JP2010022328A - Method for stabilizing coenzyme-binding enzyme, and composition, enzyme sensor and fuel cell prepared by using the stabilization method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stabilizing a coenzyme-binding enzyme improving the stability while keeping various functions held by the coenzyme-binding enzyme in itself: and provide a composition, an enzyme sensor and a fuel cell prepared by using the stabilization method. <P>SOLUTION: There are provided the method for stabilizing a coenzyme-binding enzyme by preparing a coenzyme required for the function expression of a coenzyme-binding enzyme and binding with a coenzyme-binding enzyme and adding the coenzyme to a composition containing a coenzyme-binding enzyme as a constitution component, and a stabilized coenzyme-binding enzyme composition containing the enzyme in a state stabilized by the method. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for stabilizing a coenzyme-linked enzyme and use thereof. Specifically, the present invention relates to a method for stabilizing a coenzyme-linked enzyme, a composition prepared using the stabilization method, an enzyme sensor, and a fuel cell.

  Enzymes have been applied to various applications due to their excellent molecular discrimination function. For example, it is used in techniques for analyzing and quantifying various substances in various industrial fields such as medical, food, and environmental fields. As a means for easily performing quantitative analysis, a biosensor that uses a molecular identification function of an enzyme as a sensor element has attracted attention. A biosensor analyzes and quantifies a measurement target by converting a biochemical signal generated by recognizing a substrate to be measured by an enzyme as a sensor element into an electrical signal.

  In recent years, biofuel cells using bioresources have been proposed as next-generation energy because of their high energy efficiency and low environmental load. Living organisms such as microorganisms generate high energy substances such as ATP in the in vivo metabolic process in which carbohydrates, proteins, lipids, etc. are oxidatively decomposed by biocatalysts such as enzymes, and acquire energy necessary for life activities. is doing. A biofuel cell is a power generation device that extracts energy generated in such in vivo metabolic processes as electrical energy to an electrode.

  In the practical application of enzyme biosensors and fuel cells that utilize the catalytic function of enzymes, the choice of enzyme affects the success or failure. However, since an enzyme exhibits its catalytic activity under mild conditions that allow living organisms to survive, its activity depends on temperature, etc., and generally has low thermal stability, and can be used for long-term storage. There were problems such as inactivation of the enzyme. Therefore, in application to enzyme biosensors and fuel cells, an enzyme that can stably maintain activity over a period of time in various environments is indispensable, and in particular, it has a thermal stability that is said to correlate with the enzyme lifetime. Improvement is required.

  For this reason, techniques for improving the thermal stability of enzymes have been reported. For example, in the homodimer structure of glucose dehydrogenase (hereinafter referred to as “PQQ-dependent glucose dehydrogenase”) using pyrroloquinoline quinone as a coenzyme, two subunits are linked via one or more disulfide bonds. It is described that it is modified so as to exhibit high thermal stability (for example, see Patent Document 1). This is based on the higher order structure of the PQQ-dependent glucose dehydrogenase based on the X-ray crystal structure analysis, and when the dimer enzyme is constructed, specific amino acid residues that are located close to each other on the monomer interface Is substituted with cysteine using a genetic engineering technique to form a disulfide bond between the monomers. However, since this technique is based on a higher-order structure by X-ray crystal structure analysis, there is a problem that it cannot be applied to an enzyme for which crystal structure information cannot be used. In addition, artificial alterations such as alterations at the gene level cause changes in the three-dimensional structure of the protein, so that various functions originally retained by the enzyme, such as enzyme activity, are reduced or decreased even if the thermal stability is improved. There was also a problem of deletion. Furthermore, the change in the three-dimensional structure of the protein often causes a problem that a sufficient production amount cannot be obtained at the time of enzyme production in a protein expression system using Escherichia coli or the like, which has been a major obstacle in industrial use.

  In addition, a method for stabilizing the three-dimensional structure by applying a heating treatment to the PQQ-dependent glucose dehydrogenase, thereby improving the thermal stability has been reported. However, since the three-dimensional structure of the protein is changed depending on the heat treatment, there is a problem that various functions originally held by the enzyme are reduced or deleted as in the above-described method. In addition, since this method requires delicate setting of conditions such as warming time and temperature, the application range is limited.

Therefore, it has been desired to establish a technique capable of improving stability, particularly thermal stability, while maintaining various functions inherently retained by the enzyme.
International Publication No. 2002-072839 JP 2007-129966 JP

  Therefore, in order to solve the above problems, the present invention provides a method for stabilizing a coenzyme-linked enzyme that can improve the stability while maintaining various functions originally retained by the coenzyme-linked enzyme. An object of the present invention is to provide a composition, an enzyme sensor, and a fuel cell prepared using the stabilization method.

  As a result of repeated studies to solve the above problems, the present inventors have improved the stability of the enzyme by adding a coenzyme for the enzyme to a composition comprising a coenzyme-linked enzyme as a constituent component. I found out that I can do it. Based on this finding, the present inventors have completed the present invention.

That is, in order to achieve the above object, the following inventions [1] to [7] are provided.
[1] A method for stabilizing a coenzyme-linked enzyme,
The coenzyme is added to the composition comprising the coenzyme-linked enzyme as a constituent component by adding a coenzyme required for the functional expression of the coenzyme-linked enzyme and having binding ability to the coenzyme-linked enzyme. A method for stabilizing conjugated enzymes. Here, for example, the stability is thermal stability.
[2] The method according to [1], wherein the coenzyme is at least one coenzyme selected from nicotinamide adenine dinucleotide, nicotine adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, and pyrrolopyrroline quinone. .
[3] The method of [1] or [2] above, wherein the coenzyme-linked enzyme is an oxidoreductase.
[4] The oxidoreductase is an NAD ⁺ -dependent formaldehyde dehydrogenase derived from Pseudomonas putida, a PQQ-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus , and thermus thermophilus [3] The method described above, which is selected from PPQ-dependent glucose dehydrogenase derived from ( Thermus thermophilus ).

  According to the configuration of the above [1] to [4], the stability of the enzyme including thermal stability and storage stability is maintained while maintaining various functions originally retained by the coenzyme-linked enzyme. It becomes possible to raise. Therefore, the coenzyme-linked enzyme whose stability has been improved by the stabilization method of the present invention is applied to a technical field in which functions such as a medium function of the enzyme are required in various industrial fields such as medical, food, and environmental fields. Applicable.

[5] A stabilized coenzyme-linked enzyme composition comprising the coenzyme-linked enzyme in a state stabilized by any one of the methods [1] to [4].

  According to the configuration of [5] above, a coenzyme-linked type that has improved stability, including thermal stability and storage stability, while maintaining various functions originally retained by the coenzyme-linked enzyme. A stabilized coenzyme-linked enzyme composition containing an enzyme can be provided. Accordingly, the coenzyme-linked enzyme composition of the present invention is applied to technical fields that require the function of the enzyme in various industrial fields such as medical, food, and environmental fields, such as measurement techniques that utilize the catalytic function of the enzyme. Applicable.

[6] An enzyme sensor in which a coenzyme-linked enzyme is immobilized on an electrode in a state stabilized by any one of the methods [1] to [4].

  According to the configuration of the above [6], an enzyme sensor using a coenzyme-linked enzyme whose stability has been improved by the stabilization method of the present invention can be provided, and can be used in various industrial fields such as medical, food, and environmental fields. It can be applied to technical fields where the function of the enzyme is required. In particular, since the coenzyme-linked enzyme used here is maintained in a state of high thermal stability and storage stability, the measurement accuracy can be improved without causing deterioration of the catalytic ability of the enzyme. Furthermore, since the amount of enzyme used can be reduced, a cost reduction effect can also be achieved.

[7] The coenzyme-linked enzyme is contained in a state stabilized by any of the methods [1] to [4], and receives electrons generated by the catalytic reaction of the coenzyme-linked enzyme. A fuel cell comprising: an anode electrode; a cathode electrode that holds either a catalyst capable of transferring electrons to oxygen or an enzyme; and the anode electrode and the cathode electrode are electrically coupled.

  According to the configuration of [7] above, it is possible to provide a fuel cell using a coenzyme-linked enzyme whose stability has been improved by the stabilization method of the present invention. In particular, since the coenzyme-linked enzyme used here is maintained in a state of high thermal stability and storage stability, it becomes possible to generate power continuously without causing deterioration of the catalytic ability of the enzyme. The performance of the fuel cell can be improved. Furthermore, since the amount of enzyme used can be reduced, a cost reduction effect can also be achieved.

  Hereinafter, specific embodiments of the present invention will be described. However, these are merely examples of the present invention, and do not limit the present invention.

(Method for stabilizing the coenzyme-linked enzyme of the present invention)
In the method for stabilizing a coenzyme-linked enzyme of the present invention, a coenzyme for the coenzyme-linked enzyme is added to a composition comprising a coenzyme-linked enzyme as a constituent component. Thereby, the stability of the enzyme itself is improved while maintaining various functions inherently retained by the enzyme such as a catalytic function.

“Enzyme stabilization” refers to stabilization of an enzyme against heat and storage, and means that deactivation of the catalytic function of the enzyme and reduction in activity can be suppressed against heat treatment and storage for a certain period of time. Specifically, in the present specification, by making a coenzyme-linked enzyme coexisting with a coenzyme for the enzyme, heat treatment or storage for a certain period of time rather than the state of the composition not coexisting with the coenzyme. It means that the residual ratio of the function of the enzyme maintained after being subjected to is high. For example, the catalytic activity value of the method-enzyme-binding enzyme at 25 ° C. at which the enzyme function is normally maintained is 100%, and the activity value after heat treatment and storage for a certain period is calculated as the residual rate of enzyme activity. If the residual ratio is increased compared to the composition not coexisting with the coenzyme, it can be determined that the thermal stability of the enzyme has been improved. As an example, it is preferable that a coenzyme-linked enzyme stable at room temperature can maintain a residual activity of 50% or more, particularly 80% or more by heating at 50 ° C. for 30 minutes. Further, further stabilization of a coenzyme-linked enzyme that is stable under high temperature conditions is also included as a concept of the present invention.

  Here, the target of the stabilization method of the present invention is a coenzyme-linked enzyme. A coenzyme-linked enzyme refers to an enzyme that can exert its catalytic function by binding to a coenzyme. And as long as it has such a property, all can be made into the object of this invention. Therefore, there is no restriction on the origin of the enzyme, and it may be prepared from any living organism such as naturally occurring bacteria, yeast, and animals and plants. Examples of bacteria include thermophilic bacteria and halophilic bacteria. In addition, bacteria that grow in extreme environments such as acidophilic bacteria and alkaliphilic bacteria are also included. And what was manufactured as a recombinant by the genetic engineering method, or what was synthesize | combined chemically may be used. Further, it may be a modified product obtained by artificially mutating a naturally occurring enzyme. Further, it may be modified with various labeling compounds such as fluorescent substances and radioisotopes, and in a form fused with other proteins such as antibodies and tag peptides.

  Here, the modified substance means a substance containing an amino acid sequence having a modified site where a specific amino acid of a naturally-derived enzyme is modified. “Modification” means that a modification in which at least one of one or more amino acids is deleted, substituted, inserted, or added in the amino acid sequence of the protein that is the basis of the modification has occurred. "A modification comprising at least one of deletion, substitution, insertion and addition of one or a plurality of amino acids" means a known DNA recombination technique or a point mutation introduction method for a gene encoding a protein serving as a basis for the modification. Etc. means that as many amino acids as can be deleted, substituted, inserted or added are deleted, substituted, inserted or added, including combinations thereof. For example, such a variant has an amino acid level of 70% or more, preferably 80% or more, more preferably 90% or more with respect to the amino acid sequences shown in SEQ ID NOs: 1, 2, 4, 5, 6 and the like. It can retain homology.

  The type of enzyme that is the subject of the stabilization method of the present invention is not particularly limited, but is preferably an oxidoreductase. Here, an oxidoreductase is an enzyme that catalyzes a redox reaction of a biological substance. Depending on the type, nature, donor, acceptor type, etc. of the redox reaction, a dehydrogenase (dehydrogenase), a reductase ( Reductase), oxidase (oxidase), oxygenated enzyme (oxygenase) and the like, and any of these are included. Specifically, glucose dehydrogenase, formaldehyde dehydrogenase, aldehyde dehydrogenase, cholesterol dehydrogenase, lactate dehydrogenase, pyruvate dehydrogenase, alcohol dehydrogenase, aldehyde reductase, thioredoxin reductase, glucose Examples include, but are not limited to, oxidase, pyruvate oxidase, cholesterol oxidase, lactate oxidase, ascorbate oxidase, cytochrome oxidase, xanthine oxidase, catalase, and peroxidase.

Preferably, NAD ⁺ dependent formaldehyde dehydrogenase that requires NAD ^{+ in} the expression of its catalytic activity, PQQ dependent glucose dehydrogenase that requires PQQ in the expression of its catalytic activity, and the like are exemplified. NAD ⁺ -dependent formaldehyde dehydrogenase from Pseudomonas putida (for the sequence information, the deduced amino acid sequence is shown in SEQ ID NO: 1 and GenBank Accession Number P46154), Acinetobacter calcoaceticus ( Acinetobacter calcoaceticus ) Derived PQQ-dependent glucose dehydrogenase (for the sequence information, the deduced amino acid sequence is shown in SEQ ID NO: 2 and GenBank Accession Number CAA30222), Thermos thermophilus, a thermostable enzyme derived from an extreme thermophile to (Thermus thermophilus) from the PQQ-dependent glucose dehydrogenase (sequence information We can, together show the nucleotide sequence and deduced amino acid sequence in SEQ ID NO: 3 and 4, see GenBank Accession Number YP_143836), Bacillus bacterium (Bacillus sp.) Per glucose dehydrogenase (sequence information from, the deduced amino acid sequence As well as GenBank Accession Number ZP_01173628) and Pseudomonas putida derived glucose sorbosone dehydrogenase (Sequence information shows the deduced amino acid sequence in SEQ ID NO: 6 and GenBank Accession Number YP_143836) Etc. are preferably exemplified.

  A coenzyme is a substance that binds to the protein portion of the enzyme and contributes to the expression of the catalytic function of the enzyme. Specifically, nicotinamide adenine dinucleotide (NAD), nicotine adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), pyrrolopyrroline quinone (PQQ), thiamine Although phosphoric acid (TTP) etc. are illustrated, it is not limited to this. Moreover, the analog which has the function similar to these may be sufficient.

Here, a coenzyme that is required for the functional expression of the target coenzyme-linked enzyme and has binding ability to the coenzyme-linked enzyme is added. For example, in the case of NAD ⁺ dependent formaldehyde dehydrogenase and NAD ^+, In the case of PQQ-dependent glucose dehydrogenase is added to PQQ. When the target enzyme requires two or more types of coenzymes, two or more types of coenzymes may be combined, or even one type of coenzyme can exert its effect. The above is an example and is not limited thereto.

  The ratio between the coenzyme-linked enzyme and the coenzyme is not particularly limited, but it is generally preferable to add more than the amount of coenzyme required for the coenzyme-linked enzyme to exist as a holoenzyme. The ratio of the specific coenzyme-binding enzyme and the coenzyme to be added is appropriately set in consideration of the affinity between the enzyme and the coenzyme, and the pH and salt concentration of the buffer. Preferably, the coenzyme should be added in an equimolar amount or in excess with respect to the coenzyme-linked enzyme. For example, the coenzyme molar concentration: coenzyme-linked enzyme molar concentration ratio = 1: 1 to 100. : 1. However, in consideration of cost and the like, the coenzyme may be used in an equimolar amount or less with respect to the coenzyme-linked enzyme. For example, coenzyme molar concentration: coenzyme-linked enzyme molar concentration ratio = 1: 10. It can also be added at ˜1: 1.

(Coenzyme-linked enzyme composition)
The composition of the present invention comprises a coenzyme-linked enzyme in a state stabilized by adding a coenzyme to the coenzyme-linked enzyme. The ratio of the coenzyme-linked enzyme to the coenzyme in the composition is not particularly limited, but in general, the coenzyme-linked enzyme should be added more than the amount of coenzyme required for the presence of the coenzyme-linked enzyme as a holoenzyme is required. Details are described above.

  Furthermore, the composition of the present invention may be a liquid composition or a solid composition. In addition to a coenzyme-linked enzyme and a coenzyme, a pharmaceutically acceptable carrier can be included. Such a carrier is not particularly limited as long as the biological activity of the coenzyme and the coenzyme-linked enzyme can be stably maintained regardless of whether the carrier is solid or liquid.

  Liquid compositions include solutions, suspensions, and emulsions prepared using appropriate buffers as carriers. The buffer solution is not particularly limited as long as it is generally used in this technical field. For example, buffer solutions such as tris-hydrochloric acid, phosphoric acid, boric acid, acetic acid, citric acid, phthalic acid and salts thereof may be mentioned, and those prepared at pH 5.0 to 9.0, particularly pH 6.0 to 7.0 are preferable. Furthermore, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, alcohols such as ethanol, polysorbate 80, etc. can be used as a carrier. Furthermore, auxiliary agents such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers, and solubilizing agents may be included.

  The solid preparation can be prepared, for example, by pulverizing the enzyme solution by freeze drying or vacuum drying. Furthermore, a suitable carrier can be contained, and mixed with excipients, binders, lubricants, disintegrants, etc., and tablets, powders, fine granules, granules, capsules, pills, etc. by conventional methods It can also be prepared. Here, the solid carrier is not particularly limited as long as it is generally used in this technical field. For example, as an excipient, starch, lactose, sucrose, calcium carbonate, calcium phosphate, etc., as a binder, starch, gum arabic, carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, etc., as a lubricant, stearic acid, Examples of disintegrants such as magnesium stearate and calcium stearate include carboxymethyl cellulose and talc.

  The composition of the present invention can be used as a measurement reagent or the like using the catalytic function of the coenzyme-linked enzyme that is a constituent component thereof, and the function of the enzyme in various industrial fields such as medical, food, and environmental fields. Available for required technical fields. In particular, the coenzyme-linked enzyme in the composition of the present invention is maintained in a state of high thermal stability and storage stability, and thus can be applied to a wide range of technical fields. Here, examples of the sample include biological samples derived from living organisms such as blood, urine, saliva, food samples, and environmental samples, but are not limited thereto. Moreover, the sample which performed the appropriate process to these samples as needed may also be included.

(Enzyme sensor)
The present invention provides an enzyme sensor that utilizes a coenzyme-linked enzyme in a stabilized state by adding a coenzyme to the coenzyme-linked enzyme. The enzyme sensor is configured such that a working electrode on which a coenzyme-linked enzyme with improved stability is immobilized and a counter electrode are provided on the anode electrode side on an electrode material. If necessary, a three-electrode system may be configured by providing a reference electrode. As the electrode, carbon, gold, platinum or the like can be used. The enzyme can be immobilized on the electrode material by a known method. For example, it is possible to use a carrier binding method that is immobilized through physical adsorption, ionic bond, or covalent bond. In addition, a cross-linking method in which cross-linking and fixing is performed with a reagent having a divalent functional group such as glutaraldehyde can be used. Furthermore, polysaccharides such as alginic acid and carrageenan, gels having a network structure such as polyacrylamide, and semi-permeable It is also possible to use a comprehensive method that closes and immobilizes the membrane.

  In the measurement of the substrate, for example, when a sample to be measured is brought into contact, the substrate contained in the sample reacts with the enzyme composition having improved stability according to the present invention, which is immobilized on the working electrode, and then the electron mediator is reacted. Reduced. A voltage is applied to the electrode system to oxidize the reductant of the electron acceptor, and the substrate in the sample can be detected by the change in the obtained oxidation current value. At this time, the substrate concentration can be obtained based on the obtained oxidation current value by preparing a standard curve prepared in advance with a substrate solution having a standard concentration within the target concentration range.

  Since the enzyme sensor of the present invention utilizes the catalytic ability of an enzyme with improved thermal stability and storage stability, it can also prevent degradation during the production of the enzyme sensor, and can also prevent degradation of the catalytic function of the enzyme. Measurement accuracy can be improved and continuous measurement is possible without incurring. As a result, since the amount of enzyme used can be reduced, a cost reduction effect can also be achieved. Here, examples of the sample include biological samples derived from living organisms such as blood, urine, saliva, food samples, and environmental samples, but are not limited thereto. Therefore, the enzyme sensor of the present invention can be used in the medical, food and environmental fields. Moreover, the sample which performed the appropriate process to these samples as needed may also be included.

(Fuel cell)
The present invention provides a fuel cell that uses a coenzyme-linked enzyme in a stabilized state by adding a coenzyme to the coenzyme-linked enzyme. The fuel cell of the present invention includes, for example, an anode electrode that performs an oxidation reaction and a cathode electrode that performs a reduction reaction, and includes an electrolyte layer that separates the anode and the cathode as necessary. On the anode electrode side, the coenzyme-linked enzyme with improved stability takes out electrons generated by oxidizing the substrate to the electrode and generates protons. On the other hand, on the cathode side, the proton generated on the anode side reacts with oxygen to generate water. As the electrode, carbon, gold, platinum or the like can be used. The coenzyme-linked enzyme is supplied to the anode electrode side with improved stability. At this time, it may be supplied in a form dissolved in an appropriate buffer, but it is preferably immobilized on an electrode. A catalyst and an enzyme capable of transferring electrons to oxygen may be supplied to the cathode electrode side as necessary. The enzyme can be immobilized on the electrode material by a known method. For example, it is possible to use a carrier binding method that is immobilized through physical adsorption, ionic bond, or covalent bond. In addition, a cross-linking method in which cross-linking and fixing is performed with a reagent having a divalent functional group such as glutaraldehyde can be used. Furthermore, polysaccharides such as alginic acid and carrageenan, gels having a network structure such as polyacrylamide, and semi-permeable It is also possible to use a comprehensive method that closes and immobilizes the membrane.

  With the configuration described above, a substrate that can act on the enzyme is used as a fuel, and electrons generated when the substrate is oxidized pass the electrons to the anode electrode. Then, the electrons passed to the anode electrode reach the cathode electrode through an external circuit to generate a current.

  Since the fuel cell of the present invention utilizes the catalytic function of a coenzyme-linked enzyme with improved thermal stability and storage stability, it can continuously generate power and can be constructed as a high-performance fuel cell. It is also possible to prevent degradation of the enzyme during the production of the fuel cell. As a result, the amount of enzyme used can also be reduced, so that a cost reduction effect can be achieved.

As Preparation Example 1] NAD ⁺ dependent formaldehyde coenzyme-bound enzymes for the consideration of increased stability at dehydrogenase preparation following experiments were prepared NAD ⁺ dependent formaldehyde dehydrogenase.

1. Gene cloning
A gene encoding NAD ⁺ -dependent formaldehyde dehydrogenase was cloned from the genomic DNA of Pseudomonas putida strain using PCR.

  First, Pseudomonas putida strains sold by the National Institute for Product Evaluation and Technology (Domestic Microorganisms / Gene Bank: NITE) were cultured according to the instructions of the distributors to obtain the cells. Subsequently, genomic DNA was prepared from the cells by extraction and purification using a genomic DNA extraction kit (Promega).

  The PCR reaction was performed with a PCR kit, PrimeStar (Takara Bio) according to the manufacturer's instructions. A PCR reaction solution was prepared containing 0.3 μM of each primer and 50 ng of the template DNA prepared above. In the PCR temperature program, 30 cycles of amplification reaction were carried out, with heat denaturation at 98 ° C. for 10 seconds, annealing at 55 ° C. for 5 seconds, and extension reaction at 72 ° C. for 60 seconds as one cycle.

The sequence information of the primers used here is shown.
Primer for Pseudomonas petitida
5'-GAG CA TATG T CTGGT AATCG TGGTG T-3 '(SEQ ID NO: 7 )
5′-CTA AA GCTT A GGCCG CGCTG AGGTC T-3 ′ (SEQ ID NO: 8 )
The underline indicates the restriction enzyme recognition site for cloning.

  The reaction solution after the PCR amplification reaction was purified with a DNA purification kit (manufactured by GE Healthcare) to confirm the PCR amplification product. As a result, it was confirmed that the expected DNA amplification product (about 1.2 kbp) was obtained.

2. Expression vector construction and expression in E. coli cells
A DNA fragment (about 1.2 kbp) obtained by PCR amplification was cleaved with restriction enzymes Nde-I and Hind-III, separated by agarose gel electroflow, and extracted from the gel and purified. Each DNA fragment after the restriction enzyme treatment was incorporated into restriction enzyme sites (Nde-I and Hind-III) of a pET22b vector (manufactured by Novagen), a protein expression plasmid, to construct an expression vector.

Escherichia coli BL21 (DE3) strain (manufactured by Novagen) was transformed with the obtained expression vector. E. coli was cultured at 37 ° C. in Luria-Bertani (LB) medium containing 50 mg / l ampicillin. The transformation was performed according to the method described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press). Subsequently, the obtained transformant was cultured in an LB medium containing 50 mg / l ampicillin until the absorbance of the culture solution reached OD ₆₀₀ = 0.6. Then, to induce synthesis of the recombinant protein, isopropyl-β-thiogalactopyranoside (IPTG) having a final concentration of 0.5 mM was added and further cultured for 4 hours.

  The bacterial cells collected by centrifuging 1 ml of the culture solution were suspended in 200 μl of the solubilized solution. Subsequently, after disrupting the cells by ultrasonic disruption (30-s pulses; maximum power; high intensity; 0.5-s repeating duty cycle), the cell disruption solution is centrifuged at 15,000 xg for 10 minutes. I got Qing. The supernatant was heat-treated at 95 ° C. for 3 minutes and then subjected to 12.5% acrylamide gel (Ato) electrophoresis. After electrophoresis, the protein was visualized by Coomassie Brilliant Blue (hereinafter abbreviated as “CBB”) staining (manufactured by Wako Pure Chemical Industries), thereby confirming the expression of the recombinant protein.

3. Purification of enzyme In addition, the protein expressed above was separated and purified by hydrophobic chromatography, anion exchange chromatography, and gel filtration according to a conventional method.

[Preparation Example 2] Preparation of PQQ-dependent glucose dehydrogenase PQQ-dependent type derived from Acinetobacter calcoaceticus as a coenzyme-linked enzyme to be considered for stability improvement in the following experiment Glucose dehydrogenase was prepared.

1. Gene cloning
A gene encoding PQQ-dependent glucose dehydrogenase was cloned from the genomic DNA of Acinetobacter calcoaceticus using PCR.

  For detailed procedures, refer to Yuhashi N. et al., “Development of novel glucose enzyme fuel cell system using protein engineered PQQ glucose dehydrogenase”, Biosens Bioelectron., 2005, Vol. 20, pages 2145-2150. went.

2. Construction of expression vector and expression in E. coli cells Transformation of E. coli BL21 (DE3) strain (manufactured by Novagen) was performed with the expression vector obtained above. The obtained transformant was cultured with shaking in LB medium until the absorbance of the culture solution reached OD ₆₀₀ = 0.6. Then, IPTG with a final concentration of 0.01 mM was added to induce expression of the recombinant protein. After induction of expression, the cells were further cultured overnight at 28 ° C.

  The cells recovered by centrifuging the culture solution were suspended in a phosphate buffer. Subsequently, lysozyme was added and the cells were disrupted by repeating freeze-thawing three times. An appropriate amount of benzonate was added to the cell disruption solution and allowed to stand at room temperature, followed by centrifugation to obtain a supernatant.

3. Purification of enzyme Since the His tag peptide was fused with the protein obtained here, the enzyme was separated and purified by metal ion affinity chromatography. Detailed separation and purification procedures are shown below. Specifically, TALON resin (manufactured by CLONTECH), which is a His tag purification resin, was used.

The details of the separation / purification procedure are as follows.
1. 1. Equilibrate column with phosphate buffer Add sample 3. Wash column with phosphate buffer containing 1 mM imidazole 4. Elution protein with phosphate buffer containing 100 mM imidazole

The obtained elution fraction was replaced with 30 mM MOPS buffer (pH 7.0) containing 1 mM CaCl ₂ while removing imidazole using Hitrap desalting colum (manufactured by GE Healthcare). Next, PQQ was added at a final concentration of 30 μM for enzyme hololysis and left overnight at 4 ° C. in the dark. After standing, excess PQQ was removed with Hitrap desalting colum.

[Preparation Example 3] Preparation of thermostable PQQ-dependent glucose dehydrogenase A PQQ-dependent glucose dehydrogenase derived from Thermus thermophilus was prepared as an example of a coenzyme-linked enzyme. Such an enzyme has heat resistance.

1. Gene Cloning A gene encoding a thermostable PQQ-dependent glucose dehydrogenase was cloned from the genomic DNA of Thermos thermophilus using PCR.

As a template, genomic DNA (Takara Bio: Catalog No. 3071) of Thermus thermophilus HB8 was used. Based on the base sequence (YP_143836) of the open reading frame (hereinafter referred to as “ORF”) of PQQ-dependent glucose dehydrogenase derived from Thermus thermophilus HB8, the primer has a region between the start and end codons of the ORF. Designed to be amplified. At this time, a restriction enzyme Nde I recognition sequence (CATATG) for ligating the vector was introduced into the primer tth_gdh_f1 on the start codon side. Similarly, a restriction enzyme Hind III recognition sequence (AAGCTT) for vector ligation was similarly introduced into the primer tth_gdh_r1 on the end codon side. The primer tth_gdh_r1 was designed so that the stop codon corresponds to glycine (GCC) in order to add a purification His tag label to the carboxyl terminus.

Specific sequence information is as follows.
Primer tth_gdh_f1
5'-ACATTCATATGGACCGGAGGCGCTTTCTCGT-3 '(SEQ ID NO: 9)
Primer tth_gdh_r1
5'-ATACCAAGCTTGCCAAGGAGGCGTAGCACCCGGTC-3 '(SEQ ID NO: 10)

The composition of the PCR reaction solution and the PCR program are as follows.
PCR reaction composition:
1 x KOD plus buffer (Toyobo),
0.4 mM dNTPs,
0.5 μM primer tth_gdh_f1,
0.5 μM primer tth_gdh_r1, or 0.5 μM primer tth_gdh_r1,
10% (v / v) dimethyl sulfoxide,
1.25 mM MgSO ₄ ,
0.5 unit KOD plus (Toyobo)
5 ng Thermus thermophilus HB8 genomic DNA.

Reaction program:
98 ° C. for 1 minute, [98 ° C. for 10 seconds → (65 ° C.−0.5 ° C./2 cycles) → 68 ° C. for 1 minute] × 35 cycles → 68 ° C. for 1 minute.

2. Expression vector construction and expression in E. coli cells
After PCR amplification, an amplified fragment of DNA of about 1 kbp was purified. The purified amplified fragment and the plasmid vector pET22b (Novagen) were digested with restriction enzymes NdeI and HindIII , respectively. Subsequently, the amplified fragment and the plasmid vector pET22b were mixed and ligated. For the ligation reaction, DNA ligation kit (Takara Bio) was used. The reaction solution after ligation was introduced into E. coli XL1-Blue (Stratagene) and transformed. Then, it inoculated and cultured on the plate of antibiotics ampicillin containing LB agar medium. After the culture, a single colony of XL1-Blue formed on the plate was selected, and this colony was cultured in a small amount of medium (1.5 ml). Subsequently, the plasmid was purified from the cultured cells with a plasmid mini kit (QIAGEN), and the base sequence of the fragment inserted into the plasmid was determined by the chain terminator method. The base sequence and the deduced amino acid sequence are shown in SEQ ID NOs: 3 and 4, respectively. Here, the base sequence of the amplified DNA fragment coincided with the base sequence of YP_143836, and it was confirmed that the nucleic acid molecule encoding the PQQ-dependent glucose dehydrogenase was cloned.
The expression vector obtained above was used to transform E. coli BL21 (DE3) strain in the same manner as in Preparation Example 2 to induce transgene expression.

3. Purification of enzyme In the same manner as in Preparation Example 2, the protein was purified from the cells to obtain PPQ-dependent glucose dehydrogenase.

[Example 1] Enzyme stabilization (formaldehyde dehydrogenase)
The thermal stability of the coenzyme-linked enzyme in the presence of the coenzyme was confirmed. Here, the NAD ⁺ -dependent formaldehyde dehydrogenase derived from Pseudomonas putida obtained in Preparation Example 1 was examined.

It confirmed the thermal stability of the coenzyme presence of coenzyme-bound enzyme, NAD ⁺ dependent formaldehyde dehydrogenase. NAD ⁺ was added to the NAD ⁺ -dependent formaldehyde dehydrogenase solution derived from Pseudomonas putida obtained in Preparation Example 1 above to a final concentration of 10 mM, and various in 20 mM Trisaminomethane buffer (pH 7.5). It was kept warm for 20 minutes under various temperature conditions (no heat treatment (25 ° C.), 50, 60, 70 ° C.), and the residual activity after being placed on ice for 30 minutes was confirmed.

As a comparative example, the enzyme solution to which NAD ⁺ was not added was also heat-treated in the same manner to determine the residual activity of the enzyme.

The enzyme activity was measured by quantifying NADH produced by the catalytic reaction of formaldehyde dehydrogenase by measuring the absorbance at a wavelength of 340 nm, and using this as an index to determine the formaldehyde decomposition activity. In other words, the enzymatic activity of formaldehyde dehydrogenase, formaldehyde and NAD ^+, in the catalytic reaction which produces formic acid and NADH, may be measured by quantifying the generated NADH directly.

In detail using standard reaction conditions as an example, an appropriate amount of enzyme solution was mixed with 50 mM potassium phosphate buffer (pH 8.0) containing 1 mM formaldehyde, 1 mM NAD ⁺ , and 0.01% BSA to make 200 μl. . Subsequently, the change in absorbance at 340 nm accompanying the change in NADH from NAD ⁺ in this reaction solution was measured at 37 ° C., and an activity evaluation test was performed by determining the increase in absorbance. In this example, the absorbance was measured with a fluorescent microplate reader (Molecular Device), and the activity evaluation test was performed by measuring the change in absorbance over time every 30 seconds.

The results are shown in FIGS. 1 (A) to 1 (D) and FIG.
Fig. 1 (A) shows a comparison with and without NAD ⁺ when kept at 25 ° C.
Fig. 1 (B) shows the comparison with and without NAD ⁺ when kept at 50 ° C.
Fig. 1 (C) shows a comparison with and without NAD ⁺ when kept at 60 ° C.
FIG. 1 (D) is a graph showing a comparison between addition and non-addition of NAD ⁺ when kept at 70 ° C.
2, as 100 activity when incubated at 25 ° C. without NAD ⁺ addition, a graph of NAD ⁺ addition, and the residual activity at each temperature of the enzyme solution without additives in relative activity.

In the heat treatment at 60 ° C., when NAD ⁺ was not added, the enzyme activity was 0, but it was confirmed that about 90% of the activity was retained by addition.

From the above results, it was confirmed that the thermal stability of NAD ⁺ -dependent formaldehyde dehydrogenase is significantly improved by adding NAD ⁺ which is a coenzyme of the enzyme.

[Example 2] Stabilization of enzyme (formaldehyde dehydrogenase)
The thermal stability of the coenzyme-linked enzyme in the presence of the coenzyme was confirmed by following the active structure of the enzyme.

  Formaldehyde dehydrogenase derived from Pseudomonas putida forms a tetramer as its active structure. Therefore, the thermal stability of the enzyme was confirmed by confirming whether or not the tetramer structure was maintained by heat treatment. The sample treated in Example 2 was subjected to 4-16% native acrylamide gel electrophoresis to confirm the retention of the enzyme active structure.

The results are shown in FIGS.
FIG. 3 (A) shows the electrophoresis results of the enzyme solution when NAD ^{+ is} not added. Lane 1 is 25 ° C., Lane 2 is 50 ° C., Lane 3 is 60 ° C., Lane 4 is kept at 70 ° C. Is the result of
Lane M is a molecular weight marker.
FIG. 3 (B) shows the electrophoresis results of the enzyme solution when NAD ⁺ was added. Lane 1 was 25 ° C., Lane 2 was 50 ° C., Lane 3 was 60 ° C., and Lane 4 was kept at 70 ° C. Is the result of
The arrow indicates the estimated position of the active structure of the enzyme, that is, the band signal of the tetramer structure. The estimated molecular weight of such a tetramer is 170 kDa.

3 (A) to 3 (B), it was confirmed that in the heat treatment at 50 ° C., when NAD ⁺ was not added, the active structure of the enzyme was dissociated. On the other hand, it was confirmed that by adding NAD ⁺ , the active structure can be completely retained by heat treatment up to about 60 ° C.

From the above results, it was confirmed that the NAD ⁺ -dependent formaldehyde dehydrogenase significantly improves its thermal stability by adding NAD ⁺ which is a coenzyme of the enzyme, and maintains the active structure. The result of Example 1 was confirmed also from a viewpoint.

[Example 3] Enzyme stabilization (glucose dehydrogenase)
The thermal stability of the coenzyme-linked enzyme in the presence of the coenzyme was confirmed. Here, the PQQ-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus obtained in Preparation Example 2 was examined.

We confirmed the thermal stability of PQQ-dependent glucose dehydrogenase, a coenzyme-linked enzyme, in the presence of coenzyme. The PQQ-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus obtained in Preparation Example 2 is 10 μg / g in 30 mM MOPS buffer (pH 7.0) containing 1% BSA and 1 mM CaCl _2. Prepare ml of enzyme solution, add PQQ to a final concentration of 10 mM, and keep it warm for 30 minutes under various temperature conditions (no heat treatment (25 ° C), 50, 60, 70 ° C) and then in ice for 10 minutes The remaining activity after placing was confirmed.

  As a comparative example, the enzyme solution to which PQQ was not added was similarly heat-treated to determine the residual activity of the enzyme.

  The enzyme activity was measured by measuring the reduction rate of 2,6-dichlorophenolindophenol (DCIP) using phenazine mesosulfate (PMS) as a mediator in the presence of 10 mM glucose. The reduction rate of DCIP was evaluated by the disappearance of absorbance at a wavelength of 600 nM.

The results are shown in FIG.
FIG. 4 is a graph in which the activity when kept at 25 ° C. without addition of PQQ is defined as 100, and the residual activity at each temperature of the enzyme solution with and without addition of PQQ is expressed as a relative activity.

  From FIG. 4, it was confirmed that in the heat treatment at 60 ° C., when PQQ was not added, the enzyme activity decreased by about 70%, but by adding it, about 80% activity was retained.

  From the above results, it was confirmed that the thermal stability of PQQ-dependent glucose dehydrogenase is remarkably improved by adding PQQ which is a coenzyme of the enzyme.

[Example 4] Enzyme stabilization (glucose dehydrogenase)
The thermal stability of the coenzyme-linked enzyme in the presence of the coenzyme was confirmed by following the active structure of the enzyme.

  The sample treated in Example 3 was subjected to 12.5% denaturing acrylamide gel electrophoresis to confirm the retention of the protein structure of the enzyme.

The results are shown in FIG.
Lanes 1 and 2 are the results of electrophoresis when kept at 25 ° C., lane 1 shows the results when PQQ is added, and lane 2 shows the results when PQQ is not added.
Lanes 3 and 4 are the results of electrophoresis when kept at 50 ° C., lane 3 shows the results when PQQ is added, and lane 4 shows the results when PQQ is not added.
Lanes 5 and 6 are the results of electrophoresis when kept at 60 ° C., lane 5 shows the results when PQQ is added, and lane 6 shows the results when PQQ is not added.
Lanes 7 and 8 are the results of electrophoresis when kept at 70 ° C., lane 7 shows the results when PQQ is added, and lane 8 shows the results when PQQ is not added.
Lane M is a molecular weight marker.

  From FIG. 5, it was confirmed that the protein structure was denatured in the heat treatment at 70 ° C. when PQQ was not added. On the other hand, it was confirmed that the protein structure was not denatured by heat treatment at temperatures up to about 70 ° C. by adding PQQ.

  From the above results, it was confirmed that the PQQ-dependent formaldehyde dehydrogenase significantly improves its thermal stability by adding PQQ, which is a coenzyme of the enzyme, from the viewpoint of maintaining the protein structure. The results of Example 3 were confirmed.

  In view of the results of Examples 1 to 4, it was found that the coenzyme-linked enzyme can improve its thermal stability by adding the corresponding coenzyme.

  The present invention relates to a method for stabilizing a coenzyme-linked enzyme, and can be used in various industrial fields such as medical, food and environmental fields.

(A) The graph which shows the result of Example 1 which confirmed the thermostability (25 degreeC) of the coenzyme binding type enzyme (NAD ⁺ dependence formaldehyde dehydrogenase) in presence of a coenzyme. (B) In presence of a coenzyme. The graph which shows the result of Example 1 which confirmed the thermostability (50 degreeC) of the coenzyme binding type enzyme (NAD ⁺ dependence formaldehyde dehydrogenase) (C) Coenzyme binding type enzyme (NAD ⁺ ) in presence of a coenzyme (D) A graph showing the results of Example 1 in which the thermostability (dependent formaldehyde dehydrogenase) was confirmed (60 ° C.) (D) Coenzyme-linked enzyme (NAD ⁺ dependent formaldehyde dehydrogenase) in the presence of a coenzyme The graph which shows the result of Example 1 which confirmed thermal stability (70 degreeC). The graph which shows the result of Example 1 which confirmed the thermostability of the coenzyme binding type enzyme (NAD ⁺ dependence type-formaldehyde dehydrogenase) in presence of a coenzyme. (A) complement the thermal stability of the coenzyme-linked enzyme in the enzyme presence (NAD ⁺ dependent formaldehyde dehydrogenase) results (coenzyme no addition Example 2 was confirmed by tracking the active structure of the enzyme (B) The thermostability of a coenzyme-linked enzyme (NAD ⁺ dependent formaldehyde dehydrogenase) in the presence of a coenzyme was confirmed by tracing the active structure of the enzyme of Example 2 The electrophoretic diagram which shows a result (coenzyme addition). The graph which shows the result of Example 3 which confirmed the thermostability of the coenzyme binding type enzyme (PQQ dependence glucose dehydrogenase) in presence of a coenzyme. The electrophoretic diagram which shows the result of Example 4 which confirmed the thermal stability of the coenzyme binding type enzyme (PQQ dependence glucose dehydrogenase) in coexistence presence by tracking the active structure of an enzyme.

Claims (7)

A method for stabilizing a coenzyme-linked enzyme,
The coenzyme is added to the composition comprising the coenzyme-linked enzyme as a constituent component by adding a coenzyme required for the functional expression of the coenzyme-linked enzyme and having binding ability to the coenzyme-linked enzyme. A method for stabilizing conjugated enzymes.   The method according to claim 1, wherein the coenzyme is at least one coenzyme selected from nicotinamide adenine dinucleotide, nicotine adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, and pyrrolopyrroline quinone.   The method according to claim 1 or 2, wherein the coenzyme-linked enzyme is an oxidoreductase. The redox element is a NAD ⁺ -dependent formaldehyde dehydrogenase derived from Pseudomonas putida, a PQQ-dependent glucose dehydrogenase derived from Acinetobacter calcoaceticus , and Thermus thermophilus The method according to claim 3, which is selected from PPQ-dependent glucose dehydrogenase derived from   A stabilized coenzyme-linked enzyme composition comprising the coenzyme-linked enzyme in a state stabilized by the method according to any one of claims 1 to 4.   The enzyme sensor which fix | immobilized on the electrode in the state stabilized by the method as described in any one of Claims 1-4.   An anode electrode that contains the coenzyme-linked enzyme in a state stabilized by the method according to any one of claims 1 to 4, and that receives electrons generated by a catalytic reaction of the coenzyme-linked enzyme. A fuel cell comprising a cathode electrode that holds either a catalyst capable of transferring electrons to oxygen or an enzyme, and the anode electrode and the cathode electrode are electrically coupled.