JP2004267097A - Microreactor and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new microreactor with enzyme molecule detachably bound thereto so as to easily replace a new enzyme when an enzyme bound inside a microchannel declines in potency and efficiency drops. <P>SOLUTION: The microreactor is such that an enzyme molecule is detachably bound via a nickel complex introduced onto the surface of the microchannel. This microreactor is produced by chemically modifying the microchannel of quartz or silica-based ceramic and introducing the nickel complex onto the surface thereof followed by carrying out a reaction by adding the enzyme molecule having a histidine tag to detachably bind the molecule to the surface. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a microreactor in which an enzyme is reversibly removably connected, and a method for producing the same.
[0002]
[Prior art]
Recently, in the chemical field and the electromechanical field, using a capillary or a microchannel having a diameter of 10 to 100 μm, chemical / electrical or microanalysis, microsynthesis, microbial culture, microelectrophoresis, microelectroosmosis, etc. Attempts have been made to perform mechanical operations (see Non-Patent Document 1). These methods have the advantage that the results of the respective methods can be quickly confirmed using a small amount of sample, and thus have been applied to various fields.
[0003]
By the way, among these, the device for efficiently performing the chemical reaction, the so-called microreactor, improves the controllability of the fluid chemical reaction, and is expected to improve the yield and purity of the reaction product, Since the width of the reactor can be reduced and the ratio of the surface area per volume can be increased as compared with a normal-sized reactor, it has been attracting attention as a means for improving the efficiency of a chemical reaction.
[0004]
On the other hand, in the fields of chemistry and biochemistry, a catalyst is often used in order to improve the reaction efficiency, and at this time, improving the catalyst efficiency has been an issue. For example, when using an organic molecule such as an enzyme or an organometallic complex for the asymmetric synthesis of an optically active compound, these are inevitably higher in cost than an inorganic catalyst, so that the catalyst efficiency is improved and the amount used is reduced. Reuse is necessary for practical use.
Therefore, as for a microreactor, assembling a microchannel structure for efficiently performing a catalytic reaction is a necessary requirement for practical use of the microchannel structure.
[0005]
By the way, in a microreactor in which an enzyme is immobilized on the surface of a microchannel, it is inevitable that the catalytic ability decreases over time, so in order to use the microreactor efficiently, the enzyme with the reduced ability must be appropriately replaced. Is needed. However, in conventional microreactors, if the enzyme in the microchannel is to be exchanged, the enzyme with reduced capacity must be completely removed, and then the new enzyme must be immobilized again by a complicated operation. Conversion was impossible.
[0006]
[Non-patent literature]
"Science", Volume 285, pp. 83-85
[0007]
[Problems to be solved by the invention]
The present invention has been made in view of such circumstances, and when an enzyme bound in a microchannel is reduced in efficiency due to a decrease in efficiency, the enzyme molecule is easily attached and detached so that the enzyme molecule can be easily replaced with a new enzyme. The purpose of the present invention is to provide a novel microreactor which is freely connected.
[0008]
[Means for Solving the Problems]
The present inventors have conducted various studies on a microreactor on which a catalyst is immobilized, and as a result, a nickel complex can be introduced into a microchannel surface composed of glass, quartz, or silica-based ceramics by chemical modification, It has been found that the use of this nickel complex allows the enzyme molecule to be removably bound, and the present invention has been made based on this finding.
[0009]
That is, the present invention provides a microreactor characterized in that an enzyme molecule is detachably bound via a nickel complex introduced on the surface of a microchannel, and a microreactor made of glass, quartz or silica-based ceramics. The present invention provides a method for producing a microreactor, which comprises chemically modifying a channel to introduce a nickel complex on the surface thereof, and then reacting the resultant with an enzyme molecule having a histidine tag and detachably binding the enzyme. is there.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
As the base material of the microreactor of the present invention, it is necessary to use a material that is inert to the catalyst, the reactants, the solvent, and the reaction product in the catalytic reaction. In the present invention, glass, quartz, or a silica-based ceramic, for example, a sintered body of silica or Si / SiO ₂ is used as such a material. The shape of the substrate is usually a plate, but may be an arc-shaped surface, a sphere, or a particle if desired.
[0011]
Microchannels provided on these base materials can be easily formed by processing using a microdrill or a laser or etching. The microchannel is engraved with a size of 10 to 500 μm in width, preferably 50 to 400 μm, and a depth of 10 to 500 μm, preferably 50 to 400 μm. The length of the microchannel is not particularly limited and depends on the size of the substrate to be used, but is usually selected in the range of 100 to 300 mm.
[0012]
Next, in the present invention, it is necessary to bond an enzyme molecule to the surface of the microchannel engraved as described above via a nickel complex introduced by chemical modification.
To introduce a nickel complex on the channel surface, first, the channel surface is chemically modified using a mixture of aminoalkyl trialkoxysilane and alkyl trialkoxysilane.
[0013]
As the aminoalkyl trialkoxysilane or alkyltrialkoxysilane used in this case, those in which the alkyl group is an alkyl group having 1 to 4 carbon atoms, such as aminoethyltriethoxysilane, aminopropyltriethoxysilane, and aminopropyltrialkoxysilane Preferred are aminoalkyl trialkoxysilanes such as methoxysilane and aminobutyltriethoxysilane, and alkyltrialkoxysilanes such as methyltriethoxysilane, ethyltriethoxysilane and propylethoxysilane. The mixing ratio of the aminoalkyl trialkoxy silane and the alkyl trialkoxy silane is preferably in the range of 1: 9 to 5: 5 by mass.
[0014]
This chemical modification is performed by bringing the channel surface into contact with a solution of a mixture of aminoalkyl trialkoxysilane and alkyl trialkoxysilane dissolved in a suitable solvent. Examples of the solvent used in this case include methyl alcohol, ethyl alcohol, ethyl acetate, acetone, and ethyl cellosolve. The mixture of the aminoalkyl trialkoxysilane and the alkyl trialkoxysilane is used by dissolving it in this solvent at a concentration of 1 to 5% by volume.
In this case, if desired, prior to the chemical modification, the inside of the channel can be washed with a strong oxidizing agent such as a mixture of concentrated sulfuric acid and hydrogen peroxide to remove contaminants. .
[0015]
After the chemical modification in this manner, the N-hydroxy acid is further treated with a dicarboxylic anhydride, for example, succinic anhydride, and then reacted with a mixture of 1-ethyl-3-aminopropylcarbodiimide and N-hydroxysuccinimide to give N-hydroxy A succinimide active ester is formed.
[0016]
Next, a complex compound forming agent such as N- (5-amino-1-carboxypentyl) iminodiacetic acid is reacted with the active ester, and then a solution of a soluble nickel salt such as nickel sulfate is reacted to form a nickel complex. The formation allows the nickel complex to be immobilized on the channel surface.
[0017]
The above active ester is also obtained by immersing the microchannel in a solution of a mixture of ω-hydroxyalkyl trialkoxysilane and alkyl trialkoxysilane in an organic solvent, for example, ethyl alcohol, and reacting the mixture at room temperature for 30 to 120 minutes. This is taken out, washed, and dried in an inert atmosphere to chemically modify the surface of the microchannel to introduce an ω-hydroxyalkyl group. Then, succinic anhydride is converted into an organic solvent such as N, N-dimethylformamide. The treated material is immersed in the solution dissolved in the above and reacted for 20 to 60 minutes, and then a condensing agent such as 1-ethyl-3- (3-aminopropyl) carbodiimide and N-hydroxysuccinimide are mixed with an organic solvent such as N, N- Formed by reacting a solution dissolved in dimethylformamide You can also.
[0018]
When the microchannel having the nickel complex thus obtained bound to the surface is contacted with a histidine tag, that is, an enzyme having a continuous histidine residue at the amino or carboxyl terminus, the enzyme can be easily converted via the nickel complex. Is immobilized on the surface of the microchannel.
[0019]
Examples of such an enzyme having a histidine tag include enzymes that can be prepared by constructing an expression system using gene recombination, such as casein kinase, alkaline phosphatase, and lactate dehydrogenase.
[0020]
The enzyme molecule immobilized on the surface of the microchannel of the present invention via a nickel complex can be easily detached by reduction treatment or treatment with a chelating agent such as imidazole or EDTA. Since the elimination and elimination reactions are reversible, when the catalytic activity decreases, it can be easily exchanged for a new enzyme molecule. Therefore, the microreactor of the present invention can be operated continuously for a long period of time.
In this way, the substrate provided with the microchannel on which the enzyme molecule is immobilized is covered with a transparent plate as necessary, and used as a microreactor.
[0021]
FIG. 1 is a plan view of the microreactor of the present invention obtained in this manner, and FIG. 2 is a cross-sectional view of the microchannel. The microchannel 2 is formed in a meandering manner on a substrate 1 made of an inert material. Enzyme molecules are fixed on the wall surface 3 of the microchannel 2.
[0022]
When an enzymatic reaction is carried out using a microreactor having such a structure, the reaction efficiency can be significantly increased due to the large ratio of the surface area to the volume of the microchannel, and the enzyme molecules are reversibly formed on the microchannel surface. The immobilization has the advantage that the substrate molecules can be continuously introduced into the microchannel and reacted continuously without separating and collecting enzyme molecules from the reaction solution after completion of the reaction. When the enzyme ability is reduced, there is also an advantage that the enzyme can be desorbed by flowing a reducing agent or a chelating agent into the microchannel, and the enzyme can be easily replaced with a new enzyme.
[0023]
【Example】
Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
[0024]
Example 1
Using a micro drill, a channel having a width of 400 μm, a depth of 400 μm, and a length of 200 mm is formed on a Si / SiO ₂ wafer substrate (30 × 30 × 5 mm) by bending and meandering eight times, and a glass is formed on the channel. By coating with a plate, a microreactor substrate having a microchannel structure was manufactured.
Next, this was immersed in 1 liter of a mixture of concentrated sulfuric acid and 30% hydrogen peroxide solution (volume ratio 7: 3) for 12 hours to wash the inside of the channel, and after decomposing and removing the organic substances attached to the wall surface, And washed with ultrapure water.
[0025]
Next, the mixture was treated with a 3% by volume solution prepared by dissolving a mixture of 3-aminopropyltriethoxysilane and methyltriethoxysilane at a ratio of 2: 8 in 95% ethyl alcohol. After 1 hour, the resultant was washed with ethyl alcohol, passed through nitrogen gas, and then dried in a dryer at 115 ° C. for 1 hour to perform surface modification.
[0026]
A solution obtained by dissolving succinic anhydride in N, N-dimethylformamide at a concentration of 1 mmol / L was injected into the channel of the microreactor thus obtained, and allowed to react for 30 minutes, followed by N, N-dimethylformamide. And washed. Next, a solution in which 1-ethyl-3 (3-aminopropyl) carbodiimide and N-hydroxysuccinimide were each mixed at a concentration of 1 mmol / L was passed through the channel to form an N-hydroxysuccinimide active ester.
[0027]
A solution prepared by diluting N- (5-amino-1-carboxypentyl) iminodiacetic acid with a phosphate buffer (pH 7.4) was supplied to the microchannel to be immobilized. After washing this with a phosphate buffer, an aqueous solution of nickel sulfate was allowed to flow to form a nickel complex.
[0028]
A phosphate buffer solution of the enzyme casein kinase having six consecutive histidine residues at the amino terminus, a so-called histidine tag, was passed through the microchannel to perform immobilization. Using a microreactor on which the obtained casein kinase was immobilized, casein as a substrate was phosphorylated. A Tris buffer solution of the substrate prepared at a concentration of 100 μmol / liter was injected into the microchannel with a syringe pump, and the resulting reaction product was analyzed by high performance liquid chromatography to evaluate the reaction. The reaction time was evaluated as the residence time in the channel. When the residence time was changed by changing the liquid sending speed by the syringe pump, a reaction yield as shown in FIG. 3 was obtained.
[0029]
After treating this microreactor with an EDTA solution, a nickel sulfate solution was passed again to form a nickel complex to immobilize casein kinase, and phosphorylate the substrate. When this operation was repeated again to carry out the reaction, as shown in FIG. 3, the same reaction efficiency as the first time could be obtained.
[0030]
Example 2
Using hydroxypropylsilane instead of 3-aminopropyltriethoxysilane, the inside of the microchannel was surface-treated in the same manner as in Example 1, and then succinic anhydride was reacted in pyridine to form an ester bond. And active esterification with N-hydroxysuccinimide, and N- (5-amino-1-carboxypentyl) iminodiacetic acid was introduced. A solution of alkaline phosphatase having a histidine tag on the amino terminal side was injected into the obtained microreactor, and circulated for 2 hours to fix the alkaline phosphatase.
Using a microreactor on which the alkaline phosphatase obtained by the above operation was immobilized, a hydrolysis reaction of phosphorylated coumarin as a substrate was performed, and the same results as in Examples 1 and 2 were obtained.
[0031]
Example 3
Instead of 3-aminopropyltriethoxysilane, hydroxypropylsilane was used to surface-treat the inside of the microchannel in the same manner as in Example 1, and then reacted with succinic anhydride in pyridine to form an ester bond. Thereafter, active esterification was performed using N-hydroxysuccinimide, and N- (5-amino-1-carboxypentyl) iminodiacetic acid was introduced. A solution of lactate dehydrogenase having a histidine tag on the amino terminal side was injected into the obtained microreactor, and circulated for 2 hours to fix lactate dehydrogenase.
Using a microreactor on which lactate dehydrogenase obtained by the above operation was immobilized, lactic acid was synthesized by a reduction reaction of pyruvate as a substrate, and this microreactor produced the same results as in Examples 1 and 2. Lactic acid could be produced with the reaction yield of
[0032]
【The invention's effect】
According to the present invention, by using a microreactor in which a catalyst is reversibly immobilized on the microchannel wall surface, the efficiency of an enzyme-catalyzed reaction, which is an important technology in the chemical industry, can be easily increased. In addition, it is possible to provide a next-generation microreactor overcoming the problem of degradation in enzyme performance.
[Brief description of the drawings]
FIG. 1 is a plan view of a microreactor used in Examples 1 and 2.
FIG. 2 is a cross-sectional view of a microchannel of the same microreactor.
FIG. 3 is a bar graph showing the change over time in the reaction yield obtained in Example 1 and the change in performance due to the continuous desorption / immobilization reaction of the enzyme.
[Explanation of symbols]
1 Substrate 2 Micro channel 3 Wall

Claims (5)

A microreactor, wherein an enzyme molecule is detachably bound via a nickel complex introduced to the surface of the microchannel. The microreactor according to claim 1, wherein the enzyme molecule has a histidine tag. 3. The microreactor according to claim 1, wherein the base material of the microchannel is glass, quartz or silica-based ceramic. A microchannel composed of glass, quartz, or silica-based ceramics is chemically modified to introduce a nickel complex on its surface, and then an enzyme molecule having a histidine tag is added to the mixture to cause a reaction, and the detachable bond is established. A method for producing a microreactor. The method for producing a microreactor according to claim 4, wherein the chemical modification of the microchannel is performed using a mixture of aminoalkyl trialkoxy silane and alkyl trialkoxy silane.

Images