JP2670130B2 - Method for culturing Rhodococcus bacteria and method for producing 2-ketobutyric acid using the microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application] The present invention relates to a method for culturing a bacterium of the genus Rhodococcus and a method for producing 2-ketobutyric acid using the microorganism.

According to the method of the present invention, 2-ketobutyric acid can be produced at high yield and at low cost.

2-ketobutyric acid is the most promising precursor as a raw material for the production of L-isoleucine, which is one of the essential amino acids.

(Prior Art and Problems to be Solved by the Invention) 2-Ketobutyric acid is the most promising precursor as a raw material for L-isoleucine, but the production by the chemical synthesis method is complicated and the process is inexpensive. However, it was difficult to industrially use L-isoleucine as a raw material.

The present inventors, when culturing a microorganism belonging to the genus Rhodococcus, collect the culture obtained by adding 1,2-propanediol and / or 1,2-butanediol to the medium, or recovering from the culture. The present invention has been completed based on the finding that a novel amount of 2-ketobutyric acid is produced when an enzyme reaction of 1,2-butanediol in an aqueous solvent is carried out in the presence of the treated cells or a treated product thereof. It was

[Structure of the Invention] (Means and Actions for Solving the Problems) The present invention belongs to the genus Rhodococcus,
When culturing a microorganism having the ability to produce 2-ketobutyric acid from 1,2-butanediol, Rhodococcus characterized in that 1,2-propanediol and / or 1,2-butanediol are added to the medium (Rhodococcus), a method for culturing a microorganism belonging to the genus, further comprising the step of producing 2-ketobutyric acid from 1,2-butanediol in the presence of the above-mentioned culture, the cells recovered from the culture or a treated product thereof. Is also provided.

The microorganism used in the present invention includes Rhodococcus
ococcus) and is capable of producing 2-ketobutyric acid from 1,2-butanediol, and is not particularly limited. For example, Rhodococcus eq (Rhodococcus eq.
ui) IFO 3730 can be mentioned.

The amount of 1,2-propanediol and / or 1,2-butanediol added to the medium is usually 0.1 to 10% by weight, preferably 0.2 to 4% by weight, based on the medium. The timing of adding 1,2-propanediol or 1,2-butanediol to the medium is not particularly limited, and may be before the start of the culture or during the culture. Furthermore, they may be added all at once or may be added sequentially during the culture.

The medium for culturing the above microorganisms, ethanol, glycerol, acetic acid or a salt thereof as a carbon source, succinic acid or a salt thereof, etc., ammonium chloride as a nitrogen source, ammonium sulfate, inorganic ammonium salts such as ammonium nitrate, peptone, A medium containing meat extract, corn steep liquor, an organic nitrogen source such as casamino acid, potassium phosphate, magnesium sulfate and the like as an inorganic substance, and, if necessary, various nutrients such as vitamins is preferably used. In addition, 1,2-propanediol or 1,2-butanediol added to the medium acts as a carbon source for the growth of microorganisms, in addition to acting as a production promoting substance for 2-ketobutyric acid synthase. When 1,2-propanediol or 1,2-butanediol is added to the medium before the start of culture, it is not always necessary to use ethanol, glycerol or the like as the carbon source.

The culture is carried out under aerobic conditions such as aeration and stirring, and the culture temperature is usually 20 to 40 ° C, preferably 25 to 35 ° C. The pH during the culturing is usually 5 to 10, preferably around 6 to 8,
Adjustment of the pH during the culture can be performed by adding an acid or an alkali.

The culture period is usually 1 to 7 days, preferably 2 to 5 days.
Days.

After culturing, the obtained culture, bacterial cells collected from the culture by hyperfiltration or centrifugation, disrupted products of the bacterial cells by ultrasonic waves, or treated products such as immobilized bacterial cells or their disrupted products are enzymatic agents. To use as.

The means for immobilizing the cells or the disrupted cells is not particularly limited, and for example, an entrapment method using polyacrylamide, alginic acid, κ-carrageenan, or the like is suitably used.

Each of the microorganisms used in the present invention described above may be a microorganism obtained by a technique such as mutation, gene recombination, cell fusion or the like in which each enzyme activity is improved.

This enzyme reaction system may contain at least 1,2-butanediol, and its concentration is, for example, usually 0.01 to 20% by weight, preferably about 0.05 to 10% by weight at the start of the reaction.

The amount of the culture of the microorganism that produces 2-ketobutyric acid using 1,2-butanediol as a raw material, or the amount of the bacterial cells or treated product thereof recovered from the culture is not particularly limited, but is usually 0.1 to 50. %, Preferably about 1 to 30% by weight.

This reaction is carried out at a pH of usually 5 to 10, preferably 6 to 9, and the reaction temperature is usually about 10 to 60 ° C, preferably about 20 to 45 ° C.
° C. The reaction is usually performed for about 10 to 72 hours.

The reaction solvent used in the enzymatic reaction is preferably water or a buffer solution such as phosphoric acid or Tris-hydrochloric acid.

Hereinafter, the present invention will be described in more detail with reference to Examples.
These examples do not limit the scope of the invention in any way.

In the following examples, the qualitativeness of 2-ketobutyric acid was determined by the Rf value of a paper chromatograph, and the quantification was performed by high performance liquid chromatography (Shimadzu LC-5A). Also,%
Is expressed as% by weight.

Separation and purification of 2-ketobutyric acid from the reaction-terminated liquid can be usually performed by an ether extraction method, an ion exchange resin treatment method, or the like.

Example 1 A 100 ml medium having the following medium composition was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes. Shaking culture for 2 hours (preculture)
Then, 1000 ml of the same medium composition a as described above was dispensed into a 5-volume Erlenmeyer flask and sterilized at 120 ° C for 15 minutes, inoculated with 20 ml of the above preculture, and further shake-cultured at 30 ° C for 2 days. did.

1000 ml of the culture termination solution is centrifuged (10,000 rpm, 10 minutes, 4 minutes)
° C), collect the cells, and collect the cells with a 1/15 M phosphate buffer (pH
7.0) After suspending in 50 ml, centrifuge again (10,000 rpm, 10 minutes,
The cells obtained at 4 ° C.) were used as a source of 2-ketobutyric acid-forming enzyme.

To 100 ml of the reaction liquid (1,2-butanediol 2%, 1 / 15M phosphate buffer, pH 7.0), 5 g of 2-ketobutyric acid-producing enzyme source (wet cells) prepared above was added, and the mixture was heated at 30 ° C. Perform reaction for 24 hours and centrifuge from the reaction product (10,000 rpm, 10 minutes, 4 ° C)
The supernatant liquid that had been sterilized by
-The amount of ketobutyric acid was determined. Further, 50 ml of the supernatant was adjusted to hydrochloric acid (pH 2.0), and 200 ml of ether was added to the solution for extraction. After separating the ether phase, a constant temperature water bath (50
(° C) to remove the ether by evaporation to obtain a crude purified product of 2-ketobutyric acid. The results are shown in Table 1.

0.01% medium composition basal medium yeast extract polypeptone _{0.5% (NH 4) 2 HPO} 4 0.65% KH 2 PO 4 0.35% MgSO 4 · 7H 2 O 0.04% FeSO 4 · 7H 2 O 45 ppm MnSO 4 · 4H 2 O 5 ppm _{ZnSO 4 · 7H 2 O 30 ppm} CuSO 4 · 5H 2 O 2.5 ppm NaCl 50 ppm CaCO 3 0.5% distilled water 1000 ml (pH7.0) a: ethanol 1% by volume added to the basal medium, pH 7.0 B: basal medium Ethanol 1% by volume and 1,2-butanediol 1%, pH 7.0 C: basic medium 1% glycerol, pH 7.0 D: basic medium 1% chrycerol and 1,2-butanediol 1% , PH7.0 E: ammonium succinate 1% added to the basic medium, pH7.0 F: ammonium succinate 1% and 1,2-butanediol 1% added to the basic medium, pH7.0 G: ethanol 1 added to the basic medium Volume% and 1,2-propanediol 1% added, pH 7.0 H: Glycerol 1% and 1,2-propanediol 1% added to basic medium, pH 7.0 I: 1,2-butanediene added to basic medium All 1% added, pH 7.0 J: 1,2-propanediol 1% added to the basic medium, pH 7.0 K: 1,2-propanediol 1% and 1,2-butanediol 1% added to the basic medium, pH 7.0 From Table 1 above, 1,2-butanediol or 1,2 was added to the medium.
It can be seen that the addition of -propanediol significantly increases the amount of 2-ketobutyric acid produced.

[Effects of the Invention] According to the method of the present invention, 2-ketobutyric acid can be produced from inexpensive 1,2-butanediol with good efficiency and at low cost.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication C12R 1:01)

Claims (2)

(57) [Claims] 1. A member of the genus Rhodococcus,
When culturing a microorganism having the ability to produce 2-ketobutyric acid from 1,2-butanediol, Rhodococcus characterized in that 1,2-propanediol and / or 1,2-butanediol are added to the medium A method for culturing a microorganism belonging to the genus and having the ability to produce 2-ketobutyric acid from 1,2-butanediol. 2. A member of the genus Rhodococcus,
Reaction of 1,2-butanediol in an aqueous solvent in the presence of a culture of a microorganism having the ability to produce 2-ketobutyric acid from 1,2-butanediol, or cells recovered from the culture or a treated product thereof. After generating 2-ketobutyric acid,
A method for producing 2-ketobutyric acid, which comprises collecting 2-ketobutyric acid from the reaction solution.