JPH02308796A - Method for separating l-serine - Google Patents

Abstract

PURPOSE:To elute high-purity L-serine in good yield by passing a L-serine solution containing unreacted glycine through a filling layer of specific H<+> type strongly acidic cation exchange resin, passing an alkali solution through the filling layer and then pushing out L-serine using water. CONSTITUTION:Tetrahydroleaf acid and pyridoxal-5'-phosphoric acid used as a coenzyme and glycine are added to an aqueous solution of enzyme obtained from cultured bacterium cell of Escherichia coli MT-10350 (FERM P-7437), etc., and the mixture is heated to 20-60 deg.C and pH thereof is adjusted to 6-9 by an alkali solution and then formaldehyde is continuously inserted therein and reacted therewith to provide L-serine solution containing unreacted glycine. Then the solution is passed through a filling layer of H<+> type strongly acidic cation exchange resin having 0.1 5-0.40mm effective diameter and <=1.7 uniform coefficient to retain glycine and L-serine. Then an alkali solution having 0.5-1.2M and being 1.0-1.5 times by mol based on the retained L-serine is passed through the filling layer at 2.0m/hr velocity and then pure water 1.3 times the ion exchange resin is passed through the filling layer to push out L-serine.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for separating and recovering L-serine obtained by an enzymatic method or fermentation method from the raw material glycine using glycine as a raw material.

L-serine is a compound useful as an infusion raw material, a pharmaceutical raw material, and an intermediate for amino acid synthesis such as tryptophan.

(Prior Art) As a method for producing L-serine by an enzymatic method using glycine as a raw material, a method using serine hydroxymethyltransferase (EL, 2.1.2.1) is known. Publication No. 62-19092) That is,
This is a method for producing L-serine by allowing a microorganism capable of producing the enzyme to act on glycine and formaldehyde, which are raw materials, in the presence of coenzymes pyridoxal-5'-phosphate and tetrahydrofolic acid. Since this reaction is an equilibrium reaction, the conversion rate of glycine to L-serine is 75
%, and the reaction solution always contains glycine and L-
Separation of glycine and L-serine, in which serine coexists, is necessary to obtain L-serine as a product, and it is also necessary to reuse the recovered glycine in the reaction to reduce costs. This is a technology that must be used. On the other hand, as a method for producing L-serine by a fermentation method, a method of growing microorganisms in a glycine-containing medium and accumulating L-serine in the medium (JP-A-58-31995, JP-A-56-88798) JP-A No. 55-37169) and the like are known.

Glycine and L-serine have very similar solubility in water (the saturation concentration at 20°C near neutrality is 22% for glycine and 18% for L-serine). It is not possible to isolate high-purity Shiichi-Shichirin using the selection method.

Therefore, by utilizing the solubility difference between glycine and L-serine salts of toxylene-4-sulfonic acid as described in JP-A No. 58-31995, L-
There is a method for isolating serine, but it is difficult to operate and involves a lot of loss, making it difficult to do so industrially. A method of separating glycine and L-serine using an ion exchange resin is also known, but since the isoelectric points of both are close to each other (5°97 for glycine and 5.68 for L-serine). However, it is difficult to separate the two using conventional ion exchange retention and elution methods. Therefore, JP-A-53-72893 describes a method in which glycine and L-serine are retained in a strongly acidic cation exchange resin and then fractionated and eluted while changing the pl+ of a buffer such as citric acid. There is.

However, the operation is complicated and it is difficult to say that it is industrially practicable.

Furthermore, JP-A No. 62-111953 describes a method using a strongly acidic cation exchange resin, in which the particle size is smaller than that of a general-purpose type, has a specific size, and has a particle size distribution. A method for chromatographically separating glycine and L-serine by using a narrow resin is disclosed.

[Problem to be solved by the invention]

The method described in JP-A-62-111953 is a technique that can be used industrially because it is simple in operation.
It has the following drawbacks: That is, as the elution progresses and the L-serine concentration in the eluate decreases, glycine begins to elute instead, and a fraction exists in which L-serine and glycine are eluted simultaneously. this is,
Efficiently produce high-purity L-serine crystals that do not contain glycine (
In the present invention, in order to isolate highly pure L-serine crystals, a mixed solution of glycine and L-serine is retained in a strongly acidic cation/ion exchange resin, and then
This was done to provide a simple, reliable, and efficient method for eluting only L-serine without contaminating glycine.

[Means to solve the problem]

As a result of intensive studies on the elution method of glycine and L-serine retained in a strongly acidic cation exchange resin, the present inventor found that After passing an alkaline solution at a linear velocity of 2.0 m/hr or less, we discovered that by extruding water, only L-serine could be eluted at a high recovery rate without contaminating glycine, and based on this knowledge, This led to the completion of the present invention.

That is, the present invention provides: (1) using glycine as a raw material, an L-serine solution containing unreacted glycine obtained by an enzymatic method or a fermentation method, (2) having an effective diameter of 0.15 to 0.40 mm and a uniformity coefficient of 1;
．． Glycine and L-serine are retained by passing the liquid through a packed bed of H° type strongly acidic cation exchange resin at a temperature of 7 or less. After passing a molar alkaline solution, an L-serine solution containing no glycine is eluted by extrusion with water.

L- by the enzymatic method using glycine as a raw material in the present invention
As a method for producing serine, a method that utilizes the catalytic ability of serine hydroxymethyltransferase can be used. As a source of serine hydroxymethyltransferase, Escherichia coli) I7-103
50 (FERM P-7437) and Escherichia coli' MT-1035] (FER?I P-7438). Enzymes obtained from these cultured bacteria were dissolved in water, and tetrahydrofolic acid and tetrahydrofolate were added as coenzymes. Add pyridoxal-5'-phosphate. Add the raw material Grishiji, keep it warm at 20-60'C and ply with a suitable alkaline solution.
Adjust to 116-9. To stop the alkaline solution, for example, an aqueous solution of NaOH-, KOHs KaPzOt, ammonia, etc. can be used. While stirring the reaction solution, formaldehyde, which is the other raw material, is continuously charged, and L-serine is added. Since serine hydroxymethyltransferase is inactivated by high concentrations of formaldehyde, the formaldehyde concentration during the reaction is analyzed and formaldehyde is charged to keep it below 2011M.The pH of the reaction solution is acidic. Add an alkaline solution to adjust the pH to 6-9. As the alkaline solution, for example, N a OH
Aqueous solutions such as %KO11, K4P2O7, ammonia, etc. can be used.

Since this reaction is an equilibrium reaction, the total amount of glycine as a raw material is L-
It is not converted to serine, and the reaction stops when the conversion rate reaches about 75%. After acidifying this reaction solution, activated carbon is charged, and the enzyme and bacterial components are removed by heating and filtration. The filtrate thus obtained is a mixture of L-serine and unreacted glycine. This mixed solution is passed through a column packed with a strongly acidic cation exchange resin to separate L-serine and glycine.

The strongly acidic cation exchange resin used in the present invention must have an effective diameter of 0.15 to 0.40 mm and a uniformity coefficient of 1.7 or less. Examples of such ion exchange resins include Revatit TSW-40 and Revatit TSW-40.
-40-PK, same question S-1368 (manufactured by Bayer)
, Diaion PRK-01 (manufactured by Mitsubishi Kasei), etc., and can be easily obtained.

The effective diameter as used herein is the size of the sieve mesh that leaves 90% of the entire resin on the screen, as normally defined in the industry. Further, the uniformity coefficient is an index indicating the degree of particle size distribution, and is a value obtained by dividing the size of the sieve mesh, which leaves 40% of the entire resin on the screen, by the effective diameter. That is, the closer the value of the uniformity coefficient is to 1, the sharper the particle size distribution becomes.

The strongly acidic cation exchange resin used in the present invention is made into H° type by treating with hydrochloric acid in advance.
A solution of L-serine containing unreacted glycine, obtained by an enzymatic method or a fermentation method using glycine as a raw material, is passed from the top of the column to retain the cations in the ion exchange resin. In addition to L-serine and glycine, the cough L-serine solution contains various cations such as contaminant amino acids, potassium ions, sodium ions, and ammonium ions, but the number of moles of all these cations is The amount of L-serine solution to be passed is determined so as not to exceed the total exchange capacity of the ion exchange resin. The flow rate of the L-serine solution is set to 2.0 m/hr or less in terms of linear velocity. After holding cations in the ionic phase resin, pure water is passed from the top of the column to wash away anions and electrically neutral contaminants that are not held in the ion exchange resin. is approximately the same volume as the ion exchange resin packed in the column, and the linear velocity is 2.0.
Fluid is passed at a rate of m/hr or less.

Next, an alkaline solution is passed from the top of the column to elute only L-serine from among the cations retained in the ion exchange resin. As the alkaline solution, for example, an aqueous solution of ammonia, NaOH, or KOH can be used. In either case, the concentration range is 0.5-1.2M. When L-serine is eluted, the elution of L-serine becomes broad in a low concentration alkaline aqueous solution that cannot be eluted with pure water. As the concentration of alkali increases, the elution becomes sharper, and at the same time, the elution of glycine becomes faster (and eventually, glycine and L-serine elute at the same time, making it impossible to separate them. The concentration of must be set in the range of 0.5 to 1.2M. What is important here is that the total amount of alkali passing through the solution is 1.0 If the amount of alkali exceeds this range, the elution of glycine will begin in the latter stage of the elution of L-serine even if the alkali concentration is within the specified range. , a mixture of L-serine and glycine is produced, making it impossible to achieve the object of the present invention.

On the other hand, if the amount is too low, an L-serine eluate containing no glycine can be obtained, but the amount of L-serine recovered will be small.

Since poor separation of L-serine and glycine occurs even when the flow rate is high, the elution operation is performed at a linear velocity of 2.0 m/h.
Do it below r.

At the time when the alkaline solution has finished flowing from the top of the column, the resin packed bed is still filled with alkaline solution, and the elution of L-serine has not been completed. Pure water is passed through the column to push out the alkaline solution and completely elute L-serine.The amount of pure water is preferably 1 to 3 times the volume of the ion exchange resin filled in the column.The flow rate is shall be 2.0 m/hr or less in terms of linear velocity.

After the aqueous solution of L-serine that does not contain glycine is eluted, glycine and a small amount of L-serine are still retained in the ion exchange resin. amount of L-serine can be eluted. That is, an alkaline solution is passed from the top of the column.

Examples of alkaline solutions include ammonia, NaO
Aqueous solutions such as H1KOH can be used. In either case, the concentration range is 0.5-1.2M.

However, in the next elution, the amount of alkali used is 1.0 to 1.5 times the number of moles of glycine retained. Glycine elution is possible even when the alkali is in excess, but the alkali continues to flow into the resin packed bed even after the glycine elution is completed, which is not economical. This is not desirable as it will be insufficient and the recovery rate will drop.The flow velocity is 2.0 in terms of linear velocity.
+m/hr or less. After the elution operation, pure water is passed through the top of the column to extrude water to completely elute glycine.

The amount of pure water is approximately the same volume as the ion exchange resin filled in the column. 2. Flow velocity is expressed as linear velocity. Os+/hr or less.

After the above operations are completed, the ion exchange resin is in the NH4' type, so an appropriate acid solution is passed through the top of the column to regenerate it into the ``2'' type and prepare it for the next separation operation of glycine and L-serine. . To regenerate the ion exchange resin, for example, 1 containing hydrochloric acid in an amount of 1.2 times the total exchange capacity of the ion exchange resin.
After passing a 0% by weight aqueous hydrochloric acid solution at a linear velocity of 1.1 m/hr, pure water approximately twice the volume of the ion exchange resin filled in the column was passed at a linear velocity of 1.5 m/hr. It is possible to adopt a method such as cleaning by washing.

〔Effect of the invention〕

When such an operation is performed, an L-serine solution containing no glycine is first eluted from the bottom of the column.

By subjecting this solution to a commonly used amino acid isolation operation, highly pure L-serine crystals can be obtained.

Subsequently, a mixed solution of glycine and L-serine, which is mainly composed of glycine, is eluted. This solution can be subjected to a concentration operation and reused as a part of the raw material for L-serine synthesis reaction using glycine as a raw material, and can contribute to reducing the cost of L-serine.

〔Example〕

Glucose 4g/2, citric acid 2g/2, Mg5Oa・
7Hx00.2g/l, *HPOa 10g/j
! , NaNHaHPO4-4H*00.2g/l yeast extract 0.5g/j! Escherichia coli MT grown on bouillon agar medium was placed in 10 12-volume Erlenmeyer flasks each containing 200 d of medium with the composition
-10350 (FERM P-7437) was inoculated one loop at a time and cultured with shaking at 30°C and 210 rpm for 24 hours. The obtained culture solution was centrifuged to obtain 8 wet bacterial cells.
I got g.

8 g of this wet bacterial body was added to 10 gM phosphate buffer (ρ117.5
) and then subjected to ultrasonication at 4°C for 15 minutes to obtain crude enzyme solution 20-.

A reaction solution 420d containing 117 g of glycine, 0.21 g of tetrahydrofolic acid, 3 g of pyridoxal-5'-phosphoric acid, and 20 d of crude enzyme solution was prepared, and the formaldehyde concentration in the reaction solution was adjusted to 4-amino-3-hydrazide-5-mercapto. While performing colorimetric analysis using 1,2,4-triazole,
3 while controlling its concentration not to exceed 20mM.
The synthesis reaction of L-serine was carried out by continuously supplying 7% formalin using a peristaltic pump. The reaction temperature was 50° C., and the pH was constantly controlled at 7.0 by adding 2M KaPtOq. PiL stirring is 200r
I went with p-.

Formalin was continuously supplied to the reaction solution for 42 hours, and the total amount of formalin (37% by weight formaldehyde solution) added to the reaction solution was 96.5 g.

After the reaction was completed, the PU of the reaction solution was adjusted to 4.0 using sulfuric acid, activated carbon 38 was charged, heat treatment was performed at 90°C for 30 minutes, and the residue derived from the bacterial cells was removed by hot filtration.

In this way, 705 g of L-serine solution was obtained. 246 d of this L-serine solution (containing 17.43 g/d of L-serine, 4.02 g/a of glycine, and 1.07 g/d1 of K'') was placed in a cylinder with an inner diameter of 40 cm using the strongly acidic cation exchange resin Revatit MDS. -1368 (manufactured by Bayer) was filled with 4804 cells and the liquid was passed through the resin tower which was regenerated into H3 type, with a total exchange capacity of 1k.
0.77 eq), L-serine, glycine, and K were retained in the ion exchange resin. Subsequently, 480 d of pure water was passed through to wash away impurities not retained by the ion exchange resin.
After passing 544 d of 0.88M ammonia water, pure water 9
Extrusion was performed at 00d to elute only L-serine. Furthermore, 182 m of 0.88M ammonia water was passed through, and 400 m of pure water was passed through.
A mixture of glycine and L-serine, mainly consisting of glycine, was eluted by extrusion using 1 ml. In all steps, liquid was passed at a linear speed of 1.5 m/hr.

The eluate was separated into 250i portions and analyzed by HLC to separate glycine and L.
- The results of the analysis of serine are shown in Table 1, and illustrated in Figure 1. The elution fraction of L-serine was separated from fraction 5 to Nα7, and the L-serine concentration was 4 g/
A glycine-free eluate was obtained in step a. The purity of L-serine isolated from this L-serine solution was 99.9%.
- Contained no amino acids other than serine.

(The following is a blank space) Table 1 Concentration of L-serine and glycine in each fraction [Comparative example] L-serine solution 246d prepared in Example (17.43 g/d1 for L-serine, 4.02 g/d1 for glycine) ,
1.07g/d of K4! ), and a strongly acidic cation exchange resin rebat) in a cylinder with an inner diameter of 40 mm. 1DS-136
8 (manufactured by Bayer) and passed through a resin tower (total exchange capacity 0.77 eq) that was regenerated into H4 type.
Serine, glycine, and K were retained. Impurities not retained by the ion exchange resin were washed away by passing 480 mm of pure water. The elution with alkali was not performed twice, but was performed continuously this time. That is, 816 d of 0.88M ammonia water was passed through the solution to elute L-serine and glycine. The eluate was passed through at a linear velocity of 1.5+w/hr throughout the process, and the eluate was collected in 250 d portions and analyzed for glycine and L-serine by HLC. The results are shown in Table 2, and illustrated in Figure 2. become that way.

The elution fraction of L-serine was set as fraction Nα6 to Nα7, L-serine concentration was 8.4 g/a, and glycine concentration was 0.4.
An eluate of g/a was obtained. The purity of the serif isolated from this L-serine solution was 98.5%, and the glycine content was 1.2%.
It contained.

Table 2 Concentrations of L-serine and glycine in each fraction

[Brief explanation of drawings]

FIG. 1 is a diagram plotting Table 1 in Examples of the present invention, and FIG. 2 is a diagram plotting Table 2 in Comparative Examples of the present invention. (Hereinafter, blank space) Patent applicant Mitsui Toatsu Chemical Co., Ltd. (Fraction) Figure 1 (Fraction) Figure 2

Claims (2)

[Claims] (1) From an L-serine solution containing unreacted glycine obtained by an enzyme method or a fermentation method using glycine as a raw material,
In a method for separating L-serine, the L-serine solution is passed through a packed bed of H^+ type strongly acidic cation exchange resin with an effective diameter of 0.15 to 0.40 mm and a uniformity coefficient of 1.7 or less. Then, glycine and L-serine were retained, and then an alkaline solution of 1.0 to 1.5 times the molar amount of the retained L-serine was passed through at a linear velocity of 2.0 m/hr or less. A method for separating L-serine, which comprises extruding with water in an amount of 1 to 3 times the amount of strongly acidic cation exchange resin filled in a packed bed. (2) The concentration of alkali that is passed through to elute L-serine retained in the strongly acidic cation exchange resin is 0.5-
2. The method of claim 1, wherein the amount is 1.2M.