JPH0469992B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing Rhodococcus microbial cells with high nitrile hydratase enzyme activity in high yield. Nitrile hydratase is known as an enzyme that hydrates nitriles to produce the corresponding amides, and its industrial applications include reactions to produce the corresponding amides from acrylonitrile or methacrylonitrile. important,
For example, there is a description in JP-A-62-91189. (Prior Art) In producing bacterial cells having nitrile hydratase enzyme activity by culturing bacteria belonging to the genus Rhodococcus and having the ability to produce nitrile hydratase, JP-A-61
Publication No. 162193 includes, for example, Rhodocotcus sp.S-
In the case of 6 strains, cultured in a normal nutrient medium consisting of glucose, peptone, yeast extract, and meat extract,
Active bacterial cells were obtained. On the other hand, Japanese Patent Application Laid-Open No. 62-91189, for example,
In the case of Rhodococcus sp. AK32 strain, we cultured it in a normal nutrient medium consisting of glucose, peptone, meat extract, etc. and added nitrile to obtain more highly active bacterial cells. was shown to be an enzyme inducer of Rhodococcus sp. AK32 strain. (Problems to be solved by the invention) Among the bacteria of the genus Rhodococcus that have nitrile hydratase enzyme activity,
AK32 strain, AK33 strain described in Publication No. 62-91189,
All AK3132 strains have an inducible enzyme,
In order to express high enzyme activity, it has been desired to develop a simpler and more effective enzyme inducer. (Means for Solving the Problems) The present inventor has developed an enzyme-inducing method in order to express higher enzyme activity in bacteria of the genus Rhodocotcus that have nitrile hydratase as an inducible enzyme. As a result of intensive research on substances and conditions for their use, it was discovered that Rhodococcus bacteria with extremely strong nitrile hydratase activity could be obtained by culturing them by adding an organic acid to the medium, and the present invention I was able to complete it. That is, the present invention belongs to the genus Rhodococcus,
A method for culturing bacteria of the genus Rhodococcus, which comprises adding an organic acid to a medium when culturing bacteria capable of producing nitrile hydratase to produce bacterial cells having nitrile hydratase enzyme activity. It is something. The method of the present invention will be explained in detail below. In a general embodiment of the invention, bacteria capable of producing nitrile hydratase are combined with carbon sources such as glucose, fructose, sucrose and aldose, and nitrogen sources such as ammonium sulfate, ammonium nitrate, ammonia and urea. Enzyme inducers are added to a medium containing organic nutritional sources, such as yeast extract, malt extract, meat extract and peptone, and inorganic nutritional sources, such as phosphate, sodium, potassium, iron, magnesium, manganese, zinc, etc., as appropriate. At least one of organic acids such as propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, and isovaleric acid is added as a culture agent. Alternatively, the culture may be carried out in a medium in which the organic acid as the enzyme inducer is the only carbon source, and the nitrogen source, inorganic nutrient source, and organic nutrient source described above are added as necessary.
The concentration of the enzyme inducer in the medium is usually 0.1 g/
or more, less than 20g/but preferably 0.5
Adjust so that it is at least g/ and less than 5 g/. When this concentration exceeds 20 g/g, significant inhibition of bacterial cell growth is observed and a decrease in the activity of the obtained bacterial cells is observed. On the other hand, less than 0.1g/
Enzyme activity cannot be induced sufficiently, and the activity of the bacterial cells is low. The pH of the medium is usually 5-9, preferably 6-9.
8. Temperature is usually 20-35℃, preferably 27-32℃
Then, culture is carried out aerobically for 1 to 5 days. (Effects of the Invention) According to the present invention, nitrile hydratase enzyme activity can be strongly expressed in Rhodococcus bacteria that have the ability to produce nitrile hydratase, so that amide can be produced from nitrile by an enzymatic method. In this case, it is possible to increase the amide production amount per bacterial cell and increase the amide production rate, which greatly contributes to improving productivity by downsizing equipment and reducing costs. . (Example) Next, the present invention will be explained in more detail with reference to Examples, but the scope of the present invention is not limited to the Examples. Example 1 Glucose 2% by weight, meat extract 0.1% by weight, peptone 0.1% by weight, salt 0.1% by weight, potassium phosphate 0.1% by weight, magnesium sulfate 0.05% by weight,
Ferrous sulfate 0.005% by weight, manganese sulfate 0.005% by weight, ammonium sulfate 0.1% by weight, potassium nitrate
In a medium containing 0.1% by weight (hereinafter referred to as Z medium),
After adding 0.25% by weight of isobutyric acid, the pH was adjusted to 7.0 with potassium hydroxide, sterilized at 121°C for 30 minutes, cooled to room temperature, and then incubated with Rhodococcus sp. No. 1047
No.) was inoculated from a slant using a platinum loop and cultured at 30°C for 38 hours. Next, bacteria were collected from the obtained culture solution by centrifugation at 4°C, washed with 0.05M phosphate buffer (PH7.0), and used for reaction. That is, the dry bacterial mass was 0.2% by weight, methacrylnitrile 2.0% by weight, and 0.05M phosphate buffer (PH
7.0) A 97.8% by weight reaction solution was prepared and the reaction was started at 30°C. 15 minutes after the start of the reaction, the reaction solution was analyzed by gas chromatography and found to be 2.5% by weight.
The reaction proceeded almost quantitatively and was completed, with no unreacted methacrylonitrile, methacrylic acid, or other by-products. Examples 2 to 6 Various organic acids were added to the Z medium used in Example 1.
After adding 0.25% by weight, adjust the pH with potassium hydroxide.
After adjusting the temperature to 7.0 and sterilizing it at 121℃ for 30 minutes and cooling it to room temperature,
A loopful of sp.AK32 strain (Feikoken Bibori No. 1046) was inoculated from a slant and cultured at 30°C for 38 hours. However, when the organic acids are methacrylic acid and acrylic acid, first sterilize the Z medium whose pH has been adjusted to 7.0.
After cooling, methacrylic acid or acrylic acid and potassium hydroxide in an amount necessary for pH adjustment were added aseptically and cultured at 30°C for 133 hours. next,
Collection of bacteria from the obtained culture solution, washing of the bacterial cells, and hydration reaction with methacrylonitrile were performed in the same manner as in Example 1, and the methacrylamide yields after 5 minutes of reaction time were compared. Note that gas chromatography was used for the analysis, and the results obtained are shown in Table 1.

[Table] Examples 7 to 9 After adding isobutyric acid at various concentrations to the Z medium used in Example 1, Rhodococcus sp. AK32 strain was inoculated in the same manner as in Example 1, and incubated at 30°C. Culture was carried out in For those in which bacterial growth was observed,
The reaction was carried out under the same conditions as in Example 1, and the reaction time was 5.
The methacrylamide yield after minutes was measured. The results obtained are shown in Table 2.

[Table] Example 10 Medium containing 0.1% by weight of potassium phosphate, 0.05% by weight of magnesium sulfate, 0.005% by weight of ferrous sulfate, 0.005% by weight of manganese sulfate, 0.1% by weight of ammonium sulfate, and 0.1% by weight of potassium nitrate (hereinafter referred to as After adding 0.25% by weight of isobutyric acid to the medium (referred to as B medium), Rhodococcus sp.
The AK32 strain was inoculated and cultured at 30°C for 96 hours. Next, bacterial collection from the obtained culture solution, washing of the bacterial cells, and hydration reaction with methacrylonitrile were carried out in Example 1.
When the reaction solution was analyzed after 15 minutes of reaction time, the reaction proceeded almost quantitatively, and 2.5% by weight of methacrylamide was produced. Example 11 A medium (PH
7.0) was sterilized at 121°C for 30 minutes, cooled to room temperature, and one platinum loop of Rhodococcus sp. AK33 strain was inoculated from the slant and cultured at 30°C for 40 hours. Next, the bacterial cells were collected from the obtained culture solution by centrifugation at 4°C, washed with physiological saline, and then
Added 0.25% by weight of isobutyric acid to the culture medium and added the washed bacterial cells to a solution adjusted to pH 7.0 with potassium hydroxide.
Stirred at 30°C for 8 hours. Next, the bacterial cells were separated from this bacterial cell immersion solution, and a hydration reaction with methacrylonitrile was performed in the same manner as in Example 1, and the reaction time was 15.
When the reaction solution was analyzed after minutes, 1.9% by weight of methacrylamide was produced and 0.5% by weight of methacrylonitrile was detected. Example 12 The hydration reaction of acrylonitrile was carried out under the same conditions as in Example 4, except that the reaction substrate was changed from methacrylonitrile to acrylonitrile, and the reaction was started.
After 20 minutes, the reaction solution was analyzed by gas chromatography and found to contain 2.6% by weight of acrylamide and no unreacted acrylonitrile, acrylic acid, or other by-products, indicating that the reaction proceeded almost quantitatively and was completed. was. Example 13 The hydration reaction of acrylonitrile was carried out under the same conditions as in Example 5, except that the reaction substrate was changed from methacrylonitrile to acrylonitrile, and the reaction was started.
After 20 minutes, the reaction solution was analyzed and found to be 0.4% by weight.
of acrylamide was formed and 1.7% by weight of acrylonitrile was detected. Example 14 The bacterial strain was Rhodococcus sp. AK3132 strain, and the culture was carried out under the same conditions as in Example 10, except that the culture time was 120 hours. Bacterial cells were obtained from the obtained culture solution in the same manner as in Example 1 and subjected to reaction. That is, the dry bacterial mass was 1.0% by weight, methacrylonitrile was 1.0% by weight, and 0.05M phosphate buffer (PH
7.0) A 98.0% by weight reaction solution was prepared and the reaction was started at 30°C. When the reaction solution was analyzed one hour after the start of the reaction, 0.3% by weight of methacrylamide was produced and 0.8% by weight of methacrylonitrile was detected.

Claims (1)

[Claims] 1 Belongs to the genus Rhodococcus,
1. A method for culturing bacteria of the genus Rhodococcus, which comprises adding an organic acid to a medium when culturing bacteria capable of producing nitrile hydratase to produce bacterial cells having nitrile hydratase enzymatic activity. 2. The culture method according to claim 1, wherein the organic acid is a compound selected from propionic acid, n-butyric acid, isobutyric acid, n-valeric acid, and isovaleric acid. 3 The concentration of organic acid in the medium is 0.1 g/ or more, 20
The culture method according to claim 1, wherein the amount is less than g/g/g. 4. Rhodococcus is a bacterium that belongs to the genus Rhodococcus and has the ability to produce nitrile hydratase.
sp.AK32 (Rhodococcus sp.AK32)
No. 1046, Rhodococcus sp. AK33 (Rhodococcus
sp.AK33) Microtechnical Research Institute No. 1047 or Rhodococcus erythropolis AK3132 (Rhodococcus sp.
erythropolis AK3132) The culture method according to claim 1, which is F. erythropolis AK3132).