JPS6371189A - Recovering method for amino acid from fermentation liquor - Google Patents

Abstract

PURPOSE:To recover an amino acid in high yield without diluting a fermentation liquor, by adding a specific organic solvent to an amino acid fermentation liquor containing microbial cells, stirring the resultant mixture, subjecting the mixture solution to centrifugal settling and separating the water layer containing dissolved amino acid. CONSTITUTION:An organic solvent capable of forming two layers with water without dissolving an amino acid, e.g. toluene, n-butanol or methyl isobutyl ketone, is added to an amino acid fermentation liquor containing microbial cells and the resultant mixture solution is stirred. The obtained whole microbial cell-organic solvent-water emulsion or a microbial emulsion layer prepared by allowing the emulsion to stand and separating from the water layer is subjected to centrifugal settling (60-250G centrifugal effect for 5-10min centrifugal settling time) to separate the water layer containing no microbial cell dissolving the amino acid from other layers, separate and recover the amino acid from the above-mentioned water layer. Water is then added to a microbial cell emulsion layer separated from the water layer by the centrifugal settling and the resultant mixture is further subjected to centrifugal settling. Thereby the recovery of the amino acid can be improved by using a small amount of washing water.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention separates and removes impurities such as bacterial cells and soluble proteins from an amino acid fermentation liquid containing bacterial cells, thereby producing a clear amino acid aqueous solution economically and in high yield. It is about how to get the rate.

(Prior art and its problems) It has been known that fermentation containing glutamic acid can separate the microorganisms together with the organic solvent as an emulsion layer by adding an organic solvent that forms two layers with water to the amino acid fermentation solution containing microorganisms. It is known as a method for separating bacterial cells by adding amyl alcohol in a-butanol to a solution (Patent Application Publication 1976-6).
460). In addition, there are similar reports regarding fermentation liquors using hydrocarbons as raw materials (4.
861). Incidentally, when an organic solvent that forms two layers with water is mixed with stirring, an emulsion is formed, but this emulsion is unstable and separates into the original water layer and the organic solvent layer when stirring is stopped. However, when the organic solvent and water, which form two layers with the bacteria and water, are stirred and mixed, the emulsion incorporates the bacteria and becomes stable.

Furthermore, as a method for separating impurities such as glutamic acid crystals and bacterial bodies, an organic solvent was added to the body fluid containing bacteria from which the glutamic acid crystals were precipitated, and which had a small specific gravity for the glutamic acid crystals and a large specific gravity for the crystallization mother liquor. After that, a method is known in which the crystals are sedimented by applying centrifugal force and transferred to an organic solvent layer to separate the crystals from the mother liquor containing various impurities (Patent Application Publication 1972-
12884).

However, in the above method, a small amount of water and therefore the target amino acid remain in the 2.7 layer of the separated bacterial cell emulsion. The recovery rate of glutamic acid is increased by washing the emulsion layer separated several times with equal amounts of water.

However, according to this method, the concentration of glutamic acid in the recovered aqueous layer inevitably decreases, which leads to an increase in energy consumption in the subsequent process of isolating and purifying glutamic acid from the aqueous layer. Ta.

(Means for Solving the Problems) Therefore, as a result of intensive research into a method for separating amino acids from fermentation broth that does not have these drawbacks, the present inventors added an organic solvent to the anoic acid fermentation solution containing bacteria. By centrifuging the bacterial cell-organic solvent/water emulsion (bacterial emulsion) obtained by stirring, the difference in specific gravity results in two layers: a bacterial cell emulsion 2.7 layer and an aqueous layer, or an organic solvent layer and a bacterial cell emulsion. It was discovered that the fermentation target product (acid) can be easily separated into three layers: the body emul 2.7 layer and the aqueous layer, and the fermentation target product (acid) can be obtained in high yield from the bacteria-containing fermentation liquid without diluting the fermentation liquid. We have now completed the present invention, which allows for recovery.

For example, L-phenylalanine fermentation liquid using carbohydrate-based fermentation raw materials (liquid adjusted to P)I-2,85) 100
After pouring an equal amount of toluene into the microorganism, stir and leave to stand still for 30 minutes.The amount of water layer free of bacteria obtained is 48 m, and about half of the water is included in the bacteria emulsion layer. Even if the separation time is further extended, this water does not easily separate into layers. In addition, the amount of L-phenylalanine recovered is proportional to the amount recovered in the aqueous layer, so if you want to recover L-phenylalanine with a high recovery rate, as will be shown later in Comparative Example 1, preparative separation is necessary. After adding water to the emulsion layer and stirring, it is essential to wash the emulsion layer by standing still and separating the layers again.

Therefore, in order to reduce the amount of washing water, the present inventors have conducted various studies and found that if the bacterial emulsion is subjected to centrifugal sedimentation, the I.
It was realized that it is possible to recover an aqueous layer containing a large amount of L-phenylalanine at a high concentration compared to that obtained by gravity separation of G.

Therefore, as will be shown in detail later in Examples, when the bacterial cell emulsion layer that has been separated from the water layer in a gravity field is collected and subjected to centrifugal sedimentation, the bacterial cell-free water layer will be further separated. was observed, and it was possible to realize a significant improvement in the recovery rate and concentration of L-7 engineered nylalanine without using washing water.

Normally, when a fermentation liquid that is not mixed with an organic solvent is subjected to centrifugal sedimentation, bacterial cells with a large specific gravity settle and are separated into a heavy liquid containing concentrated bacterial cells and a supernatant liquid (light liquid).

On the other hand, after stirring, for example, toluene (organic solvent), which has a lower specific gravity than water, is added to the fermentation liquid.

When subjected to centrifugal sedimentation, the bacterial cells, which should normally settle at the bottom of the aqueous layer as a heavy liquid, surprisingly remained in the emulsion layer formed by toluene and water and were separated from the aqueous layer, which was a heavy liquid. It is. If water has a high specific gravity, EDC (
It goes without saying that if an organic solvent such as ethylene dichloride is used, the bacterial emul 212 layer will settle as a heavy liquid and the aqueous layer will be recovered as a light liquid. These facts can be understood from the idea that by escaping the organic solvent, the lipophilic bacterial cell membrane and the organic solvent exhibit a strong affinity and behave in the same way.

The organic solvent that can be used in the present invention basically needs to be an organic solvent that forms two layers with water and has almost no solubility for the amino acid, such as toluene, n-hexane, and a-butanol. , MIBK (methyl isobutyl ketone), etc. can be widely used.

However, depending on the type of fermentation liquid, organic solvents are more or less suitable and unsuitable, but those skilled in the art can easily select an appropriate organic solvent. L- using carbohydrate-based fermented raw materials
As examples of the 7-engineering nylalanine fermentation liquid and the L-lysine fermentation liquid, MIBK v n-butanol, toluene, ethyl acetate, dichloromethane, oleic acid, etc. can be used.

The amount of organic solvent used will be described below. For example, L-phenylalanine fermentation liquid using carbohydrate-based fermentation raw materials (
When toluene is added to S-3), the fermentation liquid volume is 5V.
Even when 1% of toluene was added, the bacterial cells formed an emulsion layer and were separated. However, because the pre-microbial cells remained in the water layer, which should not contain microbial cells, clarity was lacking. Therefore,
In this case, the amount of toluene used is preferably 1
5Vo1% or more. For combinations of other amino acid fermentation liquids and organic solvents, the required amount of organic solvent can be determined easily in the same way.

The range of fermentation liquors suitable for the operation of the present invention will also vary depending on the fermentation liquor and the type of solvent used. For example, in a system using toluene and L-lysine fermentation liquid using carbohydrate-based fermentation raw materials, there is an optimal range where the aqueous layer recovery rate is maximized at pH -2.5-4.0, but n-butanol Using --2
, 5-5,5 shows good aqueous layer recovery. In any case, those skilled in the art can easily determine the optimum range.

Next, the centrifugal effect (
C) and the required centrifugal sedimentation time.

Figure 1 shows that an equal volume of toluene was added to the L-7 engineered nylalanine fermentation solution using carbohydrate-based fermentation raw materials adjusted to pH-2.85-3.05, stirred, and then centrifuged for 10 minutes with different centrifugal effects. The results of centrifugal sedimentation are shown.

The volume percentage of the recovered aqueous layer was 21.5% (
However, if centrifugal sedimentation is performed at 60G, an aqueous layer with a volume percentage of 41% (recovery rate of 82%) can be obtained. If the centrifugal effect is further increased, (1) The amount of water layer recovered increases, but the water layer recovery rate reaches a ceiling of 91%. ■ A toluene supernatant liquid containing no bacterial cells is formed above the light liquid, and the whole is divided into three layers: a toluene layer, a bacterial emulsion layer, and an aqueous layer. ■ It was found that when the pressure exceeds about 700G, the front of the bacterial cells appears to settle at the bottom of the water layer, which is undesirable.

In addition, the same fermentation liquid is shown in Figure 2 with constant centrifugal effect (C-250).
The results of separation with different centrifugal sedimentation times are shown. It can be seen that even with a separation time of about 5 minutes, the aqueous layer recovery rate is significantly improved.

From Figure 1.2, it can be seen that the aqueous layer recovery rate (amino acid recovery rate) can be significantly improved by a very mild centrifugal sedimentation with a centrifugal effect of 6O-250G and a centrifugal sedimentation time of 5-10 minutes.

(Action and Effect of the Invention) According to the method of the present invention, if an organic solvent is added to the amino acid fermentation liquid and stirred to form a bacterial emulsion, and the emulsion is subjected to centrifugal sedimentation, the Approximately twice the amount of water layer obtained by gravity separation can be recovered, and the amount of amino acid recovered can also be increased proportionately.

According to the method of the present invention, the amount of water layer that could be recovered in one sedimentation separation without using washing water and therefore without diluting the amino acid at all was about 91% in the case of L-phenylalanine fermentation liquid. However, if we try to obtain this recovery rate only by gravity layer,
As shown in Comparative Example 1, it was necessary to perform a comb-over stirring and layer separation operation using approximately twice the amount of washing water as the amount of fermentation liquid.

In addition, if you want to obtain an even higher amino acid recovery rate, you can add water to the bacterial cell emulsion layer that has been separated from the aqueous layer and centrifuge it again. Needless to say, it can be easily improved.

Although the present invention has been explained above using L-phenylalanine fermentation broth and L-lysine fermentation broth as examples, it goes without saying that the present invention can also be applied to other amino acid fermentation broths such as L-arginine and L-histidine.

As explained above, according to the method of the present invention, the decrease in the amino acid concentration of the bacterial cell-free amino acid solution can be minimized, and the energy consumption in the conventional isolation and purification process can be significantly reduced. There is an advantage that you can get it.

(Example) Hereinafter, the present invention will be explained in detail with reference to Examples.

However, the examples should not be considered as limiting the invention.

Moreover, the amino acid analysis used in the examples was performed by the following method.

Annoic acid analyzer analysis model: Hitachi 835-50 column: Hitachi +26174φ L-7 fermentation liquid using carbohydrate-based fermentation raw materials (100OG, centrifugal sedimentation for 30 minutes will yield 15% of the bacteria-containing liquid)
It contained Vo1% of bacterial cells. ) 250- to water 1010
Os was added and the pH was adjusted to -2.96 using 98% sulfuric acid. 175 mg of toluene was added to this liquid, thoroughly shaken in a separatory funnel, and allowed to stand still for 35 minutes to separate the layers into an upper layer of 2.7 bacterial cell emuls and a lower bacterial cell-free aqueous layer.

Separate the aqueous layer (+1 aqueous layer), add 2.7 layers of bacterial emulsion and 33 mL of water.
00- was added, and after shaking in the same manner, the mixture was allowed to stand and the aqueous layer that separated into layers (2 aqueous layers) was separated. Isolated bacterial cell emul 2.
Add 100% more water to the 7th layer and perform the same operation to form the aqueous layer (
÷3 aqueous layer) was separated.

The amount of the separated ◆1-3 aqueous layer and the amount of L-phenylalanine in the solution were as shown in Table 1.

Table 1 Water amount L-7 Nylalanine amount◆1 water layer 195mg 4.2401! 2nd water layer 294m 2.171s 3rd water layer 1131s
t 0.5191 The recovered L-phenylalanine concentration is 11.53 as the average value of the 4P1-3 water layer combined.
．． 974, and the amount of L-7 enylalanine recovered is 6.
It was 938.9. This amount corresponded to 92.5% of the amount of 9-phenyluanine contained in the fermentation liquid used.

Example 1 Using carbohydrate-based fermentation raw materials, PH-
1-phenylalanine fermentation solution adjusted to 2.96% (1
oooc, 15 Vol1 of bacteria-containing weight liquid was centrifuged for 30 minutes.
It contained % bacterial cells. ) ZoomK toluene 100-
was added, shaken in a separatory funnel, and then separated into layers using a device to obtain an aqueous layer free of bacterial cells as the lower layer.

When the 2.7 layers of the upper bacterial cell emulsion were centrifuged at 60G for 10 minutes, a further 37.2-layer aqueous layer could be recovered as a heavy liquid. The recovery rate of I,-phenylalanine by the above operation was 84.7%, and the concentration was 30.91171! ,
Met.

Furthermore, 50 mL of water was added to the 2.7 layers of this bacterial emulsion, stirred, and then centrifuged at 60 G for 10 minutes to obtain an aqueous layer of 49 td. This aqueous layer contains 0.3321 i of I,-phenylalanine, and the total amount of the above aqueous layer is 7'cL.
- The recovery rate of phenylalanine was 95.7% and the average concentration was 2L91/1. Met.

Example 2 To the same fermentation liquid 100 as in Example 1, 100 d of toluene was added and stirred, and then separated into layers by standing to form a bacterial cell-free aqueous layer 3 as the lower layer.
I got 8-. Thereafter, the bacterial cell emul 217 layer was centrifuged at 750G for 10 minutes to obtain an additional 52.7-layer of aqueous layer free of facepieces.

The total amino acid recovery was 93.4% and the concentration was 30
．． 99/J! ,Met.

Example 3 Add 30ml of toluene to 200ml of the same fermentation liquid as in Example 1, stir, and then let stand for 25 minutes to separate the layers to form a bacterial cell-free aqueous layer (÷1 aqueous layer)
was separated.

100 ml of water was added to 2.7 layers of the upper bacterial cell emulsion, stirred, and then allowed to stand for 25 minutes to separate the layers to obtain a bacterial cell-free water layer (÷2 water layer - 1). The 2.7 layers of this bacterial cell emulsion were centrifuged at 250G for 10 minutes, and a heavy liquid was obtained by further aqueous layer (+2 aqueous layer -2
) was obtained.

Table 2 shows the liquid amounts of Na1 aqueous layer, ÷2 aqueous layer-1, and Na2 aqueous layer-2 and the amount of L-phenylalanine contained therein.

Table 2 Wit L-phenylalanine amount 1 aqueous layer
88.0m 2.719F÷2 water layer-1 128.5d 2.048N◆2
Aqueous layer - 250.1 mA 0.791 The amount of L-7 enylalanine recovered was 5.566.9, and the recovery rate was 92.8%. Moreover, the average concentration of the obtained aqueous layer was 20.8 s g711.

[Brief explanation of the drawings] Figure 1 shows the layer separation situation when the centrifugal sedimentation time is constant (10 minutes) and the centrifugal effect is varied, and Figure 2 shows the layer separation situation when the centrifugal effect is constant (G-250). The following shows the layer separation situation when the centrifugal sedimentation time is changed.

Claims (1)

[Claims] An organic solvent that forms two layers when added to water and does not dissolve the amino acids is added to the amino acid fermentation liquid containing bacterial cells, and after stirring, the entire mixture or a bacterial cell emulsion layer obtained by static layer separation is added. A method for recovering amino acids from an amino acid fermentation solution, which comprises subjecting the solution to centrifugal sedimentation, separating an aqueous layer in which amino acids are dissolved therefrom from other layers, and separating and recovering amino acids from the aqueous layer by a conventional method.