JP2001046100A - Stabilized enzyme for reforming fuel and method for reforming fuel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject stabilized enzyme for desulfurization etc. of gasoline, gas oil, etc. by immmobilizing an enzyme such as a peroxidase or a sulfinase in a structural unit having a size nearly coincident with that of the enzyme and structural stability. SOLUTION: This stabilized enzyme is composed of an immobilized enzyme for reforming a fuel and obtained by immobilizing at least one kind of enzyme selected from a peroxidase, a sulfinase, a monooxygenase, a dioxygenase and an isomerase in a structural unit having a size nearly coincident with that of the enzyme and structural stability and formed as pores capable of accommodating the enzyme in a mesoporous silica porous body. The immobilized enzyme is charged into various kinds of fuels such as gasoline or gas oil and capable of carrying out the fuel reforming such as desulfurization, an improvement in the octane number of the gasoline, an improvement in the cetane number of the gas oil, a reduction of the aromatic content, lowering of the boiling point or removal of impurities by enzymic actions thereof and stably maintaining the enzymic activity even in the fuels.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a stabilized enzyme for fuel reforming and a fuel reforming method, and more particularly, to an enzyme activity related to fuel reforming, and the enzyme activity in a fuel free of water. An enzyme that is stably maintained in
It relates to an advantageous fuel reforming method using such an enzyme.
In the present invention, "fuel reforming" refers to operations that are useful for removing fuel impurities or improving performance, such as desulfurization of fuel, improvement of octane number of gasoline, improvement of cetane number of light oil, reduction of aroma content, and reduction of boiling point.

[0002]

2. Description of the Related Art Hitherto, reforming technologies for various fuels such as gasoline and light oil have been studied. In particular, the sulfur content of the fuel causes SOx in combustion exhaust gas and the activity of the exhaust purification catalyst due to sulfur poisoning. In order to reduce fuel consumption, effective desulfurization technology for fuel is being pursued.

With respect to fuel desulfurization technology, for example, in a hydrodesulfurization method typically exemplified as a chemical desulfurization method, a sulfur compound in a petroleum fraction is converted into hydrogen in the presence of a metal catalyst such as an alumina catalyst supported on molybdenum. Let it undergo a reduction reaction,
Hydrogen sulfide is easily separated and removed, and is removed by a predetermined process.

Further, research on so-called "biodesulfurization" utilizing sulfur compound-decomposing bacteria and the like has been actively conducted. In the present invention, a microorganism having the ability (enzymatic activity) to selectively cleave a CS bond of a refractory sulfur compound such as an alkylated benzothiophene or an alkylated dibenzothiophene in a fuel is used. We are trying to make it easier to decompose, separate, and remove compounds.

[0005]

However, the above-mentioned hydrodesulfurization method generally requires a high reaction temperature and pressure, so that the processing equipment and its operation become very costly, and furthermore, a specific equipment is required. There is a problem that the hardly decomposable sulfur compound is not removed so much. In the future, it is expected that the necessity of using a crude oil containing a high concentration of sulfur is expected to increase, and the presence of such a hardly decomposable sulfur compound becomes a serious problem.
Further, with respect to light oil, there is also a problem that the lubricity of light oil is lost because the polar group contributing to its lubricity is reduced by hydrogenation.

[0006] Next, in the desulfurization technique utilizing the enzymatic activity, a particularly high reaction temperature and pressure are not required, and the above-mentioned hardly decomposable sulfur compound can be decomposed by appropriate selection of the enzymatic activity having high substrate specificity. As it turns out, it may occupy an important technical position for advanced desulfurization as an alternative or at least a complementary technology to the chemical desulfurization process.

However, for example, Japanese Patent Application Laid-Open No.
The desulfurization technique utilizing the enzymatic activity of microorganisms as in the invention disclosed in Japanese Patent No. 293 has a restriction that the microorganisms are desulfurized at the water / fuel interface while mixing and stirring the fuel with water. Therefore, a system for mixing and stirring water and fuel, a system for separating fuel, water, and cells after desulfurization are indispensable, and the management of microorganisms is complicated, and considerable processing costs and complicated system operation are required. There was a problem to say.

[0008] In other words, a desulfurizing enzyme that stably acts even when directly injected into gasoline or gas oil has not been provided yet, and therefore, a microorganism-based method (using the activity of an enzyme protected by the cells of microorganisms) is used. Method). This situation has led to an increase in the octane value of gasoline,
The same applies to the case where the enzymatic activity is to be used to improve the cetane number of light oil.

In this connection, in recent years, an enzyme immobilization technique for directly immobilizing an enzyme on a resin or the like, a comprehensive immobilization method for encapsulating the enzyme in a gel, a microcapsule method for coating the enzyme with a translucent polymer film, Attention has also been paid to a surface modification method for stabilizing a protein surface by modifying it with polyethylene glycol or glycolipid. However, in any case, the structural unit for immobilizing the enzyme has a shape that does not match the enzyme molecule (for example, a simple flat surface),
Even in the case of a structural unit that matches the shape and size of an enzyme molecule, its structural stability is lacking. Therefore, in a severe environment where it is directly injected into fuel, the three-dimensional structure of the enzyme (ie, enzyme activity) Was difficult to maintain stably.

[0010] Accordingly, the present invention provides a stabilizing enzyme which exhibits an enzymatic action related to fuel reforming, and whose enzymatic action is stably maintained even in fuel, and an advantageous fuel modification utilizing such a stabilizing enzyme. Providing quality methods is an issue to be solved.

The present inventor specifically narrows down several types of enzymes related to fuel reforming such as desulfurization. These enzymes are described in Japanese Patent Application No. 11-27702 filed by the present applicant.
The present inventors have experimentally found that the enzyme action is stably maintained even in a fuel containing no water when immobilized by the means disclosed in the specification attached to the application of the present invention, and completed the present invention.

[0012]

Means for Solving the Problems (Structure of the First Invention) The structure of the first invention of the present application (the invention of claim 1) for solving the above-mentioned problems is as follows: peroxidase, sulfinase, Monooxygenase (m
onooxygenase), dioxygenase (dioxygenase),
At least one selected from isomerases
Kind Code: A1 Abstract: A stabilized enzyme for fuel reforming, comprising a kind of enzyme immobilized in a structural unit having a size substantially matching that of the enzyme and having structural stability, and used for fuel reforming.

(Structure of the Second Invention) The structure of the second invention of the present application (the invention according to claim 2) for solving the above problems is as follows.
The structural unit according to the first aspect of the present invention is a stabilized enzyme for fuel reforming, wherein the structural unit is configured as pores of the porous mesoporous silica capable of containing the enzyme.

(Structure of the Third Invention) The structure of the third invention (the invention according to claim 3) for solving the above problems is as follows.
A fuel reforming method in which the stabilizing enzyme for fuel reforming according to any one of the first invention and the second invention is charged into fuel, and a predetermined fuel reforming is performed by the enzyme action.

[0015]

[Operation and Effect of the Invention] (Operation and Effect of the First Invention) In the stabilized enzyme for fuel reforming of the first invention, at least one enzyme selected from peroxidase, sulfinase, monooxygenase, dioxygenase and isomerase is used. Are immobilized in a structural unit having a structural stability substantially conforming to the enzyme.

Each of the stabilizing enzymes for fuel reforming according to the first invention does not require a high reaction temperature or pressure, does not have the problem of impairing the lubricity in reforming gas oil, and has the enzymatic activity of microorganisms. Water / water as in the fuel reforming technology using
Since no fuel-mixed system is required, the processing system can be significantly simplified and reduced in cost.

Furthermore, since the enzyme is immobilized in a structural unit having a structural stability having a size substantially matching that of the molecule, large deformation at the molecular level of the enzyme is physically regulated, and the three-dimensional structure of the enzyme is reduced. The enzyme is hardly deactivated even in a harsh environment where it is stably maintained and is directly injected into gasoline or light oil.

From the above actions alone, the stabilized enzyme of the first invention is not always able to exhibit sufficient enzyme activity in fuel regardless of the type of the immobilized enzyme. With respect to the enzyme (1), it was confirmed that the stabilized enzyme of the first aspect of the invention actually exhibited a stable and good enzyme action even in fuel.

(Function / Effect of the Second Invention) In the second invention, the structural unit according to the first invention is constituted as pores capable of accommodating the enzyme in a porous mesoporous silica material. The structural unit for immobilizing the enzyme is provided in an efficient embodiment in which the enzyme is immobilized on a large number of pores, and the structural unit which is a porous silica material has an advantage that the structural stability is extremely high.

(Function / Effect of Third Invention) In the third invention, the stabilized enzyme for fuel reforming according to the first invention or the second invention is introduced into fuel (in fuel in which water is not mixed),
Because of the enzymatic action, predetermined fuel reforming such as desulfurization, octane number improvement, cetane number improvement, etc. is carried out.
Alternatively, the fuel reforming can be performed very advantageously while ensuring the operation and effect of the second invention.

[0021]

Next, embodiments of the first to third inventions will be described. In the following, simply "the present invention"
When it says, it points to 1st invention-3rd invention collectively.

[Fuel Reforming] In the present invention, the type of fuel is not limited, but gasoline and light oil are particularly preferred as targets for reforming. Although the content of the fuel reforming is not limited, preferable examples include desulfurization of general fuels, improvement of octane number in gasoline, improvement of cetane number in light oil, reduction of aroma content, and reduction of boiling point. The type of one or more enzymes to be immobilized in the structural unit is determined by the content of the target fuel reforming.

[Enzymes] As enzymes that can be used in the present invention, monooxygenases such as peroxidase, sulfinase, and sulfone monooxygenase, dioxygenases, and the like can be used as long as the present inventors have confirmed effective enzymatic action on fuel reforming. , Isomerase. It should be noted that any enzyme other than the above is effective for fuel reforming in some sense, and is confirmed to exhibit stable enzyme activity in fuel by the stabilization method of the present invention. Available at

In the present invention, the term "enzyme" refers to a normal enzyme protein molecule or its active unit (enzyme fragment containing an active site). In the structural unit, only one type of enzyme may be immobilized. For example, two or more types of enzymes involved in a series of reactions required for fuel reforming may be immobilized simultaneously. In the latter case, two or more types of enzymes may be fixed in separate structural units in the same porous body or the like, or may be fixed in the same structural unit.

[Stabilizing enzyme] The stabilizing enzyme according to the present invention is used for fuel reforming, and the enzyme related to fuel reforming has a structurally stable size substantially matching that of the enzyme. Fixed in a structural unit having the property.

FIG. 1 conceptually shows an example of the structure of the stabilized enzyme. The structural unit 1 has structural stability against environmental conditions such as pH, heat, and fluid flow. The enzyme 2 is a portion that expresses a predetermined enzyme activity on the substrate 4, and is composed of a normal enzyme, an enzyme fragment containing an enzyme active site, and the like. The anchor unit 3 is not an essential component in the stabilizing enzyme of the present invention, but is an element that connects the structural unit 1 and the enzyme 2, and transmits the above-mentioned structural stability of the structural unit 1 to the enzyme 2, In addition to suppressing the inactivation due to a large change in the three-dimensional structure of 2, the relatively small change in the structure of the active site required for the interaction with the substrate 4 gives an acceptable degree of freedom.

FIG. 2 conceptually shows another example of the structure of the stabilized enzyme. As shown in FIG. 2, the enzyme 2 and the structural unit 1 are bound by van der Waals force or the like without the above-mentioned anchor unit. . Each structural unit 1 contains one or a small number of enzymes 2, and the structural unit 1
Preferably approximately matches the three-dimensional shape of one or a few of these enzymes 2.

The structural unit 1 shown in FIG. 1 or FIG.
May be composed of an inorganic material or an organic material such as a polymer. In the structural unit 1 made of an organic material, a polymer forming reaction is required to coat around the enzyme and possibly via an anchor unit. The type of the monomer or polymer is not particularly limited as long as the object of the invention is not hindered.

As the structural unit made of an inorganic material,
For example, it can be composed of various metal oxides such as silicic acid (SiO ₂ ) and alumina, and a composite oxide represented by SiO ₂ -MO _{2 / n} (M is a metal such as Al). For example,
As a method for forming a structural unit composed of silicic acid, layered silicate such as kanemite, Si (OR) ₄ (R is an alkyl group), silica gel, water glass, sodium silicate, or the like can be preferably used.

In order to form a structural unit from an inorganic material, the inorganic material is mixed and reacted with a surfactant (template substance), and a surfactant / inorganic composite in which an inorganic skeleton is formed around micelles of the surfactant. After the body is formed, for example, baking at 400 ° C. to 600 ° C., or removing the surfactant by extracting with an organic solvent, the mesopore pores having the same shape as the micelle of the surfactant are formed in the inorganic skeleton. It can be formed (porous mesoporous silica).

In the above method for producing a structural unit, when a silicon-containing compound such as silicic acid is used as a starting material, a layered silicate such as kanemite is first formed, micelles are inserted between the layers, and a layer having no micelles is formed. The pores can be formed by connecting with silicate molecules and then removing the micelles. In addition, a silicon-containing substance such as water glass is used as a starting material, silica is formed by assembling and polymerizing silicate monomers around micelles,
The micelle molecules can then be removed to form pores. In this case, the micelles are usually columnar, resulting in columnar pores.

In the method of forming a structural unit through the formation of a layered silicate such as kanemite, the surface of the pores is hydrophobic and anionic. Hydrophobic surfaces are preferred for stable immobilization of unhydrated enzymes, and anionic surfaces are preferred for immobilization of enzymes having cations on the surface.

The micelles may contain various surfactants, such as cationic surfactants such as alkyltrimethylammonium, anionic surfactants such as alkylsulfonate, polyethylene glycol in a suitable medium. It is formed by dispersing a nonionic surfactant such as a system. The micelle shape includes a spherical shape, a cylindrical shape, a layered shape, and the like, and these are regularly arranged to form a liquid crystal structure having a hexagonal and cubic structure.

By changing the length of the alkyl chain of the surfactant, the diameter of the micelle can be changed, and the diameter of the formed pores can be controlled. Further, by adding relatively hydrophobic molecules such as trimethylbenzene and tripropylbenzene together with the surfactant, the micelles can be swelled and consequently larger pores can be formed. The form of the structural unit includes powder, granule, sheet, bulk, and film. The pore diameter (diameter) of the porous pores constituting the structural unit is preferably substantially equal to the diameter of the enzyme immobilized therein. This diameter is usually 1 nm
Larger, preferably larger than 2 nm. The pore size is generally smaller than 30 nm, usually smaller than 10 nm.

The molecule constituting the anchor unit desirably has basically the same structure as the structural unit. In particular, when the enzyme is an active unit, a hydroxyl group, an amide group, an amino group, a pyridine It is necessary that functional groups such as a group, a urea group, a urethane group, a carboxylic acid group, a phenol group, an azo group, a hydroxyl group, a silane derivative, and an aminoalkylene group are bonded.

In assembling the stabilized enzyme of the present invention, first, a polymer reaction may be carried out via a molecule directly linked to the enzyme to form a structural unit in a state containing the enzyme. It may be formed, and the enzyme may be introduced into this via a lysine amino group or the like of the enzyme itself. The bond between the enzyme and the structural unit may be not only a bond or a covalent bond by the van der Waals force, but also an ionic bond, a hydrogen bond, a hydrophobic bond, or the like.

[0037]

EXAMPLES Example 1 Synthesis of Porous Mesoporous Silica 5.0 g (0.028 mol) of δ-N was placed in a 100 ml beaker.
a ₂ Si ₂ O ₅ and 50 ml of ion-exchanged water are added.
The mixture was stirred at 5 ° C. for 3 hours for cation exchange, and then the aqueous solution was filtered to obtain a δ-Na _1.6 H _0.4 Si ₂ O ₅ precipitate. 50 ml of ion-exchanged water was added to the precipitate, and the mixture was stirred until a uniform dispersion was obtained to obtain a liquid A.

In a 100 ml Erlenmeyer flask, 3.0 g (0.
0082 mol) of hexadecyltrimethylammonium bromide (HDTMA-Br) and 50 ml of ion-exchanged water and stirred.
(0.025 mol) of triisopropylbenzene (TIPB)
Was added and stirred vigorously for 10 minutes. This solution was used as solution B.

The above solution A was transferred to a 250 ml three-necked round flask, and while vigorously stirring, solution B was gradually added to the solution and the temperature was raised to 80 ° C., followed by a constant temperature reaction at the same temperature for 3 hours. Next, the pH of the reaction solution was adjusted to 8 using 2N hydrochloric acid.
The mixture was adjusted to 5 ± 0.1 and continuously stirred for 3 hours. Immediately after the completion of the reaction, the mixture was filtered, washed and filtered five times with 200 ml of ion-exchanged water.

The product was air-dried at 45 ° C. for 24 hours, and then calcined in an electric furnace at 550 ° C. for 6 hours to remove the template substance to obtain about 3.5 g of a white powder.
An X-ray diffractometer (Rigaku RAD-B)
And the pore diameter, surface area, and total pore volume are determined by N
2 As a result of measurement with an adsorption device (Autosorb), it was confirmed that the white powder was a powder of a porous mesoporous silica having the structural unit described above.

Preparation of Stabilizing Enzyme Peroxidase derived from horseradish (manufactured by Sigma) was added to p.
5 mg / H5 in 50 mM sodium acetate buffer
ml. Then, 5 ml of the enzyme solution was added to 200 mg of the mesoporous silica powder synthesized above,
The mixture was gently mixed at 4 ° C for 16 hours or more.

Action of Stabilizing Enzyme Toluene, gasoline, and light oil were each prepared in an amount of 10 ml, and 1,2-diaminobenzene was dissolved in each to a concentration of 20 mM. 2 ml of a solution of -butylhydroxyperoxide dissolved in a decane solution are added, and the above stabilizing enzyme (porous mesoporous silica on which peroxidase is immobilized) is added in an amount equivalent to 0.5 mg in terms of enzyme, and the enzyme reaction is carried out at 38 ° C. Oxidation reaction).

At each reaction time (1 to 3 hours after the start of the reaction), the absorbance (absorbance) at 470 nm of 1,2-dinitrobenzene generated by the oxidation reaction was measured. The results are shown in FIG. 3, which shows that the peroxidase activity is effectively exhibited in any solvent of toluene, gasoline, and light oil, and that the enzyme activity is higher in gasoline and light oil than in toluene. I understood that.

Example 2 Into each 10 ml flask,
10 mg of the above-mentioned stabilizing enzyme and 1 mg of the above-mentioned peroxidase manufactured by Sigma were added, and n was obtained by dissolving dibenzothiophene or 2,8-dimethyldibenzothiophene.
A desulfurization reaction was attempted by adding 2 ml of tetradecane solution (sulfur concentration: about 100 ppm) and shaking reciprocally at 30 ° C. for 18 hours.

Each of these reaction solutions was prepared at 12000 rpm, 1
The mixture was centrifuged for 0 minutes, and the total sulfur content of the supernatant was measured by a pyrofluorescence method (Antech sulfur meter model 7000).
The relative desulfurization rate (%) calculated from the measurement results is 100% relative to dibenzothiophene in the example of using the stabilized enzyme.
83.4% based on 2,8-dimethyldibenzothiophene
However, in the case where the enzyme was used as it was, it was 12.3% with respect to dibenzothiophene and 3.1% with respect to 2,8-dimethyldibenzothiophene, indicating a remarkable difference.

[Brief description of the drawings]

FIG. 1 is a diagram showing a structural example of a stabilizing enzyme according to the present invention.

FIG. 2 is a diagram showing a structural example of a stabilizing enzyme according to the present invention.

FIG. 3 is a graph showing the action of a stabilizing enzyme according to an example.

[Explanation of symbols]

 1 Structural unit 2 Enzyme 3 Anchor unit 4 Substrate

Claims (3)

[Claims] 1. An enzyme comprising at least one enzyme selected from the group consisting of peroxidase, sulfinase, monooxygenase, dioxygenase, and isomerase, which is immobilized in a structural unit having a size substantially matching the enzyme and having structural stability. A stabilized enzyme for fuel reforming, which is used for fuel reforming. 2. The stabilized enzyme for fuel reforming according to claim 1, wherein the structural unit is configured as a pore in the mesoporous silica porous body capable of containing the enzyme. 3. A fuel characterized in that the stabilized enzyme for fuel reforming according to claim 1 or 2 is introduced into fuel, and a predetermined fuel reforming is carried out by the action of the enzyme. Reforming method.