JPH08322581A - Biochemical production of glyoxylic acid by oxidation of glycolic acid - Google Patents

Abstract

PURPOSE: To efficiently produce glyoxylic acid useful as a raw material, etc., for producing vanillin by reacting a microorganism, belonging to the genus Pseudomonas or Alcaligenes and capable of oxidizing glycolic acid into the glyoxylic acid or an enzyme produced thereby with the glycolic acid. CONSTITUTION: A microorganism, belonging to the genus Pseudomonas or Alcaligenes and having the ability to oxidize glycolic acid into glyoxylic acid [e.g. Pseudomonas sp. 9653 (FERM P-14931)] or an enzyme such as an oxidase and/or a dehydrogenase produced by the microorganism, belonging to the genus Pseudomonas or Alcaligenes is reacted with a substrate glycolic acid to thereby convert and accumulate the glycolic acid into the glyoxylic acid in the reactional solution. The resultant glyoxylic acid is then collected to biochemically and efficiently afford the objective glyoxylic acid useful as a raw material for producing vanillin or ethylvanillin or an ion exchange resin or as an acidic catalyst, etc., in the pharmaceutical industry.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a biochemical method for producing glyoxylic acid. More specifically, a microorganism belonging to the genus Pseudomonas or Alcaligenes and having the ability to oxidize glycolic acid to glyoxylic acid, or an enzyme produced by the microorganism acts on the substrate glycolic acid to give glycolic acid in the reaction solution. The present invention relates to a biochemical production method of glyoxylic acid, which comprises converting and accumulating into glyoxylic acid and collecting the glyoxylic acid.

Glyoxylic acid is a useful compound which is used as a raw material for the production of vanillin and ethyl vanillin, is also used for the production of ion exchange resins, and is also used as an acid catalyst in the pharmaceutical industry.

[0003]

Conventionally, glyoxylic acid has been mainly produced by a chemical method such as nitric acid oxidation of glyoxal, but recently, a biochemical production method of glyoxylic acid by a plant or animal glycolate oxidase. (Japanese Patent Publication No. 5-501800) has also been tried.

[0004]

However, when glyoxylic acid is produced by a chemical synthesis method, a large amount of by-products such as organic acids are produced, and it is necessary to remove and purify them. Therefore, complicated operation was required.

On the other hand, in the biochemical method using a plant or animal glycolate oxidase, it is necessary to extract the enzyme to be used by extracting it from the tissues of plants or animals, which makes the enzyme expensive. Since it was not obtained and there was a problem in stable supply, it was not an advantageous method for industrial implementation.

[0006]

Under these circumstances, the present inventor has been able to obtain glyoxylic acid by a biochemical method using an enzyme of microbial origin that can be supplied inexpensively and can be industrially advantageously implemented. Considered manufacturing.

Up to now, it has been presumed that glycolic acid is oxidized via glyoxylic acid in microorganisms [Applied Enbironmental Microbiol, Vol. 42, 180.
~ 183 (1981) and J. Bacteriol., 153, 134 ~ 139 (19
83)], but no report has been known that microorganisms accumulate glyoxylic acid.

[0008] However, as a result of many years of research on biochemical production of glyoxylic acid, the present inventor has found that enzymes derived from Aspergillus, Geotricum, Gluconobacter, Acetobacter, or Aspergillus that produces the enzyme. Have found that it is possible to convert and accumulate glycolic acid into glyoxylic acid by using a microorganism belonging to the genus Geotricum, the genus Gluconobacter, and the genus Acetobacter, and applied for a patent. (Japanese Patent Application No. 5-35
5324)

The present invention has been completed as a result of further broadly searching for microorganisms in the natural world. That is, in addition to the previous application, it was found that bacteria belonging to the genus Pseudomonas and the genus Alcaligenes produce an enzyme capable of converting glycolic acid to glyoxylic acid, and further the enzyme or Pseudomonas genus and Alcaligenes producing the enzyme. Converting and accumulating glycolic acid into glyoxylic acid by allowing microorganisms belonging to the genus to act on glycolic acid,
We have developed a method for biochemically producing glyoxylic acid by collecting this. According to the invention,
Glyoxylic acid can be efficiently produced under mild conditions. Next, the present invention will be described in more detail.

The chemical reaction formulas for producing glyoxylic acid from the glycolic acid of the present invention by an oxidation reaction are shown in Formulas 1 and 2.

[0011]

(Equation 1)

[0012]

(Equation 2)

The reaction represented by the above formula 1 is a case where an oxidase derived from a microorganism having the ability to convert glycolic acid to glyoxylic acid or a microorganism producing the enzyme is mediated, and the reaction represented by the formula 2 is The cases where a dehydrogenase derived from a microorganism having the ability to convert glycolic acid to glyoxylic acid or a microorganism producing the enzyme intervenes are shown.

The present inventor first established a method for quantifying the reaction product, glyoxylic acid, in order to screen for a microorganism capable of converting glycolic acid to glyoxylic acid that can be used in the above reaction.

Experimental Example 1 Glyoxylic acid was quantified by [MA Paz, OO Blumenf
eld, M. Rojikind, E. Henson, C. Furfin and P. Gallo
p. Arch. Biochem. Biophys., Vol. 109, 547-559 (1965))
The method was improved under the following conditions.

10 μl of 0.5 mM to 2.5 mM glyoxylic acid solution
Against 0.1 M glycine hydrochloride buffer (pH 4.0) 150 μl, 1%
MBTH [3-methyl-2-benzothiazolinone (3-methyl-2-benzothiazolinone
hydrazone hydrochloride)] solution (300 μl) to produce glyoxylic acid derivative-1, then 0.5 mM to 2.5
To 10 μl of mM glyoxylic acid solution, add 0.1 μM glycine hydrochloride buffer (pH 4.0) 150 μl, 1% MBTH solution 60 μl, 0.2% ferric chloride solution 750 μl to form glyoxylic acid derivative-2. , Their absorption spectra and absorbance 380n
The relationship between m, 610 nm and glyoxylic acid concentration was investigated.

As a result, the absorbance curve shown in FIG. 1 and the relationship between the absorbance and the glyoxylic acid concentration shown in FIG. 2 were obtained, and it was shown that glyoxylic acid can be quantified under these conditions.

Experimental Example 2 Glyoxylic acid derivatives 1 and 2 prepared according to Experimental Example 1 were quantified by reverse phase HPLC using a C ₁₈ column. That is,
Solutions of derivative-1 and derivative-2 before and after addition of 0.2% ferric chloride solution of glyoxylic acid were subjected to HPLC.

As a result, as shown in FIG. 3, a peak was observed around 12.1 minutes for glyoxylic acid (derivative-1) without addition of ferric chloride. In addition, in the derivative of the ferric chloride ion-added liquid, a peak of glyoxylic acid (derivative-2) was obtained at around 22.8 minutes, which enabled identification of glyoxylic acid by HPLC.

In order to obtain a microorganism having the ability to convert glycolic acid to glyoxylic acid, the present inventors have extensively screened it from the natural world, and as a result, two strains isolated from soil have the ability to convert glycolic acid to glyoxylic acid. I found it to have. That is, it was revealed that microorganisms belonging to the genus Pseudomonas and the genus Alcaligenes convert and accumulate glyoxylic acid from glycolic acid.

For each strain isolated by the present inventors, the mycological properties of each strain are described in Bergey's Manual of Systemati
c Bacteriology 8th. ed. (1974), Inspection and Technology Vol.1
7 No. 6 Special Issue (1989), Guide to Identification of Medical Bacteria (Identification of Gram-Negative Bacteria) Translated by Toshikazu Sakazaki, Canadian Journal of Micr
obiology Vol.12 (1966), International Journal of B
acteriology, Plant and Soil xx No.3 (1964), Diagn
As a result of identification with reference to ostic features of the noudule bacteria, it was identified as a microorganism belonging to the genus Pseudomonas and the genus Alcaligenes, respectively. Pseudomonas sp. 9653 (Ps
eudomonas sp. 9653), Alcaligenes SP 96
It was named 50 (Alcaligenes sp. 9650).

The mycological properties of these strains are described below. A. Pseudomonas sp. GOX-373 (1) Gram stain: Negative bacillus (2) Flagella: Polar flagella (3) Spore: Negative (4) OF: Oxidation (5) Oxygen auxotrophic: Aerobic (6) Catalase: Positive ( 7) Oxidase: Positive (8) Nitrate reduction: Positive (9) Denitrification: Negative (Those that grow in the presence of nitrate even under anaerobic conditions are positive) (10) MR reaction: Negative

(11) VP reaction: negative (12) Starch degradation: negative (13) Casein degradation: weakly positive (14) Arginine degradation (Thornleg method): weakly positive (15) Indole: negative (16) Sulfide Hydrogen: ± (by TSI agar method) (17) Urease (Christensen urea agar): Positive (18) Utilization of citrate Simmons Citrate medium: Negative Christensen Citrate medium: Positive (19) Dye formation Pyocyanin : Negative fluorescin: Negative (20) Acid from sugar (Hugh-Leifson) Glucose: Positive Lactose: Negative Sucrose: Positive Mannitol: Negative Maltose: Positive Fructose: Positive Galactose: Positive Xylose: Positive

(21) Utilization of carbon source (Mineral salt + 0.
01% Yeast Ex. Base) Lactose: Negative Malate: Positive (alkali) Dulcitol: Positive (acid production) Succinate: Positive (alkali) l-Tartarate: Negative m-Tartarate: Positive Sorbose: Positive (acid production) Mannitol: Positive ( Acid production Gluconate: Positive (alkali) Sucrose: Positive (acid production) Arabinose: Positive (acid production) Xylose: Positive (acid production) Fructose: Positive (acid production) Glucose: Positive (acid production) Ethanol: Positive (acid production) ) Lactate: Positive Adipate: Positive Srbacate: Positive (22) Growth temperature range: 15-42 ° C (23) Growth pH range: 4.5-9.6 (24) Salt growth: 0-5% (25) Colony morphology: circular , convex, translucent,
glistening, (Nutrient agar) mucoid (26) Degraded gelatin: Negative (3 days) Esculin: Negative Uric acid: Negative (27) Medium growth MacConkey Agar: Positive SS Agar: Negative

The characteristics of these results are as follows. Gram-negative bacilli, polar hair motility, aerobic, catalase-negative,
It is classified as Pseudomonas because it is oxidase positive and decomposes sugar by oxidation. This strain (Pseudomonas sp. 9653) has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry as No. 14931 (FERM P-14931).

B. Alcaligenes SP GOX-4018
About (1) Gram stain: Negative bacillus (2) Flagella: Periflagellates, but the number is small and attaches to the cell side (3) Spores: Negative (4) OF: Negative (5) Oxygen auxotrophic: Aerobic (6) Catalase: Positive (7) Oxidase: Positive (8) Nitrate reduction: Positive (9) Denitrification: Positive (Those that grow in the presence of nitrate even under anaerobic conditions are positive) (10) MR reaction: Negative

(11) VP reaction: negative (12) decomposition of starch: negative (13) decomposition of casein: negative (14) indole: negative (15) hydrogen sulfide: positive (by TSI agar method) (16) di Oxyacetone: Negative (17) Urease (Christensen urea agar): Positive (18) Utilization of citrate Simmons Citrate medium: Negative Christensen Citrate medium: Positive (19) Dye formation Pyocyanin: Negative fluorescin: Negative ( 20) Acid from sugar (Hugh-Leifson) Glucose: Negative Lactose: Negative Sucrose: Negative Mannitol: Negative Maltose: Positive Fructose: Positive Galactose: Positive Xylose: Positive

(21) Utilization of carbon source (Mineral salt + 0.
01% Yeast Ex. Base) Lactose: Negative Malate: Positive (alkali) Dulcitol: Positive (acid production) Succinate: Positive (alkali) l-Tartarate: Negative m-Tartarate: Positive Sorbose: Negative Mannitol: Positive (acid production) Gluconate : Weakly positive Sucrose: Weakly positive Arabinose: Positive (acid production) Xylose: Positive (acid production) Fructose: Positive (acid production) Glucose: Positive (acid production) Ethanol: Positive (acid production) Lactate: Positive Adipate: Positive Sebacate: Positive (22) Growth temperature range: 15-45 ° C (23) Growth pH range: 4.5-9.6 (24) Salt growth: 0-5% (25) Colony morphology: circular, convex, translucent,
glistening, (Nutrient agar) mucoid (26) Acyl amidase: Negative (27) 3-Ketolactose production: Negative (28) Degradation Gelatin: Negative (3 days) Esculin: Negative (29) Medium growth MacConkey Agar: Positive SS Agar: negative

The characteristics of these results are as follows: Gram-negative bacilli, pericardial motility, aerobic, catalase-positive,
Oxidase negative, acid not produced from glucose, denitrification positive, non-pigment production, 3-ketolactose non-production,
It is classified into the genus Alcaligenes because it grows on MacConkey agar. This strain (Alkaline Genes SP
9650) has been deposited at the Institute of Biotechnology, Institute of Technology, Ministry of International Trade and Industry as No. 14930 (FERM P-14930).

The microorganism used in the present invention can be used in any form of a cultured bacterial cell obtained by culturing or an immobilized bacterial cell obtained by immobilizing the bacterial cell. In addition, the crude enzyme obtained from the culture, the one obtained by further purifying it, or the one obtained by immobilizing these enzymes can be used in any form.

Experimental Example 3 Ammonium sulfate 0.2%, K ₂ HPO ₄ 0.1%, NaH ₂ PO ₄ 0.1%, MgSO ₄ .7H ₂
O 0.02%, yeast extract 0.1%, 1,2-propanediol 5
The bacterium that produced 100% (pH 7.0) was separated and cultured in a medium having the same composition at 30 ° C. for 2 days. The produced bacteria were collected by centrifugation, washed with physiological saline, and stored frozen.

Next, 0.2 M TES-NaOH buffer solution pH 7.0 (500
100 μl of frozen cells, 2.5 M glycolic acid (pH 7.0)
400 μl was added and reacted at 20 ° C. for 16 hours. After the reaction,
The cells were removed by centrifugation and the supernatant was used to measure the amount of glyoxylic acid produced according to Test Example 1.

As a result, Pseudomonas sp. 96
53 (FERM P-14931) is 10.8 μml / ml, Alcaligenes
It was confirmed that SP 9650 (FERM P-14930) produced 11.6 μml / ml glyoxylic acid. In addition, as a result of HPLC analysis of this reaction product according to Experimental Example 2, the peak elution time was in agreement with glyoxylic acid.

Experimental Example 4 Pseudomonas sp. 96 under the same conditions as in Experimental Example 3.
The amount of bacterial cells obtained by culturing 53 strains was changed from 50 μl to 400 μl and reacted with glycolic acid in the same manner as in Experimental Example 3.
As a result, as shown in FIG. 4, it was revealed that the amount of glyoxylic acid produced increased in proportion to the amount of cells added.

Experimental Example 5 Using 100 μl of Pseudomonas sp. 9653 strain cultured under the same conditions as in Experimental Example 3, the composition was the same as in Experimental Example 3 and the reaction temperature was changed between 5 and 30 ° C. for 16 hours. Reacted The cells were removed by centrifugation, and the concentration of produced glyoxylic acid was measured using the supernatant of the reaction.

As a result, the higher the temperature, the faster the conversion rate of glycolic acid to glyoxylic acid. (Fig. 5)

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

【Example】

Example 1 Pseudomonas sp. 9653 strain was cultured in a 500 ml melting flask containing 100 ml of medium at 30 ° C. for 3 days using the medium prepared by adding 0.5% (V / V) of CSL to the medium of Experimental Example 3 and then centrifuged. It was collected in.

After washing with physiological saline, the cells were added to 20 ml of 0.2 M TES-NaOH buffer solution (pH 7.0) containing 1 M glycolic acid and reacted at 20 ° C. for 16 hours. The amount of glyoxylic acid produced after the reaction was 207 mM. The generated glyoxylic acid was separated and purified by a conventional method.

Example 2 Using Alcaligenes sp. Strain 9650, culture was carried out in the same manner as in Example 1 to obtain bacterial cells, which were then reacted with glycolic acid to study conversion to glyoxylic acid. as a result,
Generation of 148 mM glyoxylic acid was confirmed.

Example 3 Using the cells of Alcaligenes sp. Strain 9650 obtained by culturing in the same manner as in Example 1, reaction was carried out at 20 ° C. for 67 hours. As a result, 251 mM glyoxylic acid was obtained.

[0041]

INDUSTRIAL APPLICABILITY The present invention is to bioproduce glyoxylic acid by using a microorganism having an ability to convert glycolic acid to glyoxylic acid or an enzyme produced by the microorganism. It is possible to efficiently manufacture the product under mild conditions and without producing a by-product.

[Brief description of drawings]

FIG. 1 shows absorption curves of glyoxylic acid derivative-1 (shown by a solid line) and derivative-2 (shown by a broken line). In the figure, number 1 indicates water and 2 indicates a case where an MBTH derivative is prepared using glyoxylic acid.

FIG. 2 Derivatives-1 of glyoxylic acid (measured at a wavelength of 380 nm and shown by a solid line) and derivatives-2 (wavelength of 610 n).
Measured in m, indicated by the dashed line. ) Shows the absorption curve.

FIG. 3 is a reverse phase liquid chromatograph of a derivative of glyoxylic acid. In the figure, peak 1 represents glyoxylic acid derivative-1 (measured at a wavelength of 380 nm), and peak 2 represents glyoxylic acid derivative-2 (measured at a wavelength of 610 nm).

FIG. 4 shows the relationship between the bacterial cell amount of Pseudomonas sp. 9653 strain and the production amount of glyoxylic acid.

FIG. 5 shows the effect of reaction temperature on glyoxylic acid production.

Claims (2)

[Claims] 1. A reaction solution by reacting a substrate glycolic acid with an enzyme produced by a microorganism belonging to the genus Pseudomonas or Alcaligenes and having the ability to oxidize glycolic acid to glyoxylic acid, or a microorganism belonging to the genus Pseudomonas or Alcaligenes. A biochemical production method of glyoxylic acid, characterized in that glycolic acid is converted into and accumulated in glyoxylic acid, and then collected. 2. The biochemical method for producing glyoxylic acid according to claim 1, wherein the enzyme produced by a microorganism belonging to the genus Pseudomonas or the genus Alcaligenes is oxidase and / or dehydrogenase.