JP2572567B2 - Enzyme production method - Google Patents

Description

The present invention relates to a method for culturing microorganisms, and more particularly, to a method for culturing microorganisms.
Glycine derivatives, physiologically inactive amino acids and derivatives thereof, and at least one compound selected from the group consisting of amino acid analogs and derivatives thereof are added to the medium, or these compounds and a manganese compound are added to the medium to remove microorganisms. The present invention relates to a method for culturing and increasing the production amount of a physiologically active substance produced by a microorganism, and particularly for promoting the excretion of the physiologically active substance out of the cells.

(Prior art)

In general, a method for producing a high molecular bioactive substance such as an enzyme by culturing a microorganism such as bacteria or yeast is well known. Such a high molecular weight bioactive substance is produced in the cells of the microorganism, and is generally accumulated in the cells without being excreted (secreted) outside the cells. Therefore, in order to remove the high molecular weight bioactive substance, it is necessary to crush the collected cells by ultrasonic treatment or the like, and to separate and purify the target substance by centrifugation or the like. Such a mechanical crushing operation is not preferable because it not only results in a loss of time and labor but also may deteriorate the product.

By the way, when a certain amount of product accumulates in the cells, the production naturally stops. However, if this is excreted (secreted) out of the cells without accumulating in the cells, its production should continue. For this reason, attempts have been made to discharge (secrete) products produced in the cells outside the cells, to continue production in the cells, and to accumulate a significant amount of products outside the cells. For example, Horikoshi et al. Reported that the chromosomal DNA of a bacterium belonging to the genus Bacillus responsible for the genetic information involved in the extracellular production of macromolecular substances such as penicillinase and xylanase.
Culturing a microorganism of the genus Escherichia having the ability to produce the extracellular material of the macromolecule containing the plasmid incorporating the fragment in a medium containing a Na salt or a K salt to secrete the macromolecule out of the cells; It has been successful (see Japanese Patent Publication Nos. 3-55105 and 3-75153).

[Object of the invention]

An object of the present invention is to provide a method for increasing the production of a useful bioactive substance produced by a microorganism. It is a further object of the present invention to provide a method for excreting (secreting) a useful enzyme produced in the cells of a microorganism outside the cells, thereby accumulating a significant amount of the product in the medium. is there.

[Configuration of the invention]

An object of the present invention is to provide glycylglycine, glycyl-L-
Alanine, DL-α-amino normal butyric acid, and at least one compound selected from the group consisting of DL-norvaline added to the medium, or glycylglycine, glycylglycylglycine, glycine methyl ester, glycine ethyl ester, DL -Aspartic acid, DL-alanine, DL-lysine, D-threonine, DL-methionine, glycyl-L-alanine, DL-α-amino normal butyric acid,
γ-amino normal butyric acid, DL-norvaline, and at least one compound selected from the group consisting of β-alanine and a manganese compound are added to the medium, the microorganism is cultured, the enzyme is accumulated in the medium, and this is recovered. Achieved.

 The microorganism used in the present invention is a bacterium.

Examples of the bacteria include microorganisms belonging to the genus Bacillus as Gram-positive bacteria. For example, the alkalophilic bacterium Bacillus No. A-59 (ATCC21
591) (isolated as an alkaline amylase producing bacterium).
Agricultural Biological Chemistry (Agric. Biol. Chem.) Vol. 36, p. 1819, 1972), Bacillus No. C-125 (FERMBP-469) (β-galactosidase producing bacterium, Agric. Biol. Chem. Vol. 43, p. 85 and p. 1359, 1979), Bacillus No. 2
21 (ATCC 21522), Bacillus No. A-40-
2 (ATCC 21592) and also Bacillus Subtills Marburg 168GSY1026 (ATC
C 31339). As a gram-negative bacterium, Aeromonas liquefacien
s) (IAM 12337), Pseudomonas aeruginosa (IAM 1275), Agrobacterium radiobacto
r) (IAM 12035).

Examples of intestinal bacteria include, for example, Klebsiella pneumoniae (IAM1063) and Enterobacter aerogenes.
es) (IAM1183), Serratia Marcetssense (Serrati)
a marcescens) (IAM 12142), Erwinia herbicola (IAM 1562)
Bulgaris (Proteus vulgaris) (IAM 1025), and Escherichia coli HB101 (Molecular cloning a laboratory manual: Molecular) often used for genetic engineering.
Cloning a laboratory manual CSH Lab. 1982, p504, Le
u, Pro, Thiamine requirements) can also be used.

The microorganism that can be used in the present invention is not limited to the above specific examples, and is at least one selected from the group consisting of the glycine derivative physiologically inactive amino acids and derivatives thereof and amino acid salt substances and derivatives thereof. By adding the compound to the medium, or by culturing in a medium containing these compounds and a manganese compound, the total production of the cell product is increased, and the cell product is discharged out of the cell, As long as it accumulates in a significant amount, existing culture strains, strains newly isolated from nature, or mutants of these strains, as well as new enzymes such as enzymes by gene recombination or cell fusion are useful. Any of microorganisms that have acquired the ability to produce a physiologically active substance can be used.

Glycine derivatives, physiologically inactive amino acids and derivatives thereof, and amino acid analogs and derivatives thereof used in the present invention include glycylglycine, glycylglycylglycine, glycine methyl ester, glycine ethyl ester, DL-aspartic acid, DL- Alanine, DL-lysine,
It is selected from D-threonine, DL-methionine, glycyl-L-alanine, DL-α-amino normal butyric acid, γ-amino normal butyric acid, DL-norvaline, and β-alanine.

The manganese compound used in the present invention includes, for example, inorganic acid salts such as manganese sulfate, manganese chloride, manganese nitrate, manganese carbonate, manganese silicate, manganese pyrophosphate, manganese hydrogen phosphate, manganese acetate, manganese tartrate, and manganese oxide Organic acid salts such as manganese acid and manganese citrate are exemplified.

In the present invention, the amount of the glycine derivative, physiologically inactive amino acid and its derivative and amino acid analog and its derivative is 0.1 to 10% by weight, preferably 0.2 to 7.0% by weight based on the culture medium. If it is less than this, the desired effect is insufficiently expressed, and if it is more than this, the growth inhibition of the microorganism becomes conspicuous, which is not preferable.

On the other hand, the addition amount of the manganese compound is 0.5
The concentration may be at least μM. If the amount is less than this, the desired effect is insufficiently exhibited. Since the toxicity of the manganese compound is low, no growth inhibition is observed even when the concentration is increased to a considerable concentration (for example, 100 mM).

As the culture medium of the microorganism used in the present invention, usually,
A glycine derivative, a physiologically inactive amino acid and its derivative, an amino acid analog and its derivative, or a manganese compound added thereto is used in a medium used for culturing microorganisms.

That is, any of a synthetic medium and a natural medium can be used as long as the medium appropriately contains a carbon source, a nitrogen source, an inorganic substance, and other nutrients. Any type of carbon source and nitrogen source may be used as long as the bacteria used can be used. Inorganic salts, vitamins, and the like can also be used as those usually used in microorganism culture media.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the descriptions of these Examples.

Example 1 β-galactosidase activity was examined using alkalophilic Bacillus No. C-125 (FERMBP-469).

A medium in which lactose 15 g, polypeptone 5 g, yeast extract (manufactured by Difco) 5 g, dipotassium phosphate / g, magnesium sulfate heptahydrate 0.2 g, a predetermined amount of a glycine derivative, and a manganese compound are added to 900 ml of well water. , And an aqueous sodium carbonate solution (100 g /) were separately prepared and sterilized. At the time of inoculation, a 1/10 volume of sodium carbonate solution is aseptically added to the above medium to obtain a seed medium and a main medium.

Put 50ml of medium into a 300ml 4 fold Erlenmeyer flask,
To this, 0.2 ml of the overnight culture is inoculated, shake-cultured (rotary shaker, 200 RPM) at 37 ° C. for a predetermined time, and 2 ml is sampled aseptically.
A part of the sample is directly diluted 10 to 30 times to measure the total enzyme activity. The rest of the sample is centrifuged, the supernatant is multiplied by 5 to 30 and the activity of the enzyme excreted (secreted) into the medium is measured.

Enzyme activity is measured as follows. First, O-nitrophenyl β-D-galactoside 2 mM, phosphate buffer 40 mM at pH 7.5, a total volume of 0.5 ml containing an enzyme solution 0.03-0.1 ml, a substrate was added to start the reaction, and in a thermostat, After shaking at 40 ° C for 10 minutes, the reaction is stopped by adding 0.1 ml of 1M sodium carbonate. 3 ml of distilled water was added thereto, and the mixture was diluted and stirred.The absorbance at 420 nm was measured, and from a standard curve prepared in advance,
Determine enzyme activity. The amount of the enzyme that produces 1 micromol of O-nitrophenol per minute is defined as one unit.

 Table 1 shows the results.

Example 2 β-galactosidase activity was measured using Escherichia coli HB101 strain. The medium used was the same as the medium used in Example 1 except that sodium carbonate was removed.
A 50 ml culture medium was used with a 300 ml 4-fold Erlenmeyer flask. The activity measurement method is the same as in Example 1. Table 2 shows the results when various glycine derivatives were added.

Example 3 Aeromonas liquaciens
glutamine oxaloacetate aminotransferase (GOT) activity was measured using E. coli (IAM 12337).

A standard medium containing the following components in 1 was used. Glucose or soluble starch or lactose 15 g, polypeptone 5 g, yeast extract 5 g, dipotassium phosphate 1.
0g, magnesium sulfate ・ 7H ₂ O 0.2g, manganese sulfate ・ 4
55H ₂ O 0.02 g was used. In the Erlenmeyer flask,
Add 50 ml of the medium, inoculate 0.5 ml of the preculture, and add 30-32
Rotational shaking culture (200 RPM) was carried out at an initial pH of 7.
1). For the GOT activity, a measuring reagent of Wako Pure Chemical Industries, Ltd. was used.

That is, GOT measurement is performed by measuring 0.2 ml of a substrate solution (containing 4 mM α-ketoglutaric acid, 200 mM L-aspartic acid, and 0.1 M phosphate buffer (pH 7.4)) and 0.05 ml of an enzyme solution (centrifugation after culture). Supernatant) is reacted at 40 ° C for 15 minutes or 30 minutes, 0.1m
l of color developing solution (8 mM 2,4-dinitrophenylhydrazine,
Containing 5% acetic acid and 95% dimethylformamide)
The color was developed at 40 ° C for 10 minutes, 0.4 ml of 0.5N-NaOH was added, and the absorbance at 520 nm was measured after 10 minutes at 40 ° C. The amount of the enzyme that produces 1 μmol of pyruvate per minute was defined as 1 unit.

The results obtained when various glycine derivatives were added are shown in the third section.
It is shown in the table.

Example 4 Culturing was performed on the bacterial species shown in Table 4 by using a medium and a method substantially similar to those in Example 3. However, an Erlenmeyer flask was used for the culture, and a temperature condition of 30 to 33 ° C. was set. 4th
The symbols of the media used in the table are respectively G = glucose 1.
5%, SS = soluble starch 1.5%, ME = meat extract 0.3
%. In Table 4, all manganese sulfates were 200 μM.
And DL-Norvalin = DL-2-Amino pentanoic acid, glycine ethyl ester = glycine ethyl ester hydrochloride. Extracellular enzyme activity is shown for GOT. (Note that the effect of the gram-negative strain was almost the same with or without the addition of a manganese compound.) Example 5 As in Example 1, production of xylanase by alkalophilic Bacillus No. C-125 (FERM BP-469) was carried out as an example of secretion outside the cells. The culture medium contained the compounds shown in Table 5 in 5.0, xylan 5.0 g, and polypepto.
It contained 5.0 g, yeast extract (manufactured by Difco) 5.0 g, dicalcium phosphate 1.0 g, magnesium sulfate heptahydrate 0.2 g, and sodium carbonate 10 g, and sodium carbonate was sterilized separately. 50 ml of the medium was placed in a 300 ml Erlenmeyer flask with two folds, and 0.5 ml of a preculture liquid cultured overnight was inoculated, and cultured on a rotary shaker at 37 ° C for 24 hours. The activity of the centrifuged supernatant was measured. The activity was 0.5 ml of enzyme solution (centrifuged supernatant) to 0.05 ml.
A 0.5% xylan solution (dissolved in 0.05M phosphate buffer at pH 7.0) was added and reacted at 40 ° C for 10 minutes. The DNS reagent (reducing sugar quantification method “Sakuzo Fukui”, published by The University of Tokyo Press, 1969)
Year, p. 19) Add 1.0 ml, heat at 100 ° C for 5 minutes, cool with water, and add 3.
0 ml of distilled water was added and the absorption at 510 nm was measured. In one minute
The amount of the enzyme that produced 100 μg of xylose was defined as one unit.

 The results after 24 hours are shown in Table 5.

According to the present invention, a glycine derivative, a physiologically inactive amino acid and a compound selected from the group consisting of a derivative thereof and an amino acid analog and a derivative thereof, or a manganese compound and a manganese compound are added to a medium to remove microorganisms. By culturing, the total amount of useful biologically active substances such as enzymes produced by microorganisms (sum of the inside and outside of the cells) is greatly increased, and the amount of the products discharged outside the cells is greatly increased. The ratio can be significantly improved.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12R 1:01) (C12N 9/10 C12R 1: 385) (C12N 9/10 C12R 1:37) (C12N 9/10 C12R 1:22) (C12N 9/10 C12R 1:18) (C12N 9/10 C12R 1:43) (C12N 9/10 C12R 1: 125) (C12N 9/10 C12R 1:07) (C12N 9/38 C12R 1:07) (C12N 9/38 C12R 1:19) (C12N 9/42 C12R 1:07)

Claims (2)

(57) [Claims] A bacterium is cultured by adding at least one compound selected from the group consisting of glycylglycine, glycyl-L-alanine, DL-α-aminonormalbutyric acid, and DL-norvaline to a medium, and culturing the bacterium. A method for producing oxygen, comprising accumulating an enzyme and recovering the enzyme. 2. Glycylglycine, glycylglycylglycine, glycine methyl ester, glycine ethyl ester, β-alanine lysine ethyl ester, DL-aspartic acid, DL-alanine, DL-lysine, D-threonine,
DL-methionine, glycyl-L-alanine, DL-α-amino normal butyric acid, γ-amino normal butyric acid, DL-norvaline, and at least one compound selected from the group consisting of β-alanine and a manganese compound are added to the medium. ,
A method for producing an enzyme, comprising culturing bacteria, accumulating the enzyme in a medium, and collecting the enzyme.