JP3437879B2 - Bacterial culture method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing bacterial cells having a high nitrilase enzyme activity by culturing in high yield. In recent years, there has been an active movement to utilize microorganisms or their enzymes as catalysts for various unit chemical reactions or complex chemical reactions. Nitrilases are known as enzymes that hydrolyze nitriles to produce corresponding acids, and are useful as catalysts for producing organic acids.

[0002]

The microorganisms having nitrilase activity BACKGROUND OF INVENTION Problems to Solved], for example, Bacillus (Bacil l us) genus, Bakuterijiumu (Bacteridium)
Genus Micrococcus (M icrococcus) genus and Brevibacterium (Brevibacterium) genus [Sho 58-15120
Gazette], Acinetobacter genus [see Japanese Patent Publication No. 63-2596], Corynebacterium genus [see Japanese Patent Publication No. 63-5680], and Alcaligenes genus [Japanese Patent Application Laid-Open No. 1-132392]. Gazette], Rhodococcus (Rhodo
Bacteria belonging to the genus Coccus (see JP-A-3-251192) and the like are known.

However, among these, Japanese Patent Publication No. 58-15
The method described in JP-A No. 120 has low activity and is not practical. Therefore, in order to enhance the catalytic activity of microorganisms, a method of culturing microorganisms by adding a nitrile compound, such as acetonitrile or isobutyronitrile, as a nitrilase inducer is proposed in Japanese Patent Publication No. 63-2596. Has been done. However, in such a method, in order for the microorganism to utilize these nitrile compounds added to the medium during the culture, it is necessary to add the nitrile compound to the medium according to its consumption, and it is always stable and high. It is difficult to obtain active cells. Further, some nitrile compounds have strong volatility and flammability, and some of them give off a remarkable foul odor, which makes it difficult to use in industrial culture. The above-mentioned JP-A-3-251192 proposes a method for producing bacterial cells having nitrilase enzyme activity by culturing Rhodococcus in a medium containing a lactam compound. In this method, the lactam compound in the culture solution is hardly assimilated, and is excellent in that the inducing effect of the nitrilase enzyme is stable in order to maintain a constant concentration, but the inducing effect is excellent in all Rhodococcus bacteria Not in. Therefore, it has been desired to develop an effective nitrilase enzyme inducer other than the lactam compound.

[0004]

Means for Solving the Problems The present inventor has diligently studied the substance for inducing and producing a nitrilase enzyme and the conditions of use in culturing bacteria, and as a result, added ethylene cyanohydrin to the medium and culturing. As a result, they found that bacterial cells having nitrilase enzyme activity were easily obtained, and completed the present invention.

That is, the present invention relates to a method for culturing a bacterium which has the ability to produce nitrilase, characterized by allowing ethylene cyanohydrin to be present in the culture solution.

In the present invention, ethylene cyanohydrin is used as an inducer, but it was not previously known that ethylene cyanohydrin is effective in inducing nitrilase.

[0007]

DETAILED DESCRIPTION OF THE INVENTION The bacterium to which the present invention is applied is a bacterium having a nitrilase and capable of hydrolyzing a nitrile to produce a corresponding acid. For example, a bacterium of the genus Rhodococcus, specifically, Is, for example, Rhodococcus (Rho
dococcus) sp. SK92 [Microtechnical Research Institute No. 3324], Rhodococcus rhodochrous AT
CC 33025, Rhodococcus rhodococcus
hodochrous) ATCC 33258 and the like. Of these, the Rhodococcus sp. SK92 strain was found by the present applicant, and its mycological properties are described in Japanese Patent Laid-Open No.
-192189.

The general embodiment of the present invention is as follows. That is, as a carbon source, for example, glucose, fructose, sucrose, glycerol, etc., as an organic nutrient source, for example, yeast extract, meat extract, malt extract, peptone, etc., as an inorganic salt, for example, phosphate, magnesium, potassium. Bacteria with nitrilase activity are inoculated into a medium containing, as appropriate, sodium, other trace metals, etc., and ethylene cyanohydrin as an enzyme-inducing agent is added all at once or dividedly and cultured under aerobic conditions. .

The pH of the medium is usually 5 to 10, preferably 6
~ 9, the temperature is usually 15 ~ 37 ℃, preferably 25 ~ 32
Incubate aerobically at ℃ for 1 to 7 days. Addition of ethylene cyanohydrin to the medium is 0.05% to 10% (V / V), preferably 0.5 to 5% (V / V).

[0010]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Sucrose 2% (w / v), Polypeptone 0.5% (w / v)
v), KH ₂ PO ₄ 0.1% (w / v), K ₂ HPO ₄₀ .
Prepare a medium (pH 7.2) consisting of 1% (w / v) and MgSO ₄ · 7H ₂ O 0.1% (w / v), and dispense 100 ml into a 500 ml Erlenmeyer flask and put a cotton plug on it. And sterilized in an autoclave at 120 ° C for 15 minutes. This medium was inoculated with Rhodococcus sp. 92 strain and cultured at 30 ° C for 3 days with shaking. 1 ml of this culture solution was inoculated into the same medium, 2 ml of ethylene cyanohydrin was added at the same time, and the mixture was shake-cultured at 30 ° C. for 20 hours. The cells were recovered from the culture broth by centrifugation, and the same amount of 50 mM KH ₂ as the culture broth was collected.
The cells were suspended in a PO ₄ -Na ₂ HPO ₄ buffer solution (pH 7.7).

0.5 ml of 200 mM acrylonitrile, 20
0mMKH ₂ PO ₄ -Na ₂ HPO ₄ buffer (pH 7.
7) 0.025 ml of the above-mentioned bacterial cell suspension was added to a reaction solution consisting of 0.25 ml and distilled water 0.225 ml and the reaction was carried out at 30 ° C. for 15 minutes to measure the nitrilase activity. 1
After adding 0.2 ml of N-hydrochloric acid to stop the reaction, the cells were removed by centrifugation, and the acrylic acid produced in the reaction solution was added to HP.
It was quantified by LC (manufactured by Wako Pure Chemical Industries, Ltd. Wako s il 5C8
Column use). As a result, 6.45μ in 1 ml of reaction solution
Mol acrylic acid was produced. In addition, the formation of acrylamide was not observed.

Example 2 Each of the strains shown in Table 1 was inoculated into the same medium as in Example 1,
Ethylene cyanohydrin (2 ml) was added and shake culture was carried out at 30 ° C. for 2 days. Cells were collected by the same method as in Example 1 to prepare a cell suspension, and the nitrilase activity was measured. However, the reaction time was 20 minutes. The results are shown in Table 1.

[0014]                               Table 1   ────────────────────────────────           Strain name Acrylic acid production per 1 ml of reaction solution                                           (Μmol)   ────────────────────────────────     Rodococcus Rod Crouse ATCC 33025 0.17     Rhodococcus Rhodochrous ATCC 33258 0.16   ────────────────────────────────

Example 3 Sucrose 1% (w / v), taste liquid 0.75% (w / v), yeast extract 0.1% (w / v), KH ₂ PO ₄ 0.05% (w /
v) and MgSO ₄ .7H ₂ O 0.05% (w / v) of a medium (pH 7.2) was prepared, and 100 ml of this was dispensed into a 500 ml Erlenmeyer flask and sterilized in the same manner as in Example 1.
0.5 ml of ethylene cyanohydrin was added to this medium to inoculate Rhodococcus sp. SK92 pre-cultured in the same manner as in Example 1, and the mixture was cultured at 30 ° C for 1 day, and then 1 ml of ethylene cyanohydrin was added. And further cultured for 1 day. Bacteria were collected from this culture solution in the same manner as in Example 1 and the nitrilase activity was measured.
07 μmol of acrylic acid had been produced. The concentration of ethylene cyanohydrin in the culture medium at the end of the above culture was determined by gas chromatography to be 1.4% (v /
v) (residual rate about 93%).

[0016]

EFFECT OF THE INVENTION Since ethylene cyanohydrin is hardly assimilated during culture and has low volatility, when a bacterium having a nitrilase activity is cultured, ethylene cyanohydrin, which is an inducer at a stable concentration, is contained in the culture solution. Phosphorus can be present, resulting in high yields of bacterial cells with nitrilase activity.

─────────────────────────────────────────────────── ─── Continuation of front page (56) References JP-A-3-251192 (JP, A) JP-A-60-19496 (JP, A) JP-A-53-29995 (JP, A) (58) Field (Int.Cl. ⁷ , DB name) C12N 1/00-1/38 C12N 9/78 C12P 13/00-13/02 JISST file (JOIS) BIOSIS / WPI (DIALOG) PubMed

Claims (3)

(57) [Claims] 1. Rhodococcus rhodocross (Rhodococcus rhodcross) containing ethylene cyanohydrin in the culture medium.
A method for culturing bacteria, which comprises culturing ochrous). 2. Rhodococcu Rhodococcu
s rhodochrous), but Rhodococcus rhodochrous (Rhodo
coccus rhodochrous) ATCC 33025 strain or Rhodococcus
The culture method according to claim 1, which is Rhodococcus rhodochrous ATCC 33258 strain. 3. A method for culturing a bacterium, which comprises culturing Rhodococcus sp. SK92 strain in the presence of ethylene cyanohydrin in the culture broth.