JPS60176594A - Preparation of l-tryptophan - Google Patents

Abstract

PURPOSE:To prepare L-tryptophan industrially at a low cost, by using an enzyme racemizing D-serine in enzymic reaction of indole with DL-serine. CONSTITUTION:Indole is reacted with DL-serine in the presence of tryptophan synthetase or tryptophanase to preapre L-tryptophan. In the process, serine racemase racemizing D-serine, preferably serine racemase derived from a microorganism belonging to the genus Pseudomonas is added to the reaction system when L-serine in the reaction system is consumed by the reaction of the indole with the L-serine.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-)liptophan by enzymatic action using indole and DL-serine as raw materials. More specifically, indole and L in the presence of tryptophan synthetase or tryptophanase
- In a method for producing L-)IJ butophane by reacting it with serine, indole and D
1. - A method for producing tryptophan in which serine is used and an enzyme that racemizes serine acts on the reaction system to convert at least a portion of D-serine into L-serine. The objective is to provide an improved method for industrially producing L-1 liptophan, which is important as an essential amino acid, at low cost.

An excellent method for producing tryptophan using enzymes is known to produce it from indole and serine through the action of tryptophan synthetase or tryptophanase. Indole, two of the raw materials, is industrially synthesized at low cost, but the method for producing serine, the other raw material, has the disadvantage that serine, which is synthesized by organic synthesis, is in the DL form. Furthermore, there is a serious problem inherent in organic synthesis methods in that only 5 serines are affected by tryptophan synthetase or tryptophanase, and 0 serines are not affected by these enzymes.

As an enzymatic synthesis method for L-tryptophan when indole and serine are used as raw materials, the following method is known as a synthesis method when serine is in the DL form. That is, indole and L-serine were converted to L-)IJbutophane by enzymatic action, unreacted D-serine was separated and recovered from the reaction product, and then D-serine was racemized by treating it in an aqueous solution at high temperature and high pressure. This is a method in which the enzyme reaction and racemization are alternately repeated, and then used again as a substrate for the enzyme reaction.

In this method, for example, if L-tryptophan is mixed in the reaction system during racemization reaction, not only serine but also L-tryptophan can be present in the reaction system.
-Tryptophan also racemizes.

Therefore, there are various drawbacks, such as the need to completely separate L-tryptophan and then perform a racemization reaction.

Therefore, as an improvement method,
95 As described in the publication, a method has been proposed in which L-tryptophan is synthesized while racemizing D-serine by using serine racemase, an enzyme that racemizes Seri, in the reaction system for L-tryptophan enzyme synthesis. There is.

That is, the above-mentioned publication describes serine racemase such as a bacterial aggregate collected from a culture solution of Pseudomonas putida, which is one of the producing bacteria of serine racemase, crude enzyme extracted and separated from this, and tryptophan. Tryptophan ensetase, such as a bacterial cluster collected from a culture of Escherichia coli, which is one of the synthetase-producing bacteria, and a crude enzyme extracted and separated from this.
Tryptophan enzymatic reaction is carried out by charging them in a reactor and allowing them to coexist.

According to the knowledge of the present inventors, it is true that JP-A-55-14
As described in Publication No. 8095, an enzyme that racemizes seric acid is applied to the reaction system to convert at least a portion of D-serine into L-serine.
- If the reaction is carried out while converting to serine, the yield can be improved to some extent. However, as stated in the above publication, it is difficult to keep cararacemase present while racemase cannot work effectively due to the coexistence of L-serine in the reaction system.
It was found that the yield of the desired product was limited due to the inactivation of the enzyme.

The present inventors have arrived at the present invention based on this knowledge.

Figure 1 shows the change in reaction rate over time when the L-)IJ butophane synthesis reaction was carried out by the method described in the Examples of the present invention, where the vertical axis is the reaction rate of the raw material indole, and the horizontal axis is Represents reaction time.

A in the figure shows the method of the present invention, in which indole and DL-serine are reacted in the presence of tryptophan synthetase and serine racemase, and 6 hours after L-serine is completely consumed, serine laan synthetase and serine racemase are reacted. When the reaction is carried out by the method described in JP-A-55-148095, in which the reaction is carried out with racemase coexisting from the beginning, C still remains indole, DL-serin, tryptophan and
This shows the case where the reaction was carried out using only synthetase.

As can be seen from the figure, when L-tryptophan is produced by acting on tryptophan synthetase, DL-7phosphorus, and indole in the absence of serine racemase, while KL-serine is present in the reaction system, Although the reaction rate is high, it decreases after about 6 hours, and the reaction rate at that time is about 50%.

This means that 5 serine bodies were completely consumed in a reaction that took about 6 hours. On the other hand, tryptophan fist synthetase and serine racemase were allowed to coexist, and D
When acting on L-serine and indole, the reaction rate at 6 hours was 55%, and the difference was slight, indicating that the effect of serine racemase is small up to this point. In other words, it can be concluded that serine racemase becomes effective after only five of the DL-serines are consumed. Furthermore, as shown by B in the figure, if serine racemase is allowed to coexist while L-serine is coexisting, the racemase enzyme will become inactive over time and the yield will eventually stop. I understand.

The present invention has been completed based on this knowledge. That is, the present invention provides a method for producing L-)ributophane in the presence of tryptophan synthetase or l-lyptophanase using indole and DL-serine as raw materials, in which the reaction between indole and L-serine is performed. When L-serine is consumed, D-
1) A method for producing butophane, characterized in that an enzymatic reaction is carried out by adding serine racemase that racemizes serine.

The tryptophan synthetase used in the method of the present invention is not particularly limited, and for example, those derived from microorganisms are conveniently used.
-19), microorganisms belonging to Escherichia coli such as (PERMBP-20), and other Neurospora.
Production cells or enzymes obtained from microorganisms such as Clapp (ATCC 14692) can be used. It is especially convenient to use Escherichia coli.

There are also no particular limitations on serine racemase, such as Seitomonas putida (IFO-12996).
), Pseudomonas fly (IFO-3458), and those derived from microorganisms belonging to Pseudomonas can be conveniently used.

These synthetases and racemases do not necessarily have to be pure (for example, dry or wet bacterial cells collected from the culture solution by centrifugation, or even extracts from these cells or The obtained crude enzyme may also be used.

The amount of tryptophan synthetase and serine racemase used in the reaction system varies depending on the separation and purification of the enzyme or the treatment method of the production bacteria, but there is no particular restriction, and it depends on the ratio of the amount of each substrate, the activity of the enzyme, etc. It may be changed as appropriate depending on the conditions.

Further, the amount of indole and DL-serine substrate in the reaction system is not particularly limited, and the concentration in the solution is usually 0.1 to 20% by weight, preferably 5 to 15% by weight. The reaction temperature is usually 20-60℃, preferably 30-45℃.
The pH during the reaction is preferably 6.0 to 11.0, preferably 8.0 to 9.5.

Furthermore, in the method of the present invention, for example,
As described in Publication No. 11187, an organic solvent that is miscible with the indole substrate such as toluene and immiscible with water is added to the reaction system to dissolve the indole in the organic solvent phase, and to dissolve the indole between the organic solvent phase and the aqueous phase. It is desirable to carry out the reaction by controlling the distribution ratio of the substrate so that the substrate concentration in the enzyme-containing aqueous phase is kept low below the concentration range in which the enzyme activity is substantially degraded.

Examples are shown below.

Example Tryptophan synthetase producing bacterium Escherichia coli MT-'10242 (FERM BP-2)
Table 1 Beef extract 1 (1 Polypeptone 102 NaC1!M' was dissolved in 1 t of distilled water and used. (pH 7,0) Medium 15
It was inoculated into 0 mL and cultured at 30°C for 24 hours. This culture solution (600 mA (4 flasks)) was placed in a 20 ton Jar Fermentor with a medium ioz of the composition shown in Table 2.
and cultured at 30° C. and pH 6.8 (controlled with 25% aqueous ammonia) for 40 hours. At this time, an indole-containing glucose solution was added in portions so that the glucose concentration in the culture solution was 5% by weight or less, and the indole concentration was 0.02% by weight or less. After completion of the culture, the culture solution was collected by centrifugation to obtain Neisseria gonorrhoeae cells, which were used as a source of tryptophan ginseng synthetase.

On the other hand, Sheetmonas putida (M'T-10182), which is a serine racemase-producing bacterium, was also cultured and collected in the same manner as the tryptophan synthetase-producing bacterium, and used as a source of serine racemase. However, in the jar fermentor culture in this case, the glucose solution was added in portions so that the glucose concentration in the culture solution was 5% by weight or less.

Table 2 KH2PO420f, K2HPO4209MgS0
4/7H2020i? , (NH4)2S04159 Polypeptone 20t 1 Yeast Extract 202CaCA2
112H200-4fs CuC42・2H200-
041CoC/-2" 6H200-04f, AtC7
3”6a+0 0.1ii'H3BO30-005!i
', MnSO4・5H200-1flZnSO4・
7H200,022, Na2M0O4・2H200,0
27FeSO4II7H200,42 These were dissolved in 10 tons of distilled water and used.

A tryptophan synthesis reaction with the composition shown in Table 3 was carried out using the thus obtained tryptophan synthetase and serine racemase in combination or only tryptophan synthetase.

Pyridoxal-5-phosphate 0.03L? , Indole 34.4f Toluene 137.5f, Distilled water 270
7 Tryptophan synthetase-producing bacterial wet bacterial cells, dry bacterial cells (5.6f)) Serine racemase-producing bacterial wet bacterial cells, the aqueous layer of the reaction solution with these compositions was diluted with aqueous ammonia to pH 8.
, adjusted to 5.

FIG. 1 shows the change over time in the reaction rate of each tryptophan synthesis and the yield calculated from the amount of indole remaining in the toluene layer. Curve A in the figure initially shows tryptophan
If only synthetase is added to the reaction solution to start the reaction, and serine racemase is added 6 hours after the start of the reaction when only 5 of the DL-serines in the reaction solution have been almost consumed, B is , when tryptophan synthetase and serine racemase are reacted in the coexistence from the beginning, and C is the change in reaction rate over time and yield when the reaction is performed only with tryptophan synthetase. The reaction was continued for 40 hours, and the respective yields (reaction rates of indole) were as follows.

A: 96% reaction rate when serine racemase is added after 5 of the DL-serines have been consumed B: 93% reaction rate when both enzymes are reacted together from the beginning C: Tryptophan synthetase only Reaction rate when reacting with m addition: 55%

[Brief explanation of the drawing]

Figure 1 shows the reaction time and indole reaction rate in an example of the present invention when tryptophan synthetase and serine racemase coexist or only tryptophan/ncetase is present in the L-1) butophane synthesis reaction. This is a relationship diagram. A is the method of the present invention, in which D-serine racemase was added at the time when serine in the reaction system was consumed (6 hours after the start of the reaction). B is a case where serine racemase is coexisting from the beginning. C is the case where no serine racemase was allowed to coexist. Patent applicant Mitsui Toatsu Chemical Co., Ltd.

Claims (1)

[Claims] In a method for producing L-tryptophan in the presence of tryptophan synthetase or tryptophanase using indole and DL-serine as raw materials, DL-serine in the reaction system is produced by the reaction of indole and L-serine. 1. A method for producing L-tryptophan, which comprises adding serine racemase that racemizes D-serine to the reaction system to carry out an enzymatic reaction when D-serine is consumed. 2. The method according to claim 1, wherein the tryptophan synthetase-producing bacterium is Escherichia coli≠檎≠*concept. 3. The method according to claim 1, wherein the serine racemase producing bacterium is Sheetomonas sp.