JPH01174377A - Method for cultivating microorganism - Google Patents
Method for cultivating microorganismInfo
- Publication number
- JPH01174377A JPH01174377A JP32997587A JP32997587A JPH01174377A JP H01174377 A JPH01174377 A JP H01174377A JP 32997587 A JP32997587 A JP 32997587A JP 32997587 A JP32997587 A JP 32997587A JP H01174377 A JPH01174377 A JP H01174377A
- Authority
- JP
- Japan
- Prior art keywords
- generation rate
- specific
- ratio
- nitrogen source
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 244000005700 microbiome Species 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 38
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 19
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 19
- 238000012258 culturing Methods 0.000 claims description 13
- 230000007423 decrease Effects 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 20
- 102000004169 proteins and genes Human genes 0.000 abstract description 19
- 239000001963 growth medium Substances 0.000 abstract description 9
- 230000000813 microbial effect Effects 0.000 abstract description 3
- 241000588724 Escherichia coli Species 0.000 description 16
- 239000002609 medium Substances 0.000 description 14
- 230000012010 growth Effects 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 12
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 12
- 229960005356 urokinase Drugs 0.000 description 12
- 238000004519 manufacturing process Methods 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 10
- 239000002243 precursor Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 239000000411 inducer Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 239000002184 metal Chemical class 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 108010000521 Human Growth Hormone Proteins 0.000 description 1
- 239000000854 Human Growth Hormone Substances 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 230000021332 multicellular organism growth Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は微生物の効率の良い培養方法に関するものであ
る。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an efficient method for culturing microorganisms.
（発明の背景）
従来、遺伝子組換技術を用いることで有用な蛋白質の生
産等を目標として大腸菌や酵母等の微生物の培養が行わ
れてきた。例えば、インスリン、ヒト成長ホルモン、ウ
シ成長ホルモン、ヒトウロキナーゼ、ヒト組織プラスノ
ミノーゲンアクチベータ等の異種蛋白質をこれらの遺伝
子組換をされた微生物を培養することで生産することも
可能となった。(Background of the Invention) Conventionally, microorganisms such as Escherichia coli and yeast have been cultured using genetic recombination technology with the aim of producing useful proteins. For example, it has become possible to produce heterologous proteins such as insulin, human growth hormone, bovine growth hormone, human urokinase, and human tissue plus nominogen activator by culturing these genetically modified microorganisms.
このように、微生物を使用して有用な目的蛋白質を生産
させようとする時には、使用する微生物が効率良く蛋白
質を生産するように培地中の炭素源あるいは窒素源等の
培養条件等を検討する必要がある。特に遺伝子組換され
た微生物の培養においては、生産させようとする目的蛋
白質の生産に伴い、微生物の生育が阻害されることがあ
り、微生物が目的蛋白質を効率よく生産するような培養
条件を検討する必要がある。In this way, when trying to use microorganisms to produce useful target proteins, it is necessary to consider culture conditions such as carbon sources or nitrogen sources in the culture medium so that the microorganisms used can efficiently produce proteins. There is. In particular, when culturing genetically modified microorganisms, the growth of the microorganism may be inhibited due to the production of the target protein, so consider culturing conditions that will allow the microorganism to efficiently produce the target protein. There is a need to.
本発明者らは微生物の生育あるいは目的蛋白質゛の生産
に大きな影響を与える培地中の炭素源、窒素源について
鋭意検討を行った結果、
（１）炭素源と窒素源の重量比率（以下Ｃ／Ｎ比と略す
）が一定の範囲の場合、微生物の生育あるいは目的蛋白
質の生産が良好である
下する
場合に比べて単位菌体により単位時間に発生するＣ０２
ｆｉｔ（以下比ＣＯ２発生速度とする）が大きい
（５）Ｃ／Ｎ比が一定の範囲を逸脱すると、培地のｐＨ
が急激に変化する
という他見を得、本発明を完成するに至った。すなわち
本発明は、単位菌体により単位時間に発生するＣＯ２ｆ
ｔおよび培地のｐＨ値の上昇または下降時に炭素源ある
いは窒素源を添加することを特徴とする微生物の培養方
法を提供するものであり以下本発明の詳細な説明する。The present inventors conducted intensive studies on the carbon source and nitrogen source in the culture medium, which have a large effect on the growth of microorganisms and the production of the target protein. When the N ratio (abbreviated as N ratio) is within a certain range, the growth of microorganisms or the production of the target protein is good.
fit (hereinafter referred to as specific CO2 generation rate) is large (5) When the C/N ratio deviates from a certain range, the pH of the medium
The present invention was completed based on the finding that the change occurs rapidly. In other words, the present invention aims to reduce CO2f generated per unit time by unit bacterial cells.
This invention provides a method for culturing microorganisms, which is characterized in that a carbon source or a nitrogen source is added when the pH value of the culture medium increases or decreases.The present invention will be described in detail below.
（発明の構成）
本発明は、例えば大腸菌、酵母菌、アミノ酸菌、酢酸菌
等の、好気性微生物の培養に採用されるものである。前
記のように、微生物の生育あるいは目的蛋白質の生産に
大きな影響を与えるＣ／Ｎ比がその微生物にとって適当
な範囲を逸脱すると比ＣＯ２発生速度の低下が見られる
ことから、本発明では、この比ＣＯ２発生速度の低下及
びその時のｐＨ値の変化を目安として炭素源あるいは窒
素１−一
源を添加する。好適な比ＣＯ２発生速度の範囲は、使用
する微生物を予め数種の異なったＣ／Ｎ比を有する培地
でそのＣ／Ｎ比が大きく変化しない程度に菌体を培養し
た時の、良好な菌体生育あるいは蛋白質生産を与えるよ
うなＣ／Ｎ比条件下での比ＣＯ２発生速度の範囲を採用
すればよい。適当な比ＣＯ２発生速度の範囲としてその
微生物の良好な生育を与えるような範囲を設定するか、
あるいは、目的蛋白質の良好な生産を与えるような範囲
を設定するかは適宜決定すればよい。培地の他の成分に
ついては、通常その微生物を培養するために用いる培地
であればなんら制限なく使用出来る。(Structure of the Invention) The present invention is employed for culturing aerobic microorganisms such as Escherichia coli, yeast bacteria, amino acid bacteria, and acetic acid bacteria. As mentioned above, when the C/N ratio, which has a great influence on the growth of microorganisms or the production of the target protein, deviates from the appropriate range for that microorganism, the specific CO2 generation rate decreases. A carbon source or nitrogen 1-1 source is added with the aim of decreasing the CO2 generation rate and changing the pH value at that time. The range of suitable specific CO2 generation rate is determined by culturing the microorganisms in advance in several different media with different C/N ratios to the extent that the C/N ratio does not change significantly. A range of specific CO2 generation rate under C/N ratio conditions that provides for body growth or protein production may be adopted. Set an appropriate range of specific CO2 generation rate that provides good growth for the microorganism, or
Alternatively, it may be determined as appropriate whether to set a range that provides good production of the target protein. As for the other components of the medium, any medium that is normally used for culturing the microorganism can be used without any restrictions.
比ＣＯ２発生速度は、例えば、通常の微生物の培養に用
いられるＣ０２ｍ度検出計により、例えば６００ｎｍで
の濁度当りの値等で得ればよい。The specific CO2 generation rate may be obtained as a value per turbidity at 600 nm, for example, using a CO2m degree detector used for the cultivation of ordinary microorganisms.
Ｃ／Ｎ比が低下した状態、すなわち炭素源不足の状態で
は、菌体の代謝系の影響により、培地のｐＨが上昇する
。そこで、比ＣＯ２発生速度の低下とｐＨの上昇が観察
された時には、炭素源を添Ａ −
加すればよい。一方、Ｃ／Ｎ比が上昇した状態、すなわ
ち窒素源不足の状態では、菌体の代謝系の影響により、
培地のｐＨが下降する。そこで、比ＣＯ２発生速度の低
下とｐＨの下降が観察された時には、窒素源を添加すれ
ばよい。使用する炭素源、窒素源としては、使用する微
生物の培養に通常使用されるものであればなんら制限は
ない。In a state in which the C/N ratio is decreased, that is, in a state in which a carbon source is insufficient, the pH of the medium increases due to the influence of the bacterial metabolic system. Therefore, when a decrease in the specific CO2 generation rate and an increase in pH are observed, a carbon source may be added. On the other hand, in a state where the C/N ratio is increased, that is, in a state where the nitrogen source is insufficient, due to the influence of the bacterial metabolic system,
The pH of the medium decreases. Therefore, when a decrease in the specific CO2 generation rate and a decrease in pH are observed, a nitrogen source may be added. There are no restrictions on the carbon source and nitrogen source as long as they are commonly used for culturing the microorganisms used.
培地のｐＨは、例えば通常の菌体培養で使用されるｐＨ
メーター等で測定出来る。The pH of the medium is, for example, the pH used in normal bacterial cell culture.
It can be measured with a meter etc.
他の培養条件については、使用する微生物の培養に通常
採用されるものであえば良く、本発明では特に制限はな
い。この様な条件としては、例えば、培養温度、酸素通
気量、培地のｐＨ等があげられる。本発明の一例をして
、ヒトウロキナーゼ前駆体蛋白質を大腸菌を培養するこ
とにより得る場合について述べると、Ｃ／Ｎ比は０．５
〜２．０の範囲で、そしてこの時の比ＣＯ２発生速度は
１．２５以上であった（ｐＨは６．８〜７．２の間で制
御した）。従って、比ＣＯ２発生昇または下降が観察さ
れた時に炭素源あるいは窒素源を培地に添加することに
より大腸菌の良好な生育及びヒトウロキナーゼの良好な
生産を行なわせることが出来る。Other culture conditions may be those normally employed for culturing the microorganisms used, and are not particularly limited in the present invention. Such conditions include, for example, culture temperature, oxygen aeration amount, pH of the culture medium, and the like. As an example of the present invention, when human urokinase precursor protein is obtained by culturing E. coli, the C/N ratio is 0.5.
~2.0, and the specific CO2 generation rate was at least 1.25 (pH was controlled between 6.8 and 7.2). Therefore, by adding a carbon source or a nitrogen source to the medium when an increase or decrease in specific CO2 production is observed, good growth of E. coli and good production of human urokinase can be achieved.
以下に本発明をさらに詳細に説明するため、実施例を示
すが、本発明はこれら実施例に限定されるものではない
。EXAMPLES Below, Examples will be shown to explain the present invention in more detail, but the present invention is not limited to these Examples.
（実施例）
参考例１
本発明の実施に先立って、組換大腸菌の生育および目的
蛋白質の生産に適当なＣ／Ｎ比の範囲とそのＣ／Ｎ比の
範囲において大腸菌が示す比ＣＯ２発生速度を調べた。(Example) Reference Example 1 Prior to implementing the present invention, the range of C/N ratios suitable for the growth of recombinant E. coli and the production of the target protein, and the specific CO2 generation rate exhibited by E. coli within the range of C/N ratios were determined. I looked into it.
大腸菌として、ヒトウロキナーゼ前駆体蛋白質をコード
する遺伝子を含むプラスミドで形質転換された菌（この
プラスミドは微生物工業科学技術研究所に寄託され、寄
託番号８３４１を付与されている）を用いた。この大腸
菌を、Ｃ／Ｎ比（炭素源としてはグリセロール、窒素源
としてはペプトンを用いた）を０．１．０．３．０．５
．１．０．２．０．３．０、４゜た。培養後ρそれぞれ
の培地のＣ／Ｎ比は培養前のＣ／Ｎ比と変化はなかった
。それぞれの Ｃ／Ｎ比の培地での大腸菌による蛋白質
生産量および比ＣＯ２発生速度を表１にしめす。なお、
培地は炭素源、窒素源の他に０，７％Ｎａ２ＨＰＯ４゜
０．３％Ｋ Ｈ２Ｐ Ｏ４，０，１％ＮＨ４Ｃｌ、０゜
０５％ＮａＣ１，極微量の塩、金属塩およびウロキナー
ゼ前駆体蛋白質を生産させるための発現誘導剤（イソプ
ロピルβ−Ｄ−チオガラクトピラノシド）等を含み、蛋
白質生産量は、培養後の菌体からウロキナーゼ前駆体蛋
白質を抽出し、特開昭５９−１６１３２１号に示された
様な方法で変性可溶化および復元（再活性化）し、プラ
スミン処理して得られたウロキナーゼの、合成基質Ｓ−
２４４４（第一化学薬品社製）分解活性で（Ｂ ｉ 。As Escherichia coli, a bacterium transformed with a plasmid containing a gene encoding human urokinase precursor protein (this plasmid has been deposited with the National Institute of Microbial Science and Technology and has been assigned deposit number 8341) was used. This E. coli was grown at a C/N ratio (glycerol was used as the carbon source and peptone was used as the nitrogen source) of 0.1.0.3.0.5.
．． 1.0.2.0.3.0, 4 degrees. After culture, the C/N ratio of each medium was unchanged from the C/N ratio before culture. Table 1 shows the protein production amount and specific CO2 production rate by E. coli in the culture medium with each C/N ratio. In addition,
In addition to carbon and nitrogen sources, the medium produces 0.7% Na2HPO4゜0.3%K H2P O4, 0.1% NH4Cl, 0゜05% NaCl, trace amounts of salts, metal salts, and urokinase precursor protein. It contains an expression inducer (isopropyl β-D-thiogalactopyranoside), etc. to induce urokinase, and the protein production amount is determined by extracting the urokinase precursor protein from the cultured bacterial cells and using the method shown in JP-A-59-161321. A synthetic substrate of urokinase, S-
2444 (manufactured by Daiichi Chemical Co., Ltd.) with decomposition activity (B i ).
ｔｅｃｈｎｏｌｏｇｙ、６２８． １９８４年
参照）で測定した。また、比ＣＯ２発生速度は次式より
求めた。technology, 628. 1984). Further, the specific CO2 generation rate was determined from the following equation.
比ＣＯ２発生速度＝（排気ガス中のＣＯ２濃度（％））
×空気の供給量（１／ｈｒ）÷（培養液中の菌体濃度（
ＯＤ６００ｎｍ））
排気ガス中のＣＯ２濃度は排ガス測定装置ＥＸ−１５６
２（石川製作所社製）を用いて、空気の供給量はマスフ
ローコントｏ−ラ５８５０Ｅ （ＮＥＣ社製）を用いて
、菌体濃度はオンライン懸濁針ＬＴ−２０１（小松用化
工機社製）を用いてそれぞれ測定した。Specific CO2 generation rate = (CO2 concentration in exhaust gas (%))
× Air supply amount (1/hr) ÷ (Bacterial cell concentration in culture solution (
OD600nm)) The CO2 concentration in the exhaust gas is measured using the exhaust gas measuring device EX-156.
2 (manufactured by Ishikawa Seisakusho Co., Ltd.), the air supply rate was determined using a mass flow controller 5850E (manufactured by NEC Corporation), and the bacterial cell concentration was determined using an online suspension needle LT-201 (manufactured by Kakoki Co., Ltd. for Komatsu). Each was measured using
この結果から、ウロキナーゼ前駆体蛋白質を大腸菌に効
率よく生産させるには、Ｃ／Ｎ比を０．５〜２．０維持
することが好ましく、そのための目安である比ＣＯ２発
生速度の適当な範囲として１．２５以上を設定した。尚
本発明者らが効するウロキナーゼを生産した場合である
。From this result, in order to efficiently produce urokinase precursor protein in E. coli, it is preferable to maintain the C/N ratio of 0.5 to 2.0, and the appropriate range of the specific CO2 generation rate is the standard for this purpose. It was set to 1.25 or higher. This is the case when the present inventors produce effective urokinase.
参考例２
参考例１で用いた組換大腸菌の生育に適当なＣ／Ｎ比の
範囲と、そのＣ／Ｎ比の範囲において大腸菌が示す比Ｃ
Ｏ２発生速度を調べた。参考例１の大腸菌を、Ｃ／Ｎ比
（炭素源としてはグリセロール、窒素源としてはペプト
ンを用いた）を０．１，０．３，０．５，１．０，２．
０゜３．０．４．０とした７種類の培地にそれぞれ０．
０５ｇ／ｌずつ植菌し、５時間培養してその時点の培養
液の濁度（６００ｎｍ）を測定して菌体の生育量とした
。尚、培養後の培地のＣ／Ｎ比は、培養前のＣ／Ｎ比と
変化していなかった。培地の他の成分、比ＣＯ２発生速
度等の測定等は、参考例１と同様にして行った。ただし
、本参考例では培地中に発現誘導剤を添加していない。Reference Example 2 Range of C/N ratio suitable for the growth of recombinant E. coli used in Reference Example 1 and ratio C exhibited by E. coli in that C/N ratio range
The rate of O2 generation was investigated. The E. coli of Reference Example 1 was used at a C/N ratio (glycerol was used as the carbon source and peptone was used as the nitrogen source) of 0.1, 0.3, 0.5, 1.0, 2.
0°3, 0.4.0 for each of the seven types of media.
The cells were inoculated at a rate of 0.5 g/l, cultured for 5 hours, and the turbidity (600 nm) of the culture solution at that point was measured to determine the amount of bacterial growth. Note that the C/N ratio of the medium after culture was unchanged from the C/N ratio before culture. Measurements of other components of the medium, specific CO2 generation rate, etc. were performed in the same manner as in Reference Example 1. However, in this reference example, no expression inducer was added to the medium.
この結果より、大腸菌を効率良く生育させるにはＣ／Ｎ
比を０．５〜２．０に維持することが好ましく、そのた
めの目安である比ＣＯ２発生速度の適当な範囲として１
．２５以上を設定した。尚、本発明者らが効率の良い生
育としたのはこの培養によりＯＤ６００ｎｍの濁度が３
以上となった場合である。結果を表２に示す。From this result, we found that to grow E. coli efficiently, the C/N
It is preferable to maintain the ratio between 0.5 and 2.0, and an appropriate range for the specific CO2 generation rate is 1.
．． 25 or more was set. In addition, the present inventors achieved efficient growth when the turbidity at OD600nm was 3.
This is the case when the above occurs. The results are shown in Table 2.
実施例１
参考例１．２の結果から、ウロキナーゼ前駆体蛋白質の
生産及び組換大腸菌の生育のため適切なＣ／Ｎ比の範囲
として共に０．５〜２．０を設定することとし、そのた
めの比ＣＯ２発生速度の適当な範囲として１．２５以上
を設定した。参考例と同じ大腸菌を、２．０％グリセロ
ール、３．０％ペプトン、０，７％Ｎａ２ＨＰＯ４，０
，３％ＫＨ２ＰＯ４，０，１％ＮＨ４Ｃｌ、０．０５％
ＮａＣ１及び極微量の塩、金属塩を含む培地に０．０５
ｇ／ｌ植菌し、３０℃にて１５時間培養した後、発現誘
導剤を添加した。上昇または下降したｐＨ値は、通常の
微生物の培養でｐＨの調整に用いられる酸、アルカリを
用いて６．８〜７．２の間に制御した。添加した炭素源
、窒素源は２０％ペプトン溶液、３０％グリセロール溶
液であった。その他の条件は、参考例１と同様であった
。結果を図１に示す。Example 1 Based on the results of Reference Example 1.2, it was decided to set the appropriate C/N ratio range between 0.5 and 2.0 for both the production of urokinase precursor protein and the growth of recombinant E. coli. A value of 1.25 or more was set as an appropriate range for the ratio of CO2 generation rate. The same E. coli as in the reference example was mixed with 2.0% glycerol, 3.0% peptone, 0.7% Na2HPO4,0
,3%KH2PO4,0,1%NH4Cl,0.05%
0.05 in a medium containing NaCl and trace amounts of salts and metal salts.
After inoculating g/l and culturing at 30°C for 15 hours, an expression inducer was added. The increased or decreased pH value was controlled between 6.8 and 7.2 using acids and alkalis that are commonly used to adjust pH in microbial culture. The carbon source and nitrogen source added were a 20% peptone solution and a 30% glycerol solution. Other conditions were the same as in Reference Example 1. The results are shown in Figure 1.
（発明の効果）
本発明によれば、従来経験的に決定されていた微生物の
培養の際のＣ／Ｎ比の制御を、簡単な設備により測定可
能な、比ＣＯ２発生速度及びｐＨ値の上昇または下降を
目安として行うことが出来る。このことにより、効率の
良い微生物の生育あるいは目的蛋白質の生産を行う事が
可能となる。(Effects of the Invention) According to the present invention, the control of the C/N ratio during the culture of microorganisms, which was conventionally determined empirically, can be measured with simple equipment, and the specific CO2 generation rate and pH value increase. Alternatively, you can use descending as a guide. This makes it possible to efficiently grow microorganisms or produce a target protein.
図１は、本発明の実施例１での操作を示すものである。
最上段は、ｐＨ値の変動を示し、第二段は比ＣＯ２発生
速度の変動を示し、中央は菌体の生育及び蛋白質の生産
の様子を示し、下段は炭素源及び窒素源の培地への総添
加量を示している。
ｐＨ値は、本発明の方法により炭素源あるいは窒素源を
添加した後、設定された６、８−７．２の間に復活する
ように制御されている。本発明で提供された様に、比Ｃ
Ｏ２発生速度が低下した時にｐＨ値を変化にあわせて炭
素源あるいは窒素源を添加することにより、菌体の生育
及びウロキナーゼの生産について良好な結果を得ること
が可能であった。
表１
表２FIG. 1 shows the operation in Example 1 of the present invention. The top row shows changes in pH value, the second row shows changes in specific CO2 generation rate, the middle row shows bacterial growth and protein production, and the bottom row shows changes in carbon and nitrogen sources to the medium. Shows the total amount added. The pH value is controlled to return to a predetermined range of 6, 8-7.2 after adding the carbon or nitrogen source according to the method of the present invention. As provided in the present invention, the ratio C
By adding a carbon source or a nitrogen source in accordance with the change in pH value when the O2 generation rate decreased, it was possible to obtain good results regarding bacterial growth and urokinase production. Table 1 Table 2
Claims (1)
び培地のｐＨ値の上昇または下降時に炭素源あるいは窒
素源を添加することを特徴とする微生物の培養方法。(1) A method for culturing microorganisms, which comprises adding a carbon source or a nitrogen source when the amount of Co2 generated by a unit cell per unit time and the pH value of a medium increase or decrease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32997587A JPH01174377A (en) | 1987-12-28 | 1987-12-28 | Method for cultivating microorganism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32997587A JPH01174377A (en) | 1987-12-28 | 1987-12-28 | Method for cultivating microorganism |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01174377A true JPH01174377A (en) | 1989-07-10 |
Family
ID=18227366
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP32997587A Pending JPH01174377A (en) | 1987-12-28 | 1987-12-28 | Method for cultivating microorganism |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01174377A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5284631A (en) * | 1992-01-03 | 1994-02-08 | Nkk Corporation | Crucible for manufacturing single crystals |
| JP2015509734A (en) * | 2012-03-12 | 2015-04-02 | ハンミ サイエンス カンパニー リミテッドＨａｎｍｉ Ｓｃｉｅｎｃｅｃｏ．，Ｌｔｄ． | High concentration culture method of E. coli cells |
-
1987
- 1987-12-28 JP JP32997587A patent/JPH01174377A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5284631A (en) * | 1992-01-03 | 1994-02-08 | Nkk Corporation | Crucible for manufacturing single crystals |
| JP2015509734A (en) * | 2012-03-12 | 2015-04-02 | ハンミ サイエンス カンパニー リミテッドＨａｎｍｉ Ｓｃｉｅｎｃｅｃｏ．，Ｌｔｄ． | High concentration culture method of E. coli cells |
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