salt type cationic surfactant (B4-1), laurylamine, stearylamine, triethanolamine monostearate ester, stearamide ethyldiethylamine, and condensate of stearic acid and aminoethylethanolamine are further condensed with urea. And acid neutralized products such as formic acid such as hardened beef tallow amine and rosin amine, acetic acid and hydrochloric acid.

  Examples of the quaternary ammonium salt type cationic surfactant (B4-2) include stearamide methylpyridinium chloride, lauryltrimethylammonium chloride, benzyllauryldimethylammonium chloride and cetylpyridinium bromide.

  As surfactant (B), from the viewpoint of secretion efficiency, carboxylate type amphoteric surfactant (B1-1), ether carboxylic acid (B2-1), sulfonate (B2-4), higher alcohol AO Adducts (B3-1) and polyhydric alcohol type nonionic surfactants (B3-4) are preferred, and more preferred are amidopropyl betaine laurate, betaine lauryldimethylaminoacetate, N-lauroyl-N′-carboxymethyl- N'-hydroxyethyl ethylenediamine sodium, coconut oil fatty acid amidopropyl betaine, lauric acid amidopropyl betaine, lauryl dimethylaminoacetic acid betaine, sodium cocaminopropionate, polyoxyethylene lauryl ether acetate sodium, polyoxyethylene tridecyl ether acetate sodium salt , De Sodium sill diphenyl ether disulfonic acid, polyoxyethylene alkyl ethers, polyoxyalkylene alkyl ethers and coconut oil fatty acid diethanolamide.

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

  The amount (% by weight) of the surfactant (B) contained in the culture solution in the step (a) described later is appropriately selected depending on the target bacteria, the kind of useful substance produced and the kind of secretion method. From the viewpoint of secretion efficiency and the difficulty of denaturing the useful substance produced, 0.0001 to 10 is preferable, more preferably 0.005 to 10, and still more preferably 0, based on the weight of the culture solution. .01-5.

  The surfactant (B) may be added later to a culture solution in which bacteria are inoculated and suspended, in addition to using the surfactant (B) mixed with the culture solution in advance. Mixing with the culture solution can be performed by adding the surfactant (B) to the culture solution at 4 ° C to 99 ° C and stirring with a stirring blade or a stirrer. When mixing afterwards, it can carry out by adding, stirring with a stirring blade etc.

  In using the surfactant (B), in addition to using the above surfactant alone, several types may be mixed and used.

In the present invention, the step of causing the bacteria that produce useful substances and the surfactant to coexist at the same time to secrete the useful substances outside the cells is step (a) in the extracellular secretion production method of useful substances described below.
Method of extracellular secretion production method of useful substance (a): A step of secreting a useful substance extracellularly (in the culture medium) by simultaneously existing a culture medium for cultivating bacteria producing the useful substance and a surfactant.
Step (b): A step of separating useful substances from the culture solution after the step (a).

An example of a method for producing useful substances using bacteria is shown below.
(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. Is incorporated into a phage or plasmid.
(I-2) Transform a host with the obtained recombinant phage or plasmid, and after cultivation, target DNA by hybridization with a DNA probe encoding a part of the target protein gene, or immunoassay using an antibody A phage or plasmid containing is isolated.
(I-3) It can be produced by cutting out the desired cloned DNA from the recombinant phage DNA or plasmid, and ligating the cloned DNA or a part thereof downstream of the promoter in the expression vector. DNAs encoding a signal sequence for translocating the inner membrane (a signal sequence for expressing a target substance in the periplasmic space) can be linked simultaneously.

  Examples of the plasmid include pUC series such as pUC19, pET series such as pET-22b, pBAD series, and pBR322.

(Ii) Culture (ii-1) A bacterium for producing a useful substance is transformed with an expression vector and cultured. The culture is usually performed on an agar medium at 15 to 43 ° C. for 3 to 72 hours.
(Ii-2) Autoclave sterilization is performed for the culture solution used for culture at 121 ° C. for 20 minutes, and the recombinant bacteria cultured on the agar medium is main cultured here. Kjeldahl nitrogen is contained in the culture medium used for the main culture. The amount of Kjeldahl nitrogen contained in the culture solution used for the main culture is as described above. Usually, it is carried out at 15 to 43 ° C. for 12 to 72 hours. In this step, the surfactant (B) is added. When the surfactant (B) is used from the beginning of the culture, a homogenized mixture of (B) and the culture solution is used as the culture solution. When (B) is used after the start of the culture, the surfactant (B) is added and the culture is continued 72 hours after the start of the culture immediately after the start of the culture. In (ii-2), the concentration of bacteria is preferably 1 to 10 ¹³ cells / ml, more preferably 10 ² to 10 ¹¹ cells / ml.
In (ii-2), the usage amount (% by weight) of the surfactant (B) is appropriately selected depending on the type of bacteria to be used and the useful substance to be produced, but based on the weight of the culture solution. From the viewpoint of secretion efficiency and difficulty of denaturing the useful substance to be produced, 0.0001 to 10 is preferable, 0.005 to 10 is more preferable, and 0.01 to 5 is still more preferable.

(Iii) Purification (iii-1) The protein secreted into the culture solution is separated from bacteria and bacterial residues by centrifugation, hollow fiber separation, filtration and the like.
(Iii-2) The culture solution containing protein is subjected to precipitation treatment such as ethanol precipitation, ammonium sulfate precipitation and polyethylene glycol precipitation by repeating column treatments such as ion exchange column, gel filtration column, hydrophobic column, affinity column and ultra-column. Separation and purification are carried out as necessary.

The bacteria isolated in (iii-1) can then be further cultured by returning them to the culture kettle and supplying a new culture solution. By further subjecting the culture solution and the like to the step (iii) and repeating the purification and culture, continuous production of useful substances can be performed.
In the bacterium of the present invention, since the killing or growth inhibition of the bacterium by the surfactant (B) is suppressed, the survival rate of the bacterium in such continuous production is increased. Therefore, useful substances can be produced continuously, and the production amount can be dramatically improved.

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

  Examples of the membrane in the above (iii) cell / protein separation / removal step include a hollow fiber type separation device and a flat membrane type membrane separation device. Commercially available products include CFP series such as CFP-2-E-55 (GE), UFP series such as UFP-10-E-65 (GE), MF series (Millipore), UF series (Millipore). It is available.

  Since the useful substance obtained by the production method of the present invention is secreted and produced outside the cell without destroying the bacteria, the purity is higher than before. Moreover, since the production method of the present invention is excellent in the productivity of useful substances, a high yield can be obtained.

  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 means parts by weight.

<Production Example 1>
Escherichia coli (α) transformed with a pUC19 plasmid expressing MalE having a His tag at the C-terminus was prepared by a conventional method.
Next, E. coli (α) is inoculated into 1 ml of LB culture solution (bactotryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, ampicillin 100 mg / L) with a platinum loop and shaken at 37 ° C. overnight. After culturing, the cells were collected using a centrifuge to obtain E. coli (α).

<Comparative Example 1>
The collected E. coli (α) obtained in Production Example 1 was added to a TB culture solution (Difco) containing 100 mg / L ampicillin, 0.1 mM IPTG and protease inhibitor mix (manufactured by Wako Pure Chemical Industries, Ltd.) 0.1 ml. 1) Inoculated into 1 ml and started culturing. After shaking culture at 37 ° C. for 2 hours, coconut oil fatty acid amidopropyldimethylaminoacetate betaine (manufactured by Sanyo Chemical Industries, Ltd., trade name “Levon 2000”) as a surfactant is 0.3% by weight. In addition, shaking culture was continued at 37 ° C. for 16 hours. Thereafter, the culture was centrifuged (5,000 G, 10 minutes), and the supernatant was analyzed by SDS-PAGE to quantify the band of MalE protein secreted outside the cells of E. coli. The results of quantification were set to 1.00 as the quantitative value of Comparative Example 1, and the results of Examples 1-1 to 1-5, 2 and 3 were shown as relative values based on the quantitative value of Comparative Example 1. The amount of kedar nitrogen contained in the culture solution at the start of the culture was quantified by the method described above. The results are summarized in Table 1.

<Examples 1-1 to 1-5>
In Comparative Example 1, lactalbumin was added to the TB culture solution before resuspending Escherichia coli (α) as a collected cell body, and the amount of kedar nitrogen contained in the culture solution at the start of the culture was adjusted to the amount shown in Table 1. The culture was carried out in the same manner as in Comparative Example 1 except that, and evaluation was carried out using the same method as in Comparative Example 1. Table 1 summarizes the results of relative values based on the amount of kedar nitrogen and the quantitative value of Comparative Example 1 of MalE protein.

<Example 2>
In Comparative Example 1, polypeptone is added to the TB culture solution before resuspending Escherichia coli (α) as a collected cell body so that the amount of kedar nitrogen contained in the culture solution at the start of the culture becomes the amount shown in Table 1. Was cultured in the same manner as in Comparative Example 1 and evaluated using the same method as in Comparative Example 1. Table 1 summarizes the results of relative values based on the amount of kedar nitrogen and the quantitative value of Comparative Example 1 of MalE protein.

<Example 3>
In Comparative Example 1, urea is added to the TB culture solution before resuspending Escherichia coli (α), which is a collected cell body, so that the amount of kedar nitrogen contained in the culture solution at the start of the culture becomes the amount shown in Table 1. Was cultured in the same manner as in Comparative Example 1 and evaluated using the same method as in Comparative Example 1. Table 1 summarizes the results of relative values based on the amount of kedar nitrogen and the quantitative value of Comparative Example 1 of MalE protein.

<Comparative Example 2>
In Comparative Example 1, after adding coconut oil fatty acid amidopropyldimethylaminoacetate betaine as a surfactant, SDS-PAGE was sampled by 0.1 ml over time to quantify MalE protein secreted outside the cells of E. coli. The same operation as in Comparative Example 1 was performed except that. The amount of MalE protein expressed in Escherichia coli before adding coconut oil fatty acid amidopropyldimethylaminoacetate betaine was determined by centrifuging the culture solution (5,000 G, 10 minutes), removing the supernatant and precipitating the cells. Was resuspended in Tris-HCl buffer, subjected to ultrasonic disruption (200 W, 10 minutes) and analyzed by SDS-PAGE. The result of quantifying the MalE protein was expressed as a relative value based on the quantitative value of the MalE protein expressed in E. coli before adding the palm oil fatty acid amidopropyldimethylaminoacetic acid betaine. The results are summarized in Table 2.

<Example 4>
In Example 1-2, after addition of coconut oil fatty acid amidopropyldimethylaminoacetate betaine as a surfactant, sampling for SDS-PAGE was performed over time by 0.1 ml, and MalE protein secreted outside the cells of E. coli was obtained. It implemented like Example 1-2 except quantifying. The result of quantifying the MalE protein was expressed as a relative value based on the quantitative value of the MalE protein expressed in E. coli before adding the palm oil fatty acid amidopropyldimethylaminoacetic acid betaine. Moreover, it was confirmed by measuring the turbidity of the bacterial solution with a spectrophotometer that the increase in the expression level of the MalE protein over time was not simply due to the growth of E. coli. The results are summarized in Table 2.

<Production Example 2>
E. coli BL21 (DE3) (Novagen) (β) containing the pET-26b vector was prepared by a conventional method.
Next, E. coli (β) is inoculated into 1 ml of LB culture solution (bactotryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, ampicillin 100 mg / L) with a platinum loop and shaken at 37 ° C. overnight. After culturing, the cells were collected using a centrifuge to obtain E. coli (β).

<Comparative Example 3>
E. coli (β) of the collected cells obtained in Production Example 2 was added to a TB culture solution containing 0.1 ml of 100 mg / L ampicillin, 0.1 mM IPTG and protease inhibitor mix (manufactured by Wako Pure Chemical Industries, Ltd.) Difco) After resuspending in 1 ml and shaking culture at 37 ° C. for 2 hours, coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine as a surfactant (manufactured by Sanyo Chemical Industries, Ltd., trade name “Levon 2000”) Was added at 0.3% by weight, and shaking culture was continued at 37 ° C. for 16 hours. Thereafter, the culture medium was centrifuged (5,000 G, 10 minutes), and the supernatant was analyzed with an anti-His-tag antibody to quantify the amount of peptide secreted outside the cells of E. coli. The result of the quantification was the relative value based on the quantitative value of Comparative Example 3, with the quantitative value of Comparative Example 3 being 1.00. The amount of kedar nitrogen contained in the culture solution at the start of the culture was quantified by the method described above. The results are summarized in Table 3.

<Example 5>
In Comparative Example 3, polypeptone was added to the TB culture solution before resuspending Escherichia coli (β) as a collected cell, and the total amount of kedal nitrogen contained in the culture solution at the start of the culture was adjusted to the amount shown in Table 3. The culture was performed in the same manner as in Comparative Example 3 except that the evaluation was performed, and the evaluation was performed using the same method as in Comparative Example 3. Table 3 summarizes the results of relative values based on the quantitative values of Comparative Example 3 for the amount of kedar nitrogen and the amount of peptide.

From the results of Tables 1 and 3, Examples 1-1 to 1-5, 2, 3 and 5 in which the amount of kedar nitrogen contained in the culture broth at the start of culture is 4.5 to 50 g / L are Comparative Examples 1 and 3 shows that the yield of MalE protein, which is a useful substance, is improved.
From the results in Table 2, it can be seen that in Comparative Example 2, the amount of secreted useful substance hardly changed after 4 hours from the addition of the surfactant. On the other hand, in Example 4, it turns out that the useful substance secreted continues after 4 hours progress.

The production method of useful substances of the present invention can be used when producing useful substances such as proteins. Examples of proteins include enzymes, functional proteins, hormone proteins, antibodies, vaccine proteins and peptides. When the protein to be produced is an enzyme (protease, cellulase, lipase, amylase, etc.), it can be suitably used for improving the quality of detergents and foods. When the protein to be produced is an enzyme (asparaginase, superoxide dismutase, protease, etc.), hormone protein (growth hormone, interferon α, etc.), antibody (single chain antibody, etc.), vaccine protein, etc. it can.
The surfactant of the present invention can also be used as an extraction reagent for bacterial periplasmic fractions.

Claims (1)

An extracellular secretion production method of a useful substance which comprises the following step (a) and further step (b), wherein the amount of Kjeldahl nitrogen in the culture solution at the start of culture is 4.5 to 50 g based on the volume of the culture solution / L , wherein the useful substance is a peptide or protein .
Step (a): A step of causing a culture medium for cultivating bacteria that produce useful substances and a surfactant (B) to coexist and secrete useful substances outside the cell , wherein the surfactant (B) is amphoteric. A step of being a surfactant (B1), wherein the bacterium is Escherichia coli .
Step (b): A step of separating useful substances from the culture solution after the step (a).