WO1999049068A1 - Process for producing 1-substituted-3-methoxy-4-oxybenzenes - Google Patents

Abstract

A process for economically producing on a mass scale 1-substituted-3-methoxy-4-oxybenzenes, which are expected as being useful as materials for various products including drugs, cosmetics, pesticides, food additives, feed additives and liquid crystals, comprising culturing a microorganism belonging to the genus Pseudomonas and being capable of producing 1-substituted-3-methoxy-4-oxybenzenes from clove oil in a medium containing clove oil which is a natural substance, thus forming and accumulating 1-substituted-3-methoxy-4-oxybenzenes in the medium and then taking up the same.

Description

 Specification

Technical field The present invention is expected to be widely used as a raw material for pharmaceuticals, cosmetics, agricultural chemicals, food additives, feed additives, liquid crystals, etc. 1— The present invention relates to a process for producing substituted 3-methoxy-4-oxybenzenes. Landscape technology

1 Monosubstituted 1-3-methoxy-4-methoxybenzenes, such as ferulic acid, vanillin, and vanillic acid, are naturally occurring and can be obtained by extraction from natural products, but the amount of natural products The extraction process is cumbersome and expensive, and is not suitable for mass production.

 There is also a chemical synthesis method, but the chemical synthesis method is complicated and the raw materials are expensive and difficult to obtain.

 Ferulic acid is a constituent of plant cell walls, and is a substance that is expected to be widely used as a raw material for pharmaceuticals, cosmetics, agrochemicals, food additives, feed additives, liquid crystals, and so on. Among the 3-methoxy-4-ethoxybenzenes, their production method is being studied.

 Ferulic acid is known to be obtained by the condensation reaction of vanillin and malonic acid (J. Am. Chem. Soc, vol. 74, p. 5346, 1952). is not.

 In recent years, ferulic acid has been obtained by hydrolyzing rice waste oil and the like (see Japanese Patent Publication No. 7-78032). However, even this method is still expensive, so it is widely used. Its use is being hindered while its potential is expected.

On the other hand, attempts have been made to produce vanillin-related substances by allowing eugenol to act on microorganisms (Japanese Patent Application Laid-Open Nos. 5-228790, 3-306683, and 62-19092, Agric. Biol. Chem., 41, 925-929, 197 7 and 47, 2639-2640, 1983), all of these methods can produce vanillin or vanillic acid with some efficiency, but cannot be said to be sufficiently efficient. And for ferulic acid, these methods are not very efficient.

 Under such circumstances, the present inventors have isolated the eugenol-utilizing bacterium Pseudomonas florescens E118 (FERMBP-64997) from nature, and the cells were efficiently isolated. It was often found that eugenol was converted to ferulic acid (see Japanese Patent Application Laid-Open No. Hei 9-154591).

 However, in order to produce eugenol used in the above method by chemical synthesis, it is sufficient to protect the ortho position of guaiacol aryl ether and carry out Kreisen rearrangement, but the process is complicated and costly. Also, it is difficult to isolate eugenol from essential oils such as clove oil, which is a natural product and has a high eugenol content, by rectification and extraction.

 Therefore, it has been desired to develop a method for producing 1-substituted-13-methoxy-4-methoxybenzenes inexpensively and in large quantities using readily available natural products. DISCLOSURE OF THE INVENTION The present invention has been made from the above viewpoint, and is expected to be widely used as a raw material for pharmaceuticals, cosmetics, agricultural chemicals, food additives, feed additives, liquid crystals, etc. 1-substitution — 3 It is an object of the present invention to provide a method for efficiently producing 4-methoxybenzene inexpensively and in large quantities.

 The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by culturing a specific microorganism in a medium containing clove oil itself, which is a natural product and inexpensive and easily available, 1-substitution is achieved. The inventors have found that it is possible to efficiently produce 1-3-methoxy-4-ethoxybenzenes, and have completed the present invention.

 That is, the present invention is as follows.

(1) A microorganism belonging to the genus Pseudomonas, which is capable of producing 1-substituted 13-methoxy-14-hydroxybenzenes from clove oil, is cultured in a medium containing clove oil. Monosubstituted 3- 3-methoxy 4-monobenzenes A method for producing 1-substituted-3-methoxy-14-methoxybenzenes, which is characterized by sampling.

 (2) A method for producing 1-substituted 1-3-methoxy-14-oxybenzenes of (1), wherein the microorganism belonging to the genus Pseudomonas is Pseudomonas fluorescens.

 (3) A method for producing 1-substituted 1-3-methoxy-14-oxybenzenes according to (2), wherein the pseudomonas fluorescein is pseudomonas fluorescens E118 (FERM BP-6497).

 (4) The method for producing 1-substituted-13-methoxy-14-oxybenzenes according to (1), wherein resting cells of the microorganism belonging to the genus Pseudomonas are used.

 (5) The method for producing mono-substituted 1-3-methoxy-14-oxybenzenes according to (1), wherein a microorganism belonging to the genus Pseudomonas is used in the form of immobilized cells.

 (6) The method for producing (1), wherein the 1-substituted 1-3-methoxy-14-oxybenzene is at least one compound selected from coniferyl alcohol, vanillin, and vanillic acid. Hereinafter, the present invention will be described in detail.

 The clove oil used in the present invention can be used without particular limitation as long as it is generally used as clove oil. Specifically, the essential oil obtained by extracting the buds, floral patterns or leaves of Syzygium aromaticum Merrill et Perry (Eugenia caryophyllata Thunb.) From Indonesia, which is native to Indonesia, with water or steam. It can be used regardless of the site used for extraction. In the present invention, it is possible to produce and use essential oil as clove oil by the above-mentioned method from each part of the cinnamon but the clove oil is generally commercially available and can be easily obtained. It can also be used. Since clove oil is a natural product, it is more suitable as a raw material due to the recent trend of consumers toward natural products.

Specific examples of the mono-substituted 1-3-methoxy-4-oxybenzenes to which the production method of the present invention is applied include ferulic acid, ferulaldehyde, coniferyl alcohol , Coniferyl aldehyde, diphosphorus, vanillic acid, vanillyl alcohol, etc., and also includes mixtures thereof.

 In the production method of the present invention, a microorganism belonging to the genus Pseudomonas belonging to the genus Pseudomonas, which is capable of producing 1-substituted 1-3-methoxy-4-methoxybenzenes from clove oil belonging to the genus Pseudomonas, is used as a microorganism. Any microorganism can be used as long as it is a microorganism having the ability to convert to methoxybenzenes. Pseudomonas fluorescens having the above-mentioned ability is more preferably used, and particularly preferably, Pseudomonas fluorescens having the above-mentioned high conversion ability is used. The fluorescens E118 strain is used.

 In addition, Pseudomonas fluorescens E118 strain is a strain isolated by the present inventors, and was established on September 14, 1995 by the Ministry of International Trade and Industry, (Postal code: 3 05—8 5 6 6 1-3-1, Tsukuba-Higashi, Ibaraki, Japan) Deposited as microbial accession number FERMP—15 1885, September 10, 1998 It has been transferred to the International Deposit under the Budapest Treaty and deposited under the accession number FERMBP—6497.

 In the present invention, mutants obtained from these microorganisms by natural mutation, mutation treatment with ultraviolet irradiation or a mutagen can also be used. Mutation treatment includes ultraviolet irradiation, X-ray irradiation, radiation irradiation, treatment with mutagenic agents such as N-methyl-N, 12-nitronitrosoguanidine (NTG), artificial mutation treatment such as gene recombination, cell fusion, etc. What is necessary is just to carry out using the well-known method used per se.

 In the present invention, a medium containing clove oil may be used for culturing a microorganism belonging to the genus Pseudomonas and having the ability to produce 1-substituted-3-methoxy-4-methoxybenzenes from clove oil. Culture is usually performed aerobically. As the medium, any medium containing clove oil and capable of growing the above-mentioned microorganism can be used. The clove oil contained in the medium is as described above. Medium components other than clove oil are appropriately selected according to the type of microorganism used.

For example, as a medium component other than clove oil in a medium for growing microorganisms belonging to the genus Pseudomonas, specifically, carbon sources such as maltose, lactose, fructose, and sucrose, meat dex, sodium glutamate, and peptone , Nitrogen sources such as yeast extract, various phosphates, Examples include inorganic salts such as metal salts such as sulfate, magnesium, calcium, boron, copper, potassium, iron, manganese, and sodium, and other organic trace components. Similarly, in the resting bacterial cell reaction, components other than clove oil include, specifically, potassium phosphate buffer, physiological saline and the like. In this case, the medium may further contain a surfactant or a water-insoluble organic solvent.

 In addition, in order to distinguish it from the culture of the growing cells (cells used for growth culture), the culture of the quiescent cells (cells used for the quiescent cell reaction) is referred to as “reaction” in this specification, if necessary. And the culture solution is called the “reaction solution”.

 In the present invention, the cultivation of the microorganisms for producing 1-substituted 1-3-methoxy-4-oxybenzenes from clove oil may be carried out using either a growing cell of the microorganism or a quiescent cell. However, preferably, resting cells are used. The resting cells of the microorganism used in the present invention are generally obtained, for example, by culturing the proliferating cells by the following method. In other words, by culturing the proliferating cells, it is possible to simultaneously produce resting cells and 1-substituted-3-methoxy-4-oxybenzenes from clove oil.

 For cultivation of the proliferating cells, a liquid medium is usually used in which the above-mentioned medium components and a suitable solvent such as water are mixed at an appropriate ratio depending on the microorganism used. In the present invention, for example, when Pseudomonas fluorescens E118 strain is used, a preferred liquid medium is 0.1 to 0.5% (v / v) of clove oil and 0.5 to 0.5% of sucrose. 3% (w / v;), dipotassium hydrogen phosphate 0.1 to 0.4% (w / v), peptone 0.05 to 0.2% (w / v), magnesium sulfate heptahydrate 0. In a liquid medium containing 0.1 to 0.1% (w / v), yeast extract 0.1 to 1% (w / v), and a metal salt mixture 0.05 to 0.2% (v / v) And those having a pH of 6.5 to 8.0.

When clove oil has been consumed during the culturing, it may be added to the medium successively so as to obtain the above-described concentration. In addition, the above metal salt mixed solution contains 0.4 g of calcium chloride dihydrate, 0.3 g of boric acid, 0.04 g of copper sulfate pentahydrate, and 0.1 g of potassium iodide in 1 L of deionized water. 0.2 g of ferrous sulfate heptahydrate, 0.4 g of manganese sulfate heptahydrate, 0.2 g of sodium molybdate dihydrate Is preferred.

 Culture conditions can be appropriately set according to the microorganism used.For example, preferable conditions when Pseudomonas fluorescens E118 strain is used are 18 to 72 hours at about 20 to 35 ° C. Culture conditions such as aeration and agitation culture can be mentioned. The above culture conditions can also be appropriately changed depending on the type of 1-substituted 1-3-methoxy-4-methoxybenzene to be produced. For example, in the above culture of Pseudomonas-fluorescens E118 strain, coniferyl alcohol or ferulic acid can be obtained if the culture time is as short as about 18 to 60 hours. If the culture time is as long as about 30 to 72 hours, vanillin or vanillic acid can be obtained in addition to ferulic acid. Resting cells of the microorganism are obtained by such cultivation, and mono-substituted 1-3-methoxy-4-oxybenzenes are produced and accumulated in the culture solution. Resting cells can be obtained from the culture solution by a conventional method such as centrifugation or filtration. In addition, for the separation and purification of 1-substituted 1-3-methoxy-4-monoxybenzenes, usual purification methods such as passing the culture solution or supernatant after removal of the cells through an anion exchange resin column or a silylation gel column are used. Methods can be used.

In the present invention, by culturing the resting cells obtained as described above, it is possible to further produce monosubstituted 1-3-methoxy-4-hydroxybenzenes. The reaction solution for the quiescent cell reaction may be of any composition as long as it contains quiescent cells, near-neutral buffer, and clove oil. Resting cells are usually suspended in physiological saline or an appropriate PH buffer and added to the reaction mixture in the form of a cell suspension. As the cell suspension, a suspension of quiescent cells suspended in an appropriate pH buffer solution by an ordinary method is used. Specifically, the quiescent cells obtained as described above are used. A suspension in a ratio of 0.1 to 0.8 g (wet weight) / 1 mL in physiological saline, 1 M potassium phosphate buffer (pH 7.0) or the like is used. The composition of the reaction solution is, for example, 30 to 70% (v / v) of the above cell suspension in deionized water or the like, 1 M phosphate buffer (PH 7.0) 5 to 20% (v / v;), clove oil 0.1 to 2%, and preferably 0.1 to 0.8% (w / v). When the clove oil component is completely consumed from the reaction solution during the reaction, it may be added to the reaction solution successively so that the above concentration is obtained. In addition, polyoxyethylene alkyl A surfactant such as telluric acid ester or a water-insoluble organic solvent such as n-heptane may be added to the reaction solution.

 The conditions of the quiescent cell reaction can be appropriately set according to the microorganism used.For example, preferable conditions when using Pseudomonas fluorescens E118 strain are 25 to 50 ° C. At room temperature, the reaction mixture containing resting cells, clove oil and pH buffer is allowed to stand for a period of time until the resting cells no longer consume clove oil components, preferably for 5 to 72 hours. , Shaking, or stirring. Thereafter, the reaction is stopped with a suitable reaction terminator such as methanol.

 In order to obtain the 1-substituted-3-methoxy-14-oxybenzenes formed and accumulated from the reaction solution, the cells are removed by centrifugation or filtration, and the filtrate or supernatant obtained is subjected to a conventionally known method. For example, a method of separating and purifying these by high performance liquid chromatography or the like may be employed. Alternatively, 1-substituted-3-methoxy-4-oxybenzenes may be separated and purified from the above supernatant or the like by crystallization. For example, crystallization of ferulic acid is performed as follows. Hydrochloric acid is added dropwise to the supernatant to obtain ferulic acid crude crystals. Dissolve the crude crystals in a small amount of hot water and cool to obtain recrystallization. The recrystallization is repeated to obtain purified ferulic acid.

 Further, in the production method of the present invention, the microorganisms can be used in the form of immobilized cells, which is preferable from the viewpoint of productivity.

 The microbial cells can be immobilized by any known method, as long as the enzyme system that converts clove oil in the cells into 1-substituted-1,3-methoxy-4-oxobenzenes is not inactivated. May be. For example, even if the above cells are adsorbed on a carrier such as ceramic or glass fine powder, the cells are cross-linked with a cross-linking agent such as glutaraldehyde, or the cells are wrapped with a polymer compound such as alginic acid. Good. Proliferating cells may be used as the cells used for immobilization, but normally, quiescent cells are used. Hereinafter, unless otherwise specified, immobilized cells refer to immobilized resting cells.

The reaction using the immobilized cells can be carried out by keeping the same temperature in a reaction solution having the same composition as the resting cells and shaking or stirring. The reaction time is also appropriately selected in the same manner as the quiescent cell reaction. Alternatively, the eluate may be collected by pouring the reaction solution into a column filled with immobilized cells. After stopping the reaction, fix it from the reaction solution by centrifugation or filtration. The supernatant is obtained by removing the transformed cells. From the supernatant, 1-substituted 3-methoxy-4-ethoxybenzenes such as ferulic acid are separated and purified by a known method. The separated immobilized cells can be used repeatedly. BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described specifically with reference to examples. The present invention is not limited to only these examples. Example 1 Pseudomonas fluorescens El 18 (F ERM BP-6497) was added to clove oil 0.2% (v / v), sucrose 1% (w / v), hydrogen phosphate 0.2% (w / v), 0.3% (w / v), peptone 0.03% (w / v), magnesium sulphate heptahydrate 0.05% (w / v), yeast extract 0.03% (w / v) The mixture was cultured with aeration and agitation for 24 hours in 2 L of a culture solution containing 0.1% (v / v) of a mixture of v), and a metal salt and adjusted to pH 7.0. 0.4 g of calcium chloride dihydrate in 1 L of deionized water as a metal salt mixture, 0.3 g of boric acid, 0.04 g of copper sulfate pentahydrate, 0.1 g of potassium iodide, sulfuric acid A solution prepared by dissolving 0.2 g of ferrous heptahydrate, 0.4 g of manganese sulfate heptahydrate, and 0.2 g of sodium molybdate dihydrate was used. The culture solution after the culture was centrifuged, and the supernatant was passed through an anion exchange resin (IRA) column, and then passed through a silica gel column to obtain 0.5 g of purified ferulic acid and 0.1 lg of vanillic acid. Example 2 The cells obtained by the culture in Example 1 were washed with physiological saline and suspended in physiological saline to obtain a cell suspension. The concentration of the obtained cell suspension was 0.5 g (wet weight) / mL.

 To 1 mL of this cell suspension was added 0.2 mL of 1 M potassium phosphate buffer (pH 7.0) and 4 Omg of clove oil, and the reaction solution was made up to 2 mL with deionized water. Shake at C for 4 hours.

After shaking for 4 hours, 2 mL of methanol was added to stop the reaction. afterwards, The reaction solution was centrifuged, and the separated supernatant was analyzed by high performance liquid chromatography for 1-substituted-13-methoxy-14-oxybenzenes such as ferulic acid. High-performance liquid chromatography uses an ODSC18 column with a size of 4.6 x 150 mm, and the eluate having a composition ratio of methanol: deionized water: acetic acid = 45: 52: 3 is converted to 1 per minute. The sample was developed at a flow rate of 0.0 mL, and the absorbance at 280 nm was detected. The peak around 3.0 minutes, which is the retention time of standard ferulic acid, was defined as ferulic acid, and the peak around 2.2 minutes was defined as vanillic acid.

 As a result, 5 mg of ferulic acid and 1.5 mg of vanillic acid were obtained. Example 3 The cells obtained by the culture of Example 1 were washed with physiological saline, and suspended in physiological saline to obtain a cell suspension. The concentration of the obtained cell suspension was 0.5 g (wet weight) / mL.

 10 mL of this cell suspension was adsorbed on 2 g of ceramic fine powder, and 2 mL of 1 M potassium phosphate buffer (pH 7.0) and 0.4 g of clove oil were added thereto. The reaction solution brought to 2 OmL with was shaken at 30 ° C. After shaking for 24 hours, an additional 0.4 g of clove oil was added and shaking was continued. After shaking for 24 hours after the re-addition of the clove oil, the reaction was stopped by adding 20 mL of methanol. Thereafter, the reaction solution was centrifuged, and the separated supernatant was subjected to high performance liquid chromatography to measure 1-substituted-13-methoxy-14-oxybenzenes.

 High-performance liquid chromatography uses a 003 C18 column with a size of 4.615111111, and the eluate having a component ratio of methanol: deionized water: acetic acid = 45: 52: 3 is 1.0 mL / The flow was developed at a flow rate of 1 minute, and the absorbance at 280 nm was detected. The peak around 3.0 minutes, which is the retention time of the standard ferulic acid, was ferulic acid, and the peak around 2.2 minutes was vanillic acid.

 As a result, 110 mg of ferulic acid and 33 mg of vanillic acid were obtained.

The same operation as above was repeated 10 times, but the rate of decrease of the ferulic acid production in the 10th production with respect to the first production was 12%. In addition, the rate of decrease in the production amount of vanillic acid in the 10th batch with respect to the first production volume was 15%. Chemical synthesis guaiacol 1. 2 5 g of Comparative Example 1 eugenol, placed _{Cu C 12 · H 2 0 0.} 0 1 g, chloride Natoriumu saturated aqueous 80 mL and chloride Ariru 0. 77 g to 5 0 0 mL flask, stirred While heating to 80 ° C. One hour later, 8.2 mL of a 10% (W / V) aqueous sodium acetate solution was added thereto over 3 hours. Thereafter, the reaction solution was gradually heated to 100 ° C., and then air-cooled. After reaching room temperature, 50 mL of toluene was added thereto, and the layers were separated. The toluene layer was washed three times with 50 mL of a saturated aqueous solution of sodium chloride.

 After the toluene was evaporated from the washed toluene layer, distillation was performed at 6 mmHg (800 Pa in SI unit). At 1 15—149 ° C., 0.42 mL of a fraction with a eugenol isomer content of 90% (W / V) was obtained. Of these, o-eugenol (natural type) was 11% (V / V), and 0.5 g was obtained. The yield from guaiacol to natural eugenol was 8%. Comparative Example 2 Purification of Eugenol from Clove Oil To 300 g of clove oil, 100 mL of a 5% (w / w) aqueous NaOH solution was added, and the mixture was shaken to remove other oils. Eugenol was precipitated by adding mL. Then, the mixture was washed twice with 100 mL of water, and dried by adding 30 g of anhydrous magnesium sulfate. Next, 3.2 g of eugenol (purity: 98 GC%) was obtained by fractionating a fraction at 108 ° C to 112 ° C at 5 mmHg by distillation under reduced pressure.

 In other words, purification of origenol from clove oil was inefficient with a yield of about 1%. Comparative Example 3 Production of Ferulic Acid from Eugenol Pseudomonas fluorescens El 18 (FERM BP-6497) was added to Eugenol 0.15% (v / v), sucrose 1% (w / v), peptone 0.1%

(w / v), dipotassium hydrogen phosphate 0.2% (w / v), magnesium sulfate heptahydrate 0.05% (w / v), yeast extract 0.03% (w / v) and metal Salt mixture 0.

Aerated with 1 L of a 1% (v / v) culture adjusted to pH 7.0 for 24 hours. The culture was stirred. Calcium chloride dihydrate 0.4 g, boric acid 0.3 g, copper sulfate pentahydrate 0.04 g, potassium iodide 0.1 g, sulfuric acid in 1 L of deionized water as a metal salt mixture A solution prepared by dissolving 0.2 g of ferrous heptahydrate and 0.4 g of manganese sulfate heptahydrate in 0.2 g of sodium molybdate dihydrate was used. The cells obtained by centrifuging the culture after the culture were washed with physiological saline and suspended in physiological saline to obtain a cell suspension. The concentration of the obtained cell suspension was 0.5 g (wet weight) / mL.

 To 1 mL of this cell suspension, 0.2 mL of 1 M potassium phosphate buffer (pH 7.0) and 1.6 mg of eugenol were added, and the reaction solution was made up to 2 mL with deionized water. The mixture was shaken at 30 ° C for 2 hours. Then, 2 mL of methanol was added to this reaction solution to stop the reaction, and the cells were removed by centrifugation.The separated supernatant was subjected to quantitative analysis of ferulic acid by high performance liquid chromatography. . The peak around 3.0 minutes, which is the retention time of the standard ferulic acid, was taken as ferulic acid, and 1.8 mg of ferulic acid was detected. However, no peak around 2.2 minutes, which is the retention time of vanillic acid, was observed. INDUSTRIAL APPLICABILITY By using the method of the present invention, mono-substituted 1-3-methoxy is expected to be widely used as a raw material for pharmaceuticals, cosmetics, agricultural chemicals, food additives, feed additives, liquid crystals, etc. _ 4- It will be possible to produce large quantities of 4-oxybenzene at low cost.

 Since clove oil is a natural product, it is more suitable as a raw material due to the recent trend of consumers toward natural products. If it becomes possible to process 1-substituted 3-methoxy 4-hydroxybenzenes at the site of the cinnamon production area, 1-substituted 3-methoxy 4-one-year-old xybenzenes will become less expensive, and the locality of production will increase. It can be imagined to be useful for regional promotion.

Claims

The scope of the claims 1. Culturing a microorganism belonging to the genus Pseudomonas, which is capable of producing 1-substituted 13-methoxy-14-hydroxybenzenes from clove oil, in a medium containing clove oil. A process for producing 1-substituted-3-methoxy-4-hydroxybenzenes, which comprises producing, accumulating, and collecting the substituted 1-3-methoxy-4-oxybenzenes. 2. The method for producing 1-substituted-3-methoxy-4-hydroxybenzenes according to claim 1, wherein the microorganism belonging to the genus Pseudomonas is Pseudomonas fluorescens. 3. The method for producing 1-substituted-3-methoxy-14-hydroxybenzenes according to claim 2, wherein the pseudomonas fluorescein is pseudomonas fluorescein E118 (FERMBP-6497). 4. The method for producing 1-substituted-3-methoxy-14-hydroxybenzenes according to claim 1, wherein resting cells of the microorganism belonging to the genus Pseudomonas are used. 5. The method for producing 1-substituted_3- 3-methoxy-4-hydroxybenzenes according to claim 1, wherein a microorganism belonging to the genus Pseudomonas is used in the form of immobilized cells. The method according to claim 1, wherein the 6.1-substituted 1-3-methoxy-4-oxybenzenes are one or more compounds selected from coniferyl alcohol, vanillin, and vanillic acid.