JP2006238760A - Microreacter with immobilized enzyme and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microreacter comprising hollow microchannel composed of a flexible and inactive material with immobilized enzyme molecule on an inner surface produced in a simplified producing process and enabling a microanalysis with freedom of designing. <P>SOLUTION: The invention relates to the microreacter comprising a hollow microchannel composed of the flexible and inactive material wherein the inner surface of the microchannel has enzyme molecules immobilized through a crosslinking agent and the inactive material is a fluororesin or a silicone rubber. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microreactor in which an enzyme is immobilized.

Recently, in the chemical field and electromechanical field, using a capillary or microchannel having a diameter of 10 to 100 μm, chemical / electrical or microanalysis such as microanalysis, microsynthesis, microbiological culture, microelectrophoresis, microelectroosmosis Attempts have been made to perform mechanical operations (see non-patent literature).
Since these have the advantage that the results of each method can be confirmed quickly using a small amount of sample, they are applied to various fields.

  By the way, in these devices for performing chemical reactions efficiently, so-called microreactors, the controllability of fluid chemical reactions is improved, and the yield and purity of reaction products are expected to be improved. Since the width of the reactor can be narrowed and the ratio of the surface area per volume can be increased as compared with a reactor of normal size, it has been attracting attention as a means for improving the efficiency of chemical reactions.

  On the other hand, in the fields of chemistry and biochemistry, a catalyst is often used in order to improve the reaction efficiency. In this case, improving the catalyst efficiency is a problem.

For example, in the case of asymmetric synthesis of optically active compounds, when organic molecules such as enzymes and organometallic complexes are used, these are inevitably expensive compared to inorganic catalysts, which improves catalyst efficiency and reduces the amount used. Reuse is necessary for practical use.
Accordingly, assembling a microchannel structure for efficiently performing a catalytic reaction for a microreactor is a necessary requirement for putting this into practical use.

  The conventional microreactor is a multi-step physical / chemical process such as introducing a specific residue that binds to the catalyst after engraving the microchannel as described in Patent Document 1 until the catalyst is fixed to the microchannel. Processing was necessary, and the manufacturing process of the microreactor was complicated.

“SCIENCE”, 285, 83-85 JP 2003-260351 A

  In view of such circumstances, an object of the present invention is to provide a bendable microreactor that simplifies the manufacturing process of the microreactor, has flexibility in design, and enables microanalysis.

  The present inventors have found that conventional microreactors made of an inert material such as glass and quartz have limited flexibility and are fragile, and thus have a limited field of application of the technology. As a result of the research, an enzyme immobilization method for a bendable inert material has been established and the present invention has been achieved.

  That is, the present invention resides in (1) a microreactor in which enzyme molecules are immobilized on the inner surface of a hollow microchannel made of a bendable inert material via a crosslinking agent.

  The present invention also resides in (2) the microreactor described in (1) above, wherein the bendable inert material is fluororesin or silicon rubber.

  Moreover, this invention exists in the microreactor as described in said (1) or (2) whose crosslinking agent is glutaraldehyde and paraformaldehyde (3).

  The present invention also relates to (4) a microreactor comprising a step of circulating an enzyme molecule and a crosslinking agent on an inner surface of a hollow microchannel made of a bendable inert material and immobilizing the enzyme molecule on the inner surface. Exist in the manufacturing method.

  In addition, the present invention provides (5) an enzyme immobilization that performs any one of an enzyme digestion reaction, a condensation reaction, and an antigen-antibody reaction of an organic compound using the microreactor according to any one of (1) to (3). The method of using a microreactor exists.

  According to the present invention, an enzyme can be immobilized on a chemically inert surface, and at the same time, an enzyme reaction can be carried out not only with an aqueous solution but also with a reaction solution of a contained solvent, and further, there is no need for engraving or the like. The manufacturing process of the microreactor can be simplified.

The microreactor is also called a microchannel reactor, and is a general term for a microreactor having a micro flow channel of several to several hundred μm.
In the present invention, the microchannel needs to be hollow.
The hollow shape is preferably circular, but may be oval or polygonal if desired.
Further, the inner diameter of the microchannel is preferably 170 μm or more and less than 1 mm, more preferably 170 μm or more and 500 μm or less from the viewpoint of flowability and reactivity.

  The inert material used as the microchannel of the microreactor in the present invention is a material that does not show reactivity with the reactant, solvent and product of the catalytic reaction and has flexibility.

  Any known inert material that satisfies the above requirements can be used. Preferred examples include polytetrafluoroethylene (PTFE), tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer (PFA), and tetrafluoroethylene. -Fluororesin such as hexafluoropropylene copolymer (FEP), tetrafluoroethylene / ethylene copolymer (ETFE), polyvinylidene fluoride (PVDF), polychlorotrifluoroethylene (PCTFE), silicone rubber, polyethyl ether ketone , Polyethylimide, polypropylene, polyvinyl chloride, polyurethane and the like. From the viewpoint of resistance to organic solvents, fluororesin and silicon rubber are more preferable, and fluororesin is more preferable.

The outer shape of the microchannel is preferably a cylindrical shape, but an elliptical cylinder, a polygonal column (for example, a triangular column, a quadrangular column, a pentagonal column, a hexagonal column, etc.) can be used if desired.
When immobilizing an enzyme molecule in a microchannel, if desired, the inside of the channel is washed with a strong oxidizing agent such as a mixture of concentrated sulfuric acid and hydrogen peroxide solution before the immobilization reaction, and contaminated. Things can also be removed.

In the present invention, the enzyme molecule having catalytic ability needs to be immobilized on the inner surface of the hollow microchannel via a crosslinking agent.
On the inner surface of the microchannel, a film is formed by polymerizing the enzyme with the crosslinking agent, and the enzyme is immobilized by sticking it.
Since the immobilized enzyme film can be reversibly removed by a strong oxidizing agent, the microreactor can be reused.
As the strong oxidizing agent, for example, a piranha solution (hydrogen peroxide solution: concentrated sulfuric acid = 3: 7 (volume ratio)) or the like can be used.

  The cross-linking agent preferably uses glutaraldehyde and paraformaldehyde in combination from the viewpoints of economy, cross-linking speed, and activity retention, but has a certain chain length and contains two or more linking groups (in protein). A compound having a functional group capable of binding to an amino acid or the like, for example, a compound in which a functional group is bonded to both ends of polyethylene glycol can also be used.

The concentration of glutaraldehyde and paraformaldehyde is preferably 0.25% by mass to 1% by mass for glutaraldehyde, and preferably 4% by mass to 16% by mass for paraformaldehyde.
The mixing ratio of glutaraldehyde (GA) and paraformaldehyde (PA) is preferably GA / PA = 1/100 to 1/1, more preferably 1/70 to 1/4, and even more preferably 1/16 to 1/8. .
Examples of enzyme molecules that can be immobilized on the inner surface of the hollow microchannel include amylase, protease, lipase, cellulase, hemicellulase, and pectinase.

The enzyme-immobilized microreactor of the present invention can be used for a small amount of enzyme digestion reaction, condensation reaction, antigen-antibody reaction, etc., depending on the kind of immobilized enzyme molecule.
In particular, in an enzyme digestion reaction, it can be suitably used for enzyme digestion, which is a pretreatment specifically for proteomic protein identification.

According to the enzyme-immobilized microreactor (inner product: several μl) of the present invention, not only is it effective for processing a small amount of sample, but also it is possible to prevent the enzyme itself from being mixed into the sample after processing.
It is easy to directly incorporate the enzyme-immobilized microreactor into a device such as liquid chromatography, and continuous operation is possible.

  EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited at all by these examples.

<Yield measurement>
The substrate solution was flowed into the enzyme-immobilized microreactor at a constant flow rate (1 to 10 μl / min), and the effluent was collected from the tip of the microreactor. (Implemented at 37 ° C)
Thereafter, the amount of the substrate contained in the sample was analyzed by liquid chromatography.
A: Substrate solution before flowing into the microreactor
B: Recovery solution after flowing out into microreactor In liquid chromatography, the peak area of the substrate of A: a and the peak area of the substrate of B: b were compared, and the yield was calculated by the following formula.
Yield = {(a−b) / a} × 100

[Example 1]
<Preparation of reagent>
1. Preparation of Aldehyde Solution 750 μl of a 16% by weight paraformaldehyde aqueous solution and 30 μl of a 25% by weight glutaraldehyde aqueous solution were mixed with 1500 μl of 0.1M phosphate buffer (pH 7.5), and 720 μl of water was added to prepare an aldehyde solution.
2. Preparation of enzyme solution 20 mg of chymotrypsin was dissolved in 1000 μl of 0.05 M phosphate buffer (pH 7.5) to prepare an enzyme solution.

<Immobilization of enzyme>
The aldehyde solution and the enzyme solution were each filled into two disposable syringes (inner product: 1 ml).
Next, using the three-way cock as shown in FIG. 1, the two liquids were allowed to flow into one Teflon (DuPont, USA; registered trademark) tube.
The conditions at that time were a flow rate of 1 to 5 μl / min (same rate for both liquids), 4 ° C., and 6 to 12 hours.
Thereafter, unreacted enzyme or aldehyde reagent is washed with 1000 μl of 0.05M phosphate buffer (pH 7.5) at a flow rate of 10 μl / min at 4 ° C. for 100 minutes to obtain a microreactor with immobilized chymotrypsin. It was.
The preservation was performed by filling the tube with 0.05M phosphate buffer (pH 7.5).

[Example 2]
Except that trypsin was used as the enzyme, the same operation as in Example 1 was performed, and a microreactor in which trypsin was similarly immobilized was obtained.

Example 3
Using the chymotrypsin-immobilized microreactor obtained in Example 1, a peptide digestion reaction was performed.
As a substrate, N-Glutar-Phe-pNA (glutaryl phenylalanyl paranitroanilide) was used and dissolved in a phosphate buffer (pH 7.5) to 1 mM to obtain a substrate solution.
This molecule has one peptide bond, and the enzyme cleaves the peptide part to produce two molecules, Glutar-Phe and pNA. Therefore, in liquid chromatography, the degradation product peaks at a different site from the original substrate molecule. Can be detected as
The yield at this time was 98%.
Further, when the same experiment as described above was performed after filling with 0.05 M phosphate buffer (pH 7.5) and stored for 7 days, the yield was as high as 96%.

Example 4
Using the chymotrypsin-immobilized microreactor obtained in Example 1, protein digestion reaction was performed.
Myoglobin was used as the substrate, and the substrate solution was dissolved in a phosphate buffer (pH 7.5) so as to be 70 μg / ml.
The substrate solution was flowed into the chymotrypsin-immobilized microreactor, reacted for 10 minutes, and then the effluent was collected and analyzed by reverse phase liquid chromatography. As a result, the peptide peak generated when the myoglobin was enzymatically digested was detected. It was.
The yield at this time was 68%.

Example 5
Using the chymotrypsin-immobilized microreactor obtained in Example 1, a condensation reaction of organic substances was performed.
After 50 mM acetylphenylalanine ethyl ester (Ac-L-Phe-OEt) and 200 mM lysine ethyl ester (L-Lys-OEt) were mixed with 20% N, N-dimethylformamide (DMF), the mixture was added. After flowing into the chymotrypsin-immobilized microreactor and reacting for 1 minute, the effluent was collected and analyzed by liquid chromatography. As a result, a sweet substance precursor (Ac-L-Phe-L-Lys-OEt) that is a condensation product was analyzed. )was gotten.
The yield at this time was 44%.

  The enzyme-immobilized microreactor of the present invention can be suitably used for an enzyme digestion reaction, a condensation reaction, an antigen-antibody reaction, or the like in a very small amount, depending on the kind of immobilized enzyme molecule.

FIG. 1 is a schematic diagram for explaining a microreactor production process.

Claims (5)

  A microreactor characterized in that enzyme molecules are immobilized on a hollow microchannel inner surface made of a bendable inert material via a crosslinking agent.   The microreactor according to claim 1, wherein the bendable inert material is fluororesin or silicon rubber.   The microreactor according to claim 1 or 2, wherein the crosslinking agent is glutaraldehyde and paraformaldehyde.   A method for producing a microreactor comprising a step of circulating an enzyme molecule and a crosslinking agent on an inner surface of a hollow microchannel made of a bendable inert material and immobilizing the enzyme molecule on the inner surface.   A method for using an enzyme-immobilized microreactor, wherein the microreactor according to any one of claims 1 to 3 is used to perform any one of an enzyme digestion reaction, a condensation reaction, and an antigen-antibody reaction of an organic compound.

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