JP2009089689A - Method for producing ferulic acid ester compound by enzymatic method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel method for producing an industrially advantageous and eco-friendly ferulic acid ester compound (e.g., glyceryl ferulic acid). <P>SOLUTION: The method for producing the ferulic acid compound (e.g., glyceryl ferulic acid) is to bring a ferulic acid esterase immobilized onto an immobilization support to affect a ferulic acid compound (e.g., ferulic acid) and a hydroxy compound (e.g., glycerol) in an aqueous medium. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing a ferulic acid ester compound, and more particularly to a method for efficiently producing a ferulic acid ester compound from a ferulic acid compound and a hydroxy compound by an immobilized enzyme method.

Ferulic acid ester compounds such as ferulic acid are present as esters and glycosides in cell walls and seeds of plants such as rice, wheat, vegetables and citrus. Structurally, ferulic acid has many resonance structures, can exist stably as a phenoxy radical, and acts as an antioxidant because it easily stops free radical chain reactions. Moreover, it attracts attention from the viewpoint of various physiological activities such as an anticancer effect and a blood pressure lowering effect.
However, ferulic acid ester compounds such as ferulic acid have low solubility in water, and crystals of ferulic acid precipitate at room temperature, which makes it difficult to contain them in the aqueous phase of cosmetics and foods.

In order to solve the above problem, for example, a hydrophilic group is introduced into a ferulic acid compound with glycerol or the like for the purpose of solubilization (Patent Document 1).
Ferulic acid ester compounds (for example, glyceryl ferulic acid) are known per se, and chemical synthesis methods and enzymatic methods are known as production methods thereof. For example, Non-Patent Document 1 discloses a chemical synthesis method in which ferulic acid and glycerol are subjected to a condensation reaction in a mixed solvent of dichloromethane and dimethylformamide in the presence of dicyclohexylcarbodiimide, which is a dehydrating condensation agent. Patent Document 1 discloses an enzymatic method in which ferulic acid esterase is allowed to act on ferulic acid and glycerol in a mixed solvent of water and dimethyl sulfoxide.

  However, in any of the above methods, an organic solvent must be used, which is not environmentally preferable. In addition, the enzymatic method requires the use of an organic solvent, and there are difficulties in recovering the target product (glyceryl ferulic acid) from the reaction completion solution.

Japanese Patent Laid-Open No. 2007-10 J. et al. Agric. Food Chem. , 2000, 48, 5476-5383

  An object of the present invention is to provide a method capable of producing a ferulic acid ester compound such as glyceryl ferulic acid in an industrially advantageous and environmentally friendly manner.

  In order to achieve the above object, the present inventors have conducted various studies on the production method of ferulic acid ester compounds, and as a result, ferulic acid compounds and hydroxy compounds are immobilized on an immobilizing carrier in an aqueous solvent. It has been found that if an acid esterase is allowed to act, the ferulic acid ester compound can be produced in an industrially advantageous and environmentally friendly manner, and further studies have been made to complete the present invention.

（ただし、環Ａは置換基を有していてもよいフェニル基を表す。）
で示されるフェルラ酸類化合物と一般式［ＩＩＩ］

（ただし、Ｒは一価もしくは多価アルコールまたは糖類から１つの水酸基を除いた残基を表す。）
で示されるヒドロキシ化合物に、水性溶媒中、固定化担体に固定化したフェルラ酸エステラーゼを作用させることを特徴とする一般式［Ｉ］

（ただし、環ＡおよびＲは前記と同一意味を有する。）
で示されるフェルラ酸エステル類化合物の製造方法、
［２］ 環Ａが低級アルコキシ基、低級アルキル基及び水酸基から選ばれる置換基１〜３個を有していてもよいフェニル基である前記［１］記載の製造方法、
［３］ フェルラ酸類化合物［ＩＩ］がフェルラ酸である前記［１］記載の製造方法、
［４］ ヒドロキシ化合物［ＩＩＩ］が一価もしくは多価アルコールまたは糖類である前記［１］〜［３］のいずれかに記載の製造方法、
［５］ ヒドロキシ化合物［ＩＩＩ］がグリセロールである前記［１］〜［３］のいずれかに記載の製造方法、
［６］ フェルラ酸エステラーゼが、フェルラ酸エステラーゼを含むペクチナーゼである前記［１］〜［５］のいずれかに記載の製造方法、
［７］ 固定化担体が、アミノアルキル基もしくはアミノフェニルアルキル基が導入されたキトサンである前記［１］〜［６］のいずれかに記載の製造方法、
［８］ 固定化されたフェルラ酸エステラーゼが、アミノアルキル基もしくはアミノフェニルアルキル基が導入されたキトサンとフェルラ酸エステラーゼが多官能試薬を介して共有結合されている前記［１］〜［７］のいずれかに記載の製造方法、
［９］ 多官能試薬がグルタアルアルデヒドである前記［８］記載の製造方法、
［１０］ 水性溶媒が水または緩衝液である前記［１］〜［９］のいずれかに記載の製造方法、および
［１１］ 固定化担体に固定化したフェルラ酸エステラーゼを繰返しまたは連続的に使用する前記［１］〜［１０］のいずれかに記載の製造方法
である。 That is, the present invention
[1] General formula [II]

(However, ring A represents an optionally substituted phenyl group.)
And ferulic acid compounds represented by the general formula [III]

(However, R represents a residue obtained by removing one hydroxyl group from a monohydric or polyhydric alcohol or saccharide.)
A ferulic acid esterase immobilized on an immobilization carrier in an aqueous solvent is allowed to act on the hydroxy compound represented by the general formula [I]

(However, rings A and R have the same meaning as described above.)
A method for producing a ferulic acid ester compound represented by:
[2] The production method according to the above [1], wherein the ring A is a phenyl group optionally having 1 to 3 substituents selected from a lower alkoxy group, a lower alkyl group and a hydroxyl group,
[3] The production method according to the above [1], wherein the ferulic acid compound [II] is ferulic acid,
[4] The production method according to any one of [1] to [3], wherein the hydroxy compound [III] is a monohydric or polyhydric alcohol or a saccharide.
[5] The production method according to any one of [1] to [3], wherein the hydroxy compound [III] is glycerol,
[6] The production method according to any one of [1] to [5], wherein the ferulic acid esterase is a pectinase containing ferulic acid esterase.
[7] The production method according to any one of [1] to [6], wherein the immobilization carrier is chitosan into which an aminoalkyl group or an aminophenylalkyl group is introduced,
[8] The immobilized ferulic acid esterase according to the above [1] to [7], wherein chitosan into which an aminoalkyl group or aminophenylalkyl group is introduced and ferulic acid esterase are covalently bonded via a polyfunctional reagent. The production method according to any one of the above,
[9] The production method according to the above [8], wherein the polyfunctional reagent is glutaraldehyde.
[10] The production method according to any one of [1] to [9] above, wherein the aqueous solvent is water or a buffer, and [11] ferulic acid esterase immobilized on an immobilization carrier is used repeatedly or continuously. The manufacturing method according to any one of [1] to [10].

  According to the present invention, a ferulic acid ester compound (for example, glyceryl ferulic acid) can be produced industrially and environmentally friendly from a ferulic acid compound (for example, ferulic acid) and a hydroxy compound (for example, glycerol). it can.

  In the ferulic acid compound [II], ring A is a phenyl group which may have a substituent. Examples of the substituent include a lower alkyl group (for example, a methyl group, an ethyl group, a propyl group, an isopropyl group). An alkyl group having 1 to 5 carbon atoms such as a group), a lower alkoxy group (for example, an alkoxy group having 1 to 5 carbon atoms such as a methoxy group, an ethoxy group, a propoxy group, and an isopropoxy group), and a hydroxy group. These substituents may be the same or different, and 1 to 3 substituents may be substituted on the benzene ring. Specific examples of the group containing ring A in which these substituents are substituted on the benzene ring include, for example, 3-methoxy-4-hydroxyphenyl group, 3,4-dimethoxyphenyl group, 3,5-dimethoxy-4- Examples thereof include a hydroxyphenyl group.

  Specific examples of ferulic acid compounds [II] include, for example, cinnamic acid, ferulic acid (= 3-methoxy-4-hydroxycinnamic acid), sinapinic acid (= 3,5-dimethoxy-4-hydroxycinnamic acid), dimethoxy Cinnamic acid (= 3,4-dimethoxycinnamic acid) and the like. Of these, ferulic acid is particularly preferable.

  Examples of the hydroxy compound [III] include alkanols having 1 to 20 carbon atoms, alkoxyalkanols having 2 to 20 carbon atoms, monohydric alcohols such as tocopherol, polyhydric alcohols such as glycerol, fructose, glucose, xylose, galactose, and arabinose. And saccharides containing monosaccharides such as Accordingly, R in the general formula [III] is a residue obtained by removing one hydroxyl group from these hydroxy compounds (monohydric or polyhydric alcohol or saccharide), that is, an alkyl group having 1 to 20 carbon atoms, or 2 to 20 carbon atoms. An alkoxyalkyl group, a glyceroyl group, a sugar residue, and the like. Particularly preferred as the hydroxy compound [III] is glycerol.

  The ferulic acid compound used as a substrate in the present invention can be synthesized by various methods. For example, as a method for producing ferulic acid, the production method disclosed in JP-A-5-331101 can be used, and for example, it can be easily produced by alkaline hydrolysis of crude γ-oryzanol. It can also be obtained as a commercial product.

  The enzyme used in the present invention is ferulic acid esterase (hereinafter also simply referred to as an immobilized enzyme) immobilized on an immobilization carrier. The immobilization carrier used for immobilization is not particularly limited as long as it is a water-insoluble carrier that can immobilize ferulic acid esterase. Examples of such immobilization carriers include polyvinyl alcohol, polypropylene, acrylic resins, polyester resins, ion exchange resins, water-insoluble carriers such as cellulose, chitin, and chitosan, and functional groups such as amino groups, hydroxy groups, and carboxyl groups. Examples thereof include those having a group introduced, and chitosan having an amino group introduced therein is particularly preferred. Chitosan having an amino group introduced can be produced using chitosan as a raw material, but can also be obtained as a commercial product. Examples of commercially available products include chito pearl (registered trademark, manufactured by Fuji Boseki Co., Ltd.). For example, chito pearl BCW-3001, chito pearl BCW-3003, chito pearl BCW-3005, chito pearl BCW-3010, etc. are introduced as aminoalkyl groups. Examples of the aminophenylalkyl group introduced include chitopearl BCW-3501, chitopearl BCW-3503, chitopearl BCW-3505, and chitopearl BCW-3510. Of these, the main structure of chitopearl introduced with an aminoalkyl group is as follows.

The main structure of chitopearl introduced with an aminophenylalkyl group is as follows.

  The ferulic acid esterase (hereinafter also simply referred to as an enzyme) used in the immobilized enzyme of the present invention is not particularly limited as long as it causes an esterification reaction between ferulic acid and glycerol. Examples of such enzymes include ferulic acid esterases derived from microorganisms belonging to the genus Aspergillus (for example, Aspergillus niger) and microorganisms belonging to the genus Penicillium (for example, Penicillium chrysogenum). Can be used.

  The microorganism-derived ferulic acid esterase may be a purified enzyme or a crude enzyme. Examples of the crude enzyme include commercially available pectinases such as pectinase PL (trade name: Pectinase PL “Amano” manufactured by Amano Enzyme Co., Ltd.), pectinase G (trade names: Pectinase G “Amano” manufactured by Amano Enzyme Co., Ltd.), and the like. Can be mentioned.

  In the present specification, the term “derived from” a ferulic acid esterase does not mean that the ferulic acid esterase is directly isolated from the microorganism, but also refers to the microorganism being irradiated with radiation such as genetic recombination or ultraviolet rays, or a mutagen. It means that ferulic acid esterase can be obtained by using a conventional technique such as mutagenesis by a sex substance. For example, when the Aspergillus niger ferulic acid esterase gene is cloned, the cloned gene is introduced into another host microorganism (for example, E. coli), and ferulic acid esterase is isolated from the resulting transformant. The ferulic acid esterase is said to be “derived” from Aspergillus niger. Cloning of the ferulic acid esterase gene from Aspergillus niger, and genetic recombination of the host microorganism with the cloned ferulic acid esterase gene can be performed by Molecular Cloning: A laboratory Mannual, 2nd Ed., T. Maniatis, et al., Cold It can be performed by methods described in laboratory manuals well known to those skilled in the art, such as Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989. The culture of microorganisms can be easily carried out by aerobic culture in a medium containing a carbon source and a nitrogen source according to a conventional method.

  As a method for immobilizing the above enzyme on an immobilization carrier, a crosslinking method or an activation method is preferable. In the cross-linking method, the immobilized carrier is equilibrated with a solution (buffer solution or the like) used in the enzyme adsorption treatment until the pH is stabilized, then the enzyme is adsorbed onto the carrier, and then the carrier is adsorbed onto a polyfunctional reagent ( (Crosslinking agent) is processed and washed. In the activation method, the immobilized carrier is equilibrated with a solution (buffer solution or the like) used for enzyme adsorption treatment until the pH is stabilized, and then the carrier is treated with a polyfunctional reagent (crosslinking agent). Enzyme is adsorbed on the carrier.

  Examples of the polyfunctional reagent include two selected from glycerol polyglycidyl ether, resorcinol diglycidyl ether, neopentyl glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, ethylene glycol diglycidyl ether, and polyethylene glycol diglycidyl ether. Examples include functional epoxy compounds or trifunctional epoxy compounds, polyfunctional epoxy compounds selected from sorbitol polyglycidyl ether, sorbitan polyglycidyl ether and pentaerythritol polyglycidyl ether, and aldehyde compounds such as glutaraldehyde or acetaldehyde. Preferably, it is glutaraldehyde. The immobilized ferulic acid esterase obtained by the crosslinking method and the activation method is preferably one in which the immobilization carrier and ferulic acid esterase are covalently bonded via a polyfunctional reagent (crosslinking agent).

  The enzyme reaction of the present invention is carried out in an aqueous solvent. The temperature at which the enzyme is immobilized is usually about 0 to 40 ° C., preferably about 2 to 30 ° C., so that the enzyme is not deactivated.

  The temperature of the enzyme reaction is preferably about 30 to 70 ° C, particularly preferably about 50 to 60 ° C. The pH of the enzyme reaction is preferably about 3 to 7.5, more preferably about 4 to 6.

  The enzyme reaction of the present invention is illustrated by the following scheme using ferulic acid as the ferulic acid compound [II] and glycerol as the hydroxy compound [III] as an example. The enzymatic reaction includes ferulic acid and glycerol. Is dissolved or suspended in an aqueous solvent (water or a buffer solution such as acetate buffer solution, phosphate buffer solution or Tris-HCl buffer solution), the immobilized enzyme is added to the obtained substrate solution or substrate suspension, and Or by stirring. In this case, since the substrate concentration in the reaction system changes as the enzyme reaction proceeds, in that case, the substrate solution or substrate suspension used at the beginning of the reaction is added and the enzyme reaction is carried out continuously by repeated batch methods. can do. Alternatively, the immobilized enzyme can be packed into a column and the substrate solution or substrate suspension can be continuously circulated through the column. In this case, the solution after the enzyme reaction can be circulated.

  Since ferulic acid has low solubility in water, ferulic acid is preferably supplied in a suspension state. When supplying in the state of a solution, it is preferable to implement by the said circulation system.

  After the desired amount of ferulic acid ester compound (eg, glyceryl ferulic acid) in the reaction solution has reached the desired level, the reaction solution can be purified by conventional methods (extraction, concentration, centrifugation, silica gel chromatography, ion exchange resin treatment, etc.) The ferulic acid ester compound (for example, glyceryl ferulic acid) to be collected can be collected. For example, the ferulic acid ester compound (for example, glyceryl ferulic acid) produced on the ion exchange resin Dowex 1-X2 (Muromachi Chemical Co., Ltd.) is adsorbed and then efficiently desorbed by desorption using high-concentration ethanol. can do.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited to these Examples. In the following examples, the concentrations of ferulic acid and glyceryl ferulic acid were measured by high performance liquid chromatography (HPLC) under the following conditions.
(HPLC conditions)
Analysis column: CapCell Pack 18 (4.6 mmφ × 150 mm, manufactured by Shiseido),
Pump: LC-10AD (manufactured by Shimadzu Corporation),
Mobile phase: methanol: 0.2% acetic acid = 60: 40 (v / v) mixture,
Flow rate: 0.8 ml / min,
Detector: SPD-10AVvp (manufactured by Shimadzu Corporation),
Detection wavelength: 320 nm

[Example]
(Preparation of immobilized enzyme)
Chitopearl (registered trademark) BCW2010, 3003, 3010, 3510 (manufactured by Fuji Boseki Co., Ltd.) was used as the immobilization carrier. Proteins in the solution before and after immobilization were measured using a protein quantification kit (Bio-rad) (Bradford method). The staining solution was prepared by diluting Dye Reagent 5 times with deionized water and then filtering with filter paper (125 mm, manufactured by Advantech). A calibration curve was prepared using a standard protein (bovine gamma globlin) solution (0.2, 0.5, 0.7, 1.0, 1.4 mg / ml). 5 ml of the staining solution was added to 100 μl of the sample solution and left at room temperature for 5 minutes or longer. Thereafter, the absorbance at 595 nm was measured using an ultraviolet-visible spectrophotometer.

  After removing the adhering water of chitopearl with the above filter paper, it was immersed in a 100 mM buffer (pH 4) for 24 hours. A predetermined amount of chitopearl from which adhering water had been removed with a filter paper was weighed into a 50 ml vial, 5 ml of 5 v / v% glutaraldehyde solution was added, and the mixture was shaken at 30 ° C. for about 2 hours. Next, after filtration using filter paper, the chitopearl was thoroughly washed with distilled water. After adding 10 ml of the enzyme solution (pectinase PL, concentration 20 mg / g-wet resin after mixing) to 2 g (wet weight) of the washed chitopearl, it was born for 2 hours in a low temperature room at 4 ° C. After filtering with filter paper and thoroughly washing with distilled water, 100 mM acetate buffer (pH 4) was added to obtain an immobilized enzyme. The obtained immobilized enzyme was stored frozen at 4 ° C.

(Enzyme activity measurement)
The enzyme reaction using the immobilized enzyme was performed as follows. Ferulic acid was added to a mixed solution of 98 parts by volume of glycerol and 2 parts by volume of 100 mM acetate buffer (pH 4) to give a final concentration of 5 w / v% to obtain a substrate solution. To 1 ml of the substrate solution, 0.2 g (wet weight) of the immobilized enzyme was added and reacted at 50 ° C. 0.1 ml of the reaction solution was collected at an appropriate time interval and immersed in boiling water for 5 minutes to stop the reaction. The glyceryl ferulic acid concentration in the reaction solution was measured by the HPLC. The reaction was measured up to 24 hours. Table 1 shows the amount of protein immobilized on various immobilization carriers, the initial reaction rate, and the glyceryl ferulic acid concentration after 24 hours.

  As is clear from Table 1 above, the amount of protein immobilized was the largest when immobilized on BCW-3510, but the initial reaction rate was greatest when immobilized on BCW-3003. In addition, the product concentration after 24 hours (considered to correspond to the reaction equilibrium concentration) was not much different when immobilized on any carrier. The following enzyme reaction was performed using an immobilized enzyme immobilized on BCW-3003, which had the highest initial reaction rate.

(Enzymatic reaction)
A 10 ml substrate solution (mixture of 250 mmol of ferulic acid, 98 parts by volume of glycerol and 2 parts by volume of 100 mM acetate buffer (pH 4)) was prepared. 1.56 g of immobilized enzyme was added to this solution (corresponding to 3 w / v%), and the reaction was started at 50 ° C. 0.1 ml of the reaction solution was collected at an appropriate time interval, and the concentrations of ferulic acid and glyceryl ferulic acid were measured by the HPLC.
The reaction was interrupted at 23 hours, centrifuged (424 × g, 20 minutes), and then 80% (8 ml) of the supernatant was removed. The decrease amount of ferulic acid was calculated from the concentration after 23 hours, and the decrease amount of ferulic acid was mixed with 8 ml of substrate solution (mixture of 98 parts by volume of glycerol and 2 parts by volume of 100 mM acetate buffer (pH 4)). It added to the tube which contained, and reacted again at 50 degreeC. The same operation was repeated 5 times.
FIG. 1 shows the time-dependent changes in ferulic acid and glyceryl ferulic acid concentrations during the batch reaction repeated five times, and FIG. 2 shows the glyceryl ferulic acid concentration after 23 hours in each batch reaction. No decrease in the activity of the immobilized enzyme was observed in the repeated batch reaction operation 5 times, and the concentration of glyceryl ferulic acid after 23 hours of each batch reaction was constant.

  This batch reaction is easy to operate and can be reused due to its immobilization. Therefore, it is considered to be an operation method suitable for mass production of glyceryl ferulic acid.

  According to the present invention, a ferulic acid ester compound (for example, glyceryl ferulic acid) is industrially advantageously produced from a ferulic acid compound (for example, ferulic acid) and a hydroxy compound (for example, glycerol) without using an organic solvent. It can be manufactured friendly to the environment. Moreover, since the ferulic acid ester compound (for example, glyceryl ferulic acid) obtained by the production method of the present invention is soluble in water, it can be widely applied to cosmetics and foods as an antioxidant.

FIG. 1 is a graph showing changes over time in the concentrations of ferulic acid and glyceryl ferulic acid obtained by the enzymatic reaction of the examples. The vertical axis represents concentration (mmol / L), and the horizontal axis represents time (days). FIG. 2 is a diagram showing the concentration of glyceryl ferulic acid after 23 hours in each batch reaction of the example. The vertical axis represents concentration (mmol / L), and the horizontal axis represents time (days).

Claims (11)

Formula [II]

(However, ring A represents an optionally substituted phenyl group.)
And ferulic acid compounds represented by the general formula [III]

(However, R represents a residue obtained by removing one hydroxyl group from a monohydric or polyhydric alcohol or saccharide.)
A ferulic acid esterase immobilized on an immobilization carrier in an aqueous solvent is allowed to act on the hydroxy compound represented by the general formula [I]

(However, rings A and R have the same meaning as described above.)
The manufacturing method of ferulic acid ester compound shown by these.   The production method according to claim 1, wherein the ring A is a phenyl group optionally having 1 to 3 substituents selected from a lower alkoxy group, a lower alkyl group and a hydroxyl group.   The process according to claim 1, wherein the ferulic acid compound [II] is ferulic acid.   The production method according to claim 1, wherein the hydroxy compound [III] is a monovalent or polyhydric alcohol or a saccharide.   The production method according to any one of claims 1 to 3, wherein the hydroxy compound [III] is glycerol.   The production method according to any one of claims 1 to 5, wherein the ferulic acid esterase is a pectinase containing ferulic acid esterase.   The production method according to any one of claims 1 to 6, wherein the immobilization carrier is chitosan into which an aminoalkyl group or an aminophenylalkyl group is introduced.   The production according to any one of claims 1 to 7, wherein the immobilized ferulic acid esterase is covalently bound to chitosan into which an aminoalkyl group or aminophenylalkyl group is introduced and ferulic acid esterase via a polyfunctional reagent. Method.   The production method according to claim 8, wherein the polyfunctional reagent is glutaraldehyde.   The production method according to claim 1, wherein the aqueous solvent is water or a buffer solution.   The production method according to any one of claims 1 to 10, wherein ferulic acid esterase immobilized on an immobilization carrier is used repeatedly or continuously.

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