JPH0452114B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing an enzyme, and more specifically, a method for culturing microorganisms by adding glycine and/or manganese compounds to a medium to increase the production amount of physiologically active substances produced by the microorganisms. In particular, the present invention relates to a method of promoting the excretion (secretion) of the physiologically active substance to the outside of the microbial cell. [Prior Art] Generally, methods for producing bioactive polymeric substances such as enzymes by culturing microorganisms such as bacteria and yeast are well known. Such bioactive polymer substances are produced within the cells of microorganisms, and are usually not excreted (secreted) outside the cells, but mostly accumulate within the cells. Therefore, in order to extract a bioactive polymeric substance, it is necessary to crush the collected bacterial cells by ultrasonication or the like, and then separate and purify the target product by centrifugation or the like. Such a mechanical crushing operation not only results in a loss of time and labor, but also has the risk of altering the quality of the product, and is therefore undesirable. By the way, when a certain amount of the product accumulates within the bacterial body, its production naturally stops. However, if it is excreted (secreted) from the bacterial body without being accumulated within the bacterial body, its production should continue. For this reason, attempts have been made to excrete (secrete) products produced within the microbial cells to the outside of the microbial cells, to continue production within the microbial cells, and to accumulate a significant amount of products outside the microbial cells. For example, Horikoshi et al. reported that the extracellular production of polymeric substances such as penicillinase and xylanase contains a plasmid that incorporates a chromosomal DNA fragment of a Bacillus microorganism that carries genetic information involved in the extracellular production of polymeric substances such as penicillinase and xylanase. We have succeeded in culturing a microorganism of the genus Escherichia in a medium containing Na salt or K salt and secreting the above-mentioned macromolecular substances outside the microorganism (Japanese Patent Laid-Open Nos. 59-162874 and 1983). (See Publication No. 60-126077). [Object of the Invention] An object of the present invention is to provide a method for increasing the production amount of useful physiologically active substances produced by microorganisms. A further object of the present invention is to provide a method for excreting (secreting) useful physiologically active substances produced within the microbial cells to the outside of the microbial cells, thereby accumulating a significant amount of the products in the culture medium. That's true. [Structure of the Invention] An object of the present invention is to culture microorganisms that produce enzymes in their cells using a medium containing at least one compound selected from the group consisting of manganese compounds and glycine, which contains only manganese compounds. is cultured in a medium with a content of 0.5 μM or more, and if it contains only glycine, the content is 0.5% or more, to increase the amount of enzyme produced inside the bacteria, and to increase the amount of enzyme produced outside the bacteria. This is achieved by a method for producing the enzyme, which is characterized by promoting the excretion (secretion) into the enzyme and collecting the enzyme from the culture medium. Particularly preferred microorganisms for use in the present invention are bacteria and yeast. Examples of bacteria include Bacillus No. A-59, which is a Gram-positive alkalophilic bacterium.
(ATCC21591) (Isolated as an alkaline amylase-producing bacterium. Agric.Biol.Chem.Vol.36, 1819
Page, 1972), Bacillus No. C-125 (FERM BP-
469) (β-galactosidase producing bacteria, Agric.
Biol.Chem.Vol.43, pages 85 and 1359, 1979
), Escherichia coli
HB 101 (Molecular Cloning a laboratory
manual CSH Lab.1982, 504 pages, Leu, Pro,
Examples of the yeast include baker's yeast (manufactured by Orintal Yeast Co., Ltd.). The microorganisms that can be used in the present invention are not limited to the above specific examples, but can be cultured in a medium containing glycine and/or manganese compounds to increase the total production of microbial products. , existing cultured strains, strains newly isolated from nature, or mutant strains of these strains, as well as genetically modified strains, if they excrete bacterial cell products outside the bacterial body and accumulate in significant amounts. Microorganisms that have newly acquired the ability to produce useful physiologically active substances such as enzymes through cell fusion can be used. Manganese compounds used in the present invention include:
For example, inorganic acid salts such as manganese sulfate, manganese chloride, manganese nitrate, manganese carbonate, manganese silicate, manganese pyrophosphate, manganese hydrogen phosphate, and organic acid salts such as manganese acetate, manganese tartrate, manganese oxalate, and manganese citrate. Examples include. In the present invention, the amount of glycine added is 0.5 to 10% by weight, preferably 0.5 to 3.0% by weight based on the medium.
is appropriate. If the amount is less than this, the desired effect will not be sufficiently expressed, and if it is more than this, the growth of microorganisms will be significantly inhibited, which is not preferable. On the other hand, the amount of the manganese compound added to the medium may be 0.5 μM or more. If the amount is less than this, the desired effect will be insufficiently expressed. Note that since the toxicity of manganese compounds is low, no growth inhibition is observed even when the concentration is increased to a considerable level (for example, 100 mM). The culture medium for microorganisms used in the present invention includes:
Usually, glycine and/or manganese compounds are added to the medium used for culturing microorganisms. That is, either a synthetic medium or a natural medium can be used as long as the medium contains adequate amounts of carbon sources, nitrogen sources, inorganic substances, and other nutrients. Any type of carbon source or nitrogen source may be used as long as it can be used by the bacteria used. inorganic salt,
As for vitamins and the like, those commonly used in microorganism culture media can be used. EXAMPLES Hereinafter, the present invention will be explained in more detail by way of Examples, but the present invention is not limited to the recording of these Examples. Example 1 Using alkalophilic Bacillus No. 59 (ATCC21591), α-glucosidase activity was investigated. 900ml of well water, 15g of soluble starch, 5g of polypeptone, yeast extract (manufactured by Difco)
A medium containing 5 g of dipotassium phosphate, 1 g of magnesium sulfate heptahydrate, 0.2 g of magnesium sulfate heptahydrate, and a predetermined amount of glycine and/or manganese compound, and an aqueous sodium carbonate solution (100 g/) were separately prepared and sterilized. At the time of inoculation, 1/10 volume of sodium carbonate solution is added aseptically to the above medium to form a seed medium and a main medium. Pour 50 ml of culture medium into four 300 ml pleated Erlenmeyer flasks, and add 0.2 ml of cultured medium overnight.
ml is inoculated, cultured with shaking (rotary shaker, 200 RPM) at 37°C for a predetermined time, and aseptically sampled in 2 ml portions. A portion of the sample is directly diluted 10 to 30 times to measure total enzyme activity.
The remainder of the sample is centrifuged, the supernatant is multiplied 5-30 times, and the activity of the enzyme excreted (secreted) into the medium is measured. Enzyme activity is measured as follows. First, p-
Nitrophenyl α-D-glucoside 2mM, PH7.5
Start the reaction by adding the substrate to a total volume of 0.5 ml of a solution containing 40 mM of phosphate buffer and 0.03 to 0.1 ml of enzyme solution.
After shaking reciprocally for 10 minutes at 40°C in a constant temperature bath, add 0.1ml of 1M sodium carbonate to stop the reaction. Add 3 ml of distilled water to dilute and stir, measure absorption at 420 nm, and determine enzyme activity from a standard curve prepared in advance. One unit is the amount of enzyme that produces 1 micromole of p-nitrophenol per minute. To determine the total enzyme activity, that is, the total activity of enzymes in the medium and in the bacterial cells, dilute the entire culture solution, add one drop of toluene to permeabilize the cell membrane, and start the enzyme reaction after 5 minutes. After stopping the reaction, the bacterial cells are removed by centrifugation (3000 RPM, 5 minutes), and the enzyme activity of the supernatant is determined. The excretion rate is calculated by dividing the enzyme activity in the medium by the total enzyme activity. The results are shown in Table 1.

[Table] Shows the degree of occurrence.
It can be seen that the combined use of glycine and a manganese compound increases the total enzyme activity, and that the addition of glycine increases the enzyme activity in the medium, that is, the degree of excretion. Example 2 Alkaliphilic Bacillus No.C-125 (FERMBP-
469) was used to examine β-galactosidase activity. The medium used was the same as in Example 1 except that 15 g of lactose was added instead of soluble starch. Seed culture was performed in the same manner as in Example 1, and the main culture was
The same procedure as in Example 1 was carried out by placing 400 ml of the medium in a two-fold Erlenmeyer flask and adding 1 ml of the seed culture solution. A sample of 3 ml was taken. The enzyme activity was determined by using o-nitrophenyl β-D-galactoside instead of p-nitrophenyl α-D-glucoside in Example 1.
Do the same. One unit is the amount of enzyme that produces 1 micromole of o-nitrophenol per minute. The results are shown in Table 2. In this strain, since manganese compounds increase the total activity of enzymes and promote excretion, some good results can be obtained even with the addition of manganese compounds alone. However, simultaneous addition of 0.5% glycine improves the yield of enzymes excreted from the cells, so it is desirable to use them together.

[Table] is shown below.
Example 3 β-galactosidase activity was measured using Escherichia coli. The medium used was the same as the medium used in Example 2 except that sodium carbonate was removed, and for the main culture, 50 ml of the medium was used in four 300 ml pleated Erlenmeyer flasks. Activity measurement method is Example 2
is the same as The results are shown in Table 3. Glycine not only has an effect on excretion, but also increases total enzyme activity by 3-7 times.

[Table] The numbers in the table indicate the degree of emission.
Example 4 Using baker's yeast, β-fructosidase activity was measured. The medium used was the medium of Example 1 except that sodium carbonate was removed. This medium 100
ml into a 300ml Erlenmeyer flask, inoculate, and
Incubate for 48 hours at ~32 °C. The seed culture solution was suspended in sterilized water and adjusted so that the absorption at 600 nm was around 100, and 1 ml of the suspension was inoculated into the main culture medium, and main culture was carried out in the same manner as in Example 1. Enzyme activity was measured as follows. 5% sutucarose 0.2ml, 0.2M phosphate buffer (PH7.5) 0.1
A total of 0.4 ml of sample solution containing 0.20 to 0.1 ml of enzyme solution is reacted in a constant temperature bath at 40°C for 30 minutes with shaking. Next, add 1.0 ml of DNS reagent (Sakuzo Fukui, "Determination of reducing sugars", p. 19, 1969) to stop the reaction, mix and heat at 100°C for 5 minutes, then cool with water.
Add 4 ml of distilled water and measure the absorption at 500 nm. As a control, add 1.0 ml of DNS reagent to 0.4 ml of distilled water.
Use the same treatment. The amount of enzyme that produces 1 micromole of reducing sugar as glucose per minute is defined as 1 unit. The results are shown in Table 4.

[Table] Shows the degree of emission.
Example 5 In Example 2, β-galactosidase activity was investigated using various manganese compounds instead of manganese sulfate. Culture was performed in a 300 ml Erlenmeyer flask using 50 ml of medium, and enzyme activity was measured 48 hours later. The results are shown in Table 5.

[Table] Shows the degree of occurrence.
Example 6 Extracellular production of cellulase using Escherichia coli that has acquired the ability to produce cellulase through genetic recombination The DNA of alkalophilic Bacillus No. N-4 was incorporated into Escherichia coli (E.coli.HB101) to produce cellulase. The extracellular cellulase activity of mature E. coli was measured when no glycine was added and when 1.0% glycine was added. The cellulase production of the alkaliphilic Bacillus No. N-4 used was described by Horikoshi et al. (Canadian Journal of Microbiology).
Microbiology, Vol. 30, pp. 774-779, 1984), and Sashihara et al.'s report ( Journal of Bacteriology
Bacteriology) Vol. 158, p. 503-506, 1984). The medium used was Brain Heart Infusion medium (manufactured by Difco) to which 30 μg/ml ampicillin was added. 50 ml of the medium was placed in two 300 ml pleated Erlenmeyer flasks, 1.0 ml of the culture solution that had been precultured overnight was added, and after culturing at 37°C for 24 hours, the activity of the centrifuged supernatant was compared. Activity is carboxymethylcellulose sodium salt 0.5% aqueous solution 0.25ml, M
- 0.05 ml of sodium carbonate and centrifuged supernatant of the culture.
After reacting 0.2 ml at 40℃ for 30 minutes, add DNS reagent (“Determination of reducing sugars”, edited by Sakuzo Fukui, published by the University of Tokyo Press, 1969).
2000, p. 19) was added, boiled at 100°C for 5 minutes, cooled with water, added 3.0 ml of distilled water, mixed, measured the absorption at 500 nm, and converted to the amount of glucose. The amount of enzyme that would produce 1 μmole of glucose per minute under these conditions was defined as one unit. According to a report by Sashihara et al., the total activity of pNK-1 is
95.6% and 86.1% of pNK-2 exist inside the bacterial body. It is thought that the activity of these microbial cells was excreted (secreted) into the culture medium by glycine-added culture, so that the extracellular activity in this example was increased. The results are shown in Table 6.

[Table] Activity is the enzyme unit excreted in 1 liter of culture solution. Example 7 Excretion (secretion) of transaminase A standard medium containing the following components was used as a standard medium. 15g glucose or soluble starch or lactose, 5g polypeptone, 5g yeast extract
g, dipotassium phosphate 1.0g, magnesium sulfate heptahydrate 0.2g, manganese (4 or 5) sulfate hydrate 0.02g, meat extract 3.0g if necessary for growth,
In the case of alkaliphilic bacteria, 10 g of sodium carbonate was separately sterilized and added. For the bacterial strain, put 50 ml of culture medium into a 300 ml Erlenmeyer flask, two pleated Erlenmeyer flasks, or four pleated Erlenmeyer flasks.
Inoculate 0.5 ml of preculture solution, and inoculate 37 ml of alkaliphilic bacteria and Bacillus subtilis.
℃ Others at 30 to 32℃ using a rotary shaker (200 rpm/
cultured for 1 minute). Acetobacter pasteurianus and Glconobacter roseus
The initial pH is 5.7, and the initial pH for alkaliphilic bacteria is 10.3.
Others were set to PH7.1. For glutamate-oxaloacetate transferase (aminotransferase, hereinafter referred to as GOT) and glutamate-pyruvate transaminase (GPT), measurement reagents manufactured by Wako Pure Chemical Industries, Ltd. were used. In other words, for GOT measurement, 0.2 ml of substrate solution (4mM α-
(containing ketoglutaric acid, 200mM L-aspartic acid, 0.1M phosphate buffer) and 0.05ml of enzyme solution (centrifuged supernatant after culture) were reacted at 40°C for 15 or 30 minutes, and 0.1ml of color was developed. A solution (consisting of 8mM 2,4 dinitrophenylhydrazine, 5% acetic acid, 95% dimethylformamide) was added to develop color at 40°C for 10 minutes, and 0.4ml of 0.5N sodium hydroxide was added.
Absorption at 520 nm was measured after 10 minutes at 40°C. In the case of GPT, the substrate solution is 2mM α-ketoglutaric acid,
A solution containing 200mM DL-alanine and 0.1M phosphate buffer is used, and both GOT and GPT have a pH of 7.4. The amount of enzyme that produced 1 μmol of pyrupic acid per minute under these conditions was defined as 1 unit. The results are shown in Table 7.

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【table】

【table】

〔Effect of the invention〕

According to the present invention, by culturing microorganisms with the addition of glycine and/or manganese compounds to a medium, the total production amount (sum of intracellular and extracellular substances) of useful physiologically active substances such as enzymes produced by microorganisms. can be greatly increased, and the ratio of produced substances excreted outside the microbial cells can be significantly improved. Example 8 Furthermore, by culturing with the addition of manganese compounds and/or glycine, the production amount of enzymes that are originally produced outside the bacterial cells can also be increased. Table 8 shows an example of xylanase production by alkalophilic Bacillus No. C-125 (FERMBP-469), which is possible with strains of the genus Bacillus, including alkalophilic Bacillus. The medium contains 5.0 g of xylan, 10.0 g of polypeptone, and 5.0 g of yeast extract (manufactured by Difco).
Contains 1.0 g of dipotassium phosphate, 0.2 g of magnesium sulfate heptahydrate, and 10 g of sodium carbonate.
However, sodium carbonate is sterilized separately. 2 pieces pleated
Prepare 50 ml of medium in a 300 ml Erlenmeyer flask, inoculate it with 0.5 ml of preculture that was cultured overnight,
Incubate at 37℃ for 24 hours. Measure the activity of the centrifuged supernatant. The activity is 0.5 per 0.05 ml of enzyme solution (centrifugal supernatant).
Add ml of 0.5% xylan solution (dissolved in 0.05M phosphate buffer with pH 7.0) and react at 40℃ for 10 minutes.
Add 1.0ml of DNS reagent and heat at 100℃ for 5 minutes, then cool with water.
30 ml of distilled water was added and the absorption at 510 nm was measured.
The amount of enzyme that produced 100 μg of xylose per minute was defined as 1 unit. Table 8 Additives Activity of culture supernatant (unit/ml) No addition 1.00 Manganese sulfate 200 μM 3.60 Glycine 0.5% 4.02 〃 1.0% 4.08 Manganese sulfate 200 μM + glycine 1.0% 3.46

Claims (1)

[Scope of Claims] 1. A medium containing at least one compound selected from the group consisting of manganese compounds and glycine, in which microorganisms that produce enzymes within their bodies are cultured, and in the case of containing only manganese compounds, the content thereof is 0.5μM
If the above is the case, if it contains only glycine, it is cultured in a medium with a glycine content of 0.5% or more to increase the amount of enzyme produced within the bacterial body and to inhibit the excretion (secretion) of the enzyme from the bacterial body. A method for producing the enzyme, which comprises accelerating the production of the enzyme and collecting the enzyme from a culture medium. 2. The method according to claim 1, wherein the microorganism is a bacteria or yeast.