JPH0591895A - Production of d-serine - Google Patents

Abstract

PURPOSE:To efficiently obtain D-serine in high purity by allowing a microbial cell having tyrosinase activity to act on a reaction liquid containing DL-serine and phenol and converting only L-serine into L-tyrosine. CONSTITUTION:A microorganismic cell having tyrosinase activity [preferably Escherichia coil TD-001 (FERM P-10838)] or treated product thereof is made to act on a reaction liquid containing DL-serine and phenol to convert L-serine into L-tyrosine and then unreacted D-serine is collected. The reaction is preferably carried out in a solvent such as 0.1M phosphoric acid buffer solution at 30-40 deg.C, normally for 2-72hr.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a highly efficient method for producing D-serine by an enzymatic method. According to the present invention, D-serine can be produced with high efficiency, and the co-produced L-tyrosine can be easily separated. Therefore, D-serine can be separated with high purity and high yield.

D-serine is an amino acid expected as an industrially important compound as an intermediate raw material for medicines, foods, agricultural chemicals and the like. On the other hand, co-produced L-tyrosine is also an important amino acid as a raw material for medicines, cosmetics and the like.

[0003]

The industrial production of D-serine has heretofore been largely unknown. The present inventors have conducted extensive studies on the development of a D-serine production process by an enzymatic method, and performed D-serine more efficiently than inexpensive DL-serine.
-Finding a method for producing serine, the present invention has been completed.

[0004]

[Means for Solving the Problems] The present invention allows a microbial cell having tyrosinase activity or a treated product thereof to act on a reaction solution containing DL-serine and phenol, and L-serine is converted into L-serine by an enzymatic method. It is intended to provide a method for collecting D-serine in high yield by separating L-tyrosine produced and unreacted D-serine after conversion into tyrosine.

According to the method of the present invention, due to the difference between the solubility of L-tyrosine (about 0.04 g / l, 25 ° C) and the solubility of D-serine (about 600 g / l, 25 ° C), D −
Serine can be separated.

The microbial cell used in the present invention is not particularly limited as long as it has a tyrosinase activity. For example, Escherichia coli ( Esch.
erichia coli ) TD-001 (FERM P-1083
8) is preferably used.

The medium used for preparing the above-mentioned microbial cells used in the method of the present invention is not particularly limited and may be one used for culturing general microorganisms. For example, glucose, glycerol or the like can be used as the carbon source. As the nitrogen source of the medium, for example, inorganic salts such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate and urea can be used. Further, organic nutrient sources such as peptone, yeast extract, constip liquor and casamino acid can be used as carbon sources and nitrogen sources. As the inorganic salt, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. Further, components necessary for culturing can be added depending on the characteristics of the bacterium used.

The culture is carried out under aerobic conditions such as aeration and stirring, and the culture temperature is 20 to 40 ° C., preferably 28 to 32.
Perform at ℃. The pH during the culture is 5 to 10, preferably 7 to 8.
Keeping the temperature around and adjusting the pH during culturing, add acid or alkali. The culture period is 10 hours to 4 days, and the optimum period is 1 to 3 days.

The cells are collected from the culture thus obtained, washed with water or an appropriate buffer, and subjected to the method for producing D-serine of the present invention.

In the method of the present invention, the above-prepared cultured cells can be used as they are, but the cells are crushed by ultrasonication or the like, or the crushed material is further treated with water or the like. It can also be used in the form of the extract extracted in step 1, or a crude product obtained by further treating the extract with ammonium sulfate or the like to precipitate the enzyme component. Further, the cells or their treated products can be used after being immobilized if necessary. As the immobilization method, known methods such as L. Goldstein, Method in Enzymolog
The method described in y, 19, 935 (1970) can be used, and specifically, a polymerizable monomer such as acrylamide, alginate,
There is a method of immobilizing the cells by immobilizing them on a suitable carrier such as carrageenan.

The reaction between DL-serine and phenol in the presence of the above-mentioned microbial cells or a treated product thereof is, for example, a 0.1M phosphate buffer solution (pH 6.0 to pH 6.0) in the same manner as a usual enzyme reaction.
10.0) or water (pH 6.0 to 10.0) and the like at a temperature of about 20 to 50 ° C, preferably about 30 to 40 ° C, usually for about 2 to 72 hours. The reaction is preferably carried out while giving a stirring effect.

The phenol concentration in the reaction solution is not particularly limited, but the concentration in the reaction solution is 1.0%.
(W / v) It is particularly preferable to continuously or intermittently add to the medium so that the reaction is performed so that the reaction rate is kept below. The total addition amount is not particularly limited, but is usually 0.1 to 1
It is 0%, preferably 0.5 to 5% (w / v).

The concentration of D- or DL-serine in the reaction solution is generally 0.1 to 20% (w / v), preferably 1.
It is suitable to use in the range of 0 to 10% (w / v).

[0014]

According to the manufacturing method of the present invention, the raw material DL-
L-serine in serine can be efficiently converted into L-tyrosine, and unreacted D-serine can be separated and collected in high yield.

[0015]

The present invention will be described in more detail with reference to the following examples.

Reference Example: Preparation of β-Tyrosinase-Containing Bacteria 100 ml of the medium shown in Table 1 was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes.
Escherichia coli TD-0 which is a tyrosinase-producing bacterium
01 (FERM P-10838) was inoculated and cultured with shaking at 37 ° C for 1 day.

[0017]

[Table 1]

20 ml of the culture broth was inoculated into 1000 ml of a medium prepared in the same manner, and the culture was aerated with stirring at 37 ° C.
Rotation speed 1000 rpm, ventilation volume 1 vvm, pH 7.2 (2
It was cultured in 8% ammonia water) for 20 hours.

After completion of the culture, centrifugation was carried out in 200 ml portions (80
The cells were collected by collecting the cells at 00 rpm for 15 minutes at 4 ° C. for 5
After washing the cells with 20 ml of 0 mM phosphate buffer (pH 8.0), the collected cells were frozen at -20 ° C for 1 day, and the cells were used as test cells.

Example The β-tyrosinase-containing cells prepared by collecting cells from 200 ml of the culture solution in the reference example were suspended in 50 ml of the reaction solution shown in Table 2 and then shaken in a 500 ml Erlenmeyer flask at 30 ° C. for 24 hours. And allowed to react.

[0021]

[Table 2]

After completion of the reaction, the whole reaction solution was centrifuged (8
The amount of D-serine in the supernatant after being subjected to 5,000 rpm, 15 minutes, 4 ° C.) was 4.2 mg / ml. The ratio of L-serine to D-serine (L / D) in the supernatant was measured by liquid chromatography (Shimadzu LC-5A, column: Tosoh Enantio L1), and L / D <
It was 1/1000.

Claims (1)

[Claims] 1. A microbial cell having tyrosinase activity or a treated product thereof is allowed to act on a reaction solution containing DL-serine and phenol to convert L-serine to L by an enzymatic method.
-A method for producing D-serine, which comprises converting to tyrosine and collecting D-serine from this.