JP2002233393A - Method for producing bio-diesel fuel (monoalkyl ester) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing a bio-diesel fuel (bio- diesel) that can be used in conventional petroleum-based diesel engines, can be recycled and scarcely pollutes. SOLUTION: This method for producing the bio-diesel is characterized by the esterification reaction of a fat or fatty oil with a lipase originated from a Cryptococcus genus yeast (FERM P-15155) in the presence of the fat or fatty oil and an alcohol. Thereby, the reaction can not only efficiently be carried out at one step without sequentially adding the alcohol, but the bio-diesel can efficiently be produced even when an organic solvent or water is contained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the efficient use of biodiesel fuel (hereinafter, simply referred to as "biodiesel") which can use the same engine as petroleum-based diesel fuel and can be reproduced to reduce pollution. It relates to a manufacturing method.

[0002]

2. Description of the Related Art As the air pollution caused by fine particles, sulfur oxides, nitrogen oxides, and carbon monoxide emitted from diesel engine vehicles becomes more serious today, alternative fuels that emit less polluting and cleaner gases are required. Became. On the other hand, it is certain that an energy crisis due to the depletion of fossil fuels such as petroleum will occur in the near future.
The production of fuels made from natural renewable raw materials (biomass) has become an important issue.

[0003] Under these circumstances, renewable biodiesel, which can use the same engine as petroleum-based diesel fuel and can greatly reduce exhaust gas emissions, has attracted attention.

[0004] Biodiesel refers to an alkyl ester obtained by transesterifying glycerol and an alcohol in a fat or oil, as represented by a methyl ester (ME) of a fatty acid produced by alcoholysis (methanolysis) of an animal or vegetable fat or oil. Things.

At present, most of this biodiesel is produced by chemical synthesis.
It has many problems, such as difficulty in recovering by-product glycerol and requiring a large amount of energy in the production process.

Accordingly, biodiesel production using lipase enzymes has attracted attention. Usually, lipase catalyzes a reaction of hydrolyzing fats and oils (triglycerides) to fatty acids and glycerol, but under conditions where water content is limited, it is possible to carry out a reverse reaction of forming ester bonds between fatty acids and alcohols. Furthermore, a method is also possible in which triglyceride is allowed to coexist with a lower alcohol such as methanol, and a lipase is allowed to act on the triglyceride to transesterify glycerol in the triglyceride with a lower alcohol such as methanol.

Since one molecule of triglyceride is composed of three fatty acids and one glycerol, three moles of lower alcohol are theoretically necessary to completely convert one mole of triglyceride into biodiesel (monoalkyl ester).

However, in biodiesel production using lipase, which has been attempted so far, 1 mol of a lower alcohol is mainly added to 1 mol of triglyceride for esterification, and when the reaction is almost completed, the reaction is further terminated. It has been carried out by multi-stage charging, for example, by sequentially adding 2 moles of alcohol one by one. This is due to the fact that generally used lipases tend to lose their activity with lower alcohols.
It is intended to sequentially add alcohol while suppressing the amount of alcohol added each time. Also, with known lipases,
In a system having a high water content or a system in which an organic solvent such as hexane or petroleum ether is present, there is a disadvantage that the esterification reaction is hindered, which is a problem particularly in industrialization.

[0009]

SUMMARY OF THE INVENTION The present invention has been made for the purpose of newly developing an efficient method for producing biodiesel (monoalkyl ester) without the above-mentioned disadvantages.

[0010]

Means for Solving the Problems As a result of various studies to achieve the above object, the present inventors focused on a milder enzymatic method than a chemical synthesis method in the production of biodiesel, and used a large number of yeasts in nature. From Cryptococcus sp.
S-2 produces and secretes a large amount of lipase in addition to enzymes such as α-amylase and xylanase, and the enzyme exhibits a neutral to alkaline and strong lipolytic property. It is stable in organic solvents such as hexane, and transesterifies glycerol and methanol in triglyceride in the presence of triglyceride and methanol to produce methyl esters of fatty acids, in other words, methyl esterification of fats and oils, that is, It was found for the first time to produce a bio-diesel.

[0011] The present invention has been made based on the above-mentioned new findings, and has not only confirmed that the present enzyme is a novel enzyme unknown by studying the physicochemical properties of the present enzyme, Has been successfully manufactured, and the present invention has been finally completed. Hereinafter, the present invention will be described in detail.

The basic technical idea of the present invention is to produce biodiesel by efficiently performing an esterification reaction by allowing lipase to act in the presence of an oil (triglyceride) and an alcohol. As the lipase, any lipase can be used as long as the transesterification reaction can be carried out in one step and / or the transesterification reaction can be carried out even in the presence of a large amount of water. is there. As such a lipase, a lipase derived from the genus Cryptococcus is exemplified, and its physicochemical properties are as follows.

(1) Action It is excellent in fat and oil decomposability and acts on triglyceride to hydrolyze to glycerin and fatty acid. It catalyzes the transesterification of glycerin and alcohol in triglycerides, in other words, the monoalkyl esterification of fats and oils.

(2) Substrate Specificity The substrate specificity of each fatty acid for triglyceride and oil is as shown in Table 1 below.

[0015] * The specific activity of each substrate was shown by% when the specific activity to triolein was set to 100.

The present enzyme efficiently decomposes tributyrin, tricaprylin, tripalmitin and triolein among triglycerides. On the other hand, triacetin, tricaprin, and trilaurin were moderate, and trimyristine and tristearin had a low decomposing ability. This enzyme showed good decomposition activity for all tested oils, and particularly degraded rice bran oil and sardine oil.

(3) Regiospecificity The regiospecificity of the degradation when the present enzyme acts on triolein was examined. Oleic acid and a small amount of 1,
2 (2,3) -Dolein was produced, and 1,3-diolein and monoolein were not detected.

(4) Optimum pH and stable pH range The optimal pH of the purified enzyme was 7.0. pH 8.0
Also showed high activity. In addition, the stability of pH
The lipase was stable from H5 to pH 9 and functioned over a wide range from weakly acidic to slightly alkaline.

(5) Optimal reaction temperature and deactivation conditions depending on temperature The optimal reaction temperature was 37 ° C. However, the activity was slowly deactivated by the temperature rise, and the activity of 71% was maintained even at a heat treatment of 60 ° C. for 30 minutes.

(6) Inhibition, activation and stabilization Various organic solvents are used at 0, 2.5, 5.0, 7.5, 10.
It was added at a concentration of 0% and the effect on lipase activity was investigated, and the results in Table 2 below were obtained. As a result, this lipase is stable in various organic solvents except benzene,
O (dimethylsulfoxide) and diethyl ether increased the activity rather, and showed a 64.2% increase in the activity of 7.5% DMSO.

[0021] * Specific activity (%) indicates specific activity at various solvent concentrations (%, v / v).

Thus, it should be particularly noted that the present lipase is stable in organic solvents. In transesterification and ester synthesis by lipase, the reaction proceeds only in a system in which an organic solvent and a small amount of water coexist.
In general, there has been a problem that lipase is unstable in a system in which such an organic solvent and water coexist.

However, the present lipase exhibits an activity and stability of 50% or more even in the presence of diethyl ether, chloroform and hexane, and is capable of transesterification and ester synthesis even in the presence of an organic solvent. Organic solvent used as an extraction solvent during the extraction of crude oils and fats (without purification)
Biodiesel can be produced, and the enzyme has sufficient activity even in the presence of a large amount of water,
In view of these points, the present enzyme is extremely excellent in terms of new production of biodiesel, particularly in terms of industrially efficient production.

(7) Method for measuring lipase activity The lipase activity was measured by an absorbance method at 410 nm using p-nitorophenyl laurate (p-NPL) as a reaction substrate. If necessary, lipase activity was measured by a titration method using olive oil as a substrate.

(8) Molecular weight 22 kDa (SDS-PAGE)

In view of the above-mentioned physicochemical properties, this enzyme has not been found in any of the conventionally known lipases, and has been identified as a novel substance. This enzyme was named lipase CS2 (here, CS2 is referred to as CS2). Lipase).

The novel enzyme according to the present invention is a microorganism, for example, strain S-2 belonging to the genus Cryptococcus (FERM P-
15155) (hereinafter sometimes referred to as a CS2 strain). The strains created and isolated here are extremely distinctive in that they can produce and secrete a novel enzyme (lipase CS2), which is called Cryptococcus sp. S-2 (Cryptococcus sp. S-2) was named by the National Institute of Advanced Industrial Science and Technology
Deposited as ERM P-15155.

The present enzyme can be obtained by culturing the CS2 strain according to a conventional method, extracting from the culture, and purifying it. For example, a YM medium (yeast extract 0.3
%, Malt extract 0.5%, peptone 0.5%, glucose 1%) at 25 ° C for 40 hours in preculture (5.4 to 5.8).
(× 10 ⁸ cells / ml) are used as inocula for normal main culture.

Yeast extract 0.5%, KH ₂ PO ₄ 1%, M
_{gSO 4 · 7H 2 O 1%} , the basic 1% triolein, optionally, nitrogen and carbon sources, and those of the inducer was varied as the enzyme production medium. 100 ml of enzyme production medium was inoculated with 1% (v / v) of the pre-cultured bacteria,
Enzyme production may be performed by shaking culture at 0 rpm. Triolein does not have to be added, but Cryptococcus sp.
The production of lipase by S-2 shows that secretion of lipase activity is about three times that of glucose medium by using oils and fats such as olive oil and triolein as a carbon source, and the production of lipase by using oils and fats as a carbon source Increased to an advantage. As the fat or oil, it is preferable to use at least one selected from sardine oil, soybean oil, and triglyceride in addition to the above.

The cultivation of the CS2 strain may be carried out in the same manner as the cultivation of yeast, but the use of fats and oils as a carbon source increases the production of enzymes as described above. Also, the use of yeast extract as a nitrogen source and lactose as a sugar increases the production of enzymes.

After culturing the CS2 strain, the present enzyme is separated from the culture and purified. The method may be performed according to a conventional method. For example, after concentrating a culture solution, a known purification method such as chromatography may be used in combination. In addition, the enzyme does not necessarily need to be highly purified,
For example, a concentrate obtained by ultrafiltration of a culture supernatant can be sufficiently used for producing biodiesel.

Since the enzyme (CS2 lipase) thus obtained has the above-mentioned characteristics, it can catalyze a transesterification reaction or an ester synthesis reaction even in the presence of an organic solvent or a large amount of water. production of biodiesel, in particular can be used not only in industrial production, properties possessed by conventional lipases that oil hydrolysis also of them together, pharmaceutical, cosmetic, food and drink, detergents, widely reagent other applications It goes without saying that it can be used.

In order to carry out the present invention, glycerol and alcohol in fats and oils may be transesterified with lipase in the presence of fats and alcohols.
The oil, fat, alcohol and lipase may be mixed and reacted while stirring, if necessary.Moreover, if the lipase as described above is used, there is no need to add alcohol sequentially, and the reaction can be performed in one step. In that case, even if an organic solvent or a large amount of water is mixed, the reaction proceeds smoothly and a remarkable effect is exhibited.

As described above, the present invention relates to the yeast Cryptococcus s
By using lipase produced by p.S-2, 1 mol of triglyceride can be reacted by adding 3 mol or more of lower alcohol from the beginning to simplify the lipase biodiesel production process. Was made possible without the need to add alcohol sequentially.
It is industrially outstanding.

In general, lipase causes a reaction that hydrolyzes the original triglyceride in a state of a large amount of water, and the reverse reaction, esterification, is mainly carried out only when water is severely restricted. But Cr
The lipase produced by yptococcus sp. S-2 has an excellent property that the reaction proceeds even at a water content where ordinary lipase prevents esterification. Water is inevitably generated in the esterification reaction, and the accumulation of the produced water prevents the esterification reaction by the lipase, but the Cryptococcus sp. S-2 lipase does not.

[0036] In addition, many waste oils from homes, which are used as a raw material of biodiesel in Japan, often contain a large amount of water, but Cryptococcus sp. S-2 lipase is not so concerned about the moisture in such fats and oils. It can be used as a raw material for biodiesel.

Cryptococcus sp. S-2 lipase shows particularly high ester productivity for rice bran oil among various animal and vegetable oils. Rice bran oil is a cheap oil obtained from rice bran,
It can be produced abundantly in Japan. Cryptococcus s
The ability of p.S-2 lipase to produce biodiesel from rice bran oil particularly efficiently is useful for effective use of rice bran.

Although the activity of ordinary lipase is reduced in the presence of hexane and petroleum ether, Cryptococcus sp.
Conversely, the activity of S-2 lipase increases when these organic solvents are mixed. In Cryptococcus sp. S-2 lipase, esterification efficiency is improved by the mixture of hexane and petroleum ether in the esterification reaction of triglyceride and lower alcohol. This is because hexane and petroleum used when extracting oils and fats from rice bran etc. It shows that it is possible to produce biodiesel more efficiently by using crude oil and fat that does not completely remove ether as a raw material,
This is a very advantageous point in cost and production process.

As embodiments of the present invention, the following embodiments are exemplified. (1) One-step biodiesel production method using lipase. (2) Production of biodiesel by lipase in a state of high water content. (3) A method for producing biodiesel using the lipase of yeast Cryptococcus sp. S-2. (4) Biodiesel production from rice bran oil. (5) Biodiesel production from crude oils and fats mixed with hexane, petroleum ether and various organic solvents described above.

As fats and oils on which lipase is allowed to act, one or more kinds of fats and oils derived from animals (including fish) and plants can be used, and non-limiting examples include the following. Vegetable oils such as olive oil, rapeseed oil, soybean oil, rice bran oil, walnut oil, sesame oil, camellia oil and peanut oil.
Animal oils such as butter, lard, beef tallow, sheep tallow, chicken tallow, chicken oil and the like. Fish oils such as whale oil, sardine oil, herring oil, cod liver oil.

In the present invention, the alkyl esterification reaction of fats and oils is not hindered by the presence of an organic solvent or a large amount of water. Fats and oils and mixtures of these with fats and oils can also be used. Therefore, solid fats and oils can also be used after dissolving in a solvent. Further, since the method of the present invention is not affected by an organic solvent or moisture, it is possible to appropriately use fats and oils produced by any method, for example, squeezing, extraction, elution, etc. Fats and oils can be used as appropriate without going through a special purification step.

As the alcohol, various alcohols other than lower alcohols such as methanol can be appropriately used, and non-limiting examples thereof include methyl alcohol, ethyl alcohol, propyl alcohol, isopropyl alcohol, and the like. Aliphatic alcohols such as butyl alcohol, isobutyl alcohol, s-butyl alcohol, t-butyl alcohol, amyl alcohol and hexyl alcohol; unsaturated aliphatic alcohols such as allyl alcohol and propargyl alcohol; alicyclics such as cyclohexanol and cyclopentanol Alcohols; aromatic alcohols such as benzyl alcohol and cinnamyl alcohol; and various other alcohols.

In order to carry out the present invention, it is sufficient to mix and incubate oils, fats, alcohols and lipases, and even if water and organic solvents are mixed in the reaction system, the reaction is not particularly inhibited. Since it has been confirmed that the conversion ratio may increase, the reaction conditions are not particularly limited and may be appropriately set in a wide range. Particularly preferred reaction conditions are as follows.

The amount of the enzyme is 300 to 5000 U, preferably 500 to 3000 U, more preferably 1500 to 250 U.
0 U, the amount of water is 5 to 500 wt.
%, The reaction is not inhibited, and on the contrary, the water content is 60%.
In the presence of １００100 wt%, an increase in the amount of ester was observed. When the amount of alcohol was used at a ratio of 1: 1 to 1: 4 with respect to the fat or oil, when the amount of water was small (for example, 50% or less), It was recognized that the lower the ratio of the alcohol amount was, the higher the esterification ratio was, and the higher the water amount was (for example, 50% or more), the higher the alcohol ratio was, the higher the esterification ratio was.

Regarding the reaction conditions, no particular reaction inhibition was observed at pH 5 to 9 and temperature of 25 to 40 ° C., and the reaction pH and reaction temperature can be selected in a wide range. The reaction time is also within an appropriately selected range, for example, the reaction is continued until the desired esterification rate is achieved. However, as a rough guide, the intended purpose is achieved in about 50 to 200 hours. The reaction is preferably carried out while stirring at about 50 to 300 rpm.

In the method of the present invention, the reaction is inhibited by the presence of dimethyl sulfoxide (DMSO), diethyl ether, n-hexane, petroleum ether, benzene and other organic solvents for extracting fats and oils as well as the various organic solvents described above. The reaction was not carried out, and no reaction inhibition was observed at 30% or less. On the contrary, depending on the type of the organic solvent, for example, DMSO,
When n-hexane and petroleum ether were added in an amount of 5 to 15%, an increase in the esterification rate was observed. Therefore, the esterification rate can be even increased by adding an organic solvent. Hereinafter, examples of the present invention will be described.

[0047]

Example 1 Yeast extract 0.5%, lactose 0.
5%, KH_TwoPO_Four 1%, MgSO _Four・ 7H_TwoO 1%,
Preculture on lipase production medium consisting of 1% triolein
1% of cultured CS2 bacteria (FERM P-15155)
(V / v) Inoculate, initial pH 5.6, 25 ° C, 100r
The cells were shake-cultured at pm for 120 hours.

The purification of the enzyme was carried out as follows. The culture solution from which the yeast cells had been removed was centrifuged at 8,000 rpm for 10 minutes and filtered through a 0.45 μm membrane filter.
Concentrated by ultrafiltration. Cation exchange resin TSK-gel SP
-5PW column for high performance liquid chromatography (pH 7.
The lipase was purified by a gradient elution with 0 phosphate buffer and 0.5% NaCl added thereto. The activity was 17.1 times and the yield was 11.4%. It becomes a single band on SDS-PAGE and the molecular weight is D
SS-PAGE determined 22 kDalton. The physicochemical properties of the obtained enzyme (CS2 lipase) were as described above.

[0049]

Example 2 Yeast Cryptococcus sp. S-2 was cultured in a lipase-producing medium (Example 1) for 120 hours at 25 ° C., and the resulting culture was centrifuged at 8,000 rpm for 20 minutes. The solution was concentrated using a 10,000 molecular weight cut ultrafiltration membrane (YM-10) manufactured by the company to obtain an enzyme solution.

[0050]

Example 3 A: Methyl esterification (methanolysis) of fats and oils was performed by reacting fats and oils with methanol using CS2 lipase.

(1) Lipase activity The lipase activity is determined by comparing p-nitrophenyl lauric acid (p
-NPL) as a substrate. An enzyme producing 1 micromole of p-nitrophenol was defined as one unit of the enzyme activity based on a standard activity measurement method.

(2) Methyl esterification Oil and methanol 9.65 g / 0.35 g (mol / mo)
1 = 1/1) was used as the initial substrate. 500 for this
A 4 ml enzyme solution containing U CS2 lipase was added to the reaction mixture in a 100 ml stoppered Erlenmeyer flask to obtain a reaction mixture.
The reaction was carried out by shaking at 160 rpm at 160C for 96 hours. Samples were collected every 24 hours and analyzed by capillary gas chromatography as follows.

(3) Reaction optimization study The amount of the enzyme was changed in the range of 500 to 2,500 U, the amount of water to the substrate weight was changed to 13 to 200 wt%, and the amount of methanol was changed to the oil in the range of 1: 1 to 1: 4. The reaction conditions were studied.
Further, the reaction conditions were examined at pH 5 to 9 and at a temperature of 25 to 40 ° C. The effects of adding various organic solvents (10%, w / v) were also examined.

(4) Capillary Gas Chromatographic Analysis A 300 microliter sample was independently taken three times from the reaction solution at regular intervals and centrifuged to obtain a supernatant. Fifteen
In a ml test tube, 3.0 microliters of hexane was added to 100 microliters supernatant, 60 microliters of tricaprin as an internal standard and anhydrous sodium sulfate as a dehydrating agent, followed by thorough mixing. A 1.0 microliter sample was taken therefrom and placed in a DB-5 capillary column (0.25 mm × 15 m; J & W Scienti).
ME (methyl ester) and FFA (free fatty acid content) in the reaction product were measured by a glass chromatogram (GC-17A, manufactured by Shimadzu) using Fic, Forsom, CA, USA). Injector and detector temperatures are 245 and 250 ° C., respectively. After maintaining the column at 150 ° C. for 0.5 minute, the temperature was raised to 210 ° C. at 5 ° C./min and thereafter to 300 ° C. at 10 ° C./min.

B: Yeast Cryptococcus sp. Obtained in Example 2. Using a crude purified product from S-2 as lipase, methyl ester of vegetable oil was performed.

Table 1 shows the results of examining the esterification reaction of methanol with olive oil, rapeseed oil, rice bran oil, and soybean oil using this lipase. Refer to the experimental method for the reaction conditions.
The rice bran oil showed an esterification rate of 24.9% in the reaction at 30 ° C. for 96 hours, showing the best result. From this, the subsequent experiments decided to use rice bran oil as the base oil.

(Table 1) Table 1: Cryptococcus sp. Esterification by S-2 derived lipase (a) ──────────────────────────── Ester conversion (%) (b) Oil Fat ───────────────────── 24 hours 48 hours 72 hours 96 hours ──────────────────── ──────── Olive oil 7.5 9.7 12.5 16.7 Rapeseed oil 9.4 14.3 17.6 21.1 Rice bran oil 9.8 16.6 20.2 24.9 Soybean oil 9.6 12.9 16.9 21.1 ──────────────────── ──────── (a) The reaction was carried out by using 4 ml of 9.65 g fat and oil and 0.35 g methanol (1: 1, mol / mol) containing 500 U of lipase.
One liter of water was added and performed. (B) The esterification rate was expressed as the ratio of methyl esterification to the fat used as a substrate.

(1) Effect of enzyme amount (on methyl esterification of rice bran oil) Next, the reaction was carried out at a lipase addition amount of 500 U to 2,000 U. As shown in FIG. 1, the esterification rate increased with an increase in the amount of enzyme.
Showed the esterification rate of This corresponds to the maximum value of the ester ratio obtained when 1 mol of methanol is added to an oil or fat having 3 mol of fatty acids in one molecule (FIG. 1).

(2) Influence of water (on the methyl esterification of rice bran oil) Under the conditions of a water content of 13% and 48 hours, the methyl ester content (ME) was 31.5%, and 94.6 in the reaction solution. %
Methanol had been consumed. 30 and 50% moisture
As the amount increases, the amount of ester produced at 48 hours increases to 8.
It decreased to 9 and 16.5%, but as shown in Table 2, there was almost no difference in the amount produced after 96 hours. On the other hand, the free fatty acids after 96 hours at 13% and 50% moisture were 24.0 and 42.1%, respectively.

Table 2: Effect of water content on esterification and fatty acid production (a) ａ ────────── Water content Methyl ester content (% by weight) Fatty acid content (% by weight) (% by weight) ─────────────── ────── ───────── 24 hours 48 hours 72 hours 96 hours 24 hours 48 hours 72 hours 96 hours ───────────────────────── ─────────── 13 16.4 31.5 33.3 34.0 4.7 10.3 19.9 24.0 20 22.6 31.4 33.5 34.0 12.5 21.1 30.5 33.3 30 23.0 28.7 32.4 33.3 13.5 24.2 32.4 35.4 40 23.1 26.6 30.1 33.3 18.7 31.5 38.0 40.0 50 22.5 26.3 28.5 32.4 22.5 35.4 39.1 42.1 ──────────────────────────────────── (a) 9.65 g of oil 0.35 g of methanol (1: 1,
Mol / mol) and an enzyme solution containing 2,000 U of lipase as various enzyme solutions (1.3 to 5.0 ml).
= 13 to 50% by weight of the substrate).

(3) Influence of water content and amount of methanol added (on rice bran oil methyl esterification) At least 3 mol of methanol is required to completely convert fats and oils to fatty acid esters. However, for example, in the case of Rhizopus oryzae lipase, the enzyme is inactivated in a reaction system containing 1 mol or more of methanol. Therefore, in the esterification reaction, it is necessary to take a method of sequentially adding 1 mol of methanol.

However, if the lipase to be used does not inactivate even when the amount of methanol is increased, the operation becomes easier if the amount of methanol is increased from the beginning and the number of additions is reduced. Therefore, the ratio of fats and oils to methanol is 1: 1 to 1: 4, and the water content is 2 to the substrate weight.
The reaction was carried out by a one-step method in which 0 to 200%, each of which was initially added at once. The results, as shown in FIG. 2, show that as the methanol ratio increases (1: 1 to 1: 4) and as the water content increases in the range of 60 to 100%, the amount of methyl ester increases (FIG. 2). .

From the above, it was confirmed that Cryptococcus sp. S-2 lipase can be reacted by adding a large amount of methanol at once from the beginning. Also, Ps
In general lipases such as eudomonas fluorescens and Mucor miehei, when water is added to the reaction solution, the conversion of ester is reduced, but in the case of this lipase, the conversion is better when a certain amount of water is present. An effect was observed.

(4) Influence of pH and Temperature The effect of pH on the esterification reaction was examined under the condition of 80% added water to the substrate. The results are shown in FIG. 3. In the range of pH 5 to 9 examined, the reaction decreased as the pH increased. However, as a result, those without pH adjustment showed the highest ester conversion. This is thought to be due to the presence of the salt used for pH adjustment acting in an inhibitory manner on the enzyme reaction. As a result, it was shown that there was no need to adjust the pH (FIG. 3).

FIG. 4 shows the effect of temperature. 25, 30, 3
In the short period (24 hours) of 5, 40 ° C., the lower temperature was better, but at 96 hours, 30 ° C. was slightly higher (FIG. 4).

(5) Methanol Ratio Since 1 mol of fats and oils is composed of 3 mol of fatty acids and 1 mol of glycerol, 3 mol of alcohol is theoretically required for complete esterification of fats and oils. In the esterification of fats and oils by a synthetic chemical reaction, a reaction system using 6 moles of alcohol with respect to 1 mole of fats and oils has been employed in order to increase the esterification efficiency. Therefore, in our reaction system using an enzyme, the ratio of fats and oils to alcohol is 1: 2 to 1:
The reaction was carried out while changing in the range of 6. The results are shown in FIG. 5, where the esterification ratio increased by 11.8% (at 120 hours) at 1: 4 compared to 1: 3. 1: 5 did not show an effect of 1: 4 or more, and 1: 6 reduced (FIG. 5)

On the basis of the above results, the ratio of oil and fat to methanol was 1: 4, water was 80% of the substrate, lipase activity was 2,
000 U, 120 hours, 30 ° C. is one of the best conditions
Found that it is an example.

(6) Effect of Addition of Organic Solvent DMSO, diethyl ether, n-hexane and petroleum ether were added in an amount of 10% each, and the effect on the esterification reaction of the organic solvent was observed. Table 3 shows the results. DMSO, n-
The addition of hexane and petroleum ether increased the esterification rate from 4.8% to 7.0%. This means
Organic solvents such as n-hexane are used when extracting fats and oils from plants, but by using inexpensive crude oils and fats containing some of these organic solvents as raw materials, it is possible to obtain the advantage of obtaining esters with higher efficiency. Can be

(Table 3) Table 3: Effect of organic solvent (10%, w / v) on ester (a) ── Organic solvent ME content (% by weight) ──────────────────────── No addition (b) 80.2 DMSO 85.0 Diethyl ether 70.4 n-hexane 86.1 Petroleum ether 87.2──────────────────────── (a) A mixture of 9.65 g of oil / 1.4 g (1: 4, mol / mol) of methanol, 8.84 ml of water (Lipase 2000
U containing) and a solvent at 30 ° C. for 120 hours. (B) The reaction was carried out under the same reaction conditions as above, except that the use of the solvent was stopped.

[0070]

According to the present invention, biodiesel fuel can be produced very efficiently in only one step. Further, according to the present invention, even when an organic solvent or a large amount of water is mixed in the reaction system, esterification of fats and oils proceeds smoothly, so that waste oil such as waste edible oil or a mixture thereof can be converted to biodiesel. Thus, there is no need to dispose them in a river or the like, and the present invention is excellent in terms of environmental protection as a waste liquid treatment method.

[Brief description of the drawings]

FIG. 1 shows the effect of the amount of enzyme on the methanolysis of rice bran oil.

FIG. 2 shows the cumulative effect of water content and methanol concentration on methanolysis of rice bran oil.

FIG. 3 shows the effect of pH on the methanolysis of rice bran oil.

FIG. 4 shows the effect of temperature on the methyl esterification reaction.

FIG. 5 shows the effect of the molar ratio with methanol on the methanolysis of rice bran oil.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Riki Fujii 3-7-1 Kagamiyama, Higashihiroshima City, Hiroshima Pref. National Tax Agency Brewing Research Institute (72) Inventor Takeyuki Kurosu 3-7-1 Kagamiyama, Higashihiroshima City, Hiroshima Prefecture No. National Tax Agency Brewery Research Institute (72) Naoto Okazaki Inventor 3-7-1 Kagamiyama, Higashihiroshima City, Hiroshima Prefecture National Tax Agency Brewery Research Laboratory F-term (reference) 4B050 CC01 DD04 FF05E LL05 LL10 4B064 AD85 CA21 CB30 CC03 CD06 CD07 CD30 DA20

Claims (8)

[Claims] 1. A method for producing biodiesel, comprising performing an esterification reaction of a fat or oil in a single step using a lipase in the presence of a fat or oil and an alcohol. 2. A method for producing biodiesel, wherein an esterification reaction of fats and oils is carried out using lipase in the presence of fats and oils and alcohol, despite the presence of water. 3. A method for producing biodiesel, comprising performing an esterification reaction of a fat or oil with a lipase derived from a Cryptococcus genus yeast in the presence of a fat or oil and an alcohol. 4. A yeast belonging to the genus Cryptococcus sp. S-2 (Cryptococcus sp. S-2) (FE
RM P-15155). The method for producing biodiesel according to claim 3, wherein 5. Lipase C having the following physicochemical properties:
A method for producing biodiesel, comprising using S2. (1) Action It has fat and oil decomposability, acts on triglycerides, and hydrolyzes to glycerin and fatty acids. Catalyzes the esterification of fats and oils in the presence of fats and alcohols. (2) Substrate specificity Decomposes tributyrin, tricaprylin, tripalmitin and triolein well. (3) Regiospecificity When triolein is allowed to act, oleic acid and a small amount of 1,2,2,3-diolein are formed, and 1,3-diolein and monoolein are not detected. (4) Optimum pH and stable pH range Optimum pH: 7.0 Stable pH range: 5 to 9 (5) Optimal reaction temperature and conditions of deactivation by temperature Optimal reaction temperature: 37 ° C Conditions of deactivation by temperature: The activity is slowly deactivated by the temperature rise, and the activity is maintained even after the heat treatment at 60 ° C. for 30 minutes. (6) Stability to organic solvent, activation Stable to organic solvent, furthermore, dimethyl sulfoxide,
The activity is increased by diethyl ether. 6. The method according to claim 1, wherein a vegetable oil is used as the fat or oil. 7. The method according to claim 6, wherein rice bran oil is used as the vegetable oil. 8. The method according to claim 1, wherein a waste oil or an oil mixed with an organic solvent is used as the oil.