JP2587671B2 - Purification method of L-isoleucine - Google Patents

Description

The present invention relates to a method for purifying L-isoleucine. More specifically, the present invention relates to a method for purifying isoleucine, which removes impurities mainly containing amino acids such as valine from a reaction solution containing L-isoleucine obtained by a fermentation method or an enzymatic method.

[Prior Art] L-isoleucine is one of essential amino acids, and is a useful compound used in medicines, feeds and the like.

As a method for producing L-isoleucine, a fermentation method and an enzymatic method using DL-α-aminobutyric acid, α-ketobutyric acid, D-threonine or the like as a precursor have been established, and L-produced in a reaction solution has been established.
-It is known to purify isoleucine.

L-isoleucine is obtained by fermentation or enzymatic method in the presence of cells or enzymes in an aqueous medium near neutrality, for example, using DL-α-aminobutyric acid as a precursor. The contained valine and other amino acid impurities must be removed. Therefore, non-polar porous synthetic adsorbents (JP-A-54-55519) and ion-exchange resins (JP-A-56-1)
A method for purifying L-isoleucine using S. 31550) is known. By the way, there is no example of chromatographic separation of L-isoleucine using activated carbon, and even in the report of separation of leucine and valine having similar structures, an aqueous solution of ethyl acetate or a phosphate buffer is used as an eluent (J.
P. Greenstein, M. Winitz, “Chemistry of the Amino Aci
ds, vol 2 ", p1447, John Wiley & Sons, Inc., New York (19
61) .F. Turba, M. Richter, F. Kuchar, Naturwiss., 31 , 508
(1943). ).

[Problems to be solved by the invention]

Most of the conventional non-polar porous synthetic adsorbents are styrene-divinylbenzene-based crosslinked polymers. In these generally used non-polar porous synthetic adsorbents, L-
Since the breakthrough points of isoleucine and impurities mainly composed of amino acids such as valine are almost the same, sufficient separation performance cannot be obtained, and the recovery of L-isoleucine is very low. This fact is pointed out by another inventor, Otani et al. (JP-A-61-178952, line 12 to line 19 from page 2).

Further, in the purification method using an ion exchange resin, a large amount of acid and alkali are required for regeneration of the ion exchange resin, so that the cost increases. Furthermore, when the coexisting inorganic salt is large, the exchange capacity of the ion exchange resin is exchanged with the inorganic salt, so that a large amount of the ion exchange resin is required for L-isoleucine, and there is a problem that the ion exchange resin cannot be used substantially.

In addition, in the above-mentioned purification method using activated carbon, an auxiliary agent (ethyl acetate or phosphate buffer) is mixed in the recovered solution, so that the isolation operation is complicated, and the activated carbon needs to be regenerated. I haven't become a target.

[Means for solving the problem]

The present inventors have intensively studied to solve the above-mentioned problems, and as a result, have arrived at the method of the present invention.

That is, in the purification method of the present invention, a reaction solution containing L-isoleucine obtained by a fermentation method or an enzymatic method is passed through activated carbon, then water is passed, and when the impurities are eluted, the water flow is stopped. This is a method for purifying L-isoleucine, characterized in that L-isoleucine adsorbed on activated carbon is eluted with aqueous ammonia, and L-isoleucine is isolated from the eluate according to a conventional method.

Impurities having a higher affinity for activated carbon than L-isoleucine can be chromatographed when eluted with aqueous ammonia.

The reaction solution containing L-isoleucine used in the present invention includes, for example, a solution separated from a fermentation solution or an enzyme reaction solution. In addition, the present invention can be effectively applied to an L-isoleucine-containing aqueous solution contaminated with valine and other amino acids.

The concentration of L-isoleucine in the aqueous solution of L-isoleucine, which is the starting material of the method of the present invention, may be not more than the saturation solubility of L-isoleucine.

The type of activated carbon used in the present invention is lime-based activated carbon,
Palm shell activated carbon, charcoal activated carbon, petroleum pitch activated carbon and the like are not particularly limited, and include, for example, Diamond Hope 008, S80, Diamond Hove G, W (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.), HC -30S, GL-30, 2GL, 4GL (Tsurumi Coal, Ltd.), BAC-LP, MP (Kureha Chemical Co., Ltd.), Kuraray Coal GW, GL, GLC, PK (Kuraray Chemical Co., Ltd.) ,
LH2C, W5C, KL (manufactured by Takeda Pharmaceutical Co., Ltd.) and the like can be used as appropriate.

The amount of activated carbon used is based on L-isoleucine.
About 10 to 50 times (weight basis) is sufficient.

Further, the pH of the liquid to be treated during the passage is preferably neutral, and the temperature is
It is carried out at a temperature of 80 ° C. or less and an agitation speed SV = 0.5 to 5 hr ⁻¹ .

The concentration of aqueous ammonia used for elution of L-isoleucine is not limited, and a concentration sufficient to elute L-isoleucine from activated carbon may be used, but ammonia water of about 0.1 to 10 N is preferable. In general, the use of low-concentration aqueous ammonia has the advantage of allowing chromatographic separation of impurities having a higher affinity for activated carbon than L-isoleucine during elution.
There is an advantage that isoleucine can be recovered at a high concentration and the isolation operation after that is easy.

The obtained ammonia eluate containing L-isoleucine can be easily purified at low cost and with high purity by a known isolation method, ie, unit operation such as concentration, crystallization, solid-liquid separation, and drying.
-Isoleucine can be isolated.

As described in detail above, the present invention uses activated carbon,
An object of the present invention is to provide a novel and simple L-isoleucine purification method for purifying and removing impurities mainly composed of amino acids such as valine contaminating L-isoleucine.

Example 1 L-isoleucine 19.7 g / l and L-isoleucine obtained by disinfecting an L-isoleucine fermentation solution obtained using Brevibacterium flavum using DL-α-aminobutyric acid as a precursor were used. Valine 0.7 g / l, D-α-aminobutyric acid 11.5 g / l, L-alanine 3.1 g / l, glycine 2.7 g / l,
L-isoleucine aqueous solution 200m containing L-lysine 0.2g / l etc.
l column filled with 280 ml of Diamond Hope 008 (φ3.2cm
× H35 cm). Next, water was passed at SV = 3 hr ^-1 , and when the impurities such as valine eluted, the water flow was stopped. Then, 2N ammonia water was passed to obtain an L-isoleucine solution. L-valine, D-α in L-isoleucine solution
The removal rates of -aminobutyric acid, L-alanine, glycine and L-lysine were 100%, 100%, 100%, 100% and 53%, respectively, and the recovery rate of L-isoleucine was 84%.

Example 2 L obtained using Brevibacterium flavum using α-ketobutyric acid as a precursor
-10.8 g / l of L-isoleucine and 0.3 g / l of L-valine, 0.1 g / l of L-α-aminobutyric acid, 0.2 g / l of L-alanine, 0.5 g of glycine obtained by removing the isoleucine enzyme reaction solution / l, L-
360 ml of an aqueous solution of L-isoleucine containing 0.6 g / l of lysine
It was injected into the upper part of a column (φ3.2 cm × H35 cm) packed with 280 ml of AC-LP. Next, water was passed at SV = 3 hr ^-1 and when the impurities such as valine eluted, the water flow was stopped, and the temperature of the column was lowered.
While maintaining the temperature at 60 ° C, 2N aqueous ammonia was passed through, and L-
Isoleucine was obtained. L-valine, L-α-aminobutyric acid, L-alanine, glycine in an L-isoleucine solution,
The removal rates of L-lysine were 97%, 100%, 100%, and 10%, respectively.
0% and 95%, and the recovery of L-isoleucine was 72%.

Comparative Example L-isoleucine 24.9 g / l and L-valine 1.8 g / l, DL
-30 ml of an aqueous L-isoleucine solution containing 5.3 g / l of α-aminobutyric acid, 2.8 g / l of L-alanine, 2.1 g / l of glycine and 0.6 g / l of L-lysine is Diaion, a non-polar porous synthetic adsorbent.
It was injected into the upper part of a column (φ3 cm × H88 cm) packed with 625 ml of HP-20 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.). Water was passed at SV = 1.5 hr ^-1 to obtain an L-isoleucine solution. L-valine, DL-α-aminobutyric acid, L-valine in L-isoleucine solution
100% removal rate of alanine, glycine and L-lysine
However, the recovery of L-isoleucine was as low as 7%,
84% of L-isoleucine eluted with impurities such as valine.

〔The invention's effect〕

As described above, according to the method of the present invention, L-isoleucine can be purified extremely efficiently from an aqueous solution of L-isoleucine contaminated with impurities mainly containing an amino acid such as valine. In addition, the method of the present invention is an industrially innovative method for purifying L-isoleucine, which does not require a large amount of auxiliary agent or the like.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Masaki Odagiri 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref. Inside the Central Research Laboratory of Mitsubishi Yuka Co., Ltd. (72) Inventor Shigetomo Yamamoto, Ami-cho, Inashiki-gun, Ibaraki 8-3-1, Mitsubishi Yuka Corporation Central Research Laboratory

Claims (1)

(57) [Claims] An L-isoleucine-containing aqueous solution in which L-isoleucine is contaminated with an impurity mainly containing an amino acid such as valine is brought into contact with activated carbon to adsorb L-isoleucine on activated carbon, and then water is passed through the activated carbon. And then the adsorbed L-
A method for purifying L-isoleucine, wherein isoleucine is eluted and recovered using aqueous ammonia.