JPS619294A - Production of beta-hydroxyamino acid - Google Patents

Abstract

PURPOSE:To prepare the titled compound in a short time, in high efficiency, without using expensive apparatuses, by contacting glycine with the culture liquid, etc. of microorganisms capable of producing serine hydroxymethyltransferase, and adding an aldehyde to the product to effect the reaction of the components. CONSTITUTION:beta-Hydroxyamino acid is produced from glycine and an aldehyde in the presence of the culture liquid or the cells of a microbial strain capable of producing serine hydroxymethyltransferase [e.g. Escherichia coil MT-10350 (FERM P-7473), etc.]. In the above process, the microbial cells are made to contact with glycine prior to the reaction, the pH of the reaction system is adjusted to 7.0, and formaline is added to the system in several portions.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing β-hydroxyamino acids, more specifically, the production of β-hydroxyamino acids from glycine and aldehyde in the presence of a microorganism capable of producing serine hydroxymethyltransferase. The present invention relates to a method for producing amino acids.

(Prior Art) β-hydroxyamino acids are important substances as amino acids such as serine, threonine, and phenylserine, pharmaceuticals, cosmetics, feed additives, and pharmaceutical intermediates.

Serine human waxy methyltransferase (λ1.2
．． L E, O, &+ 4 && ) is also called serine transhydroxy) tylase or serine aldolase, and is widely present in mammals, birds, higher plants, microorganisms, etc. It ferments pyridoxal phosphate and synthesizes glycine and It is already well known that it catalyzes the reaction of synthesizing β-hydroxyamino acids from aldehydes. (For example, Ad
vances in 1 zymology 53
.

83-112 (1982), ).

(Problems to be Solved by the Invention) The present inventors conducted further research on a method for producing β-hydroxyamino acids from glycine and aldehydes in the presence of microorganisms capable of producing serine hydroxymethyltransferase. If an enzymatic reaction is performed using the culture solution or cells of microorganisms as a source, the enzyme activity may be low and the reaction yield may be low due to the poor permeability of the cell membrane of the microorganisms, such as substrates. I was watching more.

Conventionally, methods for increasing the permeability of bacterial cell membranes include mechanical disruption, ultrasonic disruption, freeze-thaw treatment, pressurization and vacuum treatment, osmotic pressure treatment, autolysis, cell wall-degrading enzyme treatment, surfactant treatment, etc. was used. (For example, Agric
, Biol, Ohem, , 42.2515-2
321 (1978), J. Ferment, Tec.
hnol, ', 61°135-142 (1983
)). However, mechanical destruction, ultrasonic treatment, freeze-thaw treatment, pressurization and depressurization treatment, etc. require expensive equipment.
Moreover, it is difficult to process in large quantities. Osmotic pressure treatment, autolysis, cell wall-degrading enzyme treatment, surfactant treatment, etc. have disadvantages such as varying effectiveness depending on the type and condition of microbial cells and often requiring a long time for treatment. Another drawback is that it is difficult to remove the used organic solvent or surfactant in autolysis using an organic solvent or surfactant treatment.

(Means for solving the problem) As a result of intensive research into a method for producing β-hydroxyamino acids more efficiently than glycine and aldehyde using bacterial cells, the present inventors have found that serine hydroxymethyltransferase production ability has been achieved. When producing β-hydroxyamino acids from glycine and aldehydes in the presence of microbial cultures or microbial cells, β-hydroxyamino acids can be efficiently produced by bringing the microbial cells into contact with glycine in advance and then using them in the reaction.
- It was discovered that hydroxyamino acids are produced, and the present invention was completed.

Since the glycine used in the present invention is also a reaction substrate, there is no need to remove it after use unlike organic solvents or surfactants, and it can be used as a reaction substrate as it is. In addition, contacting the precursor with glycine (glycine treatment) does not require expensive equipment and can be carried out in a short time and in large quantities, making the method of the present invention industrially extremely superior. The result of contact between the bacteria and glycine probably affects the permeability of the bacterial cell membrane.

The microorganism used in the present invention is not particularly limited as long as it has the ability to produce serine hydroxymethyltransferase. Examples of such microorganisms include:
Fischerichia coli
li) MT-10350 (FEB No. 7437) and Escherichia coli MT-10351 (FEB No. 7468).

For culturing the microorganisms used in the present invention, any synthetic or natural medium can be used as long as it contains carbon sources, nitrogen sources, inorganic salts, organic nutrients, etc. that can be utilized by the strain used.

In the method of the present invention, the culture solution thus obtained is used as it is, or the viable bacterial cells collected by centrifugation, straining, etc. are brought into contact with glycine, and then used as an enzyme source.

As for the method of bringing microbial cells into contact with glycine (glycine treatment), if the culture solution itself is used as an enzyme source, it is sufficient to add glycine to the culture solution after completion of culture. When using the cells as an enzyme source, it is sufficient to suspend the collected cells in an appropriate glycine solution.Glycine treatment may be carried out either under static conditions or under stirring conditions, but the pH should be 6 to 9. , the temperature is 20-7
It is desirable to carry out the test within the range of °C. In this case, the glycine concentration may be as high as 4% by weight (the same applies hereinafter) for the effect to be obtained, but it is usually about 7 to 26%.

The glycine treatment time seems to vary depending on the bacterial cell concentration, glycine concentration, temperature, etc., but is usually about 60 minutes to 24 hours. The bacterial cells treated with glycine can be used as an enzyme source in a reaction by adjusting the concentration to an appropriate concentration as a glycine solution.

The reaction between glycine and aldehyde according to the present invention is carried out in the presence of the glycine-treated bacteria obtained in this manner.
It is preferable to carry out the reaction under conditions of shaking or stirring at a temperature of 6-9°C and a temperature of 20-60''C.

The use of glycine as a reaction substrate also has a wide range of concentrations.

Since the amount is usually 1 to 40%, glycine as a reaction substrate may be added in its entirety at the start of the reaction (or may be added in successive portions as the reaction progresses).

On the other hand, the aldehydes that are reaction substrates include formaldehyde, acetaldehyde, propionaldehyde,
Butyraldehyde, isobutyraldehyde, valeraldehyde, isovaleraldehyde, capronaldehyde,
Aliphatic saturated aldehydes such as capryaldehyde, dodecylaldehyde, myristic aldehyde, palmitic aldehyde, aliphatic dialdehydes such as succidinaldehyde, aliphatic unsaturated aldehydes such as acrolein and crotonaldehyde, benzaldehyde and one or one substituent
Examples of this include aldehyde acids such as benzaldehyde and formyl acetic acid having two or more, and ketoaldehydes such as methylglyoxal.

These aldehydes must be used at a concentration that does not inhibit the enzyme activity, and the entire amount may be added at the start of the reaction so that the concentration is usually about 0.001 to 10%. Alternatively, it may be added in successive portions.

Serine hydroxymethyltransferase is
Since vitamin B6 is required as a coenzyme, adding pyridoxal phosphoric acid to the reaction system may enhance the reaction more than K.

When formaldehyde is used as the aldehyde, the reaction may be enhanced by adding tetrahydrofolic acid as a coenzyme to the reaction system.

This reaction may be enhanced by the addition of a reducing agent or by conducting it under nitrogen bubbling conditions. Examples of the reducing agent in this case include ascorbic acid, dithiothreitol, 2-mercaptoethanol, dithioerythritol, reduced glutathione, cysteine, and sodium sulfite.

In order to isolate the β-hydroxyamino acid produced in the reaction solution, conventional methods such as concentration, adsorption/desorption treatment using an ion exchange resin or activated carbon can be applied.

Qualitative confirmation of the generated β-hydroxy amino acid is based on ninhydrin color development on a paper chromatogram, and quantitative determination is performed by cutting out the ninhydrin coloring spot on the paper chromatogram and colorimetrically quantifying the spot extract, and by liquid chromatography. be able to.

Quantification of i-serine was performed using Leuconostoc mesenteroides (:t, eucOnO8tOc mesenter
oides) can be carried out using a bioassay method.

(Example) Next, the present invention will be specifically explained with reference to Examples and Comparative Examples.

Example 1 1 ooml of the medium having the composition shown in Table 1 was dispensed into 500 ml shake flasks, and after sterilization, Escherichia coli MT-10 was grown on a bouillon slant at 37°C for 20 hours.
350 was inoculated one platinum loop at a time. It was cultured with shaking at 37°C for 15 hours. Bacterial cells were collected from the culture solution by centrifugation, washed with physiological saline, and centrifuged again to obtain wet bacterial cells. This wet bacterial cell No. 19 was suspended in a glycine solution No. 18 having a pH of 7.5 and gently shaken at 50° C. for 6 hours. (Glycine treatment).

Next, this bacterial cell-containing glycine solution was diluted to 10 ml with water, adjusted to pH 7.0, and then diluted with 1 ml of pyridoxal phosphate.
Then, 10q1 of tetrahydrofolic acid and 20q of formalin (37 thiformaldehyde solution) were added to start the reaction. The reaction was carried out at 50°C, pH 7.0, and about 1 ml/ml in the reaction solution.
The reaction was carried out under gentle stirring conditions while passing nitrogen gas through.

After the start of the reaction, 10η of formalin was added to the reaction solution every 30 minutes. The reaction continued for 20 hours, during which time the total amount of formalin added to the reaction solution was 420 ml, and 500 ml of L-serine was accumulated in the reaction solution.

Comparative Example 1 On the other hand, 1 g of the wet bacterial cells of Example 1 was not treated with glycine, but was directly suspended in 1 omz glycine solution (PHZo) and reacted in exactly the same manner as above. L-serine was only 98.

Table 1 G#: 7-S 1% M2O3・7H20'0.059
6 Citric acid Section 02 Yeast extract 0.05%
NaN■ (4HPO4,4I (2003% and 2HP0.
Example 2 and Comparative Example 2 Escherichia Co. IJMT-10 in the same manner as Example 1
350 wet bacterial cells were obtained. This wet bacterial body 1! 5 ml of glycine solution (adjusted to pH 7.5) with the concentration shown in Table 2 for each i
and gently shaken at 50°C for 6 hours. Next, water and glycine were added to this bacterial cell-containing glycine solution to adjust the glycine concentration to 9% and the liquid volume to 10 Tnl. Subsequently, a reaction was carried out in the same manner as in Example 1, and a total of 420 cm of formalin was added to each reaction solution.

Table 2 shows the amount of L-serine produced in the reaction solution.

Table 2 Example 6 and Comparative Example 3 Escherichia coli MT cultured in the same manner as Example 1
2.29 glycine was added to 10 mJ of the culture solution of -10350 strain, and the mixture was gently stirred at 50°C and pH 7.0 for 2 hours (glycine treatment). Next, 50 Da of aldehydes shown in Table 3 were added to this solution, and the reaction was carried out at 40° C., pH-7.0, and nitrogen gas was passed through the reaction solution at a rate of about 1 wdl/min for 5 hours with gentle stirring. The amounts of β-hydroxy amino acids corresponding to each aldehyde shown in Table 6 were accumulated in the reaction solution.

In addition, when formaldehyde was used as the aldehyde, 1 Da of tetrahydrofolic acid was added to the reaction solution.

On the other hand, Table 6 also shows the results of performing the same reaction as above using the same culture solution without glycine treatment.

Table 6 Example 4 and Comparative Example 4 Escherichia coli
i) When the same operation as in Example 1 was performed using MT-10351, 370 Da of L-serine was accumulated in the reaction solution using glycine-treated bacterial cells. On the other hand, L-serine accumulated in the reaction solution using bacterial cells that were not treated with chrysin was only 39''lF.

Experimental Example Measurement of serine hydroxymethyltransferase activity Wet bacterial cells obtained by the method of Example 1 or Example 4 were diluted to pH 7,
The cells were suspended in 50 mM potassium phosphate buffer (containing 0.5 mM pyridoxal phosphate) and sonicated at 4'°C for 5 minutes. This treated solution is centrifuged (10,000,9
, for 5 minutes, and the supernatant was used for R,T,Taylo
The method of Analytical Biochemi
Serine hydroxymethyltransferase activity was measured using the following method (Stry1], '8O-84 (1965)).

The measurement results of specific activity are shown in Table 4.

Table 4

[Brief explanation of the drawing]

The drawing is a brush showing the relationship between the glycine concentration and the amount of serine produced in the glycine treatment in Example 2.

Claims (1)

[Claims] When producing β-hydroxyamino acids from glycine and aldehyde in the presence of a culture solution or bacterial cells of a microorganism capable of producing serine hydroxymethyltransferase, the microbial cells are brought into contact with glycine in advance and then used for the reaction. A method for producing a β-hydroxyamino acid.