JPH06217762A - High-concentration and high-efficiency culture of coli bacillus - Google Patents

Abstract

PURPOSE:To provide a high-concentration and high-efficiency culture method for coli bacillus, capable of efficiently producing the objective substance in production of an enzyme, a bioactive substance, etc., by coli bacillus. CONSTITUTION:In relation to aerobic culture of coli bacillus, this high- concentration and high-efficiency culture method is carried out by adding an amino acid composition to the culture medium so that amino acids required for growth of the coli bacillus may not run short and simultaneously adding glucose to the medium so as to control pH of the medium to be a constant value in a state where pH of the medium increases.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a high-concentration efficient culturing method of Escherichia coli for efficiently producing a target substance when Escherichia coli produces an enzyme or a physiologically active substance.

[0002]

2. Description of the Related Art Conventionally, by culturing Escherichia coli, bioactive substances such as enzymes or hormones, which are metabolites thereof, have been produced. In order to increase the productivity, a culture method in which a substrate is added during the culture is performed as the culture method, but there is a phenomenon that the growth rate of Escherichia coli decreases during the culture despite the addition of the substrate. This is due to the accumulation of growth inhibitory substances secreted by E. coli, especially acetic acid, in the medium, resulting in a concentration of 17 g / L (280 m
It is said that the growth inhibition becomes remarkable at M or more.

Therefore, using the concentration of acetic acid in the culture solution as an index,
A method has been proposed in which the culture solution is intermittently or continuously withdrawn, the cells are recovered by centrifugation, and the cells are charged again in the culture tank (Japanese Patent Laid-Open No. 63-141579). Also,
A method of assimilating acetic acid, which is a major inhibitor for the growth of Escherichia coli, has been proposed by controlling the rate of substrate addition and the amount thereof with acetic acid concentration as an index [J.
rment. Technol., 66, 187-191 (1988)]. However, these culturing methods use high density cells (dry cell weight 20 g /
It takes more than 20 hours to obtain (L or more), the characteristics of Escherichia coli having a short cell cycle are not fully utilized, and the productivity cannot be said to be high.

In order to smoothly culture E. coli,
It is usually practiced to control the pH of the medium to a value suitable for the growth of Escherichia coli, but the pH is generally controlled by adding an alkali such as sodium hydroxide or an acid such as hydrochloric acid to the medium. Was there. However, it has not been disclosed or suggested that the pH of the medium can be controlled to a constant value by adding glucose to the medium during aerobic culture of Escherichia coli as in the present invention. Didn't.

[0005]

The object of the present invention is to eliminate Escherichia coli without the need of measuring acetic acid concentration during culture, solid-liquid separation treatment such as centrifugation, or pH control by addition of alkali or acid. It is an object of the present invention to provide a method for efficiently culturing at high concentration.

[0006]

In order to solve the above-mentioned problems, the present inventors have found that when Escherichia coli assimilates glucose, the secretion of organic acid, especially acetic acid, which is a growth inhibitor, becomes remarkable. Focusing attention, we diligently studied to suppress the accumulation of growth inhibitory substances by reducing the amount of glucose assimilation. As a result, by utilizing the fact that the pH of the medium is lowered by the organic acid secreted by the assimilation of glucose and the pH of the medium is increased by the basic substance secreted by the assimilation of amino acid, that is, When glucose is added to the medium to control the pH of the medium to a constant value in a state where the pH is increased, the amount of glucose assimilation per cell is reduced, the production of acetic acid during the culture is suppressed, and Escherichia coli is shortened. They have found that they can be cultivated to a high concentration in time, and have completed the present invention. The present invention, in aerobic culture for obtaining Escherichia coli, while adding an amino acid composition to the medium so as not to be deficient in the amino acids required for the growth of the bacterium, glucose is added in a state where the pH of the medium increases. The present invention relates to a high-concentration efficient culturing method of the bacterium, characterized in that the pH of the medium is controlled to a constant value.

The present invention will be described in detail below. The Escherichia coli of the present invention is not particularly limited as long as it produces an industrially useful metabolite. Preferably, Escherichia coli produced by a genetically engineered method to produce a useful enzyme or a physiologically active substance such as a hormone, and more preferably, produced by a genetically engineered method to produce human copper or zinc-type superoxide dismutase. E. coli, for example, Australian Patent Publication No. 27,461 / 84, JP-A-60-
137285, JP 61-111690 A, JP 61-111693 A, JP 61-1 A
Examples include Escherichia coli and the like disclosed in Japanese Patent No. 39390 and Japanese Patent Laid-Open No. 64-39988.

The culture of the present invention is carried out in a medium containing an inorganic salt, an amino acid composition and glucose as a substrate while controlling the temperature, pH and dissolved oxygen concentration during the culture to values preferable for the growth of these E. coli. With inorganic salts, commonly used ammonium chloride, sodium hydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, calcium chloride, manganese sulfate, aluminum iron chloride sulfate, copper chloride,
Mention may be made of zinc sulphate and boric acid. Further, the amino acid composition of the present invention means amino acids, casamino acids, soybean protein hydrolysates, and one or a mixture of two or more thereof. Examples of amino acids include methionine, tryptophan, tyrosine, arginine, proline, threonine, glycine, alanine, arginine, aspartic acid, glutamic acid, leucine, isoleucine, valine, phenylalanine and histidine.

The culture of the present invention has a glucose concentration of medium 1
It is performed in a medium containing an amino acid mixture and inorganic salts in an amount of 5 g or less per L, which is a concentration suitable for the growth of Escherichia coli.
At that time, when the pH of the medium rises above the set value, glucose is added to the medium to increase the p
H can be controlled to 6.5 to 7.4, preferably 7.2 ± 0.05, and more preferably 7.2 ± 0.025. pH
The amount of glucose to be added once for the constant value control is preferably such that the glucose concentration in the medium is 0.2 g or less per 1 L of the medium.

Further, during the culture, the amino acid composition and the inorganic salts initially charged are consumed, and these are insufficient for the conditions preferable for the growth of Escherichia coli. Therefore, the amino acid composition in the medium is adjusted to the growth of E. coli. It is possible to effectively achieve the method of the present invention by adding a substance, preferably an inorganic salt.

The medium in which a large amount of cells are efficiently obtained by the method of the present invention is separated into a liquid portion and a solid portion by a usual operation such as centrifugation, and then E. coli is treated with an enzyme or a physiologically active substance. If it is secreted outside, centrifuge the liquid part, and if it accumulates inside the cells, centrifuge the solid matter.
Ammonium sulphate salting out, gel filtration chromatography, anion exchange chromatography, cation exchange chromatography, metal chelating affinity chromatography, hydrophobic interaction chromatography by appropriately combining purification methods such as, by purification, A target physiologically active substance such as an enzyme or a hormone can be obtained.

[0012]

EXAMPLES The present invention will be described below with reference to comparative examples and examples. Note that these examples do not limit the scope of the present invention.

Comparative Example 1 1) Preculture NH_FourCl 0.1g, Na₂HPO_Four0.6g, KH₂
PO_Four0.3 g, NaCl 0.05 g, soy protein hydrolyzate
Dissolved product 0.3 g, glucose 0.4 g, MgSO _Four0.1
5g, CaCl₂・ 2H₂O1mg, MnSO_Four1m
g, AlCl₃1 mg, H₃BO₃0.05mg, Fe
SO_Four4mg, CuCl₂・ 2H₂O0.7mg, Zn
SO_Four・ 7H₂O1.25 mg and tetracycline 1.
100 mL of medium (pH 7.2) containing 25 mg was added
Recombinant E. coli 545πH in a 500 mL Erlenmeyer flask
R (pHT351) (Japanese Patent Laid-Open No. 64-39,988)
Report) and shake with a shake incubator, amplitude 7 cm, shake
The cells were cultured overnight at 37 ° C. at a strain frequency of 115 times / min.

2) Main culture K ₂ HPO ₄ 4 g, KH ₂ PO ₄ 4 g, Na ₂ HPO ₄
3 g, soy protein hydrolyzate 20 g, methionine 0.31
g, tryptophan 1.02 g, tyrosine 0.3 g, glucose 5 g, MgSO ₄ 1.5 g, CaCl ₂ .2H
₂ O 10 mg, MnSO ₄ 10 mg, AlCl ₃ 10 m
g, H ₃ BO ₃ 0.5 mg, FeSO ₄ 40 mg, Cu
_{Cl 2 · 2H 2 O8mg, ZnSO} 4 · 7H 2 O13m
g and 12.5 mg of tetracycline, 1 L (p
H7.2) was placed in a 2.6-liter culture tank containing 50 pre-cultured bacteria.
mL was added, and the culture was started at an initial volume of 1.05 L.
The temperature in the culture was maintained at 37 ° C., and the dissolved oxygen concentration was maintained at 30% or more of the saturated oxygen concentration by adjusting the stirring number and the mixing ratio of air and pure oxygen. The pH of the medium was adjusted by adding 10N NaOH to the medium by the PID method.
It was controlled to 7.2 ± 0.025.

Glucose was supplied by adding 5 g (10 mL of 50% glucose solution) when the dissolved oxygen concentration increased. For supplying other nutrients, an aqueous solution (pH 7.50) containing 7.5 g of methionine, 10.2 g of tryptophan, 2.1 g of arginine, 2.9 g of proline and 200 g of soybean protein hydrolyzate in 1 L of the solution was supplied.
2) 100 mL was added when the calculated absorbance [absorbance at measurement wavelength of 660 nm x culture solution volume (L) / initial culture solution volume (L)] was 25. The acetic acid concentration in the culture solution is
The culture solution was taken out and centrifuged, and the resulting supernatant was subjected to high performance liquid chromatography (column: ULTORON PS-
80H 8Φ × 300mm, made by Shinwa Kako Mobile phase: about 4.
It was measured with 44 mM trifluoroacetic acid, pH 2.3).
After 7 hours of culture, the absorbance of the culture solution at the measurement wavelength of 660 nm was 26 (calculated absorbance 37, dry cell weight 8.7 g).
/ L) were obtained. The amount of acetic acid produced and the amount of glucose assimilated were 1512 mg and 4.01 g per 1 g of dried E. coli cells, respectively. The acetic acid concentration after 7 hours became 200 mM or more, and the specific growth rate decreased.

Comparative Example 2 1) Preculture The same procedure as in Comparative Example 1 was carried out. 2) Main culture As for the supply of other nutrients, 50.8 g of aspartic acid, 12.5 g of threonine, and 14.7 glycine were added to 1 L of the solution.
g, alanine 8.4 g, methionine 3.1 g, tyrosine 0.4 g, tryptophan 7.4 g, arginine 3.7.
g, proline 13.6g and soy protein hydrolyzate 100g
10 mL of an aqueous solution (pH 7.2) containing
The same procedure as in Comparative Example 1 was carried out except that the addition was carried out every time the number was increased. By culturing for 7 hours, it was possible to obtain cells in which the absorbance of the culture solution at the measurement wavelength of 660 nm was 54 (calculated absorbance 63, dry cell weight 18 g / L). The amount of acetic acid produced and the amount of glucose assimilated were 681 mg and 2.03 g per 1 g of dry E. coli cells, respectively. The acetic acid concentration after 7 hours became 200 mM or more, and the specific growth rate decreased.

Example 1 1) Preculture The same procedure as in Comparative Example 1 was carried out. 2) Main culture After the glucose initially charged disappears from the medium, the glucose (50
% Glucose solution) to adjust the pH to 7.2 ± 0.
The same procedure as in Comparative Example 1 was carried out except that the PID method was used for controlling 025. After 7 hours of culture, the absorbance of the culture solution at the measurement wavelength of 660 nm is 65 (calculated absorbance: 76, dry cell weight: 21.7 g / L), and the absorbance of the culture solution at the measurement wavelength of 660 nm is 7.5 hours of culture. Was obtained (calculated absorbance: 94, dry cell weight: 25 g / L). The amount of acetic acid produced and the amount of glucose assimilated were 300 mg and 1.0, respectively, per 1 g of dried E. coli cells.
It was 7 g.

Example 2 1) Preculture The same procedure as in Comparative Example 1 was carried out. 2) Main culture The same culture as in Example 1 was performed except that glucose was not added as the initial medium. By culturing for 8 hours, it was possible to obtain cells in which the absorbance of the culture solution at the measurement wavelength of 660 nm was 56 (calculated absorbance 69, dry cell weight 18.7 g / L). The amount of acetic acid produced and the amount of glucose assimilated were 286 mg and 1.12 g per 1 g of dried E. coli cells, respectively.

[0019]

INDUSTRIAL APPLICABILITY The culturing method of the present invention does not measure the acetic acid concentration during the culturing, the solid-liquid separation treatment by centrifugation or the like, or the pH control by the addition of an alkali or an acid. It is an improved method capable of efficiently culturing Escherichia coli at a high concentration by adding glucose to control the pH of the medium to a constant value.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kazuhiro Umeda Inventor Kazuhiro Ube City, Ube City, Yamaguchi Prefecture 5 1978, Ube Research Institute, Ube Research Institute (72) Inventor, Yoshii Yamaguchi Prefecture, Ube City, 5 1978, Ube City, Kushigushi 5 Ube Kosan Co., Ltd. Ube Research Center (72) Inventor Yukio Oka 5 1978, Kozugushi, Ube City, Yamaguchi Prefecture 5 Ube Ube Research Center Co., Ltd.

Claims (1)

[Claims] 1. In aerobic culture for obtaining Escherichia coli, an amino acid composition is added to a medium so that the amino acids required for the growth of the bacterium are not deficient, and glucose is added in a state where the pH of the medium is increased. Then the pH of the medium
A high-concentration efficient culturing method of the bacterium, characterized in that the constant value control is performed.