Synthetic method of 2-ethylhexyl fatty acid esters in solvent-free system using Candida rugosa lipase

Abstract

The present invention relates to a method for synthesizing 2-ethylhexyl fatty acid esters in a solvent-free system using Candida rugosa enzymes. Specifically, the present invention relates to a method for very efficiently producing 2-ethylhexyl fatty acid esters in a solvent-free environment, comprising the steps of: (1) preparing a substrate by mixing 2-ethylhexanol and a fatty acid; and (2) adding a lipase derived from a non-genetically modified microorganism (non-Gmm) Candida rugosa to the substrate and esterifying it to synthesize 2-ethylhexyl fatty acid esters.

Description

Technology Field

[0001] The present invention relates to a method for synthesizing 2-ethylhexyl fatty acid esters in a solvent-free system using an enzyme produced from the microorganism Candida rugosa. Background Technology

[0002] Skin emollients are the second most widely used cosmetic ingredient after purified water, and they are non-miscible components such as oils. Most emollients used today, such as petroleum-based oils, are manufactured through chemical processes. However, consumers are now demanding the development of such products, driven by growing interest in environmental protection—including resource recycling and sustainability—as well as the exclusion of chemicals and the use of natural resources.

[0003] Cosmetic esters, known as emollients, can be synthesized from fatty acids and alcohols through esterification or transesterification reactions. The initial raw materials for various cosmetic esters are free fatty acids extracted from various vegetable oils. One of the most widely used emollients is 2-EHFE (2-Ethylhexyl fatty acid esters), a transparent liquid substance that is non-toxic. This substance has been approved and its safety verified by the Environmental Working Group (EWG), a U.S. non-profit environmental organization, and the Cosmetic Ingredient Review Board (CIR), and is used in the formulations of various cosmetic and personal care products. However, conventional technologies for synthesizing these substances have drawbacks, such as carbon dioxide emissions, waste generation, and the fact that they are produced through high-temperature reactions utilizing chemical catalysts. Therefore, there is a need to develop a new 2-EHFE synthesis technology through enzymatic esterification reactions that is environmentally friendly, reduces synthesis costs, and improves synthesis yield.

[0004] Currently, commercial cosmetic esters are typically synthesized by chemical reactions using acid or base catalysts at temperatures higher than 200°C. However, these chemical reactions, carried out using chemical catalysts at high temperatures, can result in the formation of many undesirable products.

[0005] Furthermore, most people today prefer cosmetics containing natural ingredients or those using biotechnology. Consequently, attempts are being made to synthesize cosmetic esters from fatty acids and alcohols through enzyme-catalyzed esterification reactions; however, since the enzymes used in conventional enzyme-catalyzed esterification reactions are mostly derived from genetically modified microorganism (GMM) strains, these methods fail to meet consumer expectations for GMO-free products regarding potential harm to the environment and health.

[0006] Therefore, there is a need to develop new technology capable of efficiently manufacturing cosmetic esters while addressing potential health hazards. Prior art literature

[0007] Korean Published Patent 10-2020-0102361 Korean Published Patent 10-2019-0125826 The problem to be solved

[0008] Accordingly, the inventors have completed the present invention by establishing an optimal synthesis method for 2-ethylhexyl fatty acid esters, which is a new synthesis method for 2-ethylhexyl fatty acid esters that can increase synthesis yield while being eco-friendly and reducing the possibility of harm to the human body, by esterifying fatty acids derived from camellia seed oil and 2-ethylhexanol using enzymes derived from non-genetically modified microorganisms (Non-GMM).

[0009] Accordingly, the object of the present invention is (1) a step of preparing a substrate by mixing 2-ethylhexanol and a fatty acid; and (2) a non-genetically modified microorganism (non-Gmm) Candida rugosa ( Candida rugosa The present invention provides a method for producing 2-ethylhexyl fatty acid esters in a solvent-free manner, comprising the step of adding a lipase derived from ) to the substrate and esterifying it to synthesize 2-ethylhexyl fatty acid esters. means of solving the problem

[0010] Therefore, the present invention comprises: (1) a step of preparing a substrate mixed with 2-ethylhexanol and a fatty acid; and (2) a non-genetically modified microorganism (non-Gmm) Candida rugosa ( Candida rugosa A method for producing 2-ethylhexyl fatty acid esters in a solvent-free manner is provided, comprising the step of adding a lipase derived from ) to the substrate and esterifying it to synthesize 2-ethylhexyl fatty acid esters.

[0011] In one embodiment of the present invention, the fatty acid may be a fatty acid derived from camellia seed oil.

[0012] In one embodiment of the present invention, the fatty acid is a hydrolyzed fatty acid of camellia seed oil and may be one or more selected from the group consisting of palmitic acid, stearic acid, oleic acid, and linoleic acid.

[0013] In one embodiment of the present invention, the lipase may be a lipase in a form immobilized on a hydrophobic carrier Lewatit VP OC 1600 or a lipase in a non-immobilized free-type form.

[0014] In one embodiment of the present invention, the 2-ethylhexanol and fatty acid may be mixed in a molar ratio of 1:1 to 2:1.

[0015] In one embodiment of the present invention, the amount of lipase may be 2 to 10 weight percent based on the weight of the substrate.

[0016] In one embodiment of the present invention, the lipase may be a lipase having a water activity of 0.2 to 1.0.

[0017] In one embodiment of the present invention, the ester reaction may be carried out at 20 to 50°C for 1 to 4 hours.

[0018] In one embodiment of the present invention, the ester reaction may be carried out in a batch reactor, a packed continuous reactor, or a recirculating packed continuous reactor.

[0019] In one embodiment of the present invention, when performing an esterification reaction in a batch reactor, a substrate is prepared by mixing 2-ethylhexanol and a fatty acid in a molar ratio of 1:1, a non-immobilized pre-type lipase with a water activity of 0.432 is added to the substrate at 4% by weight based on the total weight of the substrate, and an esterification reaction is carried out at a temperature of 20°C for 1 to 4 hours to synthesize 2-ethylhexyl fatty acid esters.

[0020] In one embodiment of the present invention, when performing an esterification reaction in a batch reactor, a substrate is prepared by mixing 2-ethylhexanol and a fatty acid in a molar ratio of 1:1, and a lipase immobilized on Lewatit VP OC 1600, a hydrophobic carrier with a water activity of 0.843, is added to the substrate at 2% by weight based on the total weight of the substrate, and an esterification reaction is carried out at a temperature of 50°C for 1 to 4 hours to synthesize a 2-ethylhexyl fatty acid ester.

[0021] In one embodiment of the present invention, when performing an esterification reaction in a packed continuous reactor, a substrate is prepared by mixing 2-ethylhexanol and a fatty acid in a molar ratio of 1:1, a lipase immobilized on a hydrophobic carrier Lewatit VP OC 1600 is added to the substrate at 6% by weight based on the total weight of the substrate, and an esterification reaction is carried out at a temperature of 50°C for 5 to 90 minutes to synthesize a 2-ethylhexyl fatty acid ester.

[0022] In one embodiment of the present invention, when performing an esterification reaction in a recirculating packed continuous reactor, a substrate is prepared by mixing 2-ethylhexanol and a fatty acid in a molar ratio of 1:1, a lipase immobilized on a hydrophobic carrier Lewatit VP OC 1600 is added to the substrate at 9% by weight based on the total weight of the substrate, and 2-ethylhexyl fatty acid esters are synthesized by performing an esterification reaction for a total reaction time of 30 to 60 minutes while fixing the residence time of the substrate in the reactor at a temperature of 50°C to 3 minutes. Effects of the invention

[0023] The method for synthesizing 2-ethylhexyl fatty acid esters according to the present invention relates to a method capable of producing 2-ethylhexyl fatty acid esters in high yield in a solvent-free system using a lipase enzyme derived from non-genetically modified Candida rugosa. The method for synthesizing 2-ethylhexyl fatty acid esters provided by the present invention has the effect of resolving environmental and health hazards associated with the use of enzymes derived from genetically modified microorganisms in the past due to the use of lipase derived from non-genetically modified Candida rugosa. Furthermore, it can provide optimal synthesis conditions for efficiently synthesizing 2-ethylhexyl fatty acid esters in batch reactors and continuous reactors, thereby having the effects of increased yield, reduced reaction time, and low production costs. Brief explanation of the drawing

[0024] Figure 1 shows a schematic diagram of batch and continuous reactors (packed continuous reactor and recirculating continuous reactor) used for the synthesis of 2-ethylhexyl fatty acid esters in one embodiment of the present invention. Figure 2 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis according to reaction time and type of enzyme catalyst in a solvent-free system through an enzyme-catalyzed esterification reaction using a batch reactor. Figure 3 shows the GC chromatograms for the initial substrate (A) and the reaction mixture (B) after 6 hours. Figure 4 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis according to the water activity of a pre-type non-GMM Candida rugosa-derived enzyme in a solvent-free system through an enzyme-catalyzed esterification reaction using a batch reactor. Figure 5 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis at different reaction temperatures using a free-type non-GMM Candida rugosa-derived enzyme in a solvent-free system through an enzyme-catalyzed esterification reaction using a batch reactor. Figure 6 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis according to the amount of enzyme added from the free-type non-GMM Candida rugosa in a solvent-free system through an enzyme-catalyzed esterification reaction using a batch reactor. Figure 7 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis according to water activity of a non-GMM Candida rugosa-derived enzyme immobilized on a carrier in a solvent-free system through an enzyme-catalyzed esterification reaction using a batch reactor. Figure 8 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis at different reaction temperatures using a non-GMM Candida rugosa-derived enzyme immobilized on a carrier in a solvent-free system via an enzyme-catalyzed esterification reaction using a batch reactor. Figure 9 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis according to the amount of enzyme derived from non-GMM Candida rugosa immobilized on a carrier in a solvent-free system through an enzyme-catalyzed esterification reaction using a batch reactor. Figure 10 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis at different reaction temperatures using a non-GMM Candida rugosa-derived enzyme immobilized on a carrier in a solvent-free system through an enzyme-catalyzed esterification reaction using a continuous reactor. Figure 11 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis over time using a non-GMM Candida rugosa-derived enzyme immobilized on a carrier in a solvent-free system through an enzyme-catalyzed esterification reaction using a recirculating continuous reactor. Figure 12 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis at different reaction temperatures using a non-GMM Candida rugosa-derived enzyme immobilized on a carrier in a solvent-free system through an enzyme-catalyzed esterification reaction using a recirculating continuous reactor. Figure 13 shows the results of analyzing the conversion rate of 2-ethylhexyl fatty acid ester synthesis according to the amount of enzyme added to a carrier in a solvent-free system via an enzyme-catalyzed esterification reaction using a recirculating continuous reactor. Specific details for implementing the invention

[0025] The present invention is characterized by providing an optimal manufacturing method that can efficiently synthesize 2-ethylhexyl fatty acid esters in a solvent-free environment.

[0026] In the conventional synthesis of 2-ethylhexyl fatty acid esters through enzymatic esterification reactions, enzymes derived from genetically modified microorganism (GMM) strains are widely used. However, as consumer interest in the environment and health gradually increases, the demand for GMO-free products is increasing due to concerns regarding the potential harm to the environment and health caused by the use of enzymes derived from genetically modified microorganisms.

[0027] Accordingly, the inventors identified the optimal synthesis conditions for 2-ethylhexyl fatty acid esters using enzymes derived from non-GMM (non-genetically modified microorganism), which are non-genetically modified microorganisms, rather than genetically modified microorganisms.

[0028] Accordingly, the present invention comprises the steps of: (1) preparing a substrate by mixing 2-ethylhexanol and a fatty acid; and (2) a non-genetically modified microorganism (non-Gmm) Candida rugosa ( Candida rugosa The present invention is characterized by providing a method for producing 2-ethylhexyl fatty acid esters in a solvent-free manner, comprising the step of adding a lipase derived from ) to the substrate and esterifying it to synthesize 2-ethylhexyl fatty acid esters.

[0029] The synthesis of 2-ethylhexyl fatty acid esters according to the present invention is an effective method that is more environmentally friendly than reactions using organic solvents and can obtain reaction products in high yield by carrying out the synthesis under solvent-free conditions.

[0030] Organic solvents have high volatility and flammability and are harmful to the environment. In the synthesis of 2-ethylhexyl fatty acid esters, organic solvents can lower the reaction temperature of the substrate during enzymatic synthesis, but they have the problem of inhibiting enzyme activity, and a separate removal process is required to remove the organic solvent after the reaction. Therefore, the use of organic solvents does not align with the current industrial direction of pursuing green chemicals, and is unsafe as a raw material for cosmetics because residual toxicity cannot be ignored. On the other hand, the method of the present invention has the effect of obtaining reaction products with a high yield without the use of such organic solvents.

[0031] The specific process of the method for producing 2-ethylhexyl fatty acid ester in a solvent-free environment according to the present invention is described as follows.

[0032] First, the above step (1) is a step of preparing a substrate, and (1) a substrate is prepared by mixing 2-ethylhexanol and a fatty acid.

[0033] In the present invention, the fatty acid may be a fatty acid derived from camellia seed oil.

[0034] Camellia seed oil is known to have the ability to protect the skin from aging and can provide cosmetic properties including antioxidant and moisturizing effects. Therefore, camellia seed oil can be very useful in the manufacture of skincare cosmetics as well as hair products.

[0035] The fatty acid derived from camellia seed oil according to the present invention is a fatty acid obtained by hydrolyzing camellia seed oil, and one or more selected from the group consisting of palmitic acid, stearic acid, oleic acid, and linoleic acid may be used.

[0036] When palmitic acid is used as the fatty acid, 2-ethylhexyl palmitate can be synthesized by the method of the present invention; when stearic acid is used as the fatty acid, 2-ethylhexyl stearate can be synthesized; when oleic acid is used as the fatty acid, 2-ethylhexyl oleate can be synthesized; and when linoleic acid is used as the fatty acid, 2-ethylhexyl linoleate can be synthesized.

[0037] In addition, in the present invention, the substrate can be prepared by mixing 2-ethylhexanol and a fatty acid, and the substrate can be prepared by mixing 2-ethylhexanol and a fatty acid in a molar ratio of 1:1 to 2:1. Preferably, the substrate can be prepared by mixing 2-ethylhexanol and a fatty acid in a molar ratio of 1:1.

[0038] When the substrate preparation of step (1) above is completed, next, a non-genetically modified microorganism (non-Gmm) Candida rugosa ( Candida rugosa) Add a derived lipase to the above substrate and perform an esterification reaction.

[0039] The above lipase is from the non-genetically modified microorganism (non-Gmm) Candida rugosa ( Candida rugosa Lipase enzymes isolated from microorganisms can be used as catalysts.

[0040] In this case, the lipase enzyme may be an immobilized form of lipase or a non-immobilized free form of lipase.

[0041] Specifically, the immobilized form of lipase may be lipase immobilized on a hydrophobic carrier, Lewatit VP OC 1600.

[0042] In addition, the above-mentioned non-immobilized free-form lipase is a free-type lipase in which lipase is not immobilized on an immobilization carrier, and is a non-genetically modified microorganism (non-Gmm) Candida rugosa ( Candida rugosa It may be lipase in powder form produced from ).

[0043] In the present invention, 'immobilized enzyme' refers to an enzyme in which lipase is physically adsorbed onto a hydrophobic carrier, meaning a form in which the lipase and the carrier are combined.

[0044] Meanwhile, when 2-ethylhexyl fatty acid esters are prepared using enzymes, the yield of the synthesized product varies significantly depending on the type of enzyme and reaction conditions. Accordingly, in order to establish optimal synthesis (preparation) conditions for 2-ethylhexyl fatty acid esters in a solvent-free environment, the inventors compared and analyzed the synthesis conversion rates of 2-ethylhexyl fatty acid esters by varying the type of enzyme, the amount of enzyme added, and the ester reaction time.

[0045] As a result, in the analysis of the conversion rate to the most superior 2-ethylhexyl fatty acid ester using various types of lipase, lipases derived from other strains ( Candida antarctica : Product name Novozym 435, Thermomyces lanuginosus Product Name Lipozyme TL IM, Rhizomucor miehei Compared to : Trade name Lipozyme RM IM), the non-genetically modified microorganism (non-Gmm) Candida rugosa used in the present invention ( Candida rugosa When using lipase derived from ), the best conversion rate to 2-ethylhexyl fatty acid ester was observed.

[0046] In addition, in the method of the present invention, the non-genetically modified Candida rugosa-derived lipase may be used in an amount of 2 to 10 weight percent based on the total weight of the substrate, and the lipase may be used having a water activity of 0.2 to 1.0.

[0047] If lipase enzyme is used in an amount of less than 2% by weight based on the total weight of the substrate, the enzyme catalytic reaction is not smooth, resulting in a problem of low synthesis conversion rate of 2-ethylhexyl fatty acid ester, whereas if it is used in an amount exceeding 10% by weight, the maximum synthesis conversion rate does not increase, resulting in a problem of reduced economic efficiency.

[0048] In addition, in the method of the present invention, it is preferable to use a non-genetically modified Candida rugosa-derived lipase having a water activity of 0.2 to 1.0, as the activity of the enzyme is known to be affected by the water content contained in the enzyme. This is because the water content bound to the enzyme significantly affects the enzyme's structure, catalytic activity, and selectivity.

[0049] Accordingly, the inventors analyzed the effect of water activity of a lipase enzyme derived from non-genetically modified Candida rugosa on the synthesis of 2-ethylhexyl fatty acid esters according to the present invention and confirmed that it is preferable to use a lipase having a water activity of 0.2 to 1.0.

[0050] If an enzyme with a water activity of less than 0.2 is used, the enzyme catalytic activity is low, which may result in insufficient esterification reaction. On the other hand, if an enzyme with a water activity greater than 1.0 is used, the esterification reaction efficiency is no longer effective, which is uneconomical.

[0051] Therefore, the use of lipase with a water activity of 0.2 to 1.0 is a suitable range for effectively shortening the reaction time in terms of enzyme activity.

[0052] In addition, the inventors analyzed the effect of reaction temperature on the esterification reaction. Since reaction temperature affects the reaction rate and the residual activity of the enzyme, it can be considered an important factor in the enzyme reaction.

[0053] The ester reaction in step (2) according to the present invention can be carried out at 20 to 50°C for 1 to 4 hours.

[0054] If the reaction is performed at a lower temperature and for a shorter time than the above range of reaction temperature and reaction time, the problem of a low synthesis yield of the desired 2-ethylhexyl fatty acid ester occurs, and if the reaction is performed at a high temperature and for a longer time, the problem of reduced enzyme activity occurs.

[0055] Therefore, it is important to perform the temperature and time of the ester reaction within the above range.

[0056] In addition, the molar ratio between substrates affecting the synthesis of 2-ethylhexyl fatty acid esters is also an important factor in the enzyme reaction.

[0057] In one embodiment of the present invention, the effect of the molar ratio between substrates during the synthesis of 2-ethylhexyl fatty acid esters was measured, and it was confirmed that the molar ratio of 2-ethylhexanol to fatty acid is preferably 1:1 to 2:1, and that using 1:1 is the most preferable.

[0058] When the molar ratio of 2-ethylhexanol to fatty acid is lower than the above molar ratio, there is a problem that the conversion rate of 2-ethylhexyl fatty acid ester decreases, whereas when it is higher than the above molar ratio, there is a problem that it is inefficient because it does not affect the conversion rate of 2-ethylhexyl fatty acid ester.

[0059] In addition, the method for producing 2-ethylhexyl fatty acid esters in a solvent-free manner according to the present invention can be carried out in a batch reactor, a packed continuous reactor, or a recirculated packed continuous reactor, and the present invention establishes optimal ester reaction conditions that can efficiently synthesize 2-ethylhexyl fatty acid esters in each of the above reactors.

[0060] Specifically, when synthesizing 2-ethylhexyl fatty acid esters in a solvent-free environment using a batch reactor, it was confirmed through experiments in the following examples that the most optimal reaction condition for synthesizing 2-ethylhexyl fatty acid esters is to prepare a substrate by mixing 2-ethylhexanol and fatty acid in a molar ratio of 1:1, add a non-immobilized, free-type lipase with a water activity of 0.432 to the substrate at 4% by weight based on the total weight of the substrate, and perform an esterification reaction at a temperature of 20°C for 1 to 4 hours.

[0061] In addition, it was confirmed that the optimal reaction condition for synthesizing 2-ethylhexyl fatty acid esters is to prepare a substrate mixed with 2-ethylhexanol and a fatty acid in a molar ratio of 1:1, add lipase immobilized on Lewatit VP OC 1600, a hydrophobic carrier with a water activity of 0.843, to the substrate at 2% by weight based on the total weight of the substrate, and perform the esterification reaction at a temperature of 50°C for 1 to 4 hours.

[0062] When the synthesis of 2-ethylhexyl fatty acid esters is carried out in a batch reactor in a solvent-free manner, lipase derived from non-genetically modified Candida rugosa can be used as immobilized lipase or non-immobilized free-type (free form) lipase, and preferably, non-immobilized free-type (free form) lipase can be used.

[0063] According to one embodiment of the present invention, the conversion rate to 2-ethylhexyl fatty acid ester in a batch reactor was found to be superior when using a non-immobilized free-type (free form) lipase compared to when using an immobilized lipase.

[0064] In addition, the synthesis of 2-ethylhexyl fatty acid esters can also be carried out in a packed continuous reactor in a solvent-free manner. In this case, it is preferable to prepare a substrate by mixing 2-ethylhexanol and a fatty acid in a molar ratio of 1:1, add lipase immobilized on a hydrophobic carrier Lewatit VP OC 1600 to the substrate at 6% by weight based on the total weight of the substrate, and synthesize 2-ethylhexyl fatty acid esters by esterifying the mixture at a temperature of 50°C for 5 to 90 minutes.

[0065] In addition, 2-ethylhexyl fatty acid esters can be synthesized in a solvent-free manner through an esterification reaction in a recirculating packed continuous reactor. In this case, it is preferable to prepare a substrate by mixing 2-ethylhexanol and a fatty acid in a molar ratio of 1:1, add lipase immobilized on a hydrophobic carrier Lewatit VP OC 1600 to the substrate at 9% by weight based on the total weight of the substrate, and synthesize 2-ethylhexyl fatty acid esters by carrying out an esterification reaction for a total reaction time of 30 to 60 minutes while fixing the residence time of the substrate in the reactor (column) at a temperature of 50°C to 3 minutes.

[0067] The present invention will be described in detail below through examples. However, these examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0069] Materials and Experimental Methods

[0070] Preparation of ingredients

[0071] 2-ethylhexyl alcohol was purchased from Sigma-Aldrich Co. LLC (St. Louis, MO, USA). Camellia seed oil was provided by Daebong LS Co., Ltd. (Incheon, Republic of Korea). Novozym 435 derived from Candida antarctica, Lipozyme RM IM derived from Rhizomucor miehei, and Lipozyme TL IM derived from Thermomyces lanuginosus were purchased from Novozymes (Novozymes Korea, Seoul, Republic of Korea). The hydrophobic carrier Lewatit VP OC 1600 was purchased from Membrana GmbH Accurel System (Obernburg, Germany). Unless otherwise specified, all other reagents used in this experiment were of special grade or higher.

[0073] Preparation of fatty acids from camellia seed oil

[0074] Camellia seed oil was converted into fatty acids by catalytic hydrolysis with Lipase OF. 500 g of camellia seed oil and 500 ml of distilled water were placed in a 1 L glass reactor, and the reactor was heated to 40°C using a circulating constant temperature water bath. Subsequently, an enzyme (0.05 wt% of Candida rugosa-derived lipase based on the oil) was added to initiate hydrolysis, and the mixture was stirred at 300 rpm. After 17 hours, the resulting lower layer was removed using a separatory funnel, and the upper layer containing fatty acids was extracted with 500 mL of n-hexane. After washing twice with 100 mL of distilled water, the remaining water was removed from the n-hexane layer containing fatty acids by passing it through anhydrous sodium sulfate. The remaining solvent was removed under vacuum using a rotary vacuum concentrator (NN series, EYELA, Japan) at 50°C. Additionally, any remaining solvent was removed using nitrogen at 60°C, and for effective solvent removal, nitrogen was injected for 20 minutes at a relatively high airflow rate. The fatty acid composition of camellia seed oil is shown in Table 1.

[0075]

[0077] Enzyme immobilization

[0078] The enzyme solution for immobilization was prepared using powdered, pre-type Candida rugosa enzyme and sodium phosphate buffer (100 mM, pH 7.0). Specifically, 80 g of non-GMM Candida rugosa-derived lipase enzyme was suspended in 800 mL of sodium phosphate buffer. The enzyme suspension was stirred at 250 rpm for 3 hours, followed by centrifugation at 3100 xg for 30 minutes at 25°C. The supernatant obtained from centrifugation was used as the enzyme solution for immobilization. The hydrophobic carrier Lewatit VP OP 1600 was pre-soaked in ethanol for 3 hours to allow the enzyme solution to physically enter the pores of the carrier in the subsequent step. Subsequently, the ethanol in the carrier was completely replaced with sodium phosphate buffer (100 mM, pH 7.0). 150 mL of the enzyme solution was added to a flask containing 15 g of the carrier. The mixture of enzyme solution and carrier was stirred in a shaking water bath at 30°C and 250 rpm for 17 hours. Subsequently, the carrier was separated from the enzyme solution by filtration and immediately washed with 750 mL of buffer solution to remove unbound or loosely bound enzymes. The enzyme-bound carrier was dried overnight at room temperature and then dried in a vacuum drying oven at 40°C for 12 hours. The immobilized lipase was stored at 4°C.

[0080] Water activity equilibrium

[0081] The enzyme was stored in a sealed container saturated with salt solutions of known water activity to reach equilibrium. The salts used were lithium chloride (aw=0.113), potassium acetate (aw=0.225), magnesium chloride (aw=0.328), potassium carbonate (aw=0.432), magnesium nitrate (aw=0.529), sodium nitrite (aw=0.654), and potassium chloride (aw=0.843). The equilibration process was carried out at 25°C for 48 hours.

[0083] Enzyme-catalyzed esterification reaction in a batch reactor

[0084] Enzyme-catalyzed esterification was carried out in a water-jacketed glass reactor (inner diameter 3.5 cm, height 8 cm) for temperature control. Fatty acids derived from camellia seed oil (3.41 g, 1.2 mmol) and 2-ethylhexyl alcohol (1.59 g, 1.2 mmol) were placed in a flask, and the reaction was initiated by adding the enzyme. The reaction mixture was preheated to the desired temperature using a circulating constant temperature water bath and stirred at 300 rpm. Individual samples were taken at appropriate times and dissolved in chloroform (0.4 mL). The samples were then filtered through a 0.45 μm GHP Acro disc syringe filter (Pall Corporation, Port Washington, NY, USA). All reactions were repeated three times.

[0086] Enzyme-catalyzed esterification reaction in a continuous reactor

[0087] To synthesize 2-ethylhexyl fatty acid esters, enzyme-catalyzed esterification was performed from camellia seed oil and 2-ethylhexyl alcohol in a continuous reactor. A schematic diagram of the continuous reactor used in this experiment is shown in Fig. 1. The continuous reactor consisted of a 265 mm long glass column (8 mm inner diameter) with a water jacket; 3 g of dried immobilized enzyme was manually filled into the glass column and secured in place using a stainless steel sieve (50 mesh). 50 g of the substrate mixture was placed into a reactor consisting of a glass vessel (120 mm height / 40 mm inner diameter) with a water jacket and stirred at 600 rpm. Prior to the reaction, the glass column and substrate reactor were preheated, and the desired temperature was maintained using a circulating constant temperature water bath. The reaction mixture was pumped up the column by a metering pump (Moder QG 6, FMI, Syosset, NY, USA), and individual reaction mixtures were collected from the sampling port at selected times and diluted in chloroform.

[0089] Enzyme-catalyzed esterification reaction in a recirculating continuous reactor

[0090] The enzyme-catalyzed esterification reaction from camellia seed oil and 2-ethylhexyl alcohol to synthesize 2-ethylhexyl fatty acid esters was carried out in a recirculating continuous reactor. A schematic diagram of the recirculating continuous reactor used in this experiment is shown in Fig. 1.

[0092] Reactant Analysis

[0093] 2-ethylhexyl fatty acid esters were analyzed using a DB-1ht column (15 m x 0.25 mm id; J&W Scientific, Folsom, CA, USA) with gas chromatography (Model 3800; Varian Inc., Palo Alto, CA, USA) equipped with a flame ionization detector (FID), and the analysis conditions were as follows. The column was maintained at an initial temperature of 80°C for 2 minutes and increased to 300°C at a rate of 20°C / min. Subsequently, the column temperature was maintained at 300°C for 3 minutes. Helium was used as the carrier gas at a flow rate of 1.5 mL / min, and the split ratio was 1 / 50. The injector and detector temperatures were set to 320°C and 330°C, respectively.

[0094] In addition, the conversion to 2-ethylhexyl fatty acid ester was calculated using the following equation:

[0095] Conversion Rate (%) = 2-Ethylhexyl Fatty Acid Ester / (Fatty Acid + 2-Ethylhexyl Fatty Acid Ester) × 100

[0097] Statistical analysis

[0098] IBM SPSS Statistics 26 was used to determine significant differences among each response condition. Analysis was performed using Analysis of Variance (ANOVA), and Duncan's notation was used to test significance within a 95% confidence interval. All experiments were repeated at least three times, and results in all tables are expressed as mean ± standard deviation. The error bars in all plots represent the standard deviation between different experiments.

[0100] <Example 1>

[0101] Establishment of Optimal Synthesis Conditions for 2-Ethylhexyl Fatty Acid Esters in a Solvent-Free System via Enzyme-Catalyzed Esterification Reaction Using a Batch Reactor

[0103] <1-1> Establishment of Optimal Conditions for Synthesis of 2-Ethylhexyl Fatty Acid Esters Using Pre-type Non-GMO Candida Lugosa Derived Enzymes

[0105] <1-1-1> Selection of Optimal Catalytic Enzyme

[0106] The inventors analyzed the synthesis efficiency of 2-ethylhexyl fatty acid esters from 2-ethylhexyl alcohol and fatty acids using the following four types of lipase enzymes to identify the most effective enzyme in the synthesis of 2-ethylhexyl fatty acid esters through enzyme-catalyzed esterification reactions. The lipases used in the experiment were Lipozyme 435, Lipozyme RM IM, Lipozyme TL IM, and Lipase OF (free type form). The Lipozyme 435, Lipozyme RM IM, and Lipozyme TL IM enzymes are commercial enzymes produced through the fermentation process of genetically modified microorganisms (GMM), and the Lipase OF (free type form) enzyme is a powder-form lipase enzyme derived from non-GMM Candida rugosa. In this experiment, the activity of the enzyme was analyzed as the degree of conversion to 2-ethylhexyl fatty acid ester, and the experiment was conducted under conditions where the enzyme's water activity, temperature, molar ratio of fatty acid to 2-ethylhexyl alcohol, and enzyme amount were maintained constant at 0.225, 40°C, 1:1, and 2% (relative to total substrate weight).

[0108] Analysis results showed that during the first 2 hours of the reaction, Lipozyme 435 exhibited the highest conversion rate and initial velocity, followed by Lipozyme RM IM, Lipozyme TL IM, and Lipase OF. However, when the reaction time increased from 2 hours to 3 hours, the conversion rate of Lipase OF was found to increase significantly from 8% to 80%. Meanwhile, the conversion rates of other enzymes were found to increase steadily as the reaction time increased. However, among the enzymes used, when Lipase OF was used as a catalyst, a maximum conversion rate of approximately 82% was achieved after 4 hours, and this effect was found to be the most superior compared to Lipozyme 435, Lipozyme RM IM, and Lipozyme TL IM (Fig. 2). Additionally, the inventors showed a chromatogram of the reaction mixture in Fig. 3 after esterification reaction for 6 hours using the initial mixture of substrates and Lipase OF.

[0109] Through these results, the inventors confirmed that it is most desirable to use Lipase OF catalyst derived from non-GMM Candida rugosa as the most optimal catalytic enzyme for the synthesis and production of 2-ethylhexyl fatty acid esters.

[0111] <1-1-2> Analysis of Enzyme Water Activity

[0112] Enzyme-catalyzed reactions on hydrophobic substrates are highly sensitive to the amount of water in the system. A minimum amount of water must be present for the enzyme to maintain its structure and achieve catalytic activity and selectivity. Therefore, having an appropriate amount of water bound to the enzyme is particularly important for ensuring enzyme activity. Accordingly, the inventors investigated the effect of initial water content on Lipase OF, the optimal catalytic enzyme selected in <1-1-1>, and controlled the initial water content of Lipase OF using a water-activated chamber. In this experiment, the temperature, molar ratio of fatty acid to 2-ethylhexyl alcohol, and enzyme amount were performed under conditions of 20°C, 1:1, and 2% (relative to total substrate weight), respectively.

[0114] As a result, as shown in Figure 4, the increase in the 2-ethylhexyl fatty ester conversion rate according to the water activity of Lipase OF was found to increase steadily up to 3 hours at a water activity of 0.113, up to 2 hours at water activities of 0.225 and 0.328, and up to 1 hour at water activities of 0.432 and 0.529, after which the conversion rate increased significantly. In addition, it was found that a maximum conversion of about 88% was achieved after 4 hours at all water activities.

[0115] Reaction equilibrium was reached after 3 hours at water activity levels of 0.432 and 0.529, and after 4 hours at water activity levels between 0.328 and 0.113. It was confirmed that increasing the enzyme's water activity to a certain level has a beneficial effect on enzyme activity and shortens the reaction time. Therefore, it was confirmed that a water activity of 0.432 is the optimal condition most suitable for the synthesis of 2-ethylhexyl fatty acid esters.

[0117] <1-1-3> Temperature Condition Analysis

[0118] Reaction temperature is a critical factor in enzyme-catalyzed esterification reactions because it affects the viscosity of the substrate mixture, reaction rate, enzyme activity, and enzyme stability. However, extremely high temperatures can lead to a decrease in enzyme activity and irreversible denaturation of the enzyme protein. Furthermore, the optimal temperature for an enzyme reaction depends on reaction conditions, the type of enzyme, the melting point of the reactants, and other factors.

[0119] Accordingly, the inventors analyzed the effect of temperature on the synthesis of 2-ethylhexyl fatty acid esters according to reaction time for the Lipase OF enzyme selected in the present invention. The temperature range investigated was analyzed in the range of 10 to 60°C, and the experiment was conducted under conditions where the enzyme water activity, the molar ratio of fatty acid to 2-ethylhexyl alcohol, and the enzyme amount were 0.432, 1:1, and 2% (relative to the total substrate weight), respectively.

[0121] As a result, as shown in Figure 5, the initial reaction rate increased significantly throughout the entire reaction as the reaction temperature increased from 10°C to 20°C. However, there was no significant difference in conversion between 20°C and 40°C. Meanwhile, the initial reaction rate decreased significantly as the temperature increased further. The lowest conversion rates were observed at 50°C and 60°C throughout the entire reaction time, whereas a maximum conversion rate of approximately 90% was achieved after 3 hours at 20°C. Consequently, it was confirmed that the optimal temperature is 20°C when considering energy costs.

[0123] <1-1-4> Analysis of Enzyme Amount Conditions

[0124] Next, the number of active sites available for the reaction is directly related to the enzyme dosage, which is one of the most critical factors affecting enzyme reaction performance. An excessive amount of enzyme can be economically inefficient and negatively impacts the selectivity level provided by the enzyme itself. Therefore, selecting the optimal enzyme dosage for the reaction is also a crucial factor to ensure the reaction reaches the highest level of efficiency.

[0125] Accordingly, the inventors analyzed the effect of the amount of enzyme used in the synthesis of 2-ethylhexyl fatty acid esters according to reaction time. At this time, the experiment was performed under conditions where the enzyme water activity, temperature, and molar ratio of fatty acid to 2-ethylhexyl alcohol were 0.432, 20°C, and 1:1, respectively.

[0127] As a result, as shown in Figure 6, it was found that the time to reach equilibrium was shortened as the enzyme amount increased from 1% to 4%. The maximum conversion obtained with enzyme amounts of 1% and 2% was significantly lower than that obtained with enzyme amounts of 2% or more. However, it was found that there was no significant difference in the maximum conversion rate between 3% and 5%.

[0128] Therefore, considering the time to reach equilibrium, maximum conversion rate, and economic feasibility, it was found that the optimal amount of enzyme added is 4% (wt%).

[0130] <1-2> Establishment of Optimal Conditions for 2-Ethylhexyl Fatty Acid Ester Synthesis Using Immobilized Non-GMO Candida Lugosa-Derived Enzymes

[0131] In <1-1> above, optimal reaction conditions were identified for synthesizing 2-ethylhexyl fatty acid esters using a batch reactor with a free type, that is, an unimmobilized non-GMM derived enzyme. Below, however, reaction conditions were analyzed for the case where an immobilized non-GMM derived enzyme is used.

[0133] <1-2-1> Analysis of Enzyme Water Activity

[0134] The effect of enzyme water activity on the synthesis of 2-ethylhexyl fatty acid esters was analyzed using an immobilized non-GMM enzyme derived from Candida rugosa. The optimal enzyme water activity can vary depending on the situation, and even when synthesizing the same product, it can differ depending on reaction conditions such as the presence of substrate, enzyme source, immobilization resin, and solvent.

[0135] In this experiment, the temperature, molar ratio of fatty acid to 2-ethylhexyl alcohol, and enzyme amount were 50°C, 1:1, and 2% (relative to total substrate weight), respectively. Additionally, the range of water activity investigated was between 0.328 and 1.000.

[0137] As a result of the analysis, as shown in Figure 7, the lowest conversion rate in the overall reaction was observed at a water activity of 1.000, except for the first hour of the reaction. Although there was no significant difference in the water activity range from 0.328 to 0.843, the conversion rate of the water activity for the overall reaction was found to be slightly higher at 0.843 than at other water activity values. Specifically, at a water activity of 0.843, a maximum conversion rate of 97% was observed at 4 hours. Considering the initial reaction rate and equilibrium time, it was found that the optimal water activity of 0.843 is most appropriate when reacting the immobilized enzyme in a batch reactor.

[0139] <1-2-2> Analysis of Temperature Effects

[0140] The effect of temperature on the synthesis of 2-ethylhexyl fatty acid esters was analyzed using an immobilized non-GMM Candida rugosa-derived enzyme. The investigated temperature range was 20°C to 60°C, and the experiment was conducted at a molar ratio of fatty acid to 2-ethylhexyl alcohol, enzyme water activity, and enzyme content of 1:1, 0.843, and 2% (relative to total substrate weight), respectively.

[0142] As a result, as shown in Figure 8, the initial reaction rate increased significantly as the temperature increased from 20°C to 30°C during the first 2 hours of the reaction. Meanwhile, the initial reaction rate increased slightly as the temperature increased from 30°C to 50°C. However, it was found that the conversion rate and the initial reaction rate decreased after 2 hours as the temperature increased from 50°C to 60°C. Consequently, the maximum conversion and equilibrium time were confirmed at 50°C after 4 hours. This indicates that in a batch reactor using an immobilized non-GMM Candida rugosa-derived enzyme, carrying out synthesis at an optimal temperature of 50°C is most effective.

[0144] <1-2-3> Analysis of Enzyme Amount Conditions

[0145] Enzyme dosage conditions for the synthesis of 2-ethylhexyl fatty acid esters were analyzed using an immobilized non-GMM Candida rugosa-derived enzyme. The amount of added enzyme was set to a range of 1–3% (relative to the total substrate weight), and the experiment was conducted under conditions where the enzyme water activity, temperature, and molar ratio of fatty acid to 2-ethylhexyl alcohol were 0.843, 50°C, and 1:1, respectively.

[0147] As a result, as shown in Figure 9, it was found that the conversion rate increased significantly as the enzyme loading increased from 1% to 2% over the entire reaction time. However, no significant difference in conversion rates was observed between 2% and 3% in the entire reaction. When using an enzyme amount of 2%, it was found that a maximum conversion of approximately 94% was achieved after 4 hours of reaction.

[0148] These results indicate that when using immobilized non-GMM Candida rugosa-derived enzymes, using an enzyme amount of 2% in a batch reaction is the optimal condition for synthesizing 2-ethylhexyl fatty esters.

[0150] <Example 2>

[0151] Establishment of Optimal Synthesis Conditions for 2-Ethylhexyl Fatty Acid Esters in a Solvent-Free System via Enzyme-Catalyzed Esterification Reaction Using a Continuous Reactor

[0152] The synthesis of 2-ethylhexyl fatty acid esters using a continuous reactor was performed using an immobilized enzyme derived from non-genetically modified Candida rugosa, and experiments were conducted to determine the optimal synthesis conditions for 2-ethylhexyl fatty acid esters in a continuous reactor and a recirculating continuous reactor.

[0154] <2-1> Establishment of Optimal Conditions for Synthesis of 2-Ethylhexyl Fatty Acid Esters Using Immobilized Non-Genetically Modified Candida Lugosa-Derived Enzymes in a Continuous Reactor

[0155] 2-ethylhexyl fatty acid esters were synthesized in a continuous reactor via an immobilized enzyme-catalyzed esterification reaction. The immobilized enzymes used in the experiment were non-GMO Candida Rugosa-derived Lipase OF (Candida Rugosa) enzymes and enzymes immobilized on a carrier using Lewatit VP OC 1600 (hydrophobic resin) as catalysts. The effect of temperature on the synthesis of 2-ethylhexyl fatty acid esters using 2-ethylhexyl alcohol and fatty acids was investigated. The experiment was conducted under conditions where the molar ratio of fatty acid to 2-ethylhexyl alcohol and the amount of enzyme loaded into the continuous reactor were 1:1 (molar ratio of fatty acid to 2-ethylhexyl alcohol) and 3g (amount of enzyme added, wt%), respectively, and the temperature range investigated was 20–60°C.

[0157] As a result, as shown in Figure 10, the conversion rate was found to increase significantly as the temperature increased from 20 to 50°C during a reaction time of 5 to 60 minutes. However, during the same period, the conversion rate was found to decrease as the temperature increased from 50°C to 60°C. The fastest equilibrium time was observed at 50°C, and the maximum conversion rate was confirmed to be 93% at a reaction time of 40 minutes. Meanwhile, after a reaction time of 90 minutes, no significant difference was observed in the conversion rates obtained at 50°C and 60°C.

[0158] Through this, the inventors found that, considering energy costs, the optimal temperature for the synthesis of 2-ethylhexyl fatty acid esters using an immobilized non-genetically modified Candida rugosa-derived enzyme in a continuous reactor is 50°C.

[0160] <2-2> Establishment of Optimal Conditions for Synthesis of 2-Ethylhexyl Fatty Acid Esters Using Immobilized Non-GMO Candida Lugosa-Derived Enzymes in a Recirculating Continuous Reactor

[0162] <2-2-1> Reaction Time Analysis

[0163] The recirculating continuous reactor (RPBR), also known as the batch mode continuous reactor, is a practical and efficient reactor that exhibits high conversion efficiency. The main advantage of the continuous reactor in immobilized enzyme-catalyzed reactions is that there is no collision between enzyme particles and the impeller, which can reduce the decrease in enzyme activity and, compared to stirred tank reactors, reduces liquid damage. To analyze the effect of reaction time on the synthesis of 2-ethylhexyl fatty acid esters in the continuous reactor, the temperature, the molar ratio of fatty acid to 2-ethylhexyl alcohol, and the amount of enzyme loaded into the reactor were 50°C, 1:1, and 3g (6% of the total substrate weight), respectively, and the substrate residence time in the reactor (column) was 1 to 9 minutes.

[0165] As a result, as shown in Figure 11, the initial reaction rate was found to increase rapidly at all temperatures during a reaction time of 30 minutes. However, during reaction times between 30 and 120 minutes, the conversion rate was found to gradually increase as the substrate residence time in the reactor decreased from 9 minutes to 3 minutes, and the fastest equilibrium times were observed at substrate residence times of 1 minute and 3 minutes, with maximum conversion rates of approximately 90% and 91%, showing no significant difference.

[0166] Based on these results, the inventors found that, considering the energy efficiency of the pump, it is most effective to perform the esterification reaction for a total reaction time of 30 to 60 minutes while keeping the residence time of the substrate in the reactor fixed at 3 minutes.

[0168] <2-2-2> Temperature Condition Analysis

[0169] Next, the inventors analyzed the effect of temperature on the synthesis of 2-ethylhexyl fatty acid esters in a recirculating continuous reactor. The reaction was carried out under conditions of a reaction time of 3 min, a molar ratio of fatty acid to 2-ethylhexyl alcohol, and an amount of enzyme loaded into the reactor of 1:1 and 3 g (6% of the total substrate weight), respectively, and the temperature range investigated was 30 to 60°C.

[0171] As a result, as shown in Figure 12, no significant difference in the initial reaction rate was observed at any temperature. However, the conversion rate was found to increase significantly during the reaction time of 30 to 120 minutes as the temperature increased from 30°C to 50°C. Meanwhile, there was no significant difference in the conversion rate between 50°C and 60°C during the entire reaction time, and the fastest equilibrium time was observed at temperatures of 50°C and 60°C.

[0172] Therefore, the inventors found that, considering energy costs, it is most appropriate to select and perform the operation at an optimal temperature of 50°C.

[0174] <2-2-3> Enzyme Amount Analysis

[0175] The effect of the amount of enzyme loaded into the reactor on the synthesis of 2-ethylhexyl fatty acid esters according to reaction time was investigated. To this end, the analysis was performed with the enzyme addition ranged from 3 to 12% (based on the total weight of the substrate), and the reaction was carried out under conditions of reaction time, temperature, and molar ratio of fatty acid to 2-ethylhexyl alcohol at 3 minutes, 50°C, and 1:1, respectively.

[0177] As a result, as shown in Figure 13, the initial reaction rate was found to increase significantly as the enzyme amount increased from 3% to 6%, while there was no significant difference in the conversion rate when the enzyme addition amount was 9% and 12% over the entire reaction time. A similar trend was also observed at the time of reaching equilibrium.

[0178] Through this, the inventors were able to determine that the optimal condition is an enzyme addition amount of 9% relative to the substrate weight.

[0180] From the above results, the inventors found that 2-ethylhexyl fatty acid esters can be synthesized more effectively when using the lipase enzyme derived from non-genetically modified microorganisms according to the present invention compared to enzymes derived from conventionally genetically modified microorganisms, and found that 2-ethylhexyl fatty acid esters can be obtained very efficiently when using the lipase enzyme derived from non-genetically modified microorganisms in batch reactors, continuous reactors, and recirculating continuous reactors, and when applying the optimal synthesis conditions identified from the results of the above examples.

[0182] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

Claim 1 (1) a step of preparing a substrate by mixing 2-ethylhexanol and a fatty acid; and (2) a step of synthesizing a 2-ethylhexyl fatty acid ester by adding a lipase derived from a non-genetically modified microorganism (non-Gmm) Candida rugosa to the substrate and esterifying it, wherein the 2-ethylhexanol and the fatty acid in step (1) are mixed in a molar ratio of 1:1 to 2:1, and the lipase in step (2) is a non-immobilized, free-type lipase with a water activity of 0.2 to 1.0 and is added in an amount of 2 to 10 weight% based on the weight of the substrate. Claim 2 A method for producing 2-ethylhexyl fatty acid ester in a solvent-free manner, characterized in that, in claim 1, the fatty acid is a fatty acid derived from camellia seed oil. Claim 3 A method for producing 2-ethylhexyl fatty acid ester in a solvent-free manner, wherein, in paragraph 2, the fatty acid is a hydrolyzed fatty acid of camellia seed oil and is one or more selected from the group consisting of palmitic acid, stearic acid, oleic acid, and linoleic acid. Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 delete Claim 8 A method for producing 2-ethylhexyl fatty acid ester in a solvent-free manner, wherein, in claim 1, the ester reaction is carried out at 20 to 50°C for 1 to 4 hours. Claim 9 A method for producing 2-ethylhexyl fatty acid ester in a solvent-free manner, characterized in that, in claim 1, when performing an esterification reaction in a batch reactor, a substrate is prepared by mixing 2-ethylhexanol and a fatty acid in a molar ratio of 1:1, a non-immobilized free-type lipase having a water activity of 0.432 is added to the substrate at 4% by weight based on the total weight of the substrate, and an esterification reaction is carried out at a temperature of 20°C for 1 to 4 hours to synthesize 2-ethylhexyl fatty acid ester. Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete

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