JPH01187092A - Production of l-isoleucine - Google Patents

Abstract

PURPOSE:To industrially improve efficiency of purification at a low cost, by bringing an L-isoleucine-containing aqueous solution containing impurities consisting essentially of an amino acid, such as valine, into contact with active carbon and carrying out treatment. CONSTITUTION:An L-isoleucine fermentation liquor or enzymic reaction solution, etc., is subjected to filtration treatment, etc., to provide an aqueous solution (A) contaminated by amino acid, such as valine, and having an L-isoleucine concentration below the saturation solubility of the L-isoleucine. The resultant solution (A) is subsequently passed through active carbon (B), such as 'Diahope 008(R)' in an amount of 10-50 times based on the L-isoleucine at a neutral pH, <=80 deg.C and 0.5-5hr<-1> superficial velocity to adsorb the L-isoleucine on the component (B). Water is then passed through the component (B) and 0.1-10N aqueous NH3 is subsequently passed through to afford an NH3 eluate (C) containing the L-isoleucine. The eluate (C) is then subjected to purification treatment, such as concentration or crystallization, to isolate the L-isoleucine.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for purifying L-isoleucine.

More specifically, the present invention relates to a method for purifying inleucine in which impurities mainly composed of amino acids such as valine are removed from a reaction solution containing L-isoleucine obtained by a fermentation method or an enzyme method.

[Conventional technology]

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

Fermentation and enzymatic methods using precursors such as DL-α-aminobutyric acid, α-ketobutyric acid, and D-threonine have been established as methods for producing L-isoleucine.
- It is known to purify isoleucine.

L-isoleucine is obtained by fermentation or enzymatic methods in the presence of bacterial cells and enzymes in a near-neutral aqueous medium using, for example, DL-α-aminobutyric acid as a precursor. It is necessary to remove the impurities of valine and other amino acids contained therein. Therefore, a non-polar porous synthetic adsorbent (Japanese Patent Application Laid-Open No. 54-55519) or an ion exchange resin (Japanese Patent Application Laid-open No. 56-131550) is used to remove L-isoleucine. lol
A method of manufacturing Ii is known. By the way, there are no examples of chromatographic separation of L-isoleucine using activated carbon, and even in reports on the separation of leucine and valine, which have similar structures, ethyl acetate aqueous solution or phosphate buffer was used as the eluent (J, P. .

GreensteinJ, 'WiniLz,'Chem
istry of the゛＾min.

Aclds+vol 2', p1447. John
Wiley & 5ons, Inc.

New Work (1961), F, Turba +
M., Richter, F., Kuchar, Na.L.
urwiss, , , 11.508 (1943), )
*[Problems to be solved by the invention] Most conventional non-polar porous synthetic adsorbents are styrene-divinylbenzene-based crosslinked polymers. These commonly used non-polar porous synthetic adsorbents cannot achieve sufficient separation performance because the breakthrough points for L-isoleucine and impurities mainly composed of amino acids such as valine are almost the same. -The recovery rate of isoleucine is very low, which was pointed out by inventor Ohkama et al. in another patent (Japanese Unexamined Patent Publication No. 61-17
8952.2 lines 12 to 19 from purchase).

In addition, the purification method using ion exchange resin requires a large amount of acid or alkali to regenerate the ion exchange resin, resulting in high costs. Further, when there are many coexisting inorganic salts, the exchange ability of the ion exchange resin is exchanged with the inorganic salt, so a large amount of ion exchange resin is required for L-isoleucine, and there is a problem that it becomes practically unusable.

In addition, in the above-mentioned purification method using activated carbon, auxiliary agents (ethyl acetate and phosphate buffer) are mixed into the recovered solution, making the isolation operation complicated and requiring regeneration of the activated carbon, which increases the cost of auxiliary agents and makes it uneconomical. I can't stand the target.

Means for Solving Problem c] The present inventors have arrived at the method of the present invention as a result of intensive studies to solve the above problems.

That is, in the purification method of the present invention, a reaction solution containing 1-inleucine obtained by a fermentation method or an enzyme method is passed through activated carbon, then water is passed through it, and when impurities are eluted, the water flow is stopped. , is a method for purifying L-isoleucine, which is characterized in that L-isoleucine adsorbed on activated carbon is eluted using aqueous ammonia, and L-isoleucine is isolated from the eluate according to a conventional method.

Impurities that have a higher affinity for activated carbon than L-isoleucine can be separated by chromatography when dissolved in aqueous ammonia.

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

L of the aqueous L-isoleucine solution which is the starting material for the method of the present invention
- The isoleucine concentration may be lower than the saturation solubility of L-isoleucine.

The types of activated carbon used in the present invention include coal-based activated carbon, palm acid-based activated carbon, charcoal-based activated carbon, petroleum pinch activated carbon, etc., and are not particularly limited. For example, Diahope 008.380. Diasoap G, W (manufactured by Mitsubishi Chemical Industries W), HC-30S, GL-30, 2GL, 4
GL (manufactured by Tsurumicol ■), BAC-LP, MP
(Manufactured by Ou Chemical Industry ■), Tararecol GW, OL, GL
C,' PK (Kuraray Chemical @161!I), LH
2C, W5C, KL (manufactured by Takeda Pharmaceutical Co., Ltd.), etc. can be used as appropriate.

The amount of activated carbon used is 1 for L-isoleucine.
About 0 to 50 times the amount (based on weight) is sufficient.

Furthermore, the pH of the liquid to be treated during passing is preferably neutral, the temperature is 80° C. or less, and the emptying speed is 5 v=o, s to 5 hr.

The concentration of ammonia water used for elution of L-isoleucine is not limited, and it is sufficient to use a sufficient concentration to elute L-isoleucine from activated carbon, but ammonia water of about 0.1 to 10N is preferable, generally. Using aqueous ammonia at a low concentration has the advantage that impurities that have a higher affinity for activated carbon than L-isoleucine can be chromatographically separated during elution;
- It has the advantage that isoleucine can be recovered at a high concentration and subsequent isolation operations are easy.

The obtained ammonia eluate containing L-isoleucine is
L-isoleucine can be easily isolated with high purity and good yield at low cost by known isolation methods, ie, unit operations such as concentration, crystallization, solid-liquid separation, and drying.

As explained in detail above, the present invention uses activated carbon and L
- To provide a new and simple L-isoleucine purification method for purifying and removing impurities mainly composed of amino acids such as valine that are present in isoleucine.

Example 1 Using DL-α-aminobutyric acid as a precursor, Brevibacterium flavum (BrevibacLerium fl.
19.7 g/j of L-isoleucine and L-isoleucine obtained by sterilizing the L-isoleucine fermentation solution obtained using
Valine 0.1g/14. l)-α-aminobutyric acid 11.5
g/J, L-alanine 3. Ig/j, glycine 2.7g
/j! , 200ml of L-isoleucine aqueous solution containing 0.2g/1 L-lysine etc. was added to Diahope 008 280r.
Column packed with n& (φ3, 2 al x H3
0-order S injected on top of 5am), V = 3
Water was passed through the tube at hr-', and the water flow was stopped when impurities such as valine were eluted, and then 2N ammonia water was passed through the tube.
- An isoleucine solution was obtained. The removal rates of L-valine, D-α-aminobutyric acid, L-alanine, glycine, and L-lysine in the L-isoleucine solution were 100% and 100%, respectively.
%, 100%, 100%, 53%, and the recovery rate of L-isoleucine was 84%.

Example 2 Using α-ketobutyric acid as a precursor, Brevibacterium flavus
10.8 g/j of L-isoleucine, 0.3 g/It of L-valine, and 0.1 g/I of L-α-aminobutyric acid obtained by sterilizing the L-isoleucine enzyme reaction solution obtained using
t, L-alanine 0.2g/l, glycine 0.5g/j
! , 360 mJ of an aqueous L-isoleucine solution containing 0.6 g/j of L-lysine, etc. was injected into the top of a column (φ3.23×H35 cm) packed with BAC-LP280mj.
Next, water was passed through the column at S V = 3 h r-1, and when impurities such as valine were eluted, the water flow was stopped, and while maintaining the column temperature at 60°C, 2N ammonia water was passed through the column. ,
L-isoleucine was obtained. L in L-isoleucine solution
- Removal rates of valine, L-α-aminobutyric acid, L-alanine, glycine, and L-lysine were 97% and 100%, respectively.
The recovery rate of L-isoleucine was 72%.

Comparative Example L-isoleucine 24°9 g/J and L-valine 1. 8 g/j! , DL-a-7t/11M5.
3 g/l, L-alanine 2.8 g/j, glycine 2
゜1 g/j, L-isoleucine aqueous solution containing 0.6 g/l L-lysine 30 m J! Diaion HP-20 (specially manufactured by Mitsubishi Chemical Industries) 6 is a non-polar porous synthetic adsorbent.
Column packed with 25 mj (φ3 cm x H88 cm
). Water was passed through it at 5V=1.5hr-1 to obtain an L-isoleucine solution.

The removal rate of L-valine, DL-α-aminobutyric acid, L-alanine, glycine, and L-lysine in the L-isoleucine solution was all 100%, but the recovery rate of L-isoleucine was as low as 7%. 84% of L-isoleucine was eluted together with impurities such as valine.

〔Effect of the invention〕

As described above, by the method of the present invention, L-isoleucine can be purified very efficiently from an aqueous solution of L-isoleucine contaminated with impurities mainly consisting of amino acids such as valine. Further, the method of the present invention is an industrially innovative method for purifying L-isoleucine without increasing the cost of auxiliary agents and the like.

Claims (1)

[Claims] (1) An aqueous solution containing L-isoleucine contaminated with impurities mainly consisting of amino acids such as valine is brought into contact with activated carbon to adsorb L-isoleucine on the activated carbon, and then water is passed through the activated carbon, A method for purifying L-isoleucine, which comprises then eluating and recovering the adsorbed L-isoleucine using aqueous ammonia.