KR100883397B1 - Method for producing bio-diesel in supercritical fluid condition using lipase - Google Patents

Abstract

The present invention relates to a biodiesel production method comprising the step of simultaneously applying a supercritical fluid to an enzyme process for producing biodiesel using lipase as a substrate with fats and alcohols.

According to the present invention, a biodiesel production method for simultaneously applying supercritical carbon dioxide to an enzymatic process using two kinds of lipases, 1.3-position specific lipase and non-position specific lipase, minimizes the production time of biodiesel and produces The efficiency can be maximized.

Fat, alcohol, lipase, biodiesel, 1.3-position specific lipase, positionless specific lipase, supercritical carbon dioxide

Description

Method for producing biodiesel using lipase in supercritical state {Method for producing bio-diesel in supercritical fluid condition using lipase}

1 is a graph showing the conversion of biodiesel with time at lab scale and bench scale using immobilized Candida anthica lipase at atmospheric pressure.

Figure 2 is a graph showing the conversion with time while changing the temperature when producing biodiesel from soybean oil using immobilized candida antarica lipase in a supercritical state.

FIG. 3 is a graph showing the conversion rate over time when biodiesel is produced from soybean oil using immobilized candida antarica lipase in a supercritical state.

FIG. 4 shows lipose oralase lipase ( R. oryzae ), candida antarica lipase ( C. antartica ), candida lugosa lipase ( C. rugosa ), Pseudomonas fluorescein lipase ( P. fluorescenes ) immobilized in a supercritical state. The productivity of biodiesel using

FIG. 5 shows lipases mixed with candida antartica lipase (lizofoss orisase lipase: candida lugosa lipase = 1 when producing biodiesel from soybean oil according to the amount of enzyme relative to soybean oil in the supercritical state. It is a graph comparing the productivity of 1).

6 is a graph comparing the conversion rate when biodiesel is produced from soybean oil while changing the ratio of Candida lugosa and lyphos oriase from 1: 3 to 3: 1 in the supercritical state.

FIG. 7 is a graph showing different conversions in time when biodiesel is produced from soybean oil using a mixture of immobilized lyphos lipase lipase and immobilized candida lugosa lipase in a 1: 1 (w: w) and supercritical carbon dioxide. to be.

 The present invention relates to a method for producing biodiesel using lipase in a supercritical fluid condition. More particularly, the present invention relates to a supercritical fluid in an enzymatic process for producing biodiesel using lipase as a substrate with fat or alcohol. It relates to a biodiesel production method comprising the step of applying simultaneously.

Global demand for oil is increasing due to the rapid increase in the use of automobiles. However, petroleum reserves are limited, and since 1997 the agreement in Kyoto has included a policy that strongly recommends mandatory use of bioenergy as an alternative to the regulation of pollution and the declaration of Johannesburg in 2002. The development of new energy resources is urgently needed. Many alternative energies are being developed, but in most cases they are difficult to use because they cannot be used. However, biodiesel is very similar to the existing petroleum diesel, and can be used as a direct fuel without changing the engine in the existing diesel vehicle, and it is spotlighted as an alternative energy because little pollution occurs. In addition, there is no problem to be exhausted by producing animal and vegetable oil as a raw material, and it is evaluated as a new renewable energy because it can be produced as waste oil. Biodiesel is regarded as a clean energy source because it can reduce the emission of soot by more than 20% and is biodegradable and less toxic than conventional petroleum diesel. In addition, biodiesel may be used as a raw material of lubricating oil, lubricating oil additives, etc. as well as alternative fuel oil, and may be used as various pollution-free solvents and biopesticides. In addition, since it can be used as a catalyst for bioremediation for beach purification contaminated by crude oil, it has been spotlighted as a clean energy that can be used in various fields. Developed countries are focusing on biodiesel as a national energy strategy commercialization. In Europe, the company is expanding investment by establishing the ERMA (Eropean Renewable Raw Material Association) and has already started using biodiesel in diesel vehicles. In the US, biodiesel is mixed with diesel, and is used as fuel for public vehicles, large trucks, and public buses. By 2020, 20% of diesel is replaced with biodiesel. have.

Biodiesel is a fatty acid methyl ester produced by transesterification of animal and vegetable oils with alcohol. Currently biodiesel is produced by synthesis method using acid-base catalyst at home and abroad. However, the process using acid-base catalyst requires a multi-stage reaction process and must be neutralized and washed in the production process by generating engine corrosion by the catalyst. Glycerol obtained as a by-product also contains a catalyst and must be separated in order to be commercialized. In addition, it is difficult to recover by-products and environmental problems due to the generation of large amounts of wastewater, which also causes treatment costs. Therefore, there is a need for a new environment-friendly process with low energy requirements that can meet the demand of large-scale biodiesel. For this reason, an enzyme process using a lipase, an enzyme, in the production process for producing biodiesel has been in the spotlight. In the enzymatic process, there is no chemical catalyst to corrode the engine, which simplifies the separation and purification process, and lowers the production cost of biodiesel by commercializing glycerol obtained as a by-product. In other words, the enzymatic process enables not only commercialization of biodiesel but also environmentally friendly processes without causing environmental pollution. However, the biodiesel production process using enzymes has a drawback in that the reaction time is longer than that of chemical catalysts.

Accordingly, the present inventors have made diligent efforts to overcome the problems of the prior arts, and when the supercritical fluid is simultaneously applied to the enzyme process for producing biodiesel using lipase, it is possible to compete in the reaction time of the chemical process. The present invention was completed, confirming that not only the shortened reaction time but also a high yield of biodiesel can be produced.

Accordingly, a main object of the present invention is to provide a method for producing biodiesel using lipase in a supercritical state so as to shorten the reaction time and obtain a high yield in an enzyme process for producing biodiesel.

According to one aspect of the invention, the present invention is a biodiesel production method characterized in that the enzyme process in the production of biodiesel using a lipase as a substrate with an oil and alcohol, the enzyme process in a supercritical state To provide.

In the present invention, biodiesel (bio-diesel) means a fuel made from animal and vegetable fats and oils. Biodiesel is a fatty acid methyl ester (FAME) produced during the transesterification reaction that produces glycerol and fatty acid methyl esters from triglycerides and alcohols in which glycerol is bound to trivalent fatty acids. Biodiesel has the advantage of being biodegradable, nontoxic, and free of carcinogenic pollutants such as sulfur or aromatics. In addition, since the fatty acid methyl ester of biodiesel may contain 10% or more of oxygen in the molecule, even if an expensive oxygen additive is not added, the fatty acid methyl ester assists in the complete combustion to reduce the emission of dust or carbon monoxide. The present invention relates to the production of biodiesel by applying supercritical carbon dioxide to an enzymatic process, characterized in that the use of supercritical carbon dioxide to minimize production time and maximize production efficiency in biodiesel manufacturing.

In the present invention, the supercritical state is defined as the state of the supercritical fluid, that is, the state in which the substance is above the critical temperature and the pressure, and the supercritical state is inferior to the incompressible solvent which does not show a large change in physical properties because the distance between molecules is hardly changed. The fluid brings about a great change in physical properties such as density, viscosity, diffusion coefficient and polarity. Therefore, supercritical fluids are attracting attention as new innovations that can solve the technical difficulties of existing reactions due to high dissolving power, fast mass transfer and heat transfer, low viscosity, high diffusion coefficient and low surface tension. Complementary can enable the commercialization of biodiesel.

In the biodiesel production method of the present invention, the enzyme process in the supercritical state is preferably reacted at a pressure of 100 to 150 bar, a temperature of 40 to 60 ℃ and a stirring speed of 100 to 300 rpm, preferably a pressure of 120 bar, temperature It is characterized in that the 50 ℃ and the stirring speed 200 rpm.

As in the embodiment of the present invention, the conversion rate of the biodiesel was increased with increasing pressure and rapidly increased after 100 bar, showing the highest conversion rate at 130 bar, and at a higher pressure, the conversion rate was slightly decreased. In addition, the conversion rate increased with increasing temperature, but the highest conversion rate was shown at 50 ° C., and the conversion rate dropped sharply at higher temperatures.

In the biodiesel production method of the present invention, the fluid for making the supercritical state may be any substance that can be introduced into the enzymatic process in the supercritical state, but is preferably supercritical carbon dioxide.

As in the present invention, the use of supercritical carbon dioxide can rapidly increase the reaction rate due to high dissolving power, fast material transfer and heat transfer, low viscosity, high diffusion coefficient and low surface tension, and thus disadvantages of biodiesel production using an enzymatic process. This can compensate for the disadvantages of slow reaction rates.

In fact, in the present inventors patent registration No. 10-0673837-0000 using the enzyme process, the reaction time is 21 hours, compared to the production process using the chemical catalyst requires a further process to compensate for this takes longer. Therefore, the use of supercritical fluid in the enzymatic process can reduce the reaction time to compensate for the disadvantages of the enzymatic process.

In the biodiesel production method of the present invention, the lipase is characterized by using 1.3-position specific lipase and positionless specific lipase at the same time.

The origin of the 1,3-position-selective lipase and non-selective lipase used in the biodiesel of the present invention is not particularly limited. For example, lipases may be lipases derived from intestine, milk, and the like, vegetable lipases (lipases derived from the inside of seeds such as wheat, soybeans, cotton, etc.), and microbial lipases (like bacteria, bacteria, yeast). Specifically, the 1,3-position-selective lipase is derived from Rhizopus , Aspergillus or Mucor , or lipase of pancreatic lipase or rice bran of the pancreas. Can be. Non-selective lipases may also be derived from Staphylococcus aureus , Penicillium cyclopium , Corynebacterium acnes , Candida rugosa , and the like.

In an embodiment of the present invention, 1,3-position-selective lipase was used as Rhizopus oryzae lipase, and positionless-selective lipase was used as Candida rugosa lipase.

Lipases can be classified into 1,3-position-selective lipases and positionless-selective lipases according to site specificity. The term “1,3-specific lipase” of the present invention refers to a lipase which exhibits reaction specificity only with a fatty acyl group at the 1-, 3-position of triglyceride. . By “non-specific lipase” in the present invention is meant a lipase that reacts with all three fatty acyl groups of all triglycerides.

It has been known that the time to reach 90% conversion in biodiesel production using immobilized general lipase alone is over 50 hours, and over 60 hours in the case of immobilized lyphosose lipase lipase. (M. Kaieda et al., J. Bioscience and Bioengineering, 88, 6, 627-631, 1999). In addition, the enzyme process using 1,3-specific lipase and non-specific lipase is known to take more than 18 hours to reach a conversion rate of more than 90%. (DH Lee et al., "Method for preparing biodiesel using 1,3-position-selective lipase and non-position-selective lipase", domestic patent, 1006738370000).

In fact, when producing the biodiesel to which the supercritical carbon dioxide of the present invention is applied, it was confirmed that the conversion rate of about 99% was reached in 3 hours after the start of the reaction and 99.8% in 4 hours. This is produced by reducing the reaction time of the enzyme process using the two existing lipases more than four times, and produced by reducing the reaction time by more than 15 times than the process using a single enzyme. Therefore, the method of the present invention using supercritical carbon dioxide simultaneously in a process using 1,3-position-specific lipase and non-position-specific lipase can shorten the reaction time to maximize biodiesel production efficiency of high conversion rate.

In the present invention, the addition ratio of the 1.3-position specific lipase and the positionless specific lipase may be variously controlled, but may be used in a weight ratio of 1: 3 to 3: 1, preferably in a 1: 1 weight ratio. It is characterized by. As shown in FIG. 6 of the present invention, in the graph comparing the productivity of biodiesel according to the mixing ratio of enzymes in the production of biodiesel, the mixing ratio of candida lugosa and lysophosphorase was 1: 1, 2: 1, 3: 1, 1: 2, 1: 3 and compared with biodiesel productivity. As a result, the productivity of the biodiesel did not show much difference, but the ratio of candida lugosa and lysophosphorase was 1: 1. When the present invention shows the highest conversion rate in the production of the biodiesel of the present invention was confirmed that the enzyme ratio of 1: 1 is most suitable in the process using a supercritical fluid.

In the biodiesel production method of the present invention, the lipase is preferably 15 to 30 parts by weight based on 100 parts by weight of the fat or oil, and preferably 20 parts by weight based on 100 parts by weight of the oil or fat. As shown in FIG. 5 of the present invention, the amount of Candida antarctica and Mixed enzyme (Kendida Lugosa, Rizofossuraize) is 5% to 30% (w / w) of the amount of oil at 5% intervals. In the experiment, the conversion rate did not increase significantly above 20%, and the most appropriate amount of enzyme was confirmed as 20% (w / w) of the oil mass.

In the biodiesel production method of the present invention, the lipase may be used in a free form or an immobilized form, but is preferably an immobilized lipase.

The method of immobilizing lipase in the present invention is not particularly limited and various applications are possible. For example, physical methods such as adsorption method, enclosing method, chemical methods such as covalent bonding method and crosslinking method can be used. In an embodiment of the present invention, a lipase solution and glutaraldehyde react with a carrier having an amino group to cause a crosslinking reaction between the lipase and the carrier to immobilize the enzyme on the membrane.

In the biodiesel production method of the present invention, the vegetable fat or oil may be used for the production of biodiesel, but vegetable fat is preferable. In addition, it may be selected from natural oils, processed oils or waste oils, specifically may be selected from soybean oil, rapeseed oil, palm oil and the like.

In the biodiesel production method of the present invention, the alcohol may be an alcohol having 2 to 8 carbon atoms, preferably an alcohol having 2 to 4 carbon atoms is preferable, and more preferably methanol, ethanol or butanol. do. For example, ethanol, methanol, 1-propanol, iso-propanol, 1-butanol, 2-butanol, iso-butanol or ter-butanol.

In the present invention, the biodiesel (methyl ester) produced by the method of the present invention in which the supercritical fluid is applied to an enzyme process for producing biodiesel using lipase based on fats and oils is a raw material of alternative fuel and lubricating oil. It can be used for oil-free solvents, paints and pesticides as well as lubricant additives. Biodiesel can also be used as an intermediate for environmentally friendly ethoxide methyl esters, sulfate methyl esters, and alcoholamine methyl esters to replace toxic surfactants used in the textile, detergent and cosmetic industries.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Reference Example 1. Preparation of Lipase

Candida rugosa lipase, an in situ specific lipase, was purchased from Sigma Co., USA. It was purchased from Novo Nordisk. Rhizopus oryzae lipase, a 1,3-position specific lipase, was purchased from Fluka Co., Germany.

Reference Example 2 Immobilization of Lipase

Two kinds of lipase solutions prepared in Reference Example 1 were reacted with soybean oil and 1: 1 (v / v) at 37 ° C. and 200 rpm for 30 minutes, respectively. It was immobilized on silica gel using the following cross-linking method. The immobilized carrier silica gel was pretreated with 35% (v / v) H ₂ O ₂ to remove impurities from the silica gel surface. The pretreated carrier was silanized with acetone as a solvent in 15% (w / v) of 3-APTES at 50 ° C. and 150 rpm for 120 minutes to introduce an amino group onto the surface of the carrier. Glutaraldehyde, a crosslinking agent, was pretreated in 0.1M phosphate buffer (pH 8.0) at 64 ° C. for 20 minutes, and silica gel with amino groups was introduced and 0.1M phosphate buffer solution (pH 7.0) at a concentration of 2% (v / v). ) Was reacted at 20 ° C. and 150 rpm for 2 hours. Thereafter, the silica gel was washed with distilled water, dried, and placed in 0.1 M phosphate buffer (pH 7.0). The prepared lipase was immobilized by reacting at 20 ° C. for 150 minutes.

Example 1 Production of Biodiesel from Soybean Oil Using Candida Antarctica Lipase at Normal Pressure

Biodiesel was produced on a lab scale and bench scale from soybean oil using immobilized candida anthica lipase (Novozyme 435), the most representative commercial enzyme that produces biodiesel under atmospheric pressure. In the lab scale, 1.8 g (2 mmol) of vegetable oil and 4.3 mL (10 mmol) of methanol were used as the reactor materials. Distilled water was added to the reactor at 10% (0.18 g) of the weight of soybean oil and reacted at 45 ° C. and 200 rpm for 24 hours. In addition, the amount of immobilized candida anthica lipase was 0.36 g corresponding to 20% (w / w) of soybean oil. On a bench scale, 50 g (57.25 mmol) and 98 mL (229 mmol) methanol of vegetable oil, soybean oil, was used. Distilled water was added to the reactor at 10% (5.0 g) of the weight of soybean oil and reacted for 24 hours at 50 ℃, 200 rpm. In addition, the amount of immobilized candida anthica lipase was 10 g corresponding to 20% (w / w) of soybean oil. The results are shown in FIG.

Figure 1 illustrates the productivity of biodiesel on a lab scale and bench scale using lipase commonly used at atmospheric pressure. As the reaction proceeded, the productivity of biodiesel increased but showed low productivity with a maximum conversion rate of 23%, indicating that the production of biodiesel under normal pressure was not suitable.

Example 2 Production of Biodiesel from Soybean Oil Using Candida Antarctica Lipase in Supercritical State

Biodiesel was produced from soybean oil using immobilized candida antarica lipase (Novozyme 435), a positionless specific lipase in the supercritical state. 50 g (57.25 mmol) of vegetable oil and 98 mL (229 mmol) of methanol were used as a reactor oil. Distilled water was added to the reactor at 10% (5.0 g) of the weight of soybean oil and reacted for 8 hours at 200 rpm while changing the temperature and pressure. In addition, the amount of immobilized candida anthica lipase was 10 g corresponding to 20% (w / w) of soybean oil. Each result is shown in FIGS. 2 and 3.

Figure 2 shows the productivity of the biodiesel with the change of temperature. As the temperature increased, the conversion rate of biodiesel increased, but the highest conversion rate (53.21%) was shown at 50 ° C, and the conversion rate rapidly dropped at higher temperatures. Up to 2 hours after the start of the reaction, the conversion rate of biodiesel increased rapidly, but the conversion rate no longer increased above a certain conversion rate.

3 shows the productivity of biodiesel according to the pressure change. As the pressure is increased, the conversion rate of the biodiesel is increased little by little, and it is rapidly increased after 100 bar, and shows the highest conversion rate (52.30%) at 130 bar. The pressure showed a slight decrease in conversion. As in FIG. 1, the conversion rate of biodiesel increased rapidly up to 2 hours after the start of the reaction, but the conversion rate did not increase any more than a certain conversion rate.

As described above in FIGS. 2 and 3, the reaction proceeds rapidly in the supercritical state when compared to the maximum conversion rate of 23% (see FIG. 1) at atmospheric pressure in Example 1, and the superconversion state is applied at a maximum conversion rate of about 50%. By performing the enzymatic process in a supercritical state showed a conversion rate three times closer than the prior art. However, the conversion rate is less than 60%, suggesting that a process using a different enzyme is more effective than biodiesel production using candida anthica lipase under supercritical conditions.

Example 3 Comparison of Biodiesel Productivity from Soybean Oil Using Lipase in Various Strains in Supercritical State

In the same manner as in Example 2, biodiesel was produced from soybean oil using lipase in various strains in a supercritical state.

As a result, the productivity of the biodiesel was illustrated in the supercritical state using lipases of various strains in FIG. 4, and it was shown that lysophos oliase lipase was more suitable for the production of biodiesel than candida anthica lipase. However, in biodiesel production using lysophosphorase lipase, the conversion rate does not exceed 80% even in the supercritical state, indicating that an additional process is required. Thus, in order to increase the efficiency of biodiesel production, Kendida Lugosa lipase, a positionless specific lipase, was added to enhance the reaction efficiency.

The resulting biodiesel was analyzed for the amount of biodiesel using gas chromatography (GC). GC used a Younglin M600D model and the column was HP-INNOWAX (19091N-133, 30m × 0.25㎛). The analysis was performed under the following conditions. The injector was 260 ° C, the oven was heated to 15 ° C / min from 150 ° C to 180 ° C and 5 ° C / min to 240 ° C and retained for 1 minute at 240 ° C. As a detector, FID was used at 260 ° C. Injection volume was 1 μl and the split ratio was 20: 1. Carrier gas was used as helium (He), the flow rate was 1 mL / min, the air (air) 280 mL / min and hydrogen (H ₂ ) was injected at 40 mL / min. Helium was used for make up flow, and the flow rate was 30 ml / min. Methyl palmitate, methyl stearate, methyl oleate, methyl linoleate, methyl linolenate, and methanol The product was analyzed by GC, and the number of moles of the above five FAMEs was calculated. The conversion rate of biodiesel was calculated by the following equation.

[Equation 1]

Conversion rate = (moles of FAMEs) / (moles of oil × 3) × 100

Example 4 Production of Biodiesel under Conditions Concurrently Using a Mixture of Immobilized Liposomal Lipase and Immobilized Candida Lugosa Lipase and Supercritical Carbon Dioxide

In the present invention, candida rugosa lipase (Candida rugosa lipase), which is a lipase without positional specificity that can accelerate the hydrolysis reaction in order to increase the production of biodiesel, has been introduced. The reaction was performed in a supercritical state by introducing Rhizopus oryzae lipase having 1,3-position specificity.

Several studies have reported that transesterification reactions for biodiesel production can occur when candida anthica lipase is used as an alcohol with eggplant as a substrate. It also reported that candida lugosa lipase causes a hydrolysis reaction but no transesterification.

Therefore, in the present invention, even though the conversion rate of candida anthica lipase in FIG. 4 was higher than that of candida lugosa lipase, candida lugosa lipase can cause a hydrolysis reaction so as to steam the biodiesel conversion rate. Lipase was chosen as Candida lugosa.

In the present invention, immobilized lyphos lipase lipase having an activity of 68 U / g prepared by the same method as immobilized candida lugosa lipase having an activity of 60 U / g by immobilization of candida lugosa lipase.

5 is a diagram comparing the productivity of biodiesel according to the dosage of enzyme in the production of biodiesel, the amount of Kendida Antarctica and Mixed enzyme (Kendida Lugosa, lysophosphorase) 5% ~ When the test was performed at 5% intervals up to 30% (w / w), the conversion rate did not increase significantly at 20% or more.

Figure 6 is a graph comparing the productivity of biodiesel according to the mixing ratio of the enzyme in the production of biodiesel, the mixing ratio of the Kendida Lugosa lipase (CRL) and lysophosphorase lipase (ROL) 1: 1, 2 The biodiesel productivity was compared by changing from 1: 1 to 3: 1 to 1: 2 to 1: 3. Productivity did not show much difference, but among them, the ratio of candida lugosa lipase (CRL) and lysophosphorase lipase (ROL) was 1: 1, indicating the highest conversion rate. In the process using a fluid, the enzyme ratio of 1: 1 was shown to be most suitable.

The amount of total immobilized lipase was fixed at 10 g (20%, w / w), and the ratio of immobilized lyphos lipase lipase and immobilized candida lugosa lipase was fixed at 1: 1 (w: w) to investigate biodiesel productivity. It was. 50 g (57.25 mmol) of vegetable oil and 98 mL (229 mmol) of methanol were used as the reactor oil. Distilled water was added to the reactor at 10% (5.0 g) of the weight of soybean oil and reacted for 5 hours at a stirring speed of 130 bar and 200 rpm at 50 ° C. The results are shown in FIG.

As described above, when biodiesel was produced in the supercritical state of the present invention, a conversion rate of 99% or more was obtained within 3 hours after the start of the reaction. As shown in FIG. 5, Kendida antartica lipase is most frequently used for the production of biodiesel. In comparison with the production method using the method and the method using the immobilized lyphos lipase lipase and immobilized Kendida Lugosa lipase, the productivity was increased by 88.12% and the production time was reduced from about 6 hours to 4 hours to produce biodiesel efficiently. It was confirmed that there is.

Enzyme processes using lipase of each strain in the supercritical state of the present invention are summarized in the following table.

Biodiesel is said to be a two-step hydrolysis reaction and esterification reaction, so that the hydrolysis reaction is a rate determining step, so that once the hydrolysis reaction occurs, the esterification reaction proceeds rapidly. The use of supercritical carbon dioxide here shortens the time of the rate determination step to hydrolyze 1,2-diglycerides so that it does not take time for acyl transfer of 1,2-diglycerides as 1,3-diglycerides. It was confirmed that the overall reaction rate is increased. More than 99% conversion was reached within 3 hours after the start of the reaction.

As described above, according to the present invention, by simultaneously applying supercritical carbon dioxide to a biodiesel production process using a lipase, a high conversion rate can be obtained in a short time, thereby maximizing the reaction efficiency and a disadvantage of the previously used enzyme process. It is a very useful invention suitable for producing biodiesel in an environmentally friendly way.

Claims (9)

The holding and alcohol as substrates and separation tank Force (Rhizopus), Aspergillus (Aspergillus), non-cor (Mucor) a lipase, pancreatic lipase derived from the genus from the group consisting of lipase of (pancreatic lipase) and rice (rice bran) Consisting of one or more selected 1,3-position-specific lipases, Staphylococcus aureus , Penicillium cyclopium , Corynebacterium acnes , and Candida rugosa In the method for producing biodiesel comprising an enzymatic process using at least one position-specific lipase selected from the group, using the enzymatic process as a supercritical carbon dioxide as a fluid pressure 115 ~ 150 bar, temperature 40 ~ 60 ℃, and Biodiesel production method characterized in that for 3 to 5 hours in a supercritical state of stirring speed 100 ~ 300 rpm . delete delete delete The method of claim 1, wherein the 1.3-position specific lipase and the positionless specific lipase are used in a weight ratio of 1: 3 to 3: 1. The method according to claim 1, wherein the lipase is 15 to 30 parts by weight based on 100 parts by weight of a fat or oil as a substrate.  The method of claim 1 wherein the lipase is an immobilized lipase. The method according to claim 1, wherein the fat or oil is natural fat, processed fat or waste fat. The method of claim 1 wherein the alcohol is methanol, ethanol or butanol.

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