JPH0698008B2 - Glycine production method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing glycine from monoethanolamine by using a microorganism.

(Prior art and its problems) As the industrial production method of glycine, the Strecker method using hydrocyanic acid, formaldehyde and ammonia as main raw materials has been known. This method has a high yield, but requires a purification step such as decolorization of the reaction solution and decolorization of the reaction solution, removal of by-products, and a large amount of salts as by-products. .

Although microorganisms Mycobacterium have reported the conversion of monoethanolamine to a part glycine (Biochemical Medicino 11 .67~70,1947) yield 0.26%
And it has little practical value.

(Means for Solving Problems) The inventors of the present invention have conducted research to find a more efficient method in which no salt is formed as a by-product with respect to such a conventional production method, and as a result, agrobacterium Glycine monoethanolamine to microorganisms of the genera Urum, Astrobacterium, Brevibacterium, Corynebacterium, Enterobacter, Flavobacterium, Klebsiella, Micrococcus, Proteus, Sarsina, and Serratia. It has been found to have the ability to convert into.

It has not been known that these microorganisms have the ability to convert monoethanolamine into glycine, and the present invention has been completed as a result of further research based on this finding.

That is, the present invention has the ability to convert monoethanolamine into glycine, Agrobacterium, Arthrobacter, Brevibacterium, Corynebacterium, Enterobacter, Flavobacterium, Klebsiella, Micrococcus. Genus, Proteus genus, Sarsina genus, culture solution of microorganisms belonging to the genus Serratia, microbial cells or a treated product of microbial cells, contact with monoethanolamine in an aqueous medium to convert into glycine Is.

As the microorganisms that can be used for the purpose of the present invention, wild strains existing in nature and microorganisms stored in public microbial preservation institutions can be used.

As a method for assaying a microorganism having the ability to convert monoethanolamine into glycine, for example, the following method can be adopted.

After collecting 2 ml of the culture solution of the test microorganism and collecting the cells by centrifugation, the collected cells were washed with the same volume of sterilized physiological food water and then added to a buffer solution (pH = 7.5) containing 3 ml / monoethanolamine. Disperse the cells and react at 32 ° C for 24 hours. Then, the supernatant obtained by separating the bacterial cells from the reaction solution by centrifugation was used as a paper chromatograph (developing solution; Bu-O
H: acetic acid: water = 4: 1: 1) and then ninhydrin color is developed,
Cut off the colored part of R value corresponding to glycine, and after extracting the colored part with 5 ml of 75% ethanol solution,
The glycine produced was quantified by measuring the absorbance at a wavelength of 570 nm.

The amino acid further produced from the strain recognized as having the ability to convert monoethanolamine into glycine as described above is isolated and confirmed by a conventional method.

Agrobacterium used in the present invention, Arthrobacter, Virebbacterium, Corinobacterium, Enterobacter, Flavobacterium, Klebsiella, Micrococcus, Proteus, Sarsina, Serratia Microorganisms belonging to the above are microorganisms that have passed the above-mentioned test.

As a method of allowing the microorganism of the present invention to act on monoethanolamine, there is a method of contacting the cells of the microorganism of the present invention and a treated product of the cells in an aqueous solution.

The medium used for culturing the above-mentioned microorganism is a usual medium containing a carbon source, a nitrogen source, and inorganic nutrients necessary for the growth of the microorganism, which are usually assimilated. Cultivation conditions may be controlled under aerobic conditions while controlling the pH to be in the range of 4 to 9 and the temperature of 25 to 45 ° C. within an appropriate range suitable for each microorganism.

The concentration of the reaction substrate in the enzyme reaction can be 0.1 to 10% by weight. The reaction temperature and pH in the reaction solution in the enzyme reaction are the optimum temperature and pH of the enzyme having the ability to convert monoethanolamine of the microorganism used to glycine, but the enzyme reaction temperature is usually 20 to 45 ° C.
In the above range, the pH of the enzyme reaction solution is usually in the range of 5-11.

Isolation of glycine produced can be carried out by utilizing known methods such as concentration, neutralization and ion exchange to obtain the desired glycine.

(Operation and Effect of the Invention) The present invention is a new and advantageous method for producing glycine because glycine can be easily obtained from monoethanolamine by using a microorganism.

(Examples) The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example-1 Glucose 5 g /, peptone 5 g /, meat extract 5 g /, (N
_{H 4) 2 2g /, KH} 2 PO 4 1g /, K 2 HPO 4 1g /, MgSO 4 · 7H 2 O
0.5g /, FeSO ₄ / 7H ₂ O 0.01g /, MnSO ₄ / 4H ₂ O 0.01g /
20ml of the medium containing (, pH = 7.0) in a 250ml Erlenmeyer flask
It was put and sterilized at 120 ° C for 15 minutes.

Micrococcus lysode (Micrococcus lysodecus) cultured in broth agar medium at 28 ° C for 24 hours
ikticus) ATCC-4698 was inoculated with 1 platinum loop and cultured at 28 ° C for 40 hours.

The cells were collected from this culture solution by centrifugation and washed once with the same amount of sterilized physiological saline as the culture solution to collect the cells.
The cells were added to 0.1 M phosphate buffer (pH = 7.5) containing 1% by weight of monoethanolamine so that the concentration was 30 g /, and 5 ml thereof was reacted at 30 ° C. for 40 hours. After the reaction,
1 ml of 12% trichloroacetic acid solution was added to 4 ml of the reaction mixture, and the total volume was 5 m.
Let l. After that, insoluble matter was removed by centrifugation (15,000 rpm, 10 mm), and the supernatant was analyzed by an amino acid analyzer (Hitachi Ltd.) to measure glycine in the reaction solution. As a result, the production of 5.5 mg / ml glycine was confirmed.

Example-2 Each of the microorganisms shown in Table 1 prepared in the same manner as in Example-1 was added to 0.1 M phosphate buffer (pH = 7.5) containing 1% by weight of monoethanolamine so that the concentration was 30 g /. 5 ml to 30
Reacted at 40 ° C for 40 hours. After the reaction, glycine was quantified in the same manner as in Example-1, and the results are also shown in Table 1.

All the microorganisms in Table 1 are known ones, and their preservation institutions, namely American Type Culture Colletio
n (ATCC) (US) and Agrucultural Research Culture
It is easily available from the Collection (NRRL) (USA). Among these microorganisms, Agrobacterium radiobacter NRRL B-11
291 is the same as described in US Pat. No. 4,332,905.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display part (C12P 13/04 C12R 1:13) (C12P 13/04 C12R 1:15) (C12P 13/04 C12R 1:20) (C12P 13/04 C12R 1:22) (C12P 13/04 C12R 1: 265) (C12P 13/04 C12R 1:39) (C12P 13/04 C12R 1: 425)

Claims (1)

[Claims] 1. Agrobacterium having an ability to convert monoethanolamine into glycine.
Genus, Arthrobacter genus, Brevibacterium genus, Corynebacterium genus, Enterobacter
ter) genus, Flavobacterium genus,
Klebsiella spp., Micrococcus (Mi
crococcus), Proteus (genus Proteus), genus Sarcina (genus Sarcina), Serratia (serratia) genus of the culture solution, fungus or bacterial cell contact with monoethanolamine in an aqueous medium to glycine A method for producing glycine, which comprises converting the glycine.