KR102074660B1 - Novel Method for Enzymatic Synthesis of Hexyl Formate - Google Patents

Abstract

The present invention relates to a method for synthesizing hexyl formate by esterifying formic acid with hexyl alcohol using an enzyme as a catalyst. The present invention is environmentally friendly because only water is produced as a by-product after the reaction, thereby not being harmful to the human body.

Description

Novel Method for Enzymatic Synthesis of Hexyl Formate

The present invention relates to a process for preparing hexyl formic acid with high conversion using lipase enzyme as a catalyst.

By-product gas is mainly generated in the steel industry and can be produced through the gasification of solid urban, industrial, agricultural, and waste resources. It mainly consists of CO, H ₂ , and CO ₂ .

If by-product gas is discharged and inhaled, there is a serious harmful problem. On the other hand, since the emitted by-product gas can generate calories through combustion, many steelworks now recover the emission gas to generate electric energy through the combined power generation. It is utilized to produce.

However, since the final gas still contains a large amount of CO ₂ , it does not contribute to the reduction of greenhouse gases, which is a problem worldwide recently. Therefore, recent studies have been conducted to synthesize fuels or chemical raw materials at room temperature and atmospheric pressure using biological conversion of by-product gas.

On the other hand, the ester can be synthesized by the esterification reaction of the carboxylic acid and the alcohol, and has a unique flavor according to the combination of the carboxylic acid and the alcohol used in the synthesis. Recently, esters are being used as such or as precursors in the food, cosmetics, pharmaceutical, and lubricant industries, and the demand is increasing day by day.

Hexyl formic acid is an ester with a sweet and scent from underripe apples and can be used in perfumes, cosmetics, food additives and detergents. However, obtaining esters from nature requires a large amount of raw materials and a demanding refining process, making mass production difficult and expensive.

In particular, the method of obtaining an ester through a fischer esterification reaction using a chemical catalyst such as an inorganic acid or a Lewis acid requires an excessive amount of alcohol, requires harsh reaction conditions, and causes many side reactions and environmental pollution problems.

Accordingly, the present inventors found that the conversion rate of hexyl formic acid was remarkably increased when the synthesis was performed by the esterification reaction under a specific reaction condition, even if the enzyme lipase was used as a catalyst in synthesizing hexyl formic acid. The present invention was first completed by identifying that it may be high.

Shieh, C. J., & Chang, S. W., Optimized synthesis of lipase-catalyzed hexyl acetate in n-hexane by response surface methodology. Journal of agricultural and food chemistry, 49 (3), 1203-1207 (2001) Diaz, M. D. R., Gσmez, J. M., Diaz-Suelto, B., & Garcia-Sanz, A., Enzymatic synthesis of short-chain esters in n-hexane and supercritical carbon dioxide: Effect of the acid chain length. Engineering in Life Sciences, 10 (2), 171-176 (2010) Wang, S. Q., & Gao, C., Study of synthesis of hexyl-formate catalyzed by strong acidic cation exchange resin. CHINA SURFACTANT DETERGENT AND COSMETICS., 34 (3; ISSU 199), 198-200 (2004) Janssen, L. M., van Oosten, R., Paul, C. E., Arends, I. W., & Hollmann, F., Lipase-catalyzed transesterification of ethyl formate to octyl formate. Journal of Molecular Catalysis B: Enzymatic, 105, 7-10 (2014)

One object of the present invention is to provide a method for producing hexyl formic acid, characterized by reacting formic acid with hexyl alcohol in the presence of lipase.

Another object of the present invention, lipase; Formic acid; And it provides a kit for the production of hexyl formic acid containing hexyl alcohol.

Still another object of the present invention is to add formic acid and hexyl alcohol in a molar ratio of 1: 3 to 1: 7 in an organic solvent of 1,2-dichloroethane or toluene; And adding a lipase at a concentration of 10 to 30 g / L to the mixture and reacting at 20 ° C. to 50 ° C. to provide a method for preparing hexyl formic acid.

In one aspect of the present invention, there is provided a method for preparing hexyl formate, which is characterized by reacting formic acid with hexyl alcohol in the presence of lipase.

Hexyl formic acid having the structure represented by Formula 1 to be synthesized in the present invention is also called hexyl formate, and is an ester compound having less ripe fruit and sweet flavor, and is mainly used as a main raw material in various industries including cosmetics, perfume, food additives, and the like. do. Moreover, hexyl formic acid has been proven stable on April 1, 2017 with permission from the US Food and Drug Administration (FDA) to add it as a flavoring substance and adjuvant.

[Formula 1]

In the present invention, an enzyme may be used as a catalyst to synthesize hexyl formic acid, and specifically, lipase may be used.

Lipases generally have regioselectivity according to their type, and can be classified into three types, including two non-selective lipases and 1,3-position-selective lipases, or 1,3-position specific lipases. Location selectivity of the lipase in the esterification reaction may be a factor that greatly affects the reaction. Regioselective lipases can advantageously act in the esterification reaction of the present invention.

In addition, lipases may be classified into immobilized lipases and unimmobilized lipases depending on whether they are immobilized in addition to classification by position selectivity. The enzyme that is not immobilized has a disadvantage in that it exhibits low activity or no activity under organic solvent conditions and high temperature conditions compared to the immobilized enzyme. However, due to their lower price, they are widely used. It is advantageous for the lipase of the present invention to be an immobilized lipase. In addition, specifically, it may be Novozym 435 ^® derived from Candida antarctica lipase B (CALB), but is not limited thereto.

Enzymes, on the other hand, selectively bind to specific substrates to produce results from enzyme-substrate complexes. According to Michaelis-Menten's equation, the initial enzyme concentration is proportional to the maximum forward rate of the reaction, so the higher the enzyme concentration is, the faster the reaction is, but the mixing rate of the reaction solution decreases due to the increase in the viscosity of the solution. Therefore, the initial reaction rate may be faster if excess enzyme is added to the reaction than the proper enzyme concentration, but the reaction rate may be reduced since the lack of substrate does not form the enzyme-substrate complex. Lipase also performs a reverse reaction as well as a forward reaction using decomposition of the ester bond. Therefore, when the amount of the product in the reaction system is excessively increased, the substrate may be used to counteract the reaction, or the substrate transfer restriction may occur in which the amount of the substrate that can enter the enzyme is limited. Therefore, searching for the optimal enzyme concentration is very important for the reaction using enzyme. In other words, the amount of enzyme in the process using the enzyme is an important factor in determining the economic cost, the maximum efficiency must be obtained using the smallest amount of enzyme.

In the present invention, the lipase has a concentration of 5 to 50 g / L, specifically 5 to 30 g / L, more specifically 10 to 30 g / L, and more specifically 10 to 20 g / L. In concentrations, even more specifically, at a concentration of about 15 g / L. If the concentration of the lipase is less than 5g / L may be too slow the reaction, if the concentration exceeds 50g / L the viscosity of the solution increases the reaction mixture is difficult or reverse reaction may occur.

Hexyl formic acid of the present invention may be produced by esterification of formic acid with hexyl alcohol. In the case of the lipase enzyme used as a catalyst in the method for preparing hexyl formic acid of the present invention, hexyl formic acid as a product and water (as a byproduct) are produced by a direct esterification reaction between formic acid as an electrophile and hexyl alcohol as a nucleophile at an active site inside the enzyme. H ₂ O) is generated (FIG. 1).

Increasing the concentration of hexyl alcohol as a reactant in the method for preparing hexyl formic acid of the present invention may inhibit the formation of tetrahedral intermediates and enzyme complexes produced by nucleophilic substitution reaction of formic acid with serine residues of the enzyme active site. . Therefore, the reaction ratio of formic acid and hexyl alcohol is an important factor because it affects the activity of the enzyme and decreases the reaction rate and consequently the conversion rate.

In the hexyl formic acid production method of the present invention, the molar ratio of the formic acid and hexyl alcohol may be 1: 1 to 1:11. Specifically 1: 3 to 1:11, more specifically 1: 3 to 1: 7, and more specifically about 1: 5. If the molar ratio is less than 1: 1, the reaction yield may be lowered. If the molar ratio is greater than 1:11, the reaction may be rather inhibited.

Synthesis reaction of hexyl formic acid in the present invention may be made at 20 to 50 ℃. Specifically at 30 to 50 ° C, more specifically at 35 to 45 ° C, more preferably at about 40 ° C. If the temperature is less than 20 ℃ reaction may occur too slowly, if it exceeds 50 ℃ there is a fear of denaturation of the enzyme and increase of the process cost. The reaction temperature is one of the important factors in the esterification reaction. As the reaction temperature increases, the reaction rate also increases because the kinetic speed of the molecules increases, increasing the effective collision frequency. However, due to its properties of protein, enzymes denature under high temperature and lose their activity.

The use of an organic solvent as the reaction solvent can increase the stability of the reaction together with the lipase activity, and obtain a higher conversion rate by shifting the thermodynamic equilibrium to the desired esterification reaction, and furthermore, the ease of removal and reuse of enzymes and solvents after the reaction. This can increase the economics of the process. However, such an organic solvent affects the solubility and activity of reactants and products, and affects the microenvironment of the enzyme, which may denature and deactivate the organic solvent.

The reaction of the present invention may be carried out under an organic solvent, and the organic solvent may be a hydrophilic and / or hydrophobic solvent. Specifically, it may be selected from the group consisting of acetonitrile, acetone, toluene, tetrahydrofuran, cyclohexane, 1,2-dichloroethane, hexane and isooctane, but is not limited thereto. More specifically, it may be selected from 1,2-dichloroethane or toluene.

Typically, the hydrophilicity / hydrophobicity of the solvent can be classified based on the logP value, where the logP value is the octanol / water partition coefficient, which indicates that the solvent contains both hydrophilic and hydrophobic tendencies but is hydrophilic and hydrophobic. The relative affinity indicates which of the two properties has a higher affinity. In general, logP <2 may be classified as a hydrophilic solvent, 2 <logP <4 may be classified as a hydrophilic / hydrophobic solvent, and logP> 4. Therefore, acetonitrile (-0.33), acetone (-0.042), tetrahydrofuran (0.49), 1,2-dichloroethane (1.48) among the solvents used in the present invention are hydrophilic solvents, toluene (2.5), cyclohexane (3.4) may be classified as hydrophilic and hydrophobic solvents, n-hexane (4.0) and isooctane (4.6) as hydrophobic solvents. In one embodiment of the present invention, the conversion was higher when using a hydrophobic solvent rather than a hydrophilic solvent, and in particular, 1,2-dichloroethane, a hydrophilic solvent, and toluene in a hydrophilic and hydrophobic solvent having both hydrophilicity and hydrophobicity. It was confirmed that the conversion rate tends to be higher when used (Experimental Example 4).

When hexyl formic acid is synthesized by the method of the present invention, it may have a high conversion rate of 90% or more, more specifically 95% or more. It can thus have a significantly higher conversion of 95% to 100%, specifically 95% to 99.5%, more specifically 97 to 99.5%.

In another aspect of the invention, a lipase; Formic acid; And it provides a kit for the preparation of hexyl formic acid containing hexyl alcohol.

The kit for preparing hexyl formic acid of the present invention is not particularly limited in kind, and a kit of a type commonly used in the art may be used.

The kit of the present invention comprises a first composition comprising a lipase; And a hexyl alcohol; Each of the third compositions comprising formic acid may be included independently. For example, each of the compositions may be packaged in a separate container, or in a container divided into one or more compartments.

The kit may also be a solvent selected from the group consisting of acetonitrile, acetone, toluene, 1,2-dichloroethane, tetrahydrofuran, cyclohexane, n-hexane and isooctane, more specifically 1,2- A solvent selected from dichloroethane or toluene, or a fourth composition comprising the solvent may be further included.

The lipase included in the kit may be positionless selector or may be an immobilized lipase.

The lipase is at a concentration of 5 to 50 g / L, specifically at a concentration of 5 to 30 g / L, more specifically at a concentration of 10 to 30 g / L, more specifically at a concentration of 10 to 20 g / L, Even more specifically, it may be present at a concentration of about 15 g / L.

In addition, formic acid and hexyl alcohol may be included in a molar ratio of 1: 1 to 1:11, specifically 1: 3 to 1:11, more specifically 1: 3 to 1: 7, more specifically And about 1: 5.

Hexyl formic acid production method of the present invention is a method using an enzyme, but because hexyl formic acid is synthesized through a direct esterification reaction of formic acid and hexyl alcohol, only water (H ₂ O) is produced as a by-product, harmless to the human body and very It is an environmentally friendly way.

In addition, the ester and water, which are the products of the present invention, have a large difference in physical properties, and thus can be easily separated and purified. Therefore, the economics of the process through the simplification of the lower process and the reliability of the desired ester synthesis are high.

Furthermore, hexyl formic acid can be obtained with a considerably high conversion rate, which is highly economical and highly valueable and commercially available.

1 is a scheme showing the synthesis of hexyl formic acid by esterification reaction between formic acid and hexyl alcohol.
Figure 2 is a diagram showing the difference between by-products according to the acetic acid derivative-based transition esterification reaction (top) and formic acid-based esterification reaction (bottom).
Figure 3 is a diagram showing the conversion of hexyl formic acid according to the concentration of the Novozym 435 enzyme.
4 is a diagram showing the conversion rate of hexyl formic acid according to the molar ratio of formic acid and hexyl alcohol.
5 is a diagram showing the conversion rate of hexyl formic acid according to the reaction temperature.
6 is a diagram showing the conversion rate of hexyl formic acid according to the type of solvent used in the reaction.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example 1: Enzymes and Reagents

The hexyl alcohol used as a reactant in the present invention was purchased from Yakuri (Kyoto, Japan), and formic acid was purchased from Daejunghwa Gold (Gyeonggi-do, Korea). Acetonitrile, acetone, acetone, cyclohexane, 1,2-dichloroethane, and isooctane, among the eight organic solvents used in the reaction of the present invention. (iso-octane) was purchased from Daejung Gold (Gyeonggi-do, Korea), and tetrahydrofuran (THF), toluene and n-hexane were purchased from Junsei (Tokyo, Japan). It was.

In the present invention, an enzyme immobilized on a polymer resin was used for easy commercial use, and Novozym 435, which is typically derived from Candida antarctica lipase B (CALB), was purchased from Novoszyme (Bagsvaerd, Denmark).

Example 2: Basic Reaction Conditions

Hexyl formic acid can be synthesized from formic acid and hexyl alcohol via lipase catalysis. In theory, one mole of formic acid reacts with one mole of hexyl alcohol to produce one mole of hexyl formic acid and one mole of water. Since lipase is also active in the hydrolysis reaction of hexyl formic acid with this esterification reaction, conditions for increasing the yield of hexyl formic acid synthesis are very important.

Specifically, 100 ml of formic acid and 100 mM of hexyl alcohol were prepared in an organic solvent, respectively, and each 50 mL volume of a reaction bottle (serum bottle) sealed with a butyl rubber stopper and an aluminum crimp seal. Put in.

Thus the final solution volume was 20 mL and the reactants formic acid and hexyl alcohol were 50 mM each. Into this solution, a fixed weight of lipase of fixed weight was added to the reaction vessel to carry out the reaction. The loading unit of the enzyme was g / L, which was weighted against the solution volume. Then, the reaction was performed at 150 rpm for 4 hours in a shaking incubator.

Example 3 Determination of Conversion to Hexyl Formic Acid-Chromatographic Analysis

To measure the conversion to hexyl formic acid, after reacting the reaction for 4 hours, a sample of about 1 ml of the reaction solution was collected by using a syringe and filtered by a filter (PALL, CR PTFE 0.2 μm,) to prepare a sample for analysis. It was.

Prepared samples were analyzed using Agilent 7890A gas chromatography. In gas chromatography, HP-INNOWax polar column (polyethylene glycol, 30.0mХ250μmХ0.25μm) was separated and analyzed by flame ionization detector (FID). 1 μl of sample was injected into the column, and nitrogen was used as a carrier gas. The column temperature was maintained for 3 minutes when the initial temperature of the oven was set at 80 ° C. and maintained for 1 minute and then increased at a rate of 10 ° C./min to reach 230 ° C. Thereafter, the mixture was heated to 250 ° C. at a rate of 20 ° C./min, and held at the final temperature (250 ° C.) for 1 minute. The temperature of the injection port and the temperature of the detector were set to 250 ° C, respectively. Detection results were analyzed using an Agilent ChemStation. Since the conversion rate of hexyl formic acid is 1: 1 molar reaction, the conversion rate of hexyl formic acid was calculated using Equation 1 below.

[Equation 1]

Experimental Example 1 Evaluation of Conversion Rate of Hexyl Formic Acid According to Enzyme Concentration

In order to evaluate the hexyl formic acid conversion according to the enzyme concentration, Novozym 435 was used as a representative lipase, and its concentration was varied (5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g). Hexyl formic acid was synthesized by reacting formic acid with hexyl alcohol in a molar ratio of 1: 1. n-hexane was used as a solvent, the reaction was carried out for 4 hours at 30 ℃, 150 rpm using a shaking incubator.

Enzyme Concentration (g / L) 5 10 15 20 25 30 % Conversion 15.50 71.87 77.33 74.34 73.32 57.92

As a result, as shown in Table 1 and FIG. 3, the lowest conversion rate was 15.50% when the enzyme concentration was 5 g / L, and the highest conversion rate was 77.33% when the concentration was 15 g / L. (FIG. 3).

Experimental Example 2: Evaluation of the conversion rate of hexyl formic acid according to the ratio of formic acid and hexyl alcohol

In order to analyze the conversion rate of hexyl formic acid according to the reaction ratio of formic acid and hexyl alcohol as reactants, the concentration of formic acid was fixed at 50 mM and formic acid and hexyl alcohol were 1: 1, 1: 3, 1: 5, and 1: 7, respectively. Hexyl formic acid was synthesized by esterification with molar ratios of 1: 9 and 1:11. The reaction was carried out in the presence of 15 g / L Novozym 435 enzyme, n-hexane was used as a solvent, the reaction was carried out for 4 hours at 30 ℃, 150rpm using a shaking incubator.

Molar ratio of formic acid and hexyl alcohol 1: 1 1: 3 1: 5 1: 7 1: 9 1:11 % Conversion 77.33 83.49 84.01 83.88 82.41 75.36

As a result, as can be seen in Table 2 and Figure 4, the conversion rate of hexyl formic acid at the molar ratio of formic acid and hexyl alcohol of 1: 1, 1: 3, 1: 5 is increased to 77.33%, 83.49%, 84.01%, respectively When the reaction was carried out at a molar ratio of 1: 7, 1: 9, and 1:11, high conversion rates of 83.88%, 82.41%, and 75.36%, respectively, were observed (FIG. 4).

Experimental Example 3: Evaluation of Conversion Rate of Hexyl Formic Acid According to Reaction Temperature

In order to evaluate the conversion rate of hexyl formic acid according to the reaction temperature, experiments with different reaction temperatures (20 ° C., 30 ° C., 40 ° C., 50 ° C.) were performed. The reaction was performed for 4 hours in the presence of 15 g / L of Novozym 435 enzyme, and the solution was put into a shaker incubator set at 20 ° C., 30 ° C., 40 ° C. and 50 ° C., respectively, before the enzyme was put into the experiment. N-hexane was used as a reaction solvent, and formic acid and hexyl alcohol were reacted at a molar ratio of 1: 5.

Reaction temperature (℃) 20 30 40 50 % Conversion 83.27 84.01 87.69 84.95

As can be seen in Table 3 and Figure 5, while showing a high conversion rate of 83.27%, 84.01%, 87.69%, 84.95% at the reaction temperature of 20 ℃ to 50 ℃, respectively, the highest hexyl at the reaction temperature of 40 ℃ Formic acid conversion (87.69%).

Experimental Example 4: Evaluation of Conversion Rate of Hexyl Formic Acid According to Organic Solvents

In order to evaluate the conversion rate to hexyl formic acid according to the type of organic solvent used in the reaction, an organic solvent having a different degree of hydrophilicity / hydrophobicity was selected. The degree of hydrophilicity / hydrophobicity is classified based on the logP value, where the logP value is the octanol / water partition coefficient, which indicates that the solvent contains both hydrophilic and hydrophobic tendencies but with hydrophilic and hydrophobic properties. Relative figures indicate whether the affinity is higher. For example, if logP <2, it may be classified as a hydrophilic solvent, if 2 <logP <4, a hydrophilic / hydrophobic solvent, and if logP> 4, it may be classified as a hydrophobic solvent.

In the present invention, acetonitrile (-0.33), acetone (-0.042), tetrahydrofuran (0.49), 1,2-dichloroethane (1.48), toluene (2.5), and cyclohexane (3.4) in the order of low logP values ), n-hexane (4.0) and isooctane (4.6) total 8 solvents were used for the reaction. In each sample, formic acid and hexyl alcohol were reacted in a molar ratio of 1: 5 in the presence of 15 g / L of Novozym 435 enzyme. The reaction was carried out for 4 hours at 40 ℃, 150rpm using a shaking incubator.

Organic solvent (logP value) % Conversion Acetonitrile (-0.33) 24.59 Acetone (-0.042) 29.97 Tetrahydrofuran (0.49) 53.57 1,2-dichloroethane (1.48) 98.28 Toluene (2.5) 95.56 Cyclohexane (3.4) 86.90 n-hexane (4.0) 87.69 Isooctane (4.6) 84.08

As a result, as shown in Table 4 and FIG. 6, the highest conversion rate (98.28%) was shown when 1,2-dichloroethane was used as a solvent, followed by high conversion rate (95.56%) when toluene was used. . The conversion was higher when the hydrophobic solvent was used rather than the hydrophilic solvent, and the conversion was higher when the organic solvent having both hydrophilicity and hydrophobicity was used in the reaction.

Claims (11)

In the presence of lipase, reacting formic acid with hexyl alcohol,
The lipase is a method for producing hexyl formate (hexyl formate), characterized in that the non-selectivity lipase.
delete The method of claim 1, wherein said non-selective lipase is an immobilized lipase.
The method of claim 1, wherein the reaction is performed under an organic solvent, wherein the concentration of the lipase is 10 to 30 g / L.
The method of claim 1, wherein the molar ratio of formic acid and hexyl alcohol is 1: 1 to 1:11.
The method of claim 1, wherein the reaction is carried out at 20 ℃ to 50 ℃.
The method of claim 1, wherein the reaction is carried out under an organic solvent selected from the group consisting of acetonitrile, acetone, toluene, tetrahydrofuran, 1,2-dichloroethane, cyclohexane, hexane and isooctane. Manufacturing method.
The method of claim 7, wherein the organic solvent is 1,2-dichloroethane or toluene.
Lipases; Formic acid; And hexyl alcohol,
The lipase is a kit for the preparation of hexyl formic acid, characterized in that the non-selectivity lipase.
10. The organic solvent of claim 9, further comprising an organic solvent selected from the group consisting of acetonitrile, acetone, toluene, tetrahydrofuran, 1,2-dichloroethane, cyclohexane, hexane and isooctane. Kit.
Adding formic acid and hexyl alcohol in a molar ratio of 1: 3 to 1: 7 to an organic solvent of 1,2-dichloroethane or toluene to form a mixture; And
To add a lipase of a concentration of 10 to 30g / L to the mixture to react at 20 ℃ to 50 ℃; includes,
The lipase is a method for producing hexyl formic acid, characterized in that the non-selectivity lipase.

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