JP6680084B2 - An enzyme electrode having a gluconic acid oxidation catalytic ability, a method for producing the enzyme electrode, a biobattery, and a biosensor. - Google Patents

Description

  The present invention relates to an enzyme electrode having a gluconic acid oxidation catalytic ability, a method for producing the enzyme electrode, and further to a biobattery and a biosensor using the enzyme electrode.

  An enzyme is a biocatalyst that promotes a chemical reaction (metabolism) in a living body, and has a basic structure of a protein composed of a chain of amino acids. As the characteristics of the enzyme, (1) it shows a catalytic action under mild conditions near room temperature and normal pressure, (2) it shows a catalytic action for a substrate specificity and a specific chemical reaction that acts only on a specific substrate, It also has a reaction specificity that does not cause side reactions. When a certain enzyme is immobilized on the surface of the electrode, only a specific reaction proceeds selectively on the electrode by the catalytic action of the enzyme, and the change of the substance due to such reaction can be converted into an electric signal by the electrode. Such an electrode is called an enzyme electrode, and is used as an electrode of a bio battery or a bio sensor.

  A bio battery is a fuel cell that applies an in-vivo energy conversion system, and is a power generation device that uses a biocatalyst as an electrode catalyst and couples a redox reaction and an electrode reaction by the biocatalyst to extract electric energy. A bio battery using a bio process has advantages that it has mild conditions and high selectivity, and that various substances existing in the environment such as sugar and alcohol can be used as a fuel. Therefore, since it is safe and has a small environmental load, further development is expected as a next-generation power source for small electronic devices such as portable devices and implantable devices. On the other hand, a biosensor is a device that detects and calibrates a substance by utilizing the excellent molecular discrimination ability of biomolecules such as enzymes.

  In the practical application of a biobattery and a biosensor that utilize the catalytic function of an enzyme, the success and failure of the selection of the enzyme and the existence of such an enzyme on the electrode so that the catalytic function can be exerted to the maximum extent depend. As a bio battery, in particular, the construction of a bio battery that uses glucose as a fuel, which is abundant in nature and has high energy conversion efficiency, is drawing attention as a highly practical technique. By constructing a complete oxidation system that decomposes glucose into carbon dioxide and water, maximum energy can be extracted from glucose. However, in constructing an artificial complete oxidation system, energy can be efficiently extracted from the substrate (fuel) by the enzyme reaction in each step, and the product produced by the enzyme reaction in the first step is a substrate for the enzyme reaction in the second step. It is necessary to appropriately combine a plurality of enzymes so that

For example, Non-Patent Document 1 reports on improvement of the performance of an enzyme electrode using glucose dehydrogenase as an electrode catalyst, which can be used as a first-stage enzyme of a glucose complete oxidation system. The enzyme electrode has flavin adenine nucleotide-dependent glucose dehydrogenase (hereinafter abbreviated as “FAD-GDH”) deglycosylated by chemical treatment with sodium periodate immobilized on a porous carbon electrode material. This is a modified bioanode (negative electrode) enzyme electrode, which is superior in current density in glucose oxidation to 5 times (100 mA / cm ² ) compared to the enzyme electrode on which FAD-GDH that is not deglycosylated is immobilized. It has been confirmed to have excellent electrode performance (see Non-Patent Document 1).

  Non-Patent Document 2 reports an enzyme electrode having an oxidation catalytic ability capable of oxidatively decomposing gluconic acid, which is a decomposition product of glucose by glucose dehydrogenase. The enzyme electrode decomposes glucose into glucuronic acid by a cascade (glucose dehydrogenase and gluconate-2-dehydrogenase) derived from a bacterium of the genus Gluconobacter, which is a type of acetic acid bacterium. In addition, Non-Patent Document 2 also reports on an enzyme electrode having a catalytic function for complete glucose oxidation. It has been reported that an enzyme electrode capable of realizing a glucose complete oxidation system could be constructed by using an enzyme such as an acetic acid bacterium or a barley-derived enzyme as an electrode catalyst.

Tsujimura S et al., "Exceptionally high glucose current on a hierarchically structured porous carbon electrode with" wired "flavin adenine dinucleotide-dependent glucose dehydrogenase.", J Am Chem Soc., 2014, 136, 41, 14432. -14 pages (Internet <URL: http: //pubs.acs.org/doi/abs/10.1021/ja5053736>) Shuai Xu and Shelley D. Minteer, "Enzymatic Biofuel Cell for Oxidation of Glucose to CO2.", ACS Catal., 2012, Volume 2, Issue 1, pp. 91-94.

  However, the enzyme used in the enzyme electrode of Non-Patent Document 1 is FAD-GDH derived from Aspergillus terreus, which has not been obtained from Ikeda Saccharification Co., Ltd. Therefore, it is considered that the technique of Non-Patent Document 1 is only an example of a special enzyme due to the type and physical properties of the sugar chain, and it is expected to be difficult to apply it to all glycosylated enzymes. Further, sodium periodate used for the deglycosylation treatment is an inorganic salt having a strong oxidizing power. Therefore, it can be easily estimated that not only deglycosylation but also the three-dimensional structure in the active site of the enzyme is affected and the enzyme is deteriorated.

  The glucose complete oxidation system reported in Non-Patent Document 2 is merely hypothetical information regarding an artificial sugar metabolism reaction system. For example, when a verification experiment was conducted on the reaction by the barley-derived oxalate oxidase that constitutes the glucose complete oxidation system, the reaction reported in Non-Patent Document 2 could not be confirmed. Therefore, even if an enzyme is selected according to the knowledge disclosed in Non-Patent Document 2, it is difficult and unrealistic to construct an enzyme electrode having the same catalytic ability for complete glucose oxidation.

  Further, in enzyme electrodes of bio-cells and biosensors, it may be necessary to select a glycosylated enzyme as an enzyme to be immobilized on the electrode material due to the problem of solubility of recombinant protein. For example, when gluconate dehydratase derived from a bacterium belonging to the genus Sulfolobus is cultivated in a host that does not have the ability to modify sugar chains, the recombinant enzyme forms an insoluble inclusion body and cannot sufficiently exhibit its original physiological activity. However, if the glycosylated enzyme is immobilized on the electrode material as it is, the sugar chain becomes an insulator and the electron transfer efficiency to the fuel → enzyme → (mediator) → electrode is significantly reduced, and the enzyme catalytic current is several milliamperes. The following problems were known to be small.

  Therefore, the present invention has an object to provide an enzyme electrode having an ability to catalyze the oxidation of gluconic acid, which is suitable for use as a negative electrode of a bio battery or a working electrode of a biosensor, and a method for producing the enzyme electrode. To do. Furthermore, another object is to provide a biobattery and a biosensor using an enzyme electrode having an oxidation catalyst ability of gluconic acid.

  As a result of repeated studies to solve the above problems, the present inventors recombinantly expressed gluconate dehydratase derived from a bacterium of the genus Sulfolobus in a state in which sugar chains were not added using a protein expression system of Escherichia coli. By immobilizing a concentrated solution of a lysate of expressed bacterial cells containing a modified gluconate dehydratase on an electrode material, it was found that an enzyme electrode capable of sufficiently exhibiting gluconate oxidation catalytic ability can be produced. . Furthermore, they have found that the enzyme electrode can function as a negative electrode of a bio battery or a working electrode of a biosensor. The present invention has been completed based on these findings.

  That is, in order to solve the above problems, the following inventions [1] to [7] are provided.

[1] An enzyme electrode having a gluconic acid oxidation catalytic ability, in which gluconic acid dehydratase derived from a bacterium belonging to the genus Sulfolobus is immobilized on an electrode material.

  According to the above configuration [1], it is possible to provide an enzyme electrode capable of sufficiently exhibiting the gluconic acid oxidation catalytic ability. Conventionally, for example, when gluconate dehydratase derived from a bacterium belonging to the genus Sulfolobus is synthesized using a protein synthesis system having no sugar chain modification pathway such as Escherichia coli in a state where no sugar chain is added, an inclusion body is formed. It was difficult because of problems such as On the other hand, in the case of synthesizing using a protein synthesis system having a sugar chain modification pathway, the addition of sugar chains reduces the association between the hydrophobic surfaces of the enzyme molecules, resulting in synthesis as a soluble enzyme. The synthesis efficiency of is improved. However, since the physical properties of the synthetic enzyme are affected by the type of sugar chain to be added and the state of addition, the handling of the enzyme to which the sugar chain is attached is often a major obstacle to industrial use. Moreover, when the glycosylated enzyme is immobilized on the electrode material as it is, the sugar chain becomes an insulator, the electron transfer efficiency to the fuel → enzyme → (mediator) → electrode is significantly reduced, and the enzyme catalytic current is several milliamperes. There are also problems such as the following being small. Therefore, conventionally, it was difficult to produce an enzyme electrode having a catalytic function of gluconic acid oxidation using gluconic acid dehydratase. With the above structure, it is possible to provide an enzyme electrode having a gluconic acid oxidation catalytic ability using gluconic acid dehydratase for the first time.

  The enzyme electrode provided by the above configuration can be used as a negative electrode of a bio battery and a working electrode of a biosensor, and can be used in industrial fields such as electronics, medical care, foods, and environmental fields.

[2] The electrode material includes a carbon base material having a porous structure with pores having an average diameter of 30 to 40 nm.

  According to the above configuration [2], it is possible to provide an enzyme electrode that can more efficiently exhibit the gluconic acid oxidation catalytic ability. When the electrode material is a carbon-based material having a porous structure, the enzyme molecules enter the pores of the carbon-based material when the enzyme is immobilized on the electrode material, so to speak, a purification effect of the enzyme can be obtained. , The association between enzyme molecules is suppressed. As a result, it is possible to provide an enzyme electrode capable of realizing an enzyme electrode even with an enzyme species that easily aggregates and efficiently exhibiting the catalytic function of gluconic acid oxidation.

[3] A method for producing an enzyme electrode having a gluconic acid oxidation catalytic ability, comprising a gluconic acid dehydratase derived from a bacterium belonging to the genus Sulfolobus immobilized on an electrode material,
(I) a step of transforming a bacterium having no sugar chain modifying ability with a gene encoding a glucose dehydratase derived from a bacterium belonging to the genus Sulfolobus, and preparing an expression cell expressing a recombinant gluconate dehydratase,
(Ii) a step of contacting the expressed bacterial cell with a surfactant,
(Iii) a step of crushing the expressed bacterial cells after the treatment with the surfactant to prepare a crushed solution of the expressed bacterial cells,
(Iv) a step of separating and removing the bacterial cell residue in the disrupted liquid of the expressed bacterial cells to prepare a lysate of the expressed bacterial cells,
(V) a step of concentrating the lysate of the expressing bacterial cells to prepare a concentrated solution of the lysing liquid of the expressing bacterial cells,
(Vi) A method for producing an enzyme electrode having a gluconic acid oxidation catalytic ability, comprising the step of immobilizing a concentrated solution of a lysate of the expressed bacterial cells on the electrode material.

  According to the configuration of the above [3], it is possible to provide a method for producing an enzyme electrode capable of sufficiently exhibiting a gluconic acid oxidation catalytic ability. Conventionally, it is difficult to produce an enzyme electrode having a gluconic acid oxidation-catalyzing ability of gluconic acid dehydratase derived from a Sulfolobus bacterium, in both a state in which a sugar chain is not added and a state in which a sugar chain is added. there were. On the other hand, according to the above-mentioned configuration, the gluconate dehydratase derived from the bacterium Sulfolobus is recombinantly expressed in a state in which sugar chains are not added using the protein expression system of Escherichia coli, and contains the recombinant gluconate dehydratase. By immobilizing the concentrated solution of the lysate of the expressed bacterial cells on the electrode material, it is possible to provide an enzyme electrode capable of sufficiently exerting the gluconic acid oxidation catalytic ability. The enzyme electrode produced by the above structure can be used as a negative electrode of a bio battery and a working electrode of a biosensor, and can be used in industrial fields such as electronics, medical care, food and environment.

[4] The electrode material includes a carbon base material having a porous structure with pores having an average diameter of 30 to 40 nm.

  According to the configuration of the above [4], it is possible to provide a method for producing an enzyme electrode that can more efficiently exhibit the gluconic acid oxidation catalytic ability. By using a carbon base material having a porous structure as the electrode material, when immobilizing the concentrated solution of the lysate of the expressing bacterial cells on the electrode material, the enzyme molecules can enter the pores of the carbon base material. In other words, the purification effect of the enzyme is obtained, and furthermore, the association between the enzyme molecules is suppressed. As a result, it is possible to provide an enzyme electrode that can achieve enzyme electrode formation even with an enzyme species that easily aggregates and can efficiently exhibit the catalytic function of gluconic acid oxidation.

[5] A step of preparing the carbon base material having the porous structure by firing a mixture of a carbon precursor and a magnesium oxide precursor and removing magnesium oxide from the obtained fired product.

  According to the above-mentioned constitution [5], by using the porous carbon base material produced by using magnesium oxide particles as a template as the electrode material, the fine carbonaceous base material in which a large number of pores of uniform size are regularly arranged is used. It is possible to stably and firmly fix the enzyme and, if necessary, the mediator, which is immobilized together with the enzyme, in the pores, and it is possible to suppress the detachment of the enzyme and the mediator from the electrode material. Thereby, the deterioration of the electrode material can be suppressed and the stability of the enzyme catalytic current can be achieved. Further, since the transfer of electrons between the enzyme and the conductive base material can be smoothly promoted, the enzyme catalytic current can be improved and the electrode performance can be improved.

[6] A biobattery having an enzyme electrode having a gluconic acid oxidation catalytic ability or an enzyme electrode having a gluconic acid oxidation catalytic ability prepared by a method for producing an enzyme electrode having a gluconic acid oxidation catalytic ability as a negative electrode.

  According to the above configuration [6], it is possible to provide a biobattery using gluconic acid or the like as a fuel. As the fuel, a substance that serves as a substrate for gluconate dehydratase such as gluconate can be used. Further, in a multi-stage glucose oxidation system, by using glucose or the like as a fuel by combining it with an enzyme such as glucose dehydrogenase which catalyzes the reaction in the preceding stage of gluconic acid oxidation to oxidize glucose to produce gluconic acid. You can By constructing a multi-stage oxidation system, the number of electrons that can be taken out from a fuel such as glucose is increased and the output of the biocell can be improved. Therefore, it is possible to construct a biobattery having a particularly high utility value in fields such as medical care, food, and the environment.

[7] A biosensor including, as a working electrode, an enzyme electrode having a gluconic acid oxidation catalytic ability or an enzyme electrode having a gluconic acid oxidation catalytic ability produced by a method for producing an enzyme electrode having a gluconic acid oxidation catalytic ability.

  According to the configuration of [7] above, it is possible to provide a biosensor for detecting a substance serving as a substrate for gluconate dehydratase such as gluconate in a sample. Further, when the enzyme electrode of the biosensor having the above-mentioned configuration forms a multi-step reaction system together with glucose dehydrogenase, etc., in the redox reaction of glucose, the glucose dehydrogenase in the preceding step of gluconate dehydratase It can also be used to detect a substance that serves as a substrate, and can be used to detect glucose such as blood glucose level. Therefore, it is possible to construct a biosensor having a particularly high utility value in the fields of medical care, food, environment and the like.

The schematic diagram which shows an example of the biosensor of this embodiment. 1 is an electrophoretogram showing the results of Example 1 in which gluconate oxidase was selected and synthesis was confirmed. The graph which shows the result of Example 2 which investigated the porous electrode material. The graph which shows the result of Example 2 which investigated the porous electrode material. 5 is a graph showing the results of Example 3 in which the electrochemical performance of an enzyme electrode having a gluconic acid oxidation catalytic ability was verified. The graph which shows the result of Example 4 which analyzed the reaction product of the enzyme electrode which has a gluconic acid oxidation catalytic ability.

  Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to this embodiment.

[Enzyme electrode]
In the enzyme electrode according to the embodiment of the present invention, gluconate dehydratase is immobilized on the electrode material. The gluconate dehydrogenase is derived from a bacterium belonging to the genus Sulfolobus belonging to the thermoacidophilic archaea. Particularly preferably, it is derived from Sulfolobus solfataricus.

  The bacterium belonging to the genus Sulfolobus is a bacterium belonging to the thermoacidophilic archaea. In archaebacteria such as Sulfolobus, the glycolysis system, which is the mechanism of glucose decomposition, does not use the Emden-Meyerhof pathway but the Entner-Doudoroff pathway and the pyrolytic system (Pyroglycolysis). Use (Ref. 1: Glycolysis, metabolic map, individual reactions / Fukuoka University Faculty of Science Department of Chemistry (http://www.sc.fukuoka-u.ac.jp/~bc1/Biochem/glyclysis.htm) ). In the pyroglycolysis system, glucose is decomposed to 2-keto-3-deoxygluconic acid via gluconic acid, and 2-keto-3-deoxygluconic acid is decomposed to pyruvic acid or glyceraldehyde. Subsequently, glyceraldehyde is decomposed into 2-phosphoglyceric acid or glycerol via glycerin, and glycerol is decomposed into glycerol 3-phosphate.

  Gluconate dehydratase is an enzyme that catalyzes a reaction that decomposes gluconic acid into 2-keto-3-deoxygluconic acid, and particularly preferably, D-glucon derived from Sulfolobus solfataricus P2 strain. Acid dehydratase (D-gluconate dehydratase, ACCESSION Number: Q97U27). The sequence information of the DNA encoding such an enzyme is shown in SEQ ID NO: 1 in the sequence listing, and the deduced amino acid sequence information is shown in SEQ ID NO: 2.

  As the gluconate dehydratase, as long as it has a catalytic function of gluconate dehydratase, one encoded by a base sequence that hybridizes with a complementary sequence of the base sequence represented by SEQ ID NO: 1 under stringent conditions, or SEQ ID NO: 2 Having an amino acid sequence having at least 80%, preferably 90%, particularly preferably 95%, or 98% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 2 and several amino acid sequences (1 to Those having an amino acid sequence in which (9) amino acid residues are substituted, deleted, and / or added can also be preferably used. The gluconate dehydratase having such a modification may be one produced by natural or artificial mutation, or a known gluconate dehydratase derived from Sulfolobus bacterium such as the base sequence shown in SEQ ID NO: 1 may be used. It can also be obtained by modifying the encoding nucleic acid molecule by a known method. For example, as a method for inserting a mutation site into a nucleic acid molecule encoding a gluconate dehydratase that is a base for modification, a site-directed mutagenesis method, a PCR sudden induction method for introducing a mutation using a PCR method, etc., Alternatively, a known mutagenesis technique such as a transposon insertion mutagenesis method can be used. Alternatively, a commercially available mutation introduction kit (for example, QuikChange (registered trademark) Site-directed Mutagenesis Kit (manufactured by Stratagene)) may be used.

  The gluconate dehydratase derived from a bacterium belonging to the genus Sulfolobus is efficiently expressed in a protein expression system such as Escherichia coli as shown in Example 1 below, but becomes insoluble when the degree of purification of the enzyme solution is increased. It is considered that this is because the recombinantly expressed gluconate dehydratase, which is a foreign substance, formed an insoluble aggregate called an inclusion body in a host such as Escherichia coli. When the inclusion body is formed, purification by chromatography and the like becomes difficult, and there arises a problem that the original physiological activity cannot be sufficiently exerted. As a result, an enzyme electrode having a catalytic function of gluconate dehydratase is formed. It was difficult to make.

  Conventionally, when inclusion bodies are formed in a protein expression system, a soluble expression system will be sought. In constructing a soluble expression system, for example, by using a eukaryote as a host, sugar chains are added by post-translational modification, and the association between the hydrophobic faces of the expressed recombinant protein molecule is reduced to synthesize it as a soluble protein. can do. When synthesizing an enzyme to which a sugar chain is added, it is known that the addition of the sugar chain at the time of synthesis greatly affects the synthesis efficiency (Reference 2: Kataoka K et al., “High-level expression of Myrothecium verrucaria bilirubin oxidase in Pichia pastoris, and its facile purification and characterization. ”Protein Expr Purif., 2005, Vol. 41, No. 1, pp. 77-83 (http://www.sciencedirect.com/science/article/pii / S1046592805000380)), and as a result, the efficiency of protein synthesis is improved. On the other hand, the protein synthesis efficiency and physical properties are affected by the type of sugar chain to be added and the state of addition, and those skilled in the art are forced to make trial and error. For example, it has been reported that the physical properties of proteins can be significantly changed by changing the state of sugar chain addition (Reference 3: Panasonic Healthcare Co., Ltd., News, 2011, Press Release: “2011 Agricultural Chemistry Technology”). Award ”(see http://www.panasonic-healthcare.com/jp/news/2011/0506)), handling of proteins with sugar chains is a major obstacle to industrial use. It is known that there are many.

  Furthermore, when gluconate dehydratase is recombinantly expressed in a protein expression system using an organism having a sugar chain modifying ability such as a eukaryote as a host, the enzyme with a sugar chain attached is used as it is as an electrode. It is assumed that when immobilized on, the sugar chain becomes an insulator, the electron transfer efficiency to the fuel → enzyme → (mediator) → electrode is remarkably lowered, and the enzyme catalytic current is as small as several milliamperes or less.

  Further, in order to construct a soluble expression system, conventionally, an appropriate vector having a sequence such that a Tag such as an appropriate hydrophilic peptide is fused to the target protein and expressed at the N-terminal or C-terminal side is used. Has also been done. In particular, by increasing the Tag of the hydrophilic peptide, it is possible that it can be synthesized as a soluble protein without forming an inclusion body, but like the sugar chain described above, it is not suitable for use as an electrocatalyst for an enzyme electrode.

  Therefore, in the enzyme electrode of the present embodiment, in order to construct an enzyme electrode that retains gluconic acid oxidation ability, a protein that utilizes an organism having no sugar chain modification ability such as a prokaryote such as Escherichia coli as a host In the expression system, an enzyme is synthesized as a protein to which a sugar chain is not added, and a concentrated solution of a lysate of the expressing bacterium containing the enzyme is immobilized on the electrode material. Here, not having the ability to modify the sugar chain includes not only the case where the pathway for modifying the sugar chain as a post-translational modification is completely absent, but also the case where the ability to modify the sugar chain is immature.

  The concentrated solution of the lysate of the expressing bacterium containing gluconate dehydratase is (i) the expression of recombinant gluconate dehydratase in the bacterium having no sugar chain modifying ability, and (ii) the expressing bacterium and the interface. Contact with an activator, (iii) disruption of the expressed bacterial cells after treatment with a surfactant, (iv) separation of a lysate of the expressed bacterial cells from the disrupted solution of the expressed bacterial cells, (v) Concentrated, prepared by

(I) Expression of recombinant gluconate dehydratase in bacterial cells of bacteria having no sugar chain modifying ability Protein expression system using bacteria having no sugar chain modifying ability of prokaryotes such as Escherichia coli as hosts Express recombinant gluconate dehydratase in.

  Specifically, a transformant is prepared by inserting a nucleic acid molecule encoding gluconate dehydratase into an appropriate expression vector and introducing this into a host. The vector that can be used is not particularly limited as long as it can incorporate a foreign DNA and can autonomously replicate in a host cell. Then, the vector may be incorporated so that the foreign gene can express its function, and may contain other known base sequences necessary for functional expression. Examples include promoter sequences, leader sequences, signal sequences, ribosome binding sequences, and the like. Further, a marking sequence or the like capable of imparting phenotypic selection in the host can be included.

  Since the nucleic acid molecule encoding gluconate dehydratase has sequence information disclosed, it can be obtained by a method known in the art based on the sequence information.

  The insertion of a foreign gene into a vector is, for example, a method in which a nucleic acid molecule encoding a desired gluconate dehydratase is cleaved with an appropriate restriction enzyme, and inserted into a restriction enzyme site of a suitable vector or a multicloning site and ligated. However, the present invention is not limited to this.

  The host cell for producing the transformant is not particularly limited as long as it is an organism having no sugar chain modification ability and can efficiently express a foreign gene. Prokaryotic cells can be preferably used, and particularly Escherichia coli can be used. In addition, Bacillus subtilis, Bacillus bacterium, Pseudomonas bacterium, etc. can also be used. As the transformation method, known methods such as calcium chloride method, electroporation method, liposome infection method, and microinjection method can be used.

  Subsequently, the obtained transformant is cultured in an appropriate nutrient medium under conditions that allow the expression of the introduced nucleic acid molecule, and recombinant gluconate dehydratase is synthesized in the host cell. Culturing can be performed according to a conventional method, and the culturing conditions may be selected in consideration of the nutritional physiological properties of host cells. The medium used is not particularly limited as long as it contains nutrients that can be assimilated by the host cell and can efficiently express the protein in the transformant. The culture form is also not particularly limited, but a liquid medium can be preferably used from the viewpoint of large-scale culture.

  The selection of transformants carrying the desired recombinant vector can be performed, for example, by the presence or absence of expression of the marking sequence. For example, when a drug resistance gene is used as the marking sequence, it can be performed by culturing in a drug-containing medium corresponding to the drug resistance gene.

(Ii) Contact of Expressed Bacteria with Surfactant The expressed bacterium expressing the gluconate dehydratase in the step (i) is brought into contact with a surfactant. The contact between the expressed bacterial cells and the surfactant may be carried out by adding a desired amount of the surfactant to the culture medium of the bacterial cells, or the expressed bacterial cells collected by centrifugation or the like may be added to an appropriate buffer solution. It may be carried out by adding a surfactant to the dispersed liquid. The contact time between the expressed bacterium and the surfactant can be appropriately changed depending on the amount of the expressed bacterium and the kind and concentration of the surfactant. The amount of the surfactant added can be appropriately changed depending on the amount of the expressed cells, the type of the surfactant, etc., but should be 0.08% to 2.0% (w / vol:% by weight) in the cell suspension. It is preferable to add.

  The surfactant may be a nonionic surfactant, an ionic surfactant, or the like. Conventionally known surfactants can be used to enhance the solubility of proteins. For example, Wako Pure Chemical Industries, Reagent Home, product information, biochemistry in general, surfactants (http: //www.wako- chem.co.jp/siyaku/info/life/article/kaimenkassei1.htm) can be used. Specifically, examples of the nonionic surfactant include polyoxyethylene (20) cetyl ether (Brij (registered trademark) 58), polyoxyethylene (23) lauryl ether (Brij (registered trademark) 35, and other polyoxyethylenes. Polyoxyethylene alkyl phenyl ethers such as ethylene alkyl ether type, poly (oxyethylene) = octyl phenyl ether (Nonidet (trademark) P-40), polyoxyethylene (10) octyl phenyl ether (Triton (registered trademark) X-100) System, N, N-bis (3-D-gluconamidopropyl) cholamide (BIGCHAP), polyoxyethylene (20) sorbitan monolaurate (Tween20), polyoxyethylene (40) sorbitan monopalmitate (Tween40) Examples of the polyoxyethylene sorbitan fatty acid ester system include ionic surfactants such as anionic surfactants and cationic surfactants. Surfactants and amphoteric surfactants are classified. For example, anionic surfactants include sodium dodecyl sulfate (SDS), and zwitterionic surfactants include 3-[(3-cholamidopropyl) dimethylammonium. O] -1-Propane sulfonate (CHAPS) and 3-[(3-Colamidopropyl) dimethylammonio] -2-hydroxy-1-propane sulfonate (CHAPSO), 3- [Dimethyl (tetradecyl) ammonio] propane -1-Sulfonate (Zwittergent (registered trademark) 3-14), 3- (N, N-dimethylpalmitylammonio) propane sulfonate (Zwittergent (registered trademark) 3-16) and other Zwittergent (registered trademark) systems Brij (registered trademark) 58 is preferable.

(Iii) Crushing of Expressed Bacteria After Treatment with Surfactant The crushed expression cells after treatment with a surfactant in the step (ii) above are performed. For crushing bacterial cells, any known method for crushing bacterial cells can be used as long as the cell structure of the bacterial cells can be destroyed by a physical method to extract the protein component from the cells. For example, use ultrasonic crushing that crushes bacterial cells by the shearing force of ultrasonic waves, or use a French press that crushes bacterial cells by shearing force by forcibly injecting the bacterial cell suspension from a small hole under high pressure. You can The cells can also be disrupted by using a homogenizer, glass beads having a polishing action, a mortar, and freezing / thawing. Ultrasonic crushing is preferable, and it can be carried out under normal crushing conditions of cells. Since heat is generated during cell disruption, it is preferable to perform cell disruption while cooling on ice or the like.

(Iv) Separation of a lysate of expressed bacterial cells from the disrupted liquid of expressed bacterial cells The disrupted liquid of bacterial cells obtained in the step (iii) is separated from bacterial cell residue by centrifugation or filtration to express the bacterial cells of expressed bacterial cells. To obtain a lysate of. Centrifugation is preferable, for example, centrifugation is performed at 5,000 to 50,000 × g for 5 to 60 minutes. In the case of filtration, gravity filtration is used, and as the filter paper, for example, cellulose filter paper (grade I: particle retention capacity 11 μm) or the like can be used. The lysate of the expressed bacterial cells on the supernatant side after centrifugation and on the filtrate side after filtration contains an enzyme solubilized by contact with a surfactant and an enzyme that is insoluble but not precipitated.

(V) Concentration of the lysate of the expressed bacterium by ultrafiltration, etc. The lysate of the expressed bacterium obtained in the step (iv) is concentrated by ultrafiltration, salting out, etc. A concentrated solution of the lysate can be obtained. Ultrafiltration is a molecular weight fractionation using an ultrafiltration membrane that is a semipermeable membrane. By pressurizing or centrifuging, low-molecular water, low-molecular substances, ions, etc. permeate the membrane surface with respect to the pore size of the ultrafiltration membrane, and target macromolecular substances permeate the membrane surface. Blocked and concentrated. The ultrafiltration membrane is a membrane capable of blocking particles and macromolecules in the range of 0.1 μm to 2 nm. For example, it can be constituted as a porous membrane having a molecular weight cut-off (NMWL) of 1 to 50K, preferably 3 to 30K, and particularly preferably 8 to 10K. The material of the ultrafiltration membrane, regenerated cellulose, triacetyl cellulose, cellulose acetate, polyacrylonitrile, polyvinyl chloride alcohol, polysulfone, polyvinylidene fluoride, aromatic polyamide, etc., it is possible to use materials known as ultrafiltration membranes. it can. The shape of the membrane is not particularly limited, and known shapes such as hollow fiber membranes, spiral membranes and flat membranes can be used.

  The ultrafiltration method may be a dead end method or a cross flow method. Ultrafiltration requires pressurization or centrifugation to allow substances on the low molecular side of the membrane pores to permeate through the membrane pores, but use a liquid delivery pump or the like for pressurization. The operating pressure can be in the range of 0.1 to 1 MPa. Further, it can be configured as a spin column suitable for centrifugation. Commercially available products can be used, and Amicon Ultra-4 10 and the like can be preferably used.

  The cell disruption solution is preferably concentrated to 2 to 20 times by ultrafiltration or the like. By ultrafiltration, water and low-molecular substances and ions are extruded to the opposite side of the membrane, increasing the concentration of glucone dehydrogenase. Through a series of steps, solubilization and reholding of gluconate dehydratase proceed, and gluconate dehydratase is in a form in which it can exert its catalytic activity.

As the electrode material, a conductive base material that can be connected to an external circuit and can transfer electrons can be used, and the material is not limited. Carbon material such as carbon cloth, carbon felt, carbon paper, graphite, and glassy carbon, metal or alloy such as aluminum, copper, gold, platinum, silver, nickel, palladium, SnO ₂ , In ₂ O ₃ , WO ₃ , TiO Examples thereof include conductive oxides such as ₂ but are not limited thereto. A conductive base material made of a conventionally known material can be used. In addition, this may be constituted by a single layer or a laminated structure of two or more kinds.

  The electrode material preferably has a porous structure, for example, a porous structure having pores having an average diameter of 20 to 50 nm, preferably 30 to 40 nm. The average diameter can be calculated as the diameter of a sphere having a projected area equivalent to the projected area of pores obtained by image analysis of a micrograph or the like. The porous structure means a structure in which a large number of pores are dispersed, and examples thereof include a sponge-like structure. The pores may be independent pores independent of other pores, or some or all of the pores may communicate with adjacent pores to form continuous pores. It is preferable to have a three-dimensional network structure.

The electrode material having a porous structure can be configured, for example, as an aggregate of conductive fibers, and examples thereof include woven and non-woven fabrics made of conductive fibers. As the material of the conductive fiber, carbon, aluminum, copper, gold, platinum, silver, nickel, metal or alloy such as palladium, SnO ₂ , In ₂ O ₃ , WO ₃ , conductive oxide such as TiO ₂ , etc., A conductive substance having a material known in the art can be used. These substances may be used alone or in combination of two or more. Preferably, a woven cloth of carbon fibers such as the above-mentioned carbon cloth, a non-woven cloth of carbon fibers such as carbon felt, etc., or a carbon base material such as glassy carbon can be used. In addition, these can be configured to have a single layer or a laminated structure of two or more kinds.

  The electrode material having a porous structure can be configured by applying the conductive carbon fine particles to the conductive base material described above, that is, the porous carbon material is laminated on the electrode base material as the current collector. Can be configured as As the carbon particles, commercially available carbon black such as Ketjen black, activated carbon powder and the like can be used, and preferably porous carbon fine particles having a porous structure can be preferably used.

  The porous carbon fine particles are configured as a porous structure, and some or all of the pores are in communication with each other. The pores are mesopores or macropores, the mesopores mean pores having an average diameter of 2 to 50 nm, and the macropores mean pores having an average diameter of 50 nm or more. .

  The porous structure means a structure in which a large number of pores are dispersed. Therefore, it is preferable that the porous carbon fine particles have a sponge-like structure in which carbon constitutes the outer portion of the pores. The pores may be independent pores independent of other pores, or some or all of the pores may communicate with adjacent pores to form continuous pores. It is preferable to have a three-dimensional network structure.

  Such porous carbon fine particles are obtained by firing a mixture of a carbon precursor and a magnesium oxide (hereinafter sometimes abbreviated as “MgO”) precursor, and removing the MgO particles from the obtained fired product. It can be configured as porous carbon fine particles (hereinafter sometimes referred to as “MgO template carbon fine particles”) using the produced MgO particles as a template. In such MgO template carbon fine particles, MgO particles serve as a template, and after the MgO particles are removed, pores having the same size and shape are formed. As a result, it is possible to form MgO template carbon fine particles in which homogeneous pores are regularly arranged. For example, it can be produced by the method described in Japanese Patent Application Laid-Open No. 2012-188309, which discloses a porous carbon having mesopores and a carbonaceous wall forming the outer periphery of the mesopores and a method for producing the same.

  The carbon precursor is not particularly limited as long as it contains carbon and is carbonized by heating. For example, carbon precursors include polyester polymers such as polyethylene terephthalate, acrylic polymers such as polyacrylamide and polyacrylonitrile, and vinyl polymers such as polyvinyl alcohol, polyvinyl acetate, polyethylene and polyvinylpyrrolidone, and hydroxypropyl cellulose. Examples thereof include cellulosic polymers, polyamide polymers, polyimide polymers, phenol polymers, epoxy polymers, and pitches such as petroleum pitch and coal pitch. Further, condensed polycyclic hydrocarbon compounds such as naphthalene, phenanthrene and anthracene, condensed heterocyclic compounds such as indole, isoindole and quinoline, and derivatives thereof can also be used.

  The MgO precursor is not particularly limited as long as it produces MgO particles by thermal decomposition by heating. In addition to MgO itself, magnesium hydroxide, magnesium carbonate, basic magnesium carbonate, and magnesium organic acid salts such as magnesium acetate, magnesium citrate, magnesium oxalate, and magnesium gluconate can be used.

  The carbon precursor and the MgO precursor can be mixed by mixing the powders with each other, or by mixing both as a solution such as an aqueous solution, or by mixing one with a powder and the other as a solution. Good. The mixing is preferably performed so that both are evenly distributed.

  The heat treatment is preferably performed in an inert gas atmosphere, for example, an argon or nitrogen atmosphere, or a reduced pressure atmosphere, for example, an atmosphere of 133 Pa or less. The heating temperature is such that elements other than carbon are desorbed from the carbon precursor to carbonize it, and the MgO precursor is heated at a temperature at which MgO particles are generated by thermal decomposition. If the heating temperature is too low, carbonization does not proceed, so heating is preferably performed at 500 to 1500 ° C, or 500 to 1000 ° C, particularly preferably 600 to 800 ° C. By calcining at such a temperature, a part of the functional group derived from the carbon precursor remains in the MgO template carbon fine particles after calcining, and a hydrophilic environment suitable for expressing the catalytic activity of the enzyme can be provided.

  Subsequently, MgO particles are removed from the obtained fired product. Removal of MgO particles can be carried out using a solution capable of dissolving MgO particles, for example, sulfuric acid, hydrochloric acid, acetic acid, citric acid, acid solutions such as formic acid, or washed or soaked in hot water, etc., MgO particles Are dissolved and removed. When using an acidic solution, it is preferable to use a weak acidic solution in order to suppress changes in the properties of carbon.

  After removing the MgO particles, an additional heat treatment may be performed to further crystallize carbon. The heat treatment here is also preferably performed in an inert gas atmosphere, for example, an argon or nitrogen atmosphere, or a reduced pressure atmosphere, for example, an atmosphere of 133 Pa or less. The heating temperature is such that amorphous carbon crystallizes. It is preferably heated at 800 ° C or higher. From the viewpoint of thermal efficiency, it is preferably performed at 2500 ° C. or lower, but in order to provide the MgO-templated carbon fine particles having a hydrophilic environment suitable for expressing the catalytic activity of the enzyme, it is particularly preferable to perform at 800 to 1000 ° C. preferable.

  The carbon after the heat treatment is preferably pulverized because it is formed into fine particles. The crushing treatment can be performed by utilizing a compressive force, a shearing force, an impact force, and a frictional force, and a plurality of these two or more forces may be combined. Specifically, a ball mill, a planetary mill, a jet mill, a mortar or the like can be used.

  By using MgO particles as a template, it is possible to obtain MgO template carbon particles in which a large number of homogeneous pores are dispersed on the surface and inside thereof. Here, the term "homogeneous pores" means that pores having a uniform pore diameter are regularly arranged.

  The average diameter of the pores of the MgO template carbon fine particles is determined by the MgO particles that are the template, and pores of the same size as the MgO particles are formed. If the average diameter of the pores is too large, the desorption of the enzyme or mediator immobilized in the pores cannot be effectively suppressed, and if it is too small, the enzyme or mediator cannot be immobilized in the pores. Therefore, the size of the pores can be appropriately set according to the types of enzymes and mediators immobilized in the pores, but the pores are of meso or macroscale size. Here, the mesopores are pores having an average diameter of 2 to 50 nm, and the macropores are pores having an average diameter of 50 nm or more. Specifically, the average diameter is preferably 30 to 100 nm, particularly preferably 30 to 40 nm. With this structure, the enzyme or mediator can be immobilized stably and firmly in the pores of the MgO template carbon fine particles.

  Also, the shape of the pores is determined according to the shape of the MgO particles that are the template. The shape is not particularly limited, but may be a substantially spherical shape, a polyhedral shape, or the like, and it does not matter whether it is an isotropic shape or an anisotropic shape. Further, the substantially spherical shape includes a perfect spherical shape, an elliptic spherical shape, a polyhedral shape close to a spherical shape, and the like. The polyhedron shape includes a polygonal pyramid shape, a polygonal column shape, and the like. Further, the pore size distribution can be appropriately adjusted by changing the mixing ratio of MgO particles as a template.

  The particle diameter of the MgO template carbon fine particles can be appropriately set according to the type of the enzyme or mediator to be immobilized and the type of the conductive substrate to be immobilized, etc., preferably 0.3 to 2 μm, particularly preferably, 0.5 to 1 μm.

  As the MgO template carbon fine particles, commercially available products can be used, and for example, it is preferable to use CNovel (registered trademark) (Mesoporous Carbon) sample kit grade: P (4) 050 available from Toyo Tanso Co., Ltd.

  The carbon fine particles may be fixed to the conductive substrate by any method known in the art. For example, it is possible to fix by coating and impregnating a conductive substrate with a dispersion liquid in which carbon fine particles are dispersed in a suitable solvent. The dispersion may be suspended as a slurry to prepare a slurry. Moreover, you may use a binder as needed. As a coating method, a screen printing method, a roll coating method, a doctor blade method, a spin coating method, a spraying method using a spray, or the like can be used. It is also possible to use an electrodeposition method, for example, by immersing the conductive base material and the counter electrode in a dispersion liquid in which carbon fine particles are dispersed in a suitable solvent, the conductive base material and the counter electrode An electric current may be passed in between to fix the carbon fine particles to the conductive base material, and a binder may be used if necessary.

  The binder can bind the carbon fine particles to the conductive base material and suppress the detachment of the carbon fine particles from the conductive base material. As the binder, any substance known in the art can be used as long as it is a substance capable of binding the carbon fine particles to the conductive base material. For example, polytetrafluoroethylene (hereinafter abbreviated as “PTFE”), a fluorine-based polymer such as polyvinylidene fluoride or polyhexafluoropropylene, a polysiloxane-based polymer, or the like can be used. The binder may be used alone or in combination of two or more.

The amount of carbon fine particles fixed to the conductive base material can be appropriately changed depending on the type of the conductive base material and the shape of the carbon fine particles, but is 0.05 to 0.5 mg per 1 cm ^{2 of the} conductive base material. It is preferable to fix the carbon fine particles. Further, when using a binder, if too much binder is not preferable because it increases the electron transfer resistance of the electrode material and hinders smooth electron transfer. It is preferable to mix the binder with.

  The size and shape of the electrode material are not particularly limited, and can be appropriately adjusted according to the purpose of use. It can be configured as a microelectrode having an electrode area reduced to the order of micrometers.

  The enzyme electrode of the present embodiment may be fixed by combining not only gluconic acid dehydratase but also another type of enzyme with the electrode material. For example, it is preferable to construct a multi-step reaction system by combining other enzymes. For example, it may be configured to construct a multi-step reaction system of glucose by combining with glucose dehydrogenase which produces gluconic acid.

  In the enzyme electrode of the present embodiment, a substance necessary for expressing the catalytic activity of an enzyme such as a mediator that mediates an enzyme reaction and electron transfer between electrodes can be fixed to the electrode material as necessary.

  A mediator is a low-molecular redox substance that is redox-reduced in response to a catalytic reaction of an enzyme, and mediates electron transfer between the enzyme and a conductive substrate. Therefore, any mediator can be used as long as it is a substance capable of exchanging electrons with the enzyme and also capable of exchanging electrons with the conductive base material. For example, the mediator is preferably a π-conjugated compound in which single bonds and double bonds are alternately arranged. Specifically, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (hereinafter abbreviated as "ABTS"), ferricyanide such as potassium ferricyanide, 5-methylphenazinium Phenazine compounds such as methylsulfate (phenazine methosulfate: PMS) and 5-ethylphenazinium methylsulfate (phenazineethosulfate: PES), phenothiazine compounds, ferrocene, dimethylferrocene, ferrocenecarboxylic acid and other ferrocenes. Examples thereof include quinone compounds such as naphthoquinone, anthraquinone, hyroquinone, pyrroloquinoline quinone, cytochrome compounds, viologen compounds such as benzyl viologen and methyl viologen, and indophenol compounds such as dichlorophenol indophenol. Naphthoquinone can be used particularly preferably.

  According to the enzyme electrode of the present embodiment, it is possible to provide an enzyme electrode capable of sufficiently exerting the gluconic acid oxidation catalytic ability. Conventionally, for example, when gluconate dehydratase derived from a bacterium belonging to the genus Sulfolobus is synthesized using a protein synthesis system having no sugar chain modification pathway such as Escherichia coli in a state where no sugar chain is added, an inclusion body is formed. It was difficult because of problems such as On the other hand, in the case of synthesizing using a protein synthesis system having a sugar chain modification pathway, the addition of sugar chains reduces the association between the hydrophobic surfaces of the enzyme molecules, resulting in synthesis as a soluble enzyme. The synthesis efficiency of is improved. However, since the physical properties of the synthetic enzyme are affected by the type of sugar chain to be added and the state of addition, the handling of the enzyme to which the sugar chain is attached is often a major obstacle to industrial use. Moreover, when the glycosylated enzyme is immobilized on the electrode material as it is, the sugar chain becomes an insulator, the electron transfer efficiency to the fuel → enzyme → (mediator) → electrode is significantly reduced, and the enzyme catalytic current is several milliamperes. There are also problems such as the following being small. Therefore, conventionally, it was difficult to produce an enzyme electrode having a catalytic function of gluconic acid oxidation using gluconic acid dehydratase. The enzyme electrode of the present embodiment makes it possible to provide an enzyme electrode having the ability to catalyze gluconate oxidation using gluconate dehydratase for the first time.

  The enzyme electrode of the present embodiment can be used as a negative electrode of a bio battery and a working electrode of a biosensor, and can be used in industrial fields such as electronics, medical care, foods and the environment.

  The enzyme electrode of the present embodiment can provide an enzyme electrode capable of efficiently exhibiting a catalytic function of gluconic acid oxidation by using an electrode material as a carbon base material having a porous structure. That is, by directly impregnating the concentrated solution of the lysate of the enzyme-expressing bacterial cell into the carbon substrate having a porous structure, the enzyme molecules enter the pores of the carbon substrate, so to speak, the enzyme purification effect is obtained. In addition, there is an advantage that the association between the enzyme molecules is suppressed. This makes it possible to realize an enzyme electrode even for an enzyme species that easily aggregates.

(Method for producing enzyme electrode)
The method for producing an enzyme electrode according to the present embodiment is a protein expression system that utilizes as a host an organism having no sugar chain modifying ability such as a prokaryote typified by Escherichia coli or the like. An enzyme electrode is produced by synthesizing an enzyme and immobilizing a concentrated solution of a lysate of expressed bacterial cells containing the enzyme on an electrode material.

  Specifically, the method for producing the enzyme electrode according to the present embodiment includes (i) transforming a bacterium having no sugar chain modifying ability with a gene encoding a glucose dehydratase from a bacterium belonging to the genus Sulfolobus to give a recombinant gluconic acid dehydration. A step of preparing expression cells expressing the enzyme; (ii) a step of contacting the expression cells with a surfactant; (iii) crushing the expression cells after the treatment with the surfactant to crush the expression cells. A step of preparing a solution, (iv) a step of separating and removing cell residues in the disrupted solution of the expressed cells to prepare a lysate of the expressed cells, and (v) concentrating the lysate of the expressed cells. A step of preparing a concentrated solution of the lysate of the expressing bacterium, and (vi) immobilizing the concentrated solution of the lysate of the expressing bacterium on an electrode material. The preparation of the concentrated solution of the lysate of the expressing bacterial cells containing gluconate dehydratase and the electrode material are described above.

  Immobilization of a concentrated solution of a lysate of expressed bacterial cells containing gluconate dehydratase onto an electrode material can be performed by any method known in the art. For example, immobilization by physical adsorption, covalent bond, ionic bond, carrier-binding method by biochemical specific binding such as antibody, cross-linking method with reagent having two or more functional groups, encapsulation method by encapsulation in gel, etc. You can Further, these may be combined, and it is desirable to appropriately select an optimal enzyme immobilization method. When carbon microparticles are used as the electrode material, the enzyme may be immobilized after the carbon microparticles are immobilized on the conductive substrate, or the enzyme may be preliminarily templated before the carbon microparticles are immobilized on the conductive substrate. It may be fixed to carbon fine particles.

  Particularly preferred is physical adsorption, which can be carried out by bringing a concentrated solution of a lysate of expressed bacterial cells in which an enzyme is dissolved or dispersed in a suitable solvent into contact with the surface of the electrode material. The contact can be performed by a known method such as spraying, dropping, impregnation and the like. Further, a coating method such as spin coating, spraying method, screen method, dip coating or blade method can be used.

  When a substance necessary for expressing the catalytic activity of an enzyme such as a mediator that mediates an enzyme reaction and electron transfer between electrodes is immobilized on the electrode material, the mediator can be immobilized by mixing with the enzyme. The enzyme may be fixed before or after the enzyme is fixed.

  According to the method for producing an enzyme electrode of the present embodiment, it is possible to produce an enzyme electrode capable of sufficiently exhibiting the gluconic acid oxidation catalytic ability. Conventionally, it is difficult to produce an enzyme electrode having a gluconic acid oxidation-catalyzing ability of gluconic acid dehydratase derived from a Sulfolobus bacterium, in both a state in which a sugar chain is not added and a state in which a sugar chain is added. there were. In the present embodiment, a gluconate dehydratase derived from a bacterium belonging to the genus Sulfolobus is recombinantly expressed in a state in which a sugar chain is not added using a protein expression system of Escherichia coli, and a lysate of a bacterium containing the recombinant gluconate dehydratase is lysed. By immobilizing the enzyme on an electrode material, it is possible to provide a method for producing an enzyme electrode capable of sufficiently exerting a gluconic acid oxidation catalyst ability. The enzyme electrode produced by the method for producing the enzyme electrode of the present embodiment can be used as a negative electrode of a bio battery and a working electrode of a biosensor, and is used in industrial fields such as electronic, medical, food, and environmental fields. It is available.

  In particular, when the electrode material is a carbon-based material having a porous structure, the enzyme molecules enter into the pores of the carbon-based material when the concentrated solution of the lysate of the expressing bacterial cells is immobilized on the electrode material. In other words, it is possible to provide a method for producing an enzyme electrode that can obtain a purification effect of an enzyme and that can suppress the association of enzyme molecules with each other, thereby efficiently exhibiting the catalytic function of gluconic acid oxidation. . Furthermore, by preparing a carbon substrate having a porous structure using magnesium oxide particles as a template, a large number of pores of uniform size are regularly aligned in the pores of the carbon substrate to stabilize the enzyme and mediator. It can be firmly fixed, and desorption of the enzyme and the mediator from the electrode material can be suppressed. Thereby, the deterioration of the electrode material can be suppressed and the stability of the enzyme catalytic current can be achieved. In addition, the transfer of electrons between the enzyme and the conductive base material can be smoothly promoted, whereby the produced enzyme catalytic current can be improved and the electrode performance can be improved.

(Bio battery)
The biobattery of the present embodiment is configured to include a biofuel cell, which includes one set of a positive electrode, a negative electrode, and an electrolyte layer. A cell is a minimum unit of a biofuel cell, and a cell stack can be formed by stacking the cells, whereby a biofuel cell can be constructed.

  In the biobattery of the present embodiment, for example, the negative electrode and the positive electrode are arranged so as to face each other with the electrolyte layer in between, and the negative electrode and the positive electrode are connected by an external circuit. A fuel tank is attached to the negative electrode side so that fuel can be supplied to the negative electrode, and an air intake port or the like is formed on the positive electrode side so as to take in oxygen in the atmosphere. For example, the cells are laminated in the order of a separator, a fuel tank, a negative electrode, an electrolyte layer, a positive electrode, and a separator.

  The negative electrode is configured to perform a fuel oxidation reaction, and is configured using the enzyme electrode of the present embodiment. The positive electrode is configured to carry out a reduction reaction of oxygen. For the positive electrode, it is preferable to select an enzyme such as oxidase that performs a reduction reaction of oxygen. The positive electrode may be configured as a biological electrode containing a biocatalyst on at least a part of an appropriate electrode material, or a metal electrode such as platinum utilizing a catalytic reaction of metal. Here, the biocatalyst is a substance existing in a living body and having a substance converting ability. In addition to natural substances derived from living organisms, artificial substances imitating them are also included. Examples include enzymes, microorganisms, organelles and cells. When the positive electrode is used as the biological electrode, it is preferable to use an enzyme electrode having an enzyme immobilized on a suitable electrode material. As the electrode material, the same conductive base material as that described for the negative electrode can be used.

  As the enzyme, any enzyme known in the art can be used, as long as it is an enzyme that is catalyzed by a reduction reaction of oxygen. It is preferable to use redox enzymes such as bilirubin oxidase, pyruvate oxidase, laccase, and ascorbate oxidase, and the enzymes can be used alone or in combination. Here, bilirubin oxidase is a multi-copper oxidase having a copper ion as an active center and is an enzyme that catalyzes the oxidation reaction of bilirubin to viruverdin. It is an enzyme with a high utility value as an electrode catalyst for the positive electrode of a biobattery because it has a property of catalyzing the reaction of electron-reducing molecular oxygen by using electrons taken out from a substrate to generate a water molecule. There is no particular limitation on the presence or absence of coenzyme and cofactor requirements.

  When an enzyme is used as the catalyst for the positive electrode, the origin of the enzyme is not particularly limited. Therefore, it may be of natural origin, which is purified from a naturally occurring organism by an appropriate protein isolation / purification technique, or may be recombinantly produced by a genetic engineering technique or chemically synthesized. There may be things. Furthermore, commercially available products can also be used. For example, Myrothecium verrucaria-derived bilirubin oxidase (MvBOD) available from Amano Enzyme can be used. Particularly preferably, bilirubin oxidase having the amino acid sequence disclosed in UniProtKB / Swiss-Prot: Q12737 (http://www.ncbi.nlm.nih.gov/protein/Q12737) can be used.

  In the case of producing by a genetic engineering method, a method known in the art can be used. Specifically, by a hybridization method using a DNA prepared based on the nucleotide sequence of a desired enzyme gene as a probe, a desired enzyme is obtained from a genomic DNA derived from an organism, a cDNA synthesized by reverse transcription from total RNA, or the like. A nucleic acid molecule encoding the can be prepared. The probe used here is an oligonucleotide containing a sequence complementary to the desired enzyme and can be prepared according to a conventional method. For example, in addition to the chemical synthesis method, when the target nucleic acid has already been obtained, its restriction enzyme fragment or the like can be used.

  Similarly, a nucleic acid molecule encoding a desired enzyme can be prepared by using a genomic DNA or cDNA derived from an organism as a template by PCR using a primer prepared based on the nucleotide sequence of the desired enzyme gene. The primer used when PCR is used is an oligonucleotide containing a sequence complementary to the nucleic acid sequence encoding the desired enzyme, and can be prepared in the same manner as the above-mentioned probe based on a conventional method. When the probe or primer is prepared based on the chemical synthesis method, the probe or primer is designed based on the sequence information of the target nucleic acid prior to the synthesis.

  Here, “complementary” means that the probe or primer and the target nucleic acid molecule can specifically bind to each other according to the base pairing rule to form a stable double-stranded structure. Here, not only complete complementarity, but only some nucleobases are subject to the rules of base pairing as long as the probe or primer and the target nucleic acid molecule are sufficient to be able to form a stable duplex structure with each other. Even partial complementarity that is compatible with The length of the probe or primer is determined depending on many factors such as sequence information of the target nucleic acid such as GC content and the hybridization reaction conditions such as reaction temperature and salt concentration in the reaction solution.

  Further, a transformant is prepared by inserting a nucleic acid molecule encoding a desired enzyme into an appropriate expression vector and introducing this into a host. Here, the available vectors and the insertion of the foreign gene into the vectors have been described above.

  There is no particular limitation on the host cell for producing the transformant as long as it can efficiently express the foreign gene. Prokaryotic cells can be preferably used, and particularly Escherichia coli can be used. In addition, Bacillus subtilis, Bacillus bacterium, Pseudomonas bacterium, etc. can also be used. Furthermore, it is possible to utilize eukaryotic cells without being limited to prokaryotes. The transformation method, the method of culturing the obtained transformant and the method of selection are described above.

  In order to isolate and purify the desired enzyme from the culture of the transformant, ordinary protein isolation and purification methods can be used. For purification, a method similar to a general protein isolation and purification method may be applied from the culture of the above transformant, depending on the fraction in which the desired enzyme is present. Specifically, when the desired enzyme is produced outside the host cells, the culture solution is used as it is, or the host cells are removed by means such as centrifugation or filtration to obtain a culture supernatant. Subsequently, the culture supernatant can be isolated and purified by appropriately selecting a protein purification method known in the art.

  Further, when the desired enzyme is produced in the host cells, the host cells are recovered by means such as centrifugation or filtration of the culture. Then, the host cells are disrupted by an enzymatic disruption method, a physical disruption method such as ultrasonic treatment, freeze-thawing, or osmotic shock. After crushing, the solubilized fraction is collected by means such as centrifugation or filtration. The obtained solubilized fraction can be isolated and purified by treating it in the same manner as in the case where it can be produced extracellularly. Further, a concentrated solution of the lysate of the expressing bacterial cells may be used as in the case of gluconate dehydratase.

  In addition, an enzyme whose amino acid sequence is known in the art can also be produced by a chemical synthesis technique. For example, it can be also prepared by synthesizing all or part of the amino acid sequence of the desired enzyme using a peptide synthesizer and reconstructing the obtained polypeptide under appropriate conditions. At least 80%, 90%, 95%, or 98, such as those having an amino acid sequence in which one or several amino acids are modified by deletion, substitution, or addition in the amino acid sequence of an enzyme whose amino acid sequence is known Variants having% sequence identity can also be used as long as they have the desired activity.

  Further, the organism itself such as microorganisms, organelles and cells may be used as long as it can catalyze the reduction reaction of oxygen. Further, it may be a crudely purified product from these organisms.

  A substance necessary for the enzyme to exert its catalytic activity may be immobilized on the electrode material together with the enzyme. For example, a mediator that mediates electron transfer between the catalytic reaction of the enzyme and the electrode may be immobilized together with the enzyme. The usable mediators are described above. When bilirubin oxidase is used as the enzyme on the positive electrode side, ABTS is preferably used as the mediator. Further, in the case of an enzyme that requires a coenzyme or the like to exhibit the catalytic activity of the enzyme, a substance required for activation can be immobilized on the electrode material together with the enzyme. Regarding the enzyme that requires a cofactor, the immobilized enzyme may be of the apo form or the holo form, but it must be constructed so that it can exhibit activity during the electrode reaction. Therefore, when the apo form is retained on the electrode material, a means for supplying a cofactor to the enzyme immobilized on the electrode material is provided to convert the apo form enzyme into the active holo form. It is necessary to provide means.

  The electrolyte layer of the biobattery of the present embodiment has ion conductivity capable of penetrating protons and the like, and a negative electrode constituent other than ions such as protons, a positive electrode constituent, and as long as it has a property of not allowing electrons to pass, its material There is no limitation on the shape and the like. For example, a solid electrolyte membrane can be used to form a diaphragm. Examples of the solid electrolyte membrane include a strong acid group such as a sulfone group, a phosphoric acid group, a phosphon group, and a phosphine group, a weak acid group such as a carboxyl group, and a solid membrane having an ion exchange function such as an organic polymer having a polar group. Although illustrated, it is not limited thereto. Specifically, a cellulose membrane and Nafion (registered trademark) which is a copolymer of tetrafluoroethylene and perfluoro [2- (fluorosulfonylethoxy) propylvinyl ether]: tetrafluoroethylene and perfluoro [2- (fluorosulfonylethoxy) propylvinyl ether]. The perfluorocarbon sulfonic acid (PFS) resin film can be used.

  The fuel tank is filled with fuel. The fuel is not particularly limited as long as it is a substance that can release electrons by the redox reaction which is promoted by the catalytic enzyme of the negative electrode. Therefore, the fuel is a substance that can serve as a substrate for gluconate dehydratase, and preferably gluconate can be used. When a multi-step reaction system is constructed by combining with another enzyme, it can be appropriately selected according to the kind of the other enzyme. For example, when a multistage glucose oxidation system is constructed by combining with glucose dehydrogenase, a substance serving as a substrate for glucose dehydrogenase, particularly glucose fuel, can be used.

  The fuel is supplied in the form of being dissolved in an appropriate solvent, or the fuel is encapsulated in a gel, and the supply form is appropriately selected according to the structure of the biofuel cell. The solvent is an aqueous medium, and may be an appropriate buffer solution in addition to distilled water. Examples of the buffer component contained in the buffer include imidazole, carbonate, phosphate, borate, tartrate, citrate, tris (hydroxymethyl) aminomethane (TRIS), 4- (2-hydroxy). Examples thereof include ethyl) -piperazine-1-ethanesulfonic acid (HEPES) and 3-morpholinopropanesulfonic acid (MOPS). These may be used alone or in combination of two or more. Phosphate buffer is preferred. As the gel, natural polymers such as agarose, agaropectin, gum arabic, color ginnan, collagen and gelatin, synthetic polymers such as polyacrylamide polymers, polyvinyl alcohol polymers, polyvinylpyrrolidone polymers and polyvinyl ether polymers. Etc. can be used suitably. The fuel may be provided with a means for supplying a substance necessary for activation to the enzyme immobilized on the electrode material. If necessary, a mediator, a coenzyme and the like can be included.

  In the biofuel cell of this embodiment, a known buffer solution can be used as the electrode buffer solution that fills the biofuel cell. The buffer solution component contained in the buffer solution is as described above, but the phosphate buffer solution is preferable. Since the enzyme that is the electrode catalyst is very sensitive to the pH of the solution, it is preferable to control it to a pH around the pH at which the enzyme easily functions by the electrode buffer. Therefore, most of the enzymes show maximum activity in the vicinity of neutrality, and therefore it is preferable to prepare the enzyme in the vicinity of neutrality.

  The power generation mechanism of the biofuel cell of the present embodiment will be described. When the fuel is supplied to the negative electrode side, the fuel is oxidized by the catalytic action of the enzyme on the negative electrode. The electrons generated by the oxidation of the enzyme are transferred to the conductive base material. The transfer of electrons from the enzyme to the conductive base material may be performed via a mediator.

  Then, the electrons are transmitted from the negative electrode through the external circuit to the positive electrode, and a current is generated. On the other hand, the protons generated together with the electrons due to the oxidation reaction of the fuel by the enzyme move to the positive electrode side through the electrolyte layer. On the positive electrode, the protons produce water by the reaction between oxygen taken from the atmosphere and electrons that have moved through an external circuit.

  According to the biofuel cell of this embodiment, a biocell using gluconic acid or the like as a fuel can be provided. As the fuel, a substance that serves as a substrate for gluconate dehydratase such as gluconate can be used. Further, in a multi-stage glucose oxidation system, by using glucose or the like as a fuel by combining it with an enzyme such as glucose dehydrogenase which catalyzes the reaction in the preceding stage of gluconic acid oxidation to oxidize glucose to produce gluconic acid. You can By constructing a multi-stage oxidation system, the number of electrons that can be taken out from a fuel such as glucose is increased and the output of the biocell can be improved. According to the biobattery of the present embodiment, it is possible to construct a biobattery having a particularly high utility value in the fields of medical care, food, environment and the like.

(Biosensor)
The biosensor A of this embodiment includes the enzyme electrode 1 of this embodiment and can be used as an electrode of the biosensor A, preferably a working electrode. In the biosensor A of the present embodiment, the enzyme electrode 1 of the present embodiment may be configured by a two-electrode system by providing a working electrode and a counter electrode thereof, and from the viewpoint of increasing the reliability of measurement accuracy, silver-silver chloride is used. It may be configured as a three-electrode system having a reference electrode such as.

  An example of the biosensor A of this embodiment is schematically shown in FIG. In the enzyme electrode 1 of the biosensor A shown in FIG. 1, the enzyme 11 and the mediator 12 are immobilized on the electrode material 10 in which the porous carbon material 10b is laminated on the electrode base material 10a as the current collector. The measurement is performed by bringing the measurement sample into contact with the biosensor A of the present embodiment, and detecting the current generated by the redox reaction of the enzyme 11 on the electrode and the substrate (detection target 100) with the detector 2. Thereby, the presence or absence or the concentration of the substrate (detection target 100) in the sample can be measured. For example, it can be measured using a known method such as chronoamperometry or coulometry for measuring an oxidation current or a reduction current, and a cyclic voltammetry method.

  According to the biosensor A of the present embodiment, it is possible to provide a biosensor for detecting a substance (detection target 100) that is a substrate of gluconate dehydratase (enzyme 11) such as gluconate in a sample. . Further, when the enzyme 11 of the enzyme electrode 1 of the biosensor A of the present embodiment forms a multi-step reaction system together with glucose dehydrogenase and the like, the step before the gluconate dehydratase in the redox reaction of glucose is performed. It can also be used for detection of a substance (detection target 100) that is a substrate of the glucose dehydrogenase, and can be used for detection of glucose such as blood glucose level. According to the biosensor A of the present embodiment, it is possible to construct the biosensor A having a particularly high utility value in the fields of medical care, food, environment and the like.

  Hereinafter, the present embodiment will be described in detail with reference to examples, but the present invention is not limited to these examples.

  Hereinafter, detailed description will be given with reference to examples.

Example 1: Selection of gluconate oxidase and confirmation of synthesis In this example, gluconate oxidase used in the following examples was selected, and verification was performed to establish a method for synthesizing the gluconate oxidase. .

(Method)
1. Selection of Gluconate Oxidase In this example, D-gluconate dehydratase (ACCESSION Number: Q97U27) derived from Sulfolobus solfataricus P2 strain was selected as the gluconate oxidase. . The bacterium belonging to the genus Sulfolobus is a bacterium belonging to the thermoacidophilic archaea. In archaebacteria such as Sulfolobus, the glycolysis system, which is the mechanism of glucose decomposition, does not use the Emden-Meyerhof pathway but the Entner-Doudoroff pathway and the pyrolytic system (Pyroglycolysis). To use. (Reference 3: See http://www.sc.fukuoka-u.ac.jp/~bc1/Biochem/glyclysis.htm). In the pyrolysis system, glucose is decomposed into 2-keto-3-deoxygluconic acid via gluconic acid.

2. Confirmation of synthesis of gluconate oxidase In order to synthesize the D-gluconate dehydratase from the Sulfolobus solfataricus P2 strain selected as the gluconate oxidase above in the Escherichia coli protein synthesis system, the above-mentioned enzyme was coded The gene was cloned into an expression plasmid vector (pET22b) to transform E. coli BL21 (DE3) pLysS strain. E. coli after transformation was pre-cultured (37 ° C, absorbance OD ₆₀₀ = 0.2), IPTG (0.2 mM isopropyl thio-β-galactoside) was added, and main culture (37 ° C, 4 hours) was performed (25 ml). . The expressed bacterial cells were suspended in a buffer solution (20 mM Tris-HCl pH7.4), a surfactant (0.4% Brij-58) was added, and the mixture was allowed to stand on ice for 30 minutes. The cells were ultrasonically disrupted, and the supernatant after centrifugation (42,000 × g, 30 minutes) was collected as a lysate of enzyme-expressing cells. The enzyme was then purified using an affinity carrier. Specifically, TALON (Clontech), a metal affinity carrier for purifying histidine-tagged proteins, was packed in an appropriate amount in an open column, pre-washed with 50 mM sodium phosphate, 0.3 M NaCl solution, and then crushed to 0.3 in the disrupted solution of expressed cells. M NaCl was added and applied to the column. After washing with 50 mM sodium phosphate, 5 mM imidazole, 0.3 M NaCl solution, the enzyme was eluted with 50 mM sodium phosphate, 150 mM imidazole, 0.3 M NaCl solution. It was dialyzed against a buffer (25 mM Tris-HCl pH 7.4) to remove the salts used for elution. Acrylamide gel for enzyme-expressing bacterial cells after treatment with surfactant (Brij (registered trademark) 58), disrupted solution of enzyme-expressing bacterial cells after ultrasonic disruption, supernatant fraction after centrifugation, and precipitation fraction after centrifugation Electrophoresis was performed. Further, the lysate of the Escherichia coli-expressing cells was subjected to ultrafiltration (Amicon Ultra-4 10K) for molecular weight fractionation, and acrylamide gel electrophoresis was also performed in the same manner.

(result)
The results are shown in Figure 2. Lane 1 in FIG. 2 is an enzyme-expressing bacterial cell after treatment with a surfactant (Brij (registered trademark) 58), lane 2 is an enzyme-expressing bacterial cell lysate after ultrasonic disruption, and lane 3 is an enzyme-expressing bacterial cell. The supernatant fraction after centrifugation of the disrupted solution (lane 2) (lysate of enzyme-expressing bacterial cells), lane 4 is the precipitate fraction after centrifugation of the disrupted solution of enzyme-expressing bacterial cells (lane 2), and lane 5 is , A lysate of enzyme-expressing bacterial cells (lane 3) concentrated 20 times by ultrafiltration, a concentrated solution of lysate of enzyme-expressing bacterial cells (enzyme solution 1), lane 6 is a lysate of enzyme-expressing bacterial cells (lane) 3) was concentrated 10 times by ultrafiltration, diluted 20 times with water, and was further concentrated 20 times. The result of electrophoresis of a concentrated solution (enzyme solution 2) of a lysate of enzyme-expressing bacterial cells, lane M indicates a molecular weight marker. Indicates. In addition, electrophoresis was also performed on the affinity-purified sample, but no enzyme-derived band could be confirmed (results not shown). From these results, gluconate dehydratase derived from Sulfolobus solfataricus P2 strain is efficiently expressed in the Escherichia coli protein expression system. However, it can be understood that the enzyme solution becomes insoluble as the degree of purification increases. In addition, heat treatment of purified enzyme (70 ℃) and low temperature expression of E. coli were tried, but the same result was obtained.

Embodiment 2. FIG. Examination of Porous Electrode Material In this example, a lysate of bacterium expressing gluconate dehydratase is used as an enzyme member of an enzyme electrode, and thus a porous carbon electrode material having pores capable of encapsulating an enzyme molecule is used. It was investigated. In addition, the electrode material of porous carbon and the electrode buffer solution were examined using glucose dehydrogenase as the enzyme molecule.

(Method)
1. Production of Battery Cell A negative electrode and a positive electrode of the battery cell were produced by the method described below.

1-1. Preparation of Negative Electrode Side As an electrode material on the negative electrode side, an electrode material obtained by modifying a carbon electrode material with porous carbon fine particles (hereinafter sometimes referred to as “porous carbon electrode material”) and a modification with porous carbon fine particles are used. A carbon electrode material which was not made was used as it was as an electrode material.

1-1-1. Negative electrode using porous carbon electrode Porous carbon particles (0.4 g), 20% PVDF solution (0.8 g, KF polymer, Kureha Battery Materials Japan), N-methyl-2-pyrrolidone (8 ml) ) Was mixed and sonicated (15 W, 5 minutes) to prepare a carbon ink. The electrode material (Nippon Carbon GF-20, 2 cm × 2 cm) was dipped in carbon ink, lifted, and dried on a glass plate by dry heat (60 ° C.). A mediator (1,4-naphthoquinone solution, 8 mg in acetonitrile) was added dropwise to the electrode material and dried in a vacuum desiccator. A solution prepared by adding glucose dehydrogenase (2 to 10 mg, measured 2 mg) in 1 M potassium phosphate pH 7.0 was added dropwise to the electrode material thus prepared, and dried in a vacuum desiccator. It was The glucose dehydrogenase used here is a PQQ-dependent soluble glucose dehydrogenase derived from Acinetobacter calcoaceticus NBRC12552 strain. A method for producing such an enzyme and sequence information are disclosed in JP-A-2013-45647.

1-1-2. Negative Electrode on the Negative Electrode Side Utilizing Carbon Electrode Material Unmodified with Porous Carbon Fine Particles Except that the porous carbon fine particles were not used, it was prepared as in 1-1-1 above.

1-2. Preparation of positive electrode
MgO porous carbon fine particles (0.4 g: average pore size 35 nm, Toyo Tanso), PTFE (0.4 g, PTFE 6J, Mitsui DuPont Fluorochemicals), isopropanol (8 ml) were mixed and sonicated (15 W, 5 minutes) to prepare a carbon slurry. Porous carbon fine particles were applied by immersing carbon cloth (Nippon Carbon GF-20, 2 cm x 2 cm) as an electrode substrate in carbon slurry. This was dried on a glass plate by dry heat (60 ° C., 6 hours). The electrode material prepared in this way was added to the enzyme (MvBOD, 2-10 mg, measured 5 mg: Amanoenzyme BO “Amano” 3) and mediator (ABTS: 2,2 ′) in 1 M potassium phosphate pH 7.0. -Azino-
A solution prepared containing bis (3-ethylbenzothiazoline-6-sulfonic acid) and Milli-Q water (5 mg) was added dropwise and dried in a vacuum desiccator.

1-3. Battery cell
1-3-1. Battery cell (negative electrode material: porous carbon electrode material + electrode buffer: imidazole)
Using the negative electrode (porous carbon electrode material) prepared in 1-1-1 above and the positive electrode prepared in 1-2 above, glucose solution (1M, 400 μl) on the negative electrode, and electrode on the positive electrode A buffer solution (1 M imidazole, 200 μl) was added, and both electrodes were sandwiched between cellulose membranes and placed in the electrode part of the battery cell casing.

1-3-2. Battery cell (negative electrode material: carbon electrode material that does not use porous fine particles + electrode buffer solution: imidazole)
The negative electrode was prepared in the same manner as 1-3-1 above except that the carbon electrode material prepared in 1-1-2 that was not modified with the porous carbon fine particles was used (Comparative Example).

1-3-3. Battery cell (negative electrode material: porous carbon electrode material + electrode buffer: phosphate buffer)
It was prepared in the same manner as 1-3-1 above except that a phosphate buffer (1 M potassium phosphate pH 7.0) was used as the electrode buffer (Comparative Example).

2. Measurement method Using an electronic load device, a constant current (1 mA / cm ² ) was applied to the battery cell prepared above and the voltage change was measured.

(result)
The results of examining the discharge characteristics of the battery cell manufactured above are shown in FIGS. 3 and 4. FIG. 3 shows the results of using imidazole as the electrode buffer solution for both waveform 1 and waveform 2, and waveform 1 is a battery prepared by using the negative electrode material modified with porous carbon fine particles. The cell (1-3-1) and the waveform 2 show the results of the battery cell (1-3-2) prepared by using the negative electrode material prepared without modification with the porous carbon fine particles. . As a result, the waveform 1 was able to obtain higher electrical conversion efficiency. It can be understood that the efficiency of electron transfer to glucose → enzyme → mediator → electrode material was improved by encapsulating the enzyme component in the pores of the porous carbon.

  FIG. 4 shows the results of the battery cell prepared by using both the waveform 3 and the waveform 4 prepared by modifying the negative electrode material with the porous carbon fine particles, and the waveform 3 represents the waveform 1 of FIG. The same result as in the battery cell (1-3-1) using imidazole as the electrode buffer solution, waveform 4 is the result in the battery cell (1-3-3) using phosphate buffer solution as the electrode buffer solution. Indicates. As a result, the waveform 4 was able to obtain higher electric conversion efficiency. Phosphate buffer solution improved the electrical conversion efficiency of glucose fuel by 1.3 times compared with imidazole which has been widely used as an electrode buffer solution for biocells.

Example 3 Electrochemical Performance of Enzyme Electrode Having Gluconic Acid Oxidation Catalytic Activity In this example, a measurement system using the discharge characteristics of battery cells as an index can be constructed as the enzyme electrode performance measurement system in Example 2. Therefore, using this measurement system, the concentrated solution of the lysate of Escherichia coli expressing gluconate dehydrogenase was immobilized on the negative electrode material, and the discharge characteristics of the battery cell were examined.

(Method)
1. Preparation of Battery Cell Two types of battery cells were prepared as an electrode catalyst for the negative electrode, a battery cell using only glucose dehydrogenase and a battery cell using glucose dehydrogenase and gluconate dehydratase.

1-1. Battery cell (negative electrode catalyst: glucose dehydrogenase only)
A battery cell was produced in the same manner as in 1-3-1 of Example 2 except that a glucose solution (1M, 200 μl) was used as the glucose solution added to the negative electrode side.

1-2. Battery cell (negative electrode catalyst: glucose dehydrogenase + gluconate dehydratase)
Glucose dehydrogenation was performed on the negative electrode material by subjecting the lysate of Escherichia coli-expressing bacterium of gluconate dehydratase prepared in Example 1 to ultrafiltration (Amicon Ultra-4 10K) for molecular weight fractionation. It was prepared in the same manner as in 1-1 above except that it was immobilized together with the enzyme. The lysate of Escherichia coli expressing bacterial cells of gluconate dehydratase used here was the total amount of the disrupted bacterial cells (supernatant after centrifugation) obtained from the culture solution (25 ml) of the enzyme expressing bacterial cells. Yes, this was concentrated 20 times by ultrafiltration (corresponding to enzyme solution 1 of Example 1).

2. Measurement Method In the same manner as in Example 2, a constant current was applied to the battery cell to measure the voltage change.

(result)
Results are shown in FIG. Waveform 1 in FIG. 5 shows the result (same condition as waveform 4 in FIG. 4 showing the result of Example 2) in the battery cell (1-1) using only glucose dehydrogenase as the negative electrode catalyst. Waveform 2 of 5 shows the result in the battery cell (1-2) using two enzymes, glucose dehydrogenase and gluconate dehydratase, as the negative electrode catalyst. From these results, it is about 12,300 seconds (when the cell voltage is 0.1 V lower) with only one enzyme of glucose dehydrogenase, whereas it is about 2300 with glucose dehydrogenase and gluconate dehydratase. It became 24,500 seconds (same voltage), and it was revealed that the duration of power generation was doubled with the two enzymes. This is presumed to be due to the electron transfer reaction when gluconic acid is oxidized in the negative electrode on which two kinds of enzymes are immobilized, in combination with the mediator (1,4-naphthoquinone).

  Since the additional current stops when the voltage drops by 0.1 V, the voltage of the battery cell thereafter can be confirmed to return to the initial value, and it can be understood that the electrode manufactured in this example also has stability. .

Example 4: Analysis of reaction product of enzyme electrode having gluconic acid oxidation catalytic ability In Example 3, the enzyme electrode using glucose dehydrogenase and gluconic acid dehydratase as an electrocatalyst has a two-step glucose oxidation system functioning. It was confirmed that In response to this, in this example, the reaction product by the gluconate dehydratase used in Example 3 was analyzed by gas chromatography (GCMS).

(Method)
1． Enzymatic reaction
25 mM potassium phosphate pH 7.0, 1 mM gluconic acid (substrate), enzyme solution, prepare an enzyme reaction solution to be 0.1 mM DCIP, react at 40 ° C for 30 minutes, add equal volume of chloroform and stir The reaction was stopped. As the enzyme, a concentrated solution of a lysate of the same expressing bacterial cell as that immobilized on the electrode material in Example 3 was used. A control was prepared in the same manner as above except that the enzyme was not added.

2． Preparation of GCMS sample 10 μL of the supernatant of the enzyme reaction solution after the reaction was stopped was collected in a glass vial and dried to dryness by blowing N ₂ gas. Then, 50 μL of methoxyamine hydrochloride / pyridine (20 mg / mL) was added, and the mixture was incubated at 30 ° C. for 90 minutes. After adding 50 μL of MSTFA and incubating at 37 ° C. for 30 minutes, analysis was performed by GCMS.

3． GCMS conditions
The GCMS conditions are as follows.
GCMS equipment: SHIMADZU OP-2010 Ultra
Auto Sampler: SHIMADZU AOC-5000 Plus
Column used: Agilent Technologies DB-5 30m 0.250mm 1.00 μm
Vaporization chamber temperature: 280 ℃
Oven temperature: 100 ℃ (Duration 4min) -Rate 4 ℃ / min-320 ℃ (Duration 8min)
Connection temperature: 280 ℃
Ion source temperature: 200 ℃
Ionization method: EI
Sample introduction method: Splitless
Flow Rate: 39cm / sec (1.1mL / min)
Scan Speed: 2000u / sec
MassRange: m / z = 45-600
Sample injection volume: 1mL

(result)
FIG. 6 shows the results. FIG. 6 shows the result of GCMS analysis of the reaction product. In the control to which no enzyme was added, only gluconic acid as the added substrate was detected. On the other hand, the addition of gluconate oxidase confirmed the detection of 2-keto-3-deoxygluconate, which is the reaction product of gluconate dehydratase. From these results, the enzyme electrode having a gluconic acid oxidation catalytic ability catalyzes the oxidation reaction of gluconic acid, and the two-step oxidation system of glucose confirmed in Example 3 is based on glucose dehydrogenase and gluconate dehydratase. You can prove that.

  INDUSTRIAL APPLICABILITY The present invention relates to an enzyme electrode, a method for producing an enzyme electrode, a biobattery, and a biosensor, and can be used in all fields in which these are required, particularly in the industrial fields such as electronics, medical care, food, and the environment.

Biosensor A
Enzyme electrode 1
Electrode material 10
Electrode substrate (collector) 10a
Porous carbon material 10b
Enzyme 11
Mediator 12
Detector 2
Object to be detected 100

Claims (7)

  An enzyme electrode having a gluconic acid oxidation catalytic ability, which comprises gluconic acid dehydratase derived from a bacterium belonging to the genus Sulfolobus immobilized on an electrode material.   The enzyme electrode having a gluconic acid oxidation catalytic ability according to claim 1, wherein the electrode material includes a carbon substrate having a porous structure having pores having an average diameter of 30 to 40 nm. A method for producing an enzyme electrode having a gluconic acid oxidation catalytic ability, which comprises fixing a gluconic acid dehydratase derived from a sulforobas bacterium to an electrode material,
(I) a step of transforming a bacterium having no sugar chain modifying ability with a gene encoding a glucose dehydratase derived from a bacterium belonging to the genus Sulfolobus, and preparing an expression cell expressing a recombinant gluconate dehydratase,
(Ii) a step of contacting the expressed bacterial cell with a surfactant,
(Iii) a step of crushing the expressed bacterial cells after the treatment with the surfactant to prepare a crushed solution of the expressed bacterial cells,
(Iv) a step of separating and removing the bacterial cell residue in the disrupted liquid of the expressed bacterial cells to prepare a lysate of the expressed bacterial cells,
(V) a step of concentrating the lysate of the expressing bacterial cells to prepare a concentrated solution of the lysing liquid of the expressing bacterial cells,
(Vi) A method for producing an enzyme electrode having a gluconic acid oxidation catalytic ability, comprising the step of immobilizing a concentrated solution of a lysate of the expressed bacterial cells on the electrode material.   The method for producing an enzyme electrode having a gluconic acid oxidation catalytic ability according to claim 3, wherein the electrode material includes a carbon substrate having a porous structure having pores having an average diameter of 30 to 40 nm.   The carbon substrate having the porous structure is prepared by calcining a mixture of a carbon precursor and a magnesium oxide precursor, and removing magnesium oxide from the obtained calcined product. A method for producing an enzyme electrode having the ability to catalyze gluconic acid oxidation.   Glucon produced by the method for producing the enzyme electrode having the gluconic acid oxidation catalytic ability according to claim 1 or 2, or the enzyme electrode having the gluconic acid oxidation catalytic ability according to any one of claims 3 to 5. A biobattery comprising an enzyme electrode having an acid oxidation catalytic ability as a negative electrode.   Glucon produced by the method for producing the enzyme electrode having the gluconic acid oxidation catalytic ability according to claim 1 or 2, or the enzyme electrode having the gluconic acid oxidation catalytic ability according to any one of claims 3 to 5. A biosensor comprising an enzyme electrode having an acid oxidation catalytic ability as a working electrode.

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