JPH012594A - Method for producing D-serine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for industrially producing monoserine, which is useful as a pharmaceutical intermediate, by a preselective method.

<Prior art> A method is already known in which DL-5-hydroxymethylhydantoin is converted to N-carbamyl-D-serine using microorganisms and then hydrolyzed to obtain D-serine (Japanese Patent Laid-Open No. 152291/1983). Public bulletin).

<Problems to be Solved by the Invention> The above method is excellent as a method for obtaining D-serine from DL-5-hydroxymethylhydantoin.

However, the reaction to obtain N-carbamyl-D-serine from DL-5-hydroxymethyl-dantoin was 40%.
The yield is low (less than 50 t/J!), and the enzyme activity is low, and when using live cells, the yield is less than 50 t/J! This method cannot be said to be advantageous as an industrial production method, as it requires isolated bacterial cells.

Means and action for solving the above problems> Therefore, the present inventors cultivated microorganisms in a medium containing DL-serine to selectively assimilate only L-serine.
- A method for H-forming serine was investigated.

According to this method, since serine is consumed as energy for the growth of microorganisms, only D-serine is accumulated in the medium when the entire amount of L-serine is assimilated, and other by-products are will almost never exist.

In addition, whether the selected microorganism does not possess racemase or
Alternatively, even if D-serine is retained, by culturing it under conditions where it is inactivated, D-serine contained in DL-serine can be accumulated in a substantially stoichiometric amount.

The present inventors searched for microorganisms that meet these objectives.

That is, we investigated microorganisms that have the ability to selectively assimilate only L-serine and not substantially assimilate D-serine in a medium containing inexpensive L-serine as a carbon source and nitrogen source. By culturing specific microorganisms in DL-serine medium, tens of g/j! D at the accumulated concentration above
- It was discovered that serine can be obtained, and the present invention was completed.

That is, the present invention provides, in a medium containing DL-serine,
It belongs to yeasts such as the genus Candida, Torulopsis, Cryptococcus, Satucharomycobsis, and Hansenula, or bacteria such as Escherichia, Klebsiella, Providencia, Microbacterium, and Serratia, and - A method for producing D-serine, which comprises culturing a microorganism that has the ability to assimilate serine but not substantially assimilate D-serine, and then isolating and collecting D-serine from the culture solution.

Hereinafter, the configuration of the present invention will be explained in detail.

The microorganisms used in the present invention include yeasts such as the genus Candida, Torulopsis, Cryptococcus, Satucharomycobsis, and Hansenula; Examples include microorganisms belonging to the bacterial genus, such as the genus Umbrella and the genus Serratia.

Among these microorganisms, those that can grow in a medium containing substantially DL-serine as a carbon source and nitrogen source and have the ability to assimilate L-serine and substantially assimilate D-serine. Microorganisms that do not contain any of the following are used in the present invention.

Here, microorganisms that do not substantially assimilate D-serine are:
It also includes microorganisms that assimilate D-serine in small amounts to the extent that the effects of the present invention are not substantially inhibited, or microorganisms that assimilate D-serine in the absence of L-serine after assimilating L-serine. It will be done.

For example, Candida Rugoza 1candida
rugosa) ATCC10571,'? Yandida・
Candida l 1polytic
a) l F 00717, Torulopsis candida I FO
O380, Cryptococcus lowrench 4 tcry
ptococcus Iaurentii) ATCC3
6832, Satucharomycobsis ribolytica (Sa
ccharonycopsis l1polytica
) ATCC20306, Hansenula anomala (Ha
nsenula anoiala) I F OO12
0, Escherichia coli
coli) I A M 1268, Klebsiella pnaumonia
ae) ATCC21316, Providencia rattgeri A
T CC9886, Microbacterium ammonia film
philun) F ERM-P2408, Serratia marcescens (Serratia narcescens)
) I AM 12142, etc.

In the present invention, culture is performed in a medium containing DL-serine as a carbon source and nitrogen source, but may contain other carbon sources and/or nitrogen sources.

Preferably, the culture is carried out in a medium containing DL-serine as the sole carbon and nitrogen source.

The concentration of DL-serine in the medium is 1 (1 to 150 g, preferably 10 to 100 g. , the growth of microorganisms tends to be inhibited.

You can add the entire amount of DL-serine to the culture solution from the beginning, but if the concentration becomes too high, the growth of microorganisms will slow down, and supersaturated DL-serine will precipitate, so the initial concentration should be 1 to 40.
g/i! A fed-batch culture method is preferred, in which the remaining DL-serine is added in portions.

Cultivation is preferably carried out under acidic conditions. The culture solution is usually adjusted to pH 5-6 at the start of culture. As the culture progresses, the pH increases. If cultured as is, the growth rate of the microorganisms will be slow and the recovery rate of D-serine will tend to be low, so it is necessary to make the pH acidic and pool the contents.

The pH during culturing is usually 4 to 7, preferably 4.5 to 6.5.
Adjust to. The adjusting acid is preferably an aqueous inorganic acid solution such as phosphoric acid, sulfuric acid, or hydrochloric acid.

Culture temperature is usually 20-40°C, preferably 25-35°C
It is.

The culture is stirred with aeration. Airflow rate is usually 0.5-2
．． Ovvm, preferably 0.6-1.2vvn.

If the amount of aeration is too small, the growth of microorganisms will be inhibited, and the assimilation rate of L-serine will also be slowed down.

Further, even if the amount is large, the effect remains the same, but rather the concentration of the culture solution becomes high because it promotes evaporation of the culture solution, and foaming becomes intense, which is not preferable.

The culture is terminated when all L-serine is assimilated.

The total assimilation of L-serine can be determined by monitoring DO, by analyzing serine paste and L, or by monitoring the amount of acid added.

If the culture is continued after all the serine has been assimilated, D-serine may also be gradually assimilated, so it is preferable to clearly know the end point of the culture.

After removing the bacterial cells from the thus obtained culture solution by centrifugation, D-serine can be isolated by a conventional method. After being adsorbed on the resin, the resin is thoroughly washed, then eluted with an aqueous ammonia solution, and the eluate is concentrated. If the crude serine obtained here is recrystallized with water, purified D-serine can be obtained.

<Example> Hereinafter, the present invention will be specifically explained with reference to Examples.

In the examples, the optical purity analysis of serine was performed by methyl esterifying concentrated and dried D-serine-containing powder with methanol-hydrochloric acid, reacting it with 3,5-dinitrophenyl isocyanate, and then HPLCing it under the following conditions.
The analysis was carried out using the method described in C.

Column: 0A-1000 (Order Chemical) Transfer l: n-hexane: dichloromethane: ethanol (
4:2:1) Flow rate: 0.7 ml/II in detection: U
V254nm Example 1 Dry broth 30 t/1. , Dispense 50 ml of medium containing (pH 6,0) into 11 Erlenmeyer flasks,
The mixture was sterilized at ℃ for 20 minutes and used as a seed culture medium. A loopful of Candida rugosa ATCC 10571 was inoculated into this, and cultured with shaking at 30°C for one day. On the other hand, DL-serine 8
0 g/It, potassium phosphate 2 g/J!
A medium (pH 5, 6) IJ containing 065 g/J2 of magnesium phosphate and 1.0 g/L of powdered yeast extract was placed in 34 mini-jar fermenters and sterilized to serve as the main culture medium. This was inoculated with the previous seed culture solution and heated at 30°C. 1.
Ov v ra aerated agitation culture was performed. In addition, during cultivation, 4
The pH was adjusted to 5.5±0.1 using N sulfuric acid. The total amount of serine in the medium was assimilated in about 70 hours, and the culture solution containing 37 g of D-serine and 25 g of ammonium sulfate was used.
．． I got 11.

This culture solution was centrifuged at 10,000 rpm for 10 minutes to remove bacterial cells, and then passed through a column filled with ion exchange resin 5K-IB (H type) to adsorb D-serine. After thoroughly washing this column with water, D-serine was eluted with 4% aqueous ammonia. This eluate was concentrated under reduced pressure to dryness to obtain 35 g of D-serine. When analyzed by HPLC, the optical purity was 9.6%ee or higher.

Chemical purity was 99.4%.

Example 2 DL-serine 10 g/O, glucose 5 g/'l, potassium phosphate 2 g/l, magnesium sulfate 0.5 t
/ J! , medium containing powdered yeast extract 0.5g/R (
pH5,0) 50ml 1j! Dispense into an Erlenmeyer flask,
It was sterilized and used as a seed culture medium. A loopful of Candida rugosa ATCC 10571 was inoculated thereto, and cultured at 30° C. for about 20 hours.

On the other hand, among the above medium composition, 30 g of DL-serine
/1, and medium 11 from which glucose was removed was placed in a 3-size mini jar fermentor and sterilized to serve as the main culture medium. This was inoculated with the previous seed culture solution and heated at 30°C. 1. Ov
Aerated agitation culture was performed using VTS. During the culture, the pH was adjusted to 5.6±0.1 using 2N sulfuric acid. After about 20 hours, when L-serine in the medium has been assimilated, DL-
Serine 30Iz was added and cultured for an additional 30 hours.

Thereafter, D-serine 23°3'' was obtained in the same manner as in Example 1. The optical purity and chemical purity were almost the same as in Example 1.

Examples 3-15 Seed medium consisting of 30 g/1 dry broth (PH5
,0) Dispense 5ml into a 18x180min test tube,
Sterilized. A platinum loop of the microorganisms shown in Table 1 was inoculated into this, and cultured with shaking at 30°C for 1 to 2 days. On the other hand, DL-Serine 10'sr/J! , potassium phosphate 2g/J! , magnesium sulfate 0.5g/l, powdered yeast extract 1.0g
18X 5ml of main culture medium (pH 5, 6) consisting of /l
The mixture was dispensed into 180 nmφ test tubes and sterilized. Seed culture f ahead of this! The solution was inoculated with 5% seeds and cultured with shaking at 30°C.

After 24 hours, the cells were removed by centrifugation, concentrated under reduced pressure, and dried. About the solid content obtained
The residual rates of 1 and 9 serines remaining were determined by LC.

The results are shown in Table 1.

<Effect of the invention> The present invention exhibits the following effects.

(1) By culturing microorganisms using DL-serine as a carbon source and nitrogen source, L-serine can be assimilated with high selectivity.

(2) Furthermore, the accumulation concentration is high, and D-serine can be obtained in a short time.

(3) In addition, most of the consumed D-serine is converted to carbon dioxide gas and water, and there are virtually no by-products other than D-serine in the culture solution.

(4) Therefore, isolation and purification of D-serine is easy.

Claims (1)

[Claims] Candida (Ca
ndida), Torulopsis
) genus, Cryptococcus
Genus, Saccharomycopsis
Yeasts such as the genus Psis, genus Hansenula, or Escherichia
ia) genus, Klebsiella genus,
Cultivating a microorganism that belongs to the genus Providencia, the genus Microbacterium, the genus Serratia, etc., and has the ability to assimilate L-serine but not substantially assimilate D-serine, A method for producing D-serine, which comprises isolating and collecting D-serine from a culture solution.