JPH11225787A - Production of 3,4-dihydroxybenzoic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing 3,4-dihydroxybenzoic acid at low cost in high conversion efficiency from a raw material by the use of microorganisms. SOLUTION: This method for producing 3,4-dihydroxybenzoic acid comprises contacting vanillin or vanillic acid with microorganisms which belong to the genus Sphingomonas, have enzymatic activity to catalyze the demethylation of vanillic acid, but have no cleavage enzyme activity to 3,4,-dihydroxybenzoic acid; wherein the microorganisms are viable with syringaldehyde or syringic acid as a sole carbon source.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a process for producing 3,4-dihydroxybenzoic acid, which is useful as a pharmaceutical intermediate.

[0002]

2. Description of the Related Art 3,4-Dihydroxybenzoic acid is
Various compounds having a 4-dihydroxybenzoyl group,
It is particularly useful as an intermediate for pharmaceuticals and the like.

As a method for producing this 3,4-dihydroxybenzoic acid, there are a method based on a chemical synthesis reaction and a method based on an enzymatic reaction using a microorganism. Among them, the method using microorganisms is a more energy-saving and safer method, as compared with the method using a chemical synthesis reaction, in that the reaction proceeds under mild conditions of normal temperature and normal pressure, and no harmful catalyst is used. . As a method for producing 3,4-dihydroxybenzoic acid using a microorganism, a method of causing glucose to act on a bacterium of the genus Corynebacterium, a method of causing isophthalic acid or vanillin to act on a bacterium of the genus Pseudomonas, and the like are known.

[0004]

However, the method using these microorganisms has a low yield of 3,4-dihydroxybenzoic acid and requires an expensive nutrient source in addition to the raw material. It was not available.
Accordingly, an object of the present invention is to provide a method for producing 3,4-dihydroxybenzoic acid using microorganisms, which has high conversion efficiency from raw materials and is inexpensive.

[0005]

The present inventors have searched for microorganisms that utilize vanillin or vanillic acid as a carbon source and studied the metabolic processes thereof, and found that they catalyze the methyl group elimination reaction of vanillic acid. Enzyme produces 3,4-dihydroxybenzoic acid from vanillic acid and then 3,4
-Dihydroxybenzoic acid was found to have its benzene ring cleaved by a cleavage enzyme. As a result of further studies, it was found that a microorganism belonging to the genus Sphingomonas, which has an enzymatic activity to catalyze the methyl group elimination reaction of vanillic acid, was able to use a recombinant DNA technique to cleave the enzyme activity of 3,4-dihydroxybenzoic acid. Succeeded in making it disappear,
Using such a novel microorganism, it has been found that 3,4-dihydroxybenzoic acid can be obtained extremely efficiently from vanillin or vanillic acid, and the present invention has been completed.

That is, the present invention relates to a microorganism belonging to the genus Sphingomonas and having an enzymatic activity for catalyzing a methyl group elimination reaction of vanillic acid and having no 3,4-dihydroxybenzoic acid cleaving enzyme activity. A process for producing 3,4-dihydroxybenzoic acid, comprising contacting vanillin, vanillin or vanillic acid.

Further, the present invention relates to a microorganism belonging to the genus Sphingomonas and having an enzymatic activity for catalyzing a methyl group elimination reaction of vanillic acid and having no enzymatic activity for cleaving 3,4-dihydroxybenzoic acid. To provide.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The microorganism used in the present invention belongs to the genus Sphingomonas as described above, has an enzymatic activity for catalyzing the methyl group elimination reaction of vanillic acid, and cleaves 3,4-dihydroxybenzoic acid. As long as it is a microorganism having no enzyme activity, it is not particularly limited, whether it is a wild strain or a mutant strain,
Among microorganisms belonging to the genus Sphingomonas and having an enzymatic activity of catalyzing a methyl group elimination reaction of vanillic acid, recombinant microorganisms in which 3,4-dihydroxybenzoic acid cleaving enzyme activity has been eliminated by recombinant DNA technology are particularly preferred.

[0009] Such a recombinant microorganism is preferably a microorganism that can grow using syringaldehyde or syringic acid as a single carbon source. Because these compounds are present in pulp manufacturing wastewater, if this microorganism is cultured in a medium containing the wastewater, syringaldehyde or syringic acid is used as an energy source, and vanillin or vanillic acid is converted to 3,4-dihydroxy. This is because benzoic acid is generated.

[0010] That is, while effective utilization of resources is required, effective utilization of unused biomass is strongly desired.
Among them, lignin, which is present in a large amount as biomass, is released in a large amount as a residue containing various aromatic compound resources from a pulp production process. These pulp manufacturing effluents contain a large amount of vanillin, vanillic acid, syringaldehyde, syringic acid, etc., depending on the tree species and pulping method used. As a method of using pulp production waste liquid, culture of yeasts and the like using saccharides contained in the waste liquid is performed, but aromatic compound resources are further released as waste liquid. A method of producing only vanillin from this fermentation waste liquid is also known, but most of the concentrated liquid is simply incinerated after concentration. There is a long-felt need for a method for producing a useful substance using vanillin, vanillic acid, syringaldehyde, syringic acid, etc., which are present in a pulp manufacturing waste liquid. From such a viewpoint, microorganisms having the above properties are particularly preferable.

A wild strain for producing such a recombinant microorganism belongs to the genus Sphingomonas, has an enzymatic activity for catalyzing the methyl group elimination reaction of vanillin, and cleaves 3,4-dihydroxybenzoic acid. The microorganism is not particularly limited as long as it is a microorganism having an enzymatic activity.
A strain that can grow using vanillic acid, syringaldehyde, or syringic acid as a single carbon source is preferred. Strains having such properties include Sphingomonas sp.
The CR-0300100 strain can be exemplified. The CR-0300100 strain can be obtained by, for example, enrichment culture from soil in a medium containing vanillin, vanillic acid, syringaldehyde, and syringic acid as a carbon source. Among the microorganisms that can grow using vanillin, vanillic acid, syringaldehyde, and syringic acid as a single carbon source isolated from the soil, a microorganism that metabolizes 3,4-dihydroxybenzoic acid by cleavage is selected. Such a microorganism is determined as follows.

A microorganism capable of growing using vanillin, vanillic acid, syringaldehyde, or syringic acid as a single carbon source is cultured in a suitable medium, collected, suspended in a suitable buffer, and sonicated. Crush the cells using a method such as The supernatant obtained by centrifugation is added to a buffer containing 0.01 to 10 mM, preferably 0.1 to 2 mM 3,4-dihydroxybenzoic acid. After incubation for 1 to 30 minutes at 30 ° C., 2M sodium hydroxide is added and the presence of yellow color development is examined. The yellow color is 3, 4
-Means the formation of dihydroxybenzoic acid cleaving substances, in particular meta-cleaving substances.

In order to produce a strain from such a strain which has lost the enzyme activity of cleaving 3,4-dihydroxybenzoic acid by recombinant DNA technology, first, a DNA is prepared from the above strain.
Is extracted, the restriction enzyme fragment of this DNA is ligated to a plasmid vector, and transformed into a microorganism such as Escherichia coli,
A plasmid having a 3,4-dihydroxybenzoic acid cleavage enzyme gene is selected. The plasmid can be treated with a restriction enzyme and religated to select a plasmid that has lost the cleavage activity of 3,4-dihydroxybenzoic acid, and the plasmid is incorporated into a microorganism of the genus Sphingomonas. More specifically, it can be performed as follows. (1) First, for example, DNA from CR-03001100 strain
And cut with an appropriate restriction enzyme. This is ligated to a plasmid vector such as pUC18 or pKT230, and transformed into Escherichia coli. The recombinant Escherichia coli thus constructed can be obtained as an aggregate having various fragments of the genome of the CR-03001100 strain in a plasmid vector. The aggregate of Escherichia coli is spread on an agar plate or the like, separated into individual colonies, and containing an appropriate medium.
Culture in a 6-well microplate or the like. Next, an enzyme such as lysozyme that gently dissolves the cell wall is added to the culture solution,
The cell contents leak out of the cells. Lysozyme and the like added here may be of any concentration as long as it dissolves the cell wall. Next, an aqueous solution containing 3,4-dihydroxybenzoic acid is added, incubated for a while, and an aqueous sodium hydroxide solution is added. Here, 3, 4
-Dihydroxybenzoic acid is 0.01 to 10 mM, preferably about 0.1 to 2 mM, and the incubation time is:
One minute to several hours, preferably 3 minutes to 30 minutes is appropriate.
In addition, the aqueous sodium hydroxide solution to be added
Should be 10 or more.

(2) As described above, when 3,4-dihydroxybenzoic acid is added and then the pH is adjusted to 10 or more by adding an aqueous solution of sodium hydroxide, a yellow color is produced from the E. coli aggregate. Things are distinguished. The yellow coloration indicates the formation of a meta-cleavable substance of 3,4-dihydroxybenzoic acid. The plasmid of Escherichia coli thus distinguished contains a meta-cleaving enzyme gene of 3,4-dihydroxybenzoic acid. An example of the plasmid thus obtained is pPX150.

(3) Next, the plasmid thus obtained is cleaved with an appropriate restriction enzyme and re-ligated, so that the meta-cleaving enzyme activity of 3,4-dihydroxybenzoic acid (yellow color development) disappears. A plasmid is prepared. PPX151 can be exemplified as the plasmid thus obtained.

(4) Next, a new plasmid is prepared by ligating an antibiotic-resistant gene to which CR-0300100 strain is susceptible with the above-mentioned restriction enzyme-cleaved plasmid. pPX152 can be exemplified as a plasmid prepared using kanamycin resistance gene as an antibiotic resistance gene based on pPX151.

(5) Next, the vector part and the inserted part are separated from the plasmid prepared above using an appropriate restriction enzyme. The DNA of the inserted insertion was separated into CR-0
The 300,100 competent cells are mixed and the DNA is incorporated into the cells. An example of such a method is an electroporation method. The cells having the DNA incorporated therein are cultured in an appropriate medium for several hours and spread on an agar plate containing an antibiotic (in this case, kanamycin) to obtain colonies resistant to kanamycin. The kanamycin resistant strain thus obtained is
The meta-cleaving enzyme activity of 3,4-dihydroxybenzoic acid (yellow color development) has completely disappeared.

An example of a strain obtained by performing the above operation using strain CR-03001100 is CR-031020.
1 strain (FERM P-16624). This CR-0310201 strain belongs to the genus Sphingomonas, has an enzymatic activity of catalyzing a methyl group elimination reaction of vanillic acid, and has no meta-cleaving enzyme activity of 3,4-dihydroxybenzoic acid, And it can grow using syringaldehyde or syringic acid as a single carbon source.

In order to produce 3,4-dihydroxybenzoic acid from vanillin or vanillic acid using the above strain,
The strain may be brought into contact with vanillin or vanillic acid, but it is preferable that the strain be cultured in a medium containing vanillin or vanillic acid, in addition to vanillin or vanillic acid, containing syringaldehyde or syringic acid. , By collecting 3,4-dihydroxybenzoic acid from the culture.

Culture conditions are not particularly limited as long as the strain can grow, but the pH is 5 to 8, and the temperature is 2 to 37 ° C.
5 to 32 hours are preferred. In addition to the above components, a nitrogen source, vitamins, and the like can be added to the medium. In order to separate 3,4-dihydroxybenzoic acid from the culture, for example, extraction with an organic solvent is performed.

[0021]

The present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the present invention.

Example 1 (1) Table 1 containing 0.2% syringic acid in a 21 mm test tube
10 ml of a medium having the composition of
And cultured at 30 ° C. for 3 days.

[0023]

[Table 1]

(2) Next, the above was allowed to stand for about 5 minutes, and 0.1 ml of the slightly turbid supernatant was transferred to a new test tube having the same composition as (1) and cultured at 30 ° C. for 3 days. (3) The above operation was repeated once or twice, and the final supernatant was appropriately diluted in sterilized distilled water, developed on an agar medium having the composition shown in Table 1 containing 0.2% syringic acid, and heated at 30 ° C. After culturing for 3 days, colonies were formed. (4) Table 1 containing the colonies containing 0.2% vanillic acid
Using a toothpick to inoculate an agar medium having the following composition, a growing strain was obtained. (5) The strain grown with vanillic acid was again inoculated into 10 ml of a medium having the composition shown in Table 1 containing 0.2% syringic acid.
The cells were cultured at 30 ° C. for 3 days. The culture was appropriately diluted in sterile distilled water, spread on an LB agar medium, and cultured at 30 ° C. for 1 day to form a colony. (6) For the same colony, 0.2
% Of vanillic acid and syringic acid were used to inoculate two types of agar medium having the composition shown in Table 1 using a toothpick to obtain a growing strain. (7) One strain capable of growing on both types of agar medium containing vanillic acid and syringic acid was obtained. This strain is CR-0
When the mycological properties were examined based on 300100, the results were as follows.

A, Morphological characteristics ・ Size of cell 0.5 × 1.1 to 1.3 μm Bacillus ・ Presence or absence of spores None ・ Flagella flagella flagella ・ Motility +

B, Growth condition on agar medium ・ In the medium of Table 1, circular colony, glossy, yellow

C, physiological properties-Gram stainability--Reduction of nitrate--Gelatin solubility + weak-OF test O; +, F;--Growth range pH 5.0 to 7.5, 10 ° C To 40 ° C ・ Utilization of carbon source glucose + sucrose + lactose-glycerol-starch-vanillin + vanillic acid + syringaldehyde + syringic acid + • cytochrome oxidase + • catalase +

D, Chemical taxonomic properties-G / C content of DNA (mol%) 68-Quinone type Q-10-Dye + (hydrophobic yellow pigment)-Cell wall lipid Sphingolipid e, Other properties-3, 4-dihydroxybenzoic acid oxidase meta-cleavage

From the above-mentioned properties, the strain CR-03100100 was identified as a genus Sphingomonas .

Example 2 (1) Sphingomonas sp. CR-0300100 was added to 100 ml of LB medium (10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride,
(1 g / L of glucose) with shaking at 30 ° C. for 24 hours. After collecting cells by centrifugation, the method of Marmur (Marmur, J. 1961. J. Mol. Bio) is used.
l. 3: 208-218), and total DNA was extracted and purified by a conventional method. (2) Sphingomonas sp. CR obtained in (1)
0300100 Total DNA was digested with restriction enzymes XhoI and Sp
hI, completely digested with the restriction enzymes SalI and SphI, mixed with the vector plasmid pUC19, and
Treated with 4 DNA ligase to form a hybrid plasmid. (3) The hybrid plasmid obtained in (2) was
The B101 strain was transformed into an LB medium containing 100 mg / L ampicillin, and 200 to 300
The cells were spread so that colonies appeared, and cultured at 37 ° C. for 18 hours. (4) 96 colonies per plate were subcultured on an LB medium containing 100 mg / L ampicillin to obtain a master plate. By this operation, a total of 2000 colonies were obtained. 100mg each
A 0.2-ml LB medium containing / L of ampicillin was inoculated into a 96-well microplate. Shaking culture was performed at 37 ° C. using a microplate shaker. (5) After culturing for 18 hours, lysozyme was added to each well so that the final concentration was 50 μg / ml.
Incubated for 2 hours at ° C. Next, 2 μl of a 20 mM solution of 3,4-dihydroxybenzoic acid was added to each well and incubated at 30 ° C. for 5 minutes. (6) When 10 μl of 2 M sodium hydroxide was added to each well subjected to the above treatment, 1 out of 2000 strains was added.
Yellow color development was observed in the strain. (7) extracting the plasmid from the strain that had developed yellow color,
When the cleavage patterns of some restriction enzymes were examined, it was found that they had the structure shown in FIG. This plasmid was named pPX150.

Example 3 (1) pPX150 was cut with a restriction enzyme ApaI to prepare a self-ligated plasmid pPX151. Escherichia coli HB101 strain transformed with this plasmid and Escherichia coli HB101 strain transformed with pPX150 were cultured in 10 ml of LB medium containing 100 mg / L ampicillin.
The cells were cultured for 8 hours and collected. The cells were suspended in 1 ml of a 50 mM phosphate buffer, and the cells were disrupted using an ultrasonic disrupter.
This was centrifuged at 5000 g for 20 minutes and the supernatant 20 obtained.
μl was added to 1 ml of phosphate buffer containing 0.2 mM 3,4-dihydroxybenzoic acid. After 5 minutes incubation at 30 ° C., 2 M sodium hydroxide was added to 0.1 ml.
When 1 ml was added, yellow color was observed in the E. coli HB101 strain transformed with pPX150.
No yellow color was observed in the E. coli HB101 strain transformed in No. 51. (2) Plasmid pUC4K (Pharmacia Biotech) was digested with restriction enzyme EcoRI and bluntin was digested.
The resultant was blunted using g kit (Takara Shuzo) and separated by 1% agarose gel electrophoresis. (3) A 1.3 kbp fragment was excised from the gel, phenol extracted three times, and DNA was precipitated by ethanol precipitation.
Was purified. (4) pPX151 is cut with a restriction enzyme ApaI and bl
Smoothing was performed using unting kit (Takara Shuzo). This is mixed with the DNA obtained in the above (3) and mixed.
A ligation reaction was carried out using Gation kit (Takara Shuzo) to transform Escherichia coli strain HB101. From the transformed Escherichia coli HB101 strain, a strain resistant to kanamycin and ampicillin was selected, and pPX152 was obtained as a plasmid carried by the strain (FIG. 1).

Example 4 (1) Plasmid pPX152 obtained in the previous experiment was digested with restriction enzyme XbaI, separated by 1% agarose electrophoresis, and a 2.3 kbp fragment was cut out. Next, DNA was extracted by freeze-thaw method, phenol was sufficiently extracted, and purified by ethanol precipitation. (2) Sphingomonas sp. CR-03001
The 00 strain was cultured in 500 ml of LB liquid medium (containing 25 mg / L of nalidixic acid) at 28 ° C. for 23 hours and cooled in ice for 15 minutes. (3) Collect cells by centrifugation at 10,000 rpm at 4 ° C for 10 minutes.
After gentle washing with 00 ml of 0 ° C. distilled water, the cells were collected by centrifugation again. Subsequently, the cells were gently washed with 250 ml of 0 ° C. distilled water and centrifuged. Further, after gently washing with 125 ml of 0 ° C. distilled water, the cells were collected by centrifugation. (4) The cells were suspended in distilled water containing 10% glycerol and kept at 0 ° C. (5) 4 μm of distilled water containing about 0.05 μg of the DNA of (1)
in a 0.2 cm cuvette, and add 40 μl of the competent cell of (4), and add (25 μF, 2500 V, 12 V).
(msec). (6) All the cells treated above were inoculated into 10 ml of LB liquid medium and cultured at 30 ° C. for 6 hours. After the culture, the cells were collected by centrifugation, spread on an LB plate containing 25 mg / L kanamycin, and cultured at 30 ° C. for 48 hours. (7) Very strong kanamycin resistant strain under the above conditions, 1
Got the strain. This strain is transformed into Sphingomonas sp.
-0310201 strain. (8) Sphingomonas sp. CR-03102
The 01 strain and the CR-0300100 strain were cultured in 10 ml of LB medium for 24 hours and collected. The cells were suspended in 1 ml of a 50 mM phosphate buffer, and the cells were disrupted using an ultrasonic disrupter. This was centrifuged at 5000 g for 20 minutes, and 100 µl of the supernatant obtained was added to 1 ml of a phosphate buffer containing 0.2 mM 3,4-dihydroxybenzoic acid. After incubating for 5 minutes at 30 ° C., 0.1 ml of 2M sodium hydroxide was added, and yellow color was observed in CR-0300100 strain, but no yellow color was observed in CR-0310201 strain. Was. CR-03102
The 01 strain exhibited exactly the same mycological properties as the CR-03100100 strain except for the above-mentioned properties and the resistance to kanamycin. This strain was deposited as FERM P-16624 with the National Institute of Bioscience and Biotechnology, Institute of Industrial Science and Technology.

Example 5 (1) 25 mg of kanamycin was added to the CR-0310201 strain.
/ L of LB medium, and 6% inoculated into a medium having a composition of Table 1 containing 2 g / L of syringic acid (SA). (2) After culturing at 30 ° C. for 24 hours, syringic acid and vanillic acid were added at a concentration of 2 g / L each, and further added for 24 hours.
Culture was continued for hours. HPLC analysis of syringic acid, vanillic acid, and 3,4-dihydroxybenzoic acid in the culture solution (column: BONDASPHERE 5 μC ₁₈ 3.9 × 150)
mm, MILLIPORE; mobile phase: 2% aceticacid and
10% methanol; flow rate: 0.7 ml / min; detecti
on 295 nm). The bacterial growth was determined by optical density at 660 nm (OD ₆₆₀ ). (3) As shown in FIG. 2, the CR-0310201 strain quantitatively produced 3,4-dihydroxybenzoic acid with respect to vanillic acid with a decrease in syringic acid and vanillic acid,
The conversion yield was 90%.

Example 6 (1) Kanamycin (25 mg) was used for the CR-0310201 strain.
And LB medium containing 1 g / L of syringic acid (SA). (2) After culturing for 24 hours at 30 ° C., syringaldehyde,
Vanillin was added to each at a concentration of 1 g / L, and the culture was further continued for 24 hours. HPLC analysis of syringaldehyde, vanillin, and 3,4-dihydroxybenzoic acid in the culture solution (column: BONDASPHERE 5 μC ₁₈ 3.
9 × 150mm, MILLIPORE; mobile phase: 2% acetic acid
acid and 10% methanol; flow rate: 0.7ml / mi
n; detection 295 nm). The bacterial growth was determined by optical density at 660 nm (OD ₆₆₀ ). (3) As shown in FIG. 3, the CR-0310201 strain quantitatively produced 3,4-dihydroxybenzoic acid with respect to vanillin with a decrease in syringaldehyde and vanillin, and the conversion yield was 90%. Met.

[0035]

According to the present invention, 3,4-dihydroxybenzoic acid can be produced very efficiently from vanillin or vanillic acid.

[Brief description of the drawings]

FIG. 1. Plasmid pPX150 to plasmid pPX
152 shows the construction strategy to 152 and the restriction maps of these plasmids.

FIG. 2: Vanillic acid, syringic acid and 3,4-
3 shows the change over time in the concentration of dihydroxybenzoic acid.

FIG. 3 shows changes over time in the concentrations of vanillin, syringaldehyde, and 3,4-dihydroxybenzoic acid in a medium.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:01) (72) Inventor Koji Hotta 1134-2 Gongendo, Satte City, Saitama Prefecture Cosmo Research Institute R & D Center Co., Ltd.

Claims (4)

[Claims] 1. A microorganism belonging to the genus Sphingomonas and having an enzymatic activity for catalyzing a methyl group elimination reaction of vanillic acid and having no enzymatic activity for cleaving 3,4-dihydroxybenzoic acid, comprising vanillin or vanillin. A process for producing 3,4-dihydroxybenzoic acid, which comprises contacting an acid. 2. The method according to claim 1, wherein the microorganism is a microorganism that can grow using syringaldehyde or syringic acid as a single carbon source. 3. A microorganism belonging to the genus Sphingomonas, having an enzymatic activity for catalyzing a methyl group elimination reaction of vanillic acid, and having no 3,4-dihydroxybenzoic acid cleaving enzyme activity. 4. Sphingomonas sp. CR-0
The microorganism according to claim 3, which is 310201 (FERM P-16624).