JPH05227980A - Production of vanillin and its related compound by fermentation - Google Patents

Abstract

PURPOSE:To efficiently produce vanillin or its related compound from eugenol as the raw material by culturing a specified mutant of Pseudomonas in the presence of eugenol. CONSTITUTION:A mutant of a microorganism belonging to Pseudomonas, capable of utilizing only eugenol as the carbon source for its growth, entirely or almost incapable of growing in vanillin (vanillic acid) or vanillyl alcohol and capable of producing a compound selected from vanillin, coniferyl alcohol, coniferyl aldehyde, ferulic acid and vanillyl alcohol by oxidative decomposition of eugenol is cultured in the presence of eugenol so as to synthesize and accumulate the compound selected from the above-mentioned vanillin and its related compounds in the culture medium. In addition, Pseudomonas TK-2101 (FERM P-12689) is exemplified as the above-mentioned microorganism.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to one or more compounds selected from the group consisting of vanillin and its related compounds, coniferyl alcohol, coniferyl aldehyde, ferulic acid and vanillyl alcohol, obtained by fermentation using a mutant belonging to the genus Pseudomonas. To a method for producing the compound.

[0002]

2. Description of the Related Art Vanillin is a compound used in large amounts in food flavors and also useful as a raw material for pharmaceuticals. Coniferyl alcohol, coniferyl aldehyde and vanillyl alcohol are also used as a fragrance or a raw material thereof, and ferulic acid is used as a pharmaceutical raw material.

[0003] These compounds exist in nature, but extraction from natural products is too expensive. Therefore, at present, they are produced by chemical synthetic means on an industrial scale. As a production method by fermentation using a conventionally known microorganism, genus Ceresia, genus Klebsirea,
Alternatively, a method for obtaining vanillin from eugenol and / or isoeugenol using a microorganism belonging to the genus Enterobacter (JP-A-3-30683), an aldehyde such as vanillin from a styrene derivative utilizing a specific microorganism belonging to the genus Pseudomonas JP-A-2-200192) and a method for obtaining coniferyl aldehyde from n-eugenol utilizing Arthrobacter globiformis (JP-A-62-190092) are disclosed. In addition, K. Tadasa et al. Reported that microorganisms belonging to the genus Corynebacterium and microorganisms belonging to the genus Pseudomonas were
Eugenol is decomposed oxidatively, and the decomposition process is that eugenol sequentially passes through coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillic acid, and protocatechuic acid as shown in Formula 1. [Agric.Biol.Chem.,
Volume 41 (6) pp. 925-929 (1977) and Agric. Biol. Che
m., 47 (11) 2639-2640 (1983)].

[0004]

[Chemical 1]

The inventor of the present invention reported that the microorganism belonging to the genus Pseudomonas, which is a strain having a stronger ability to assimilate eugenol among the two strains reported by K. Tadasa and others, was treated with soil by the method described in the above document. The eugenol conversion by the obtained strain was measured by high performance liquid chromatography (abbreviated as HPLC) over time. As a result, it was possible to confirm the above pathway, but the conversion rate was very fast, and most of them were immediately converted from coniferyl alcohol to vanillic acid to obtain useful compounds such as vanillin in the decomposition process. I couldn't do that.
Further, this strain has a drawback that it cannot grow in the presence of 0.2% or more of eugenol, and the amount of eugenol as a raw material cannot be increased in order to obtain a large amount of product.

Furthermore, the present inventor searched for the ability to assimilate eugenol also in some other microorganisms existing in the natural world, and it was found that any of the microorganisms was immediately eugenol-like as the above-mentioned microorganisms belonging to the genus Pseudomonas. It was not possible to find a microorganism capable of efficiently accumulating vanillin or the like because it was converted to vanillic acid or protocatechuic acid or the benzene ring was opened.

[0007]

Therefore, the present invention can efficiently produce and accumulate vanillin and its related compounds coniferyl alcohol, coniferyl aldehyde, ferulic acid and vanillyl alcohol by oxidatively decomposing eugenol. An object of the present invention is to provide a method for preparing vanillin and its related compounds from eugenol as a raw material by a fermentation method using a mutant strain prepared and the obtained mutant strain.

[0008]

Means for Solving the Problems As a result of earnest studies for solving the above problems, the present inventor has found that K. Tadasa described above.
Isolate from the soil according to the document described by the above, as a parent strain a microorganism belonging to the genus Pseudomonas confirmed, to prepare a mutant strain having desired properties by subjecting it to a mutation induction operation,
It was found that the above problems can be solved by adopting a fermentation method using the mutant strain.

That is, the present invention belongs to the genus Pseudomonas, which can grow with eugenol as the sole carbon source, cannot grow with vanillin, vanillic acid, or vanillyl alcohol or has extremely poor growth, and oxidatively decomposes eugenol, A mutant strain having the property of producing one or more compounds selected from vanillin, coniferyl alcohol, coniferyl aldehyde, ferulic acid, and vanillyl alcohol is cultured in the presence of eugenol, and vanillin and coni Ferryl alcohol,
Generates one or more compounds selected from coniferyl aldehyde, ferulic acid, and vanillyl alcohol,
The present invention provides a method for producing one or more compounds selected from vanillin, coniferyl alcohol, coniferyl aldehyde, ferulic acid, and vanillyl alcohol by a fermentation method, which is characterized by collecting them and accumulating them. ..

The mutant strain used in the present invention belongs to the genus Pseudomonas as described above, can grow with eugenol as the sole carbon source, and cannot grow with vanillin, vanillic acid, or vanillyl alcohol, or has very poor growth. Further, it has the property of oxidatively decomposing eugenol to produce one or more compounds selected from vanillin and related compounds such as coniferyl alcohol, coniferyl aldehyde, ferulic acid and vanillyl alcohol. Further, it has the following mycological properties. (A) Morphology (1) Cell shape and size: bacillus, 0.6 × 1.1 μm (2) Flagella: None (3) Gram stainability: Negative (b) Physiological properties (1) Reduction of nitrate: Positive (2) ) Indole formation: negative (3) Glucose acidification: negative (4) Urease: positive (5) Oxidase: positive (6) Arginine dihydrase: positive (7) β-Glucosidase: negative (8) Protease: positive (9) β-galactosidase: negative (c) assimilation of carbon compounds (1) L-arabinose:-(2) D-glucose: + (3) D-mannose:-(4) D-mannite: + (5) N-acetyl-D-glucosamine: + (6) Maltose:-(7) Potassium gluconate: + (8) n-Capric acid: + (9) Adipic acid: + (10) dl-malic acid: + (11 Sodium citrate: + (12) phenyl acetate: - This mutant, K.Tadasa et al. Method shown earlier [Agric.Bi
ol. Chem., 41 (6) 925-929 (1977) and Agric.
Biol. Chem., 47 (11) 2639-2640 (1983)], it can be obtained by performing mutagenesis using a microorganism belonging to the genus Pseudomonas that utilizes eugenol, which is isolated and confirmed from soil, as a parent strain. .. The soil from which the parent strain can be separated is not particularly limited, and may be ordinary soil, but if the soil contains a plant containing eugenol, for example, clove (Syzygium aromaticum), the objective is The parent stock is separated with a high probability. Then, the obtained parent strain is subjected to a normal mutagenesis operation, for example, ultraviolet irradiation, or N-methyl-N'-nitro-N-
A mutant having a desired property can be induced by treatment with a chemical agent such as nitrosoguanidine (abbreviated as NTG), proflavin hemisulfate, and dimethyl sulfate alone or in combination. In addition, Site-Specific Mutagenesis
enesis) [Proc.Natl.Acad., 82, 5662-5666 (198
It is also possible to induce the mutant strain by modifying the specific site of the gene of the parent strain by making full use of genetic engineering techniques such as 4)]. Then, the mutant strain used in the method of the present invention can be obtained by applying selective pressure according to the desired properties of the mutant strain to the mutated bacterial cells. Of course, it is preferable that such selection pressure is reliable and efficient in isolating the mutant strain as much as possible. For example, the mutated cells are cultured on a minimal medium containing eugenol as a sole carbon source, and replicated on a minimal medium containing eugenol or vanillin as the sole carbon source. Then, the mutant strain can be obtained by isolating a colony that can grow with eugenol as the sole carbon source but cannot grow with vanillin or has extremely poor growth. In the present exemplary method, vanillin, ferulic acid, or vanillyl alcohol may be used as the only carbon source contained in the minimal medium, in addition to vanillin. These carbon sources should be selected depending on whether the compound ultimately intended to be manufactured is vanillin and its related compounds. Although coniferyl alcohol and coniferyl aldehyde can be used as the sole carbon source, these compounds are very expensive, and therefore it is preferable to use the other compounds described above.

Next, the method for inducing mutation of the mutant strain used in the present invention will be specifically shown in the following experimental examples. [Experimental Example] (1) The present inventor isolates a microorganism belonging to the genus Pseudomonas, which immediately oxidatively decomposes eugenol to vanillic acid from general soil by the method disclosed in the document described by K. Tadasa et al. The microorganism was used as a parent strain of the mutant strain used in the present invention. This parent strain TK-1112 was obtained from the Institute of Microbial Science and Technology, the Institute of Industrial Science and Technology, by the Microtechnology Research Institute No. 12776 (F
It has been deposited as ERN P-12776). This parent strain was cultivated on a minimal agar plate medium containing eugenol as the sole carbon source, and the grown bacterial cells were collected and suspended in sterile water so that the petri dish contained 10 ⁵ to 10 ⁶ bacterial cells / ml. After adjusting to, the mixture was irradiated with ultraviolet rays for 20 seconds while being stirred and separated from the germicidal lamp of about 255 nm by 23 cm. After irradiation, 40 μl of the bacterial cell suspension was applied to a minimal agar plate medium containing eugenol as the sole carbon source, and cultured at 30 ° C. for 2 days. The colonies that appeared were replicated on minimal agar plates containing eugenol, vanillin, vanillic acid, or vanillyl alcohol as the only carbon sources. After culturing at 30 ° C. for 2 days, colonies that grew on the minimal agar plate medium containing eugenol but did not grow on the other minimal medium were selected and separated. The composition of the minimal agar plate medium used in this experimental example is shown in Table 1.

Table 1 Minimum agar plate medium composition (per liter) ──────────────────────────────── phosphoric acid Monopotassium 2.0 g Ammonium sulfate 1.0 g Magnesium sulfate (heptahydrate) 0.05 g Ferrous sulfate (heptahydrate) 0.01 g Eugenol (or vanillin, vanillic acid, 1.0 ml vanillyl alcohol) Agarose HGS (manufactured by Nacalai Tesque, Inc.) 7.5 g ──────────────────────────────── The above composition is dissolved in 1 liter of distilled water and water is added. The pH was adjusted to 7.2 to 7.3 with sodium oxide, and sterilized at 121 ° C for 10 minutes before use.

After repeating the above ultraviolet irradiation treatment three times,
It was subjected to the next treatment step (2). (2) A microbial cell obtained by repeating the ultraviolet irradiation treatment three times was prepared in the same manner as in the above (1) so as to contain 10 ⁵ to 10 ⁶ microbial cells / ml, and 1 ml of this microbial cell suspension was added with 50 ~ 20
An NTG aqueous solution was added so as to have a concentration of several steps of 0 μg / ml, and each was kept at room temperature for 30 to 60 minutes. This NT
The G-treated cells were thoroughly washed with water, suspended in 1 ml of distilled water, and 50 μl of this was applied to the same minimal agar plate medium as (1) containing eugenol as the sole carbon source, and the suspension was incubated at 30 ° C. It was cultured for 2 days. From the emerged colonies, those which grew on the minimal medium containing eugenol but did not grow on other minimal medium were selected in the same manner as in (1). This NT
After the G treatment was repeated twice, the same dimethyl sulfate treatment as above was performed once except that dimethyl sulfate was used instead of NTG, and the ultraviolet irradiation treatment of (1) was further performed twice. Then, the obtained mutant strain was shake-cultured in a minimal medium containing eugenol as a sole carbon source using a Sakaguchi flask, and conversion of eugenol by the obtained mutant strain was carried out by HP.
Strains were selected that had a slow rate of conversion to vanillic acid as measured by LC over time and produced and accumulated vanillin and its related compounds coniferyl alcohol, coniferyl aldehyde, ferulic acid and vanillyl alcohol.

Among these, strain TK capable of assimilating the highest concentration of eugenol and having high vanillin-producing ability
I chose -2102. This strain was produced by the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Micro Engineering Research Institute No. 12689 (FERM P
-12689). The results of time-course measurement of eugenol conversion by this strain by HPLC are shown in FIG. In addition, as a comparative example, the result of measuring the conversion of eugenol by the parent strain in the same manner is shown in FIG.

In FIG. 2, although coniphenyl alcohol in the medium was temporarily increased in synchronization with the consumption of eugenol by the parent strain, vanillin and other useful related compounds, coniferyl aldehyde, ferula The acid and vanillyl alcohol could not be detected by HPLC (not shown), only a considerable amount of vanillic acid and a trace amount of protocatechuic acid were detected. In contrast, in FIG. 1 using the mutant strain, not only coniphenyl alcohol but also vanillin, coniferyl aldehyde, and ferulic acid could be detected by HPLC in a considerable amount in synchronization with the consumption of eugenol.

From the above results, it is revealed that a mutant strain which produces and accumulates one or more compounds selected from the group consisting of vanillin and its related compounds, coniferyl alcohol, coniferyl aldehyde, ferulic acid and vanillyl alcohol by mutagenesis operation. It became clear that it was obtained.
The method for producing vanillin and its related compounds of the present invention is carried out by culturing the mutant strain thus obtained in the presence of eugenol, producing and accumulating the target compound in the medium, and collecting it. To be done.

Preferably, a nutrient medium containing eugenol as a carbon source and a nitrogen source, inorganic salts and, if necessary, organic micronutrients such as amino acids, vitamins and nucleic acids is used. As a carbon source, in addition to eugenol, saccharides such as glucose, organic acids such as acetic acid, and alcohols such as ethanol can be used together, but in order to maximize the ability to assimilate eugenol. It is preferable to use only Eugenol as the carbon source. Eugenol can be added to the medium at a high concentration of about 1.5% by weight, but when the yield of vanillin and its related compounds is taken into consideration, the concentration is about 0.1 to 0.8% by weight.
Is preferred.

As the nitrogen source, for example, an inorganic nitrogen source such as ammonium salts and nitrates, or an organic nitrogen source such as urea is used. As the inorganic salts, sodium, potassium, magnesium, calcium or iron salts can be used. The culture conditions are sufficient aeration, the temperature is 15 to 45 ° C, particularly preferably 25 to 40 ° C, and the pH of the medium is
Is in the range of 5.5 to 7.5, and is preferably carried out in a rotary or reciprocal shaking device or in a fermentation device equipped with a stirring and aeration device. Culturing can be carried out batchwise, semi-continuously, or continuously. The culture can also be performed by other known methods, for example, by using the immobilized cells entrapped with a natural polymer or the like. As for the culture time, it is sufficient to select the time at which the target compound reaches the maximum amount of vanillin and its related compounds coniferyl alcohol, coniferyl aldehyde, ferulic acid and vanillyl alcohol, for example, until vanillin reaches the maximum amount. The time depends on the culture conditions, particularly the amount of eugenol and the culture temperature, but in the case of the batch system, it is 8 to 100 hours from the start of the culture.

The method for collecting vanillin and its related compounds from the culture broth may be carried out according to a known method. After separating and removing the bacterial cells from the culture broth, the cells are extracted with an organic solvent such as ethyl acetate, It can be performed by separating an acidic compound and a neutral compound by chemical resolution, and further separating each compound by silica gel column chromatography. Further, the culture solution can be easily collected by directly adsorbing it through an adsorption resin and then separating it with an organic solvent and water.

[0020]

EXAMPLES Next, the present invention will be specifically described by way of examples, but the present invention is not limited to these examples.

[0021]

Example 1 Distilled water 1 in a Sakaguchi flask with a capacity of 500 ml
00 ml, eugenol 0.1 ml (eugenol concentration in the medium 0.1 v / v%, eugenol specific gravity = 1.
Approximately 0.11 w / v%), 0.1 g ammonium sulfate, 0.2 g monopotassium phosphate, 0.005 g magnesium sulfate and 0.001 g ferrous sulfate are added, and the pH is 7.2 with sodium hydroxide. After adjusting to ˜7.3, it was sterilized at 121 ° C. for 10 minutes. After cooling, the mutant strain T
K-2102 was inoculated and cultured at 37 ° C. on a reciprocal shaker set at 150 times / min. The content of vanillin in the culture broth was measured with HPLC over time, and 9 hours after culturing, vanillin 318 mg / l, vanillyl alcohol 102
It was confirmed that mg / liter and ferulic acid 51 mg / liter were produced. 1 liter of the culture solution containing vanillin and the like thus obtained was centrifuged to remove the cells, and then 50 g of the adsorption resin DAIAION H
It was poured into a column packed with P20 (manufactured by Mitsubishi Kasei). Ferulic acid did not adsorb and flowed out as it was. Then, after washing with 1 liter of distilled water, 1 liter of a 10% aqueous ethanol solution was poured to recover vanillyl alcohol. Further, 1.5 liters of 30% ethanol aqueous solution was flowed to collect vanillin. The resin after the recovery of each compound was washed with 1 liter of ethanol and 1 liter of distilled water prescribed by the Japanese Pharmacopoeia and repeatedly used. Ethanol and water were removed from the solution in which each compound was recovered to obtain 280 mg of vanillin, 100 mg of vanillyl alcohol, and 48 mg of ferulic acid.

[0022]

Example 2 Distilled water 5 in a Sakaguchi flask with a capacity of 2 liters
00 ml, eugenol 1.5 ml (eugenol concentration 0.3 v / v% in the medium, eugenol specific gravity = 1.
Approximately 0.32 w / v%), 0.5 g ammonium nitrate, 0.5 g monopotassium phosphate, 5.0 g disodium phosphate, 0.1 g magnesium sulfate, 0.05 g calcium chloride, first sulfate Iron 0.05g, manganese sulfate 0.3mg and sodium molybdate 0.3
After adding mg and adjusting the pH to 7.2 to 7.3 with sodium hydroxide, it was sterilized at 121 ° C. for 10 minutes. After cooling, the mutant strain TK-2102 was inoculated and cultured with a reciprocal shaker set at 37 ° C and 150 times / min. The vanillin content in the culture broth was measured with HPLC over time, and 72 hours after culturing, coniferyl alcohol 1050 mg / liter,
Vanillin 460 mg / liter, vanillyl alcohol 4
40 mg / liter, coniferyl aldehyde 270 m
It was confirmed that g / liter was produced. 1 liter of the culture broth containing vanillin and the like thus obtained was centrifuged to remove the cells, followed by extraction with 1 liter of ethyl acetate to obtain an aqueous sodium hydrogen carbonate solution (pH 8).
After washing with 100 ml, the ethyl acetate layer was concentrated. The concentrated solution was subjected to silica gel column chromatography to cause 100 ml of a mixed solution of pentane: ether = 7: 3 (volume ratio) as a developing solvent to flow, and then 200 ml of ether to flow to obtain a crude vanillin solution. After removing the ether, dissolve in a small amount of ethanol and gradually cool to 350 m of vanillin crystals.
g was obtained. The ethanol solution from which vanillin crystals were removed was poured into a column packed with 50 g of an adsorption resin DAIAION HP20 (manufactured by Mitsubishi Kasei Co., Ltd.). 1 liter of a 5% ethanol aqueous solution was poured into this product to collect vanillyl alcohol. Next, 1 liter of a 25% ethanol aqueous solution was flown to recover coniferyl alcohol, and further 1 liter of a 40% ethanol aqueous solution was flowed to recover coniferyl aldehyde. The solution in which each compound was recovered was subjected to removal of ethanol and water, respectively, to give 780 mg of coniferyl alcohol and 32 mg of vanillyl alcohol.
0 mg and coniferyl aldehyde 190 mg were obtained.

[0023]

According to the method of the present invention, one or more compounds selected from the group consisting of eugenol as a raw material, vanillin and its related compounds, coniferyl alcohol, coniferyl aldehyde, ferulic acid, and vanillyl alcohol are efficiently prepared. It can be manufactured.

[Brief description of drawings]

FIG. 1 is a mutant strain TK-210 as an eugenol-converting bacterium.
Change of eugenol when 2 was used

FIG. 2: Parent strain TK-1112 as eugenol-converting bacterium
Change of eugenol with time

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical display location C12R 1:38) (72) Inventor Tsuneyoshi Kanizawa 5-36-31 Kamata, Ota-ku, Tokyo Takasago Fragrance Industry Co., Ltd.

Claims (1)

[Claims] 1. A genus of Pseudomonas, which can grow with eugenol as the sole carbon source, cannot grow with vanillin, vanillic acid, or vanillyl alcohol or has extremely poor growth, and oxidatively decomposes eugenol to cause vanillin or coniferyl. A mutant strain having the property of producing one or more compounds selected from alcohol, coniferyl aldehyde, ferulic acid, and vanillyl alcohol is cultured in the presence of eugenol, and vanillin, coniferyl alcohol, coni Ferryl aldehyde, ferulic acid, and one or more compounds selected from vanillyl alcohol are produced and accumulated, and vanillin, coniferyl alcohol, coniferyl aldehyde, ferulic acid by fermentation, which is characterized by collecting it, And vanillyl A method for producing one or more compounds selected from alcohols.