WO2014119516A1

WO2014119516A1 - Diaphorase composition

Info

Publication number: WO2014119516A1
Application number: PCT/JP2014/051680
Authority: WO
Inventors: 幸夫岩井; 誠嗣竹嶋; 柳谷　周作
Original assignee: 東洋紡株式会社
Priority date: 2013-01-29
Filing date: 2014-01-27
Publication date: 2014-08-07
Also published as: JP6160094B2; JP2014143942A

Abstract

[Problem] To provide a diaphorase composition, in which a stabilizing agent that is readily available, has uniform quality and does not affect the appearance, performance and quality of an enzyme preparation or an enzyme-containing composition is used as a stabilizing agent for diaphorase. [Solution] A composition characterized by comprising (a) diaphorase and (b) at least one compound selected from the group consisting of a sugar, a sugar alcohol and an amino acid.

Description

Diaphorase composition

The present invention relates to a composition containing diaphorase. *

Diaphorase [EC. 1.6.99. -] Plays an important role in the electron transport system in vivo.
Diaphorase has been studied for in vitro use in various technical fields, and a part of it has been put into practical use. Such technical fields include production of useful substances, production of energy-related substances, measurement or analysis, environmental conservation, medical care, and the like. For example, in the field of clinical diagnostics, diaphorase takes advantage of the property that it uses reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate. Used in diagnostic reagents. Diaphorase is also used in an enzyme battery which is a kind of fuel cell (Non-patent Document 1).

As diaphorase, those isolated and purified from microorganisms belonging to the genus Clostridium (Non-patent Document 2) or the genus Bacillus (Patent Document 1, Patent Document 2) are commercially available. The Bacillus stearothermophilus as producing diaphorase described in Patent Document 1 and Patent Document 2 was reclassified as Geobacillus stearothermophilus in 2001 (Geobacillus stearothermophilus). Non-patent document 3). Known from Geobacillus stearothermophilus, or modified ones thereof, and their gene sequence, amino acid sequence and physicochemical properties have been investigated (Patent Document 2, Patent Document 3, Non-Patent Document 1).

On the other hand, most of the raw material enzymes used for reagents and sensors are distributed as dried products (for example, powders) (dry products). The reason for this is that the product is light and small in volume, so that it can be easily stored and transported, and since it is dry, there is no risk of spoilage due to microbial contamination. In addition, the dissolution concentration of the enzyme can be freely adjusted according to the purpose of use, and the type of buffer solution for dissolution can be arbitrarily selected, so that it can be developed for various uses. Furthermore, in general, the enzyme activity can be stably maintained for a long time in a dry state rather than in a solution state. *

There are various means for drying the enzyme. For example, a method of precipitating the target enzyme from a solution containing enzyme protein with an organic solvent such as acetone or alcohol and collecting it to obtain a dry powder. A spray drying method of spraying a solution containing the enzyme and applying hot air to dry the solution. There is a freeze drying method in which a solution containing an enzyme is frozen and dried under reduced pressure. *

Regardless of the conditions, if the enzyme is inadvertently dried, problems such as loss of activity due to protein denaturation and turbidity generation during re-dissolution often occur. It is essential to add a stabilizer to prevent this. The stabilizer added to the enzyme product must not only prevent denaturation and deactivation of the enzyme protein due to drying, but also have the ability to prevent loss of activity during storage and distribution. *

Sho 60-156381 Japanese Patent No. 3953578 JP2007-143493

An object of the present invention is a diaphorase that uses a stabilizer that is easily available, has a uniform quality, and does not affect the appearance, performance, and quality of an enzyme product or a composition containing an enzyme, as a stabilizer for diaphorase. It is to provide a composition. *

The present inventors examined various substances in order to improve the storage stability of a diaphorase composition in a dry state. As a result, the inventors have found that the stability of diaphorase is improved by coexisting one or more compounds of saccharides, sugar alcohols, and amino acids, and the present invention has been completed.

That is, the present invention relates to the following.
Item 1. (A) A composition containing any one or more compounds selected from the group consisting of diaphorase, (b) saccharides, sugar alcohols and amino acids.
Item 2. Item 2. The composition according to Item 1, wherein the compound (b) is at least one selected from the group consisting of mannitol, inositol, xylitol, trehalose, sorbitol, and sodium L-glutamate.
Item 3. Item 3. The composition according to Item 1 or Item 2, wherein the compound (b) is contained in an amount of 30 to 70% (w / w) based on the amount of diaphorase protein.
Item 4. Item 4. The composition according to any one of Items 1-3, wherein the diaphorase is any one of the following proteins:
(A) a protein having the amino acid sequence described in SEQ ID NO: 1 (b) a protein having an amino acid sequence in which one or several amino acids are deleted, inserted, added or substituted in the amino acid sequence described in SEQ ID NO: 1 A protein having diaphorase activity
(C) a protein having the amino acid sequence of SEQ ID NO: 2 (d) a protein having an amino acid sequence in which one or several amino acids are deleted, inserted, added or substituted in the amino acid sequence of SEQ ID NO: 2 A protein having diaphorase activity
Item 5. Item 10. A product comprising the composition according to any one of Items 1-4.
Item 6. (A) A method for stabilizing diaphorase in a diaphorase composition, wherein any one or more compounds selected from the group consisting of diaphorase, (b) saccharides, sugar alcohols and amino acids are allowed to coexist.

According to the present invention, the stability of a dried diaphorase product, particularly a freeze-dried product, can be secured, and inactivation of the enzyme can be prevented even during long-term storage. *

Geobacillus thermodenitificans NG80-2 derived wild-type diaphorase (Sequence Listing 1) and Geobacillus sp. Amino acid sequence alignment of wild type diaphorase derived from Y4.1MC1 (Sequence Listing 2)

Hereinafter, the present invention will be described in detail.
1. Diaphorase 1-1. Diaphorase activity “Diaphorase” is an activity that catalyzes a reaction of oxidizing NADH or NADPH with a dye such as potassium ferricyanide, methylene blue, 2,6-dichloroindophenol (DCPIP), tetrazolium salt (ie, diaphorase activity). It is an enzyme with a wide distribution from microorganisms such as bacteria and yeast to mammals. This diaphorase plays an important role in the in vivo electron transport system, and NADH or NADPH produced by the dehydrogenation reaction from the substrate by NAD or NADP-dependent dehydrogenases by this diaphorase is an electron acceptor. The electron acceptor becomes a reduced form.

Diaphorase activity can be measured by a known method. For example, using DCPIP as an electron acceptor, the activity can be measured using the change in absorbance of the sample at a wavelength of 600 nm before and after the reaction as an index. More specifically, the activity can be measured using the following reagents and measurement conditions.

Method for measuring diaphorase activity <Reagent>
Distilled water 200 mM Tris-HCl buffer pH 7.5
6.0 mM NADH aqueous solution 1.2 mM 2,6-dichlorophenolindophenol (DCPIP) solution enzyme diluted solution 200 mM Tris-HCl buffer pH 7.5 containing 0.1% bovine serum albumin
<Procedure 1>
The diaphorase solution is diluted to 0.4 to 0.8 U / ml with the above enzyme-diluted solution that has been ice-cooled in advance, and the solution that has been ice-cooled is used as the enzyme solution.
<Procedure 2>
The reaction mixture is prepared by mixing 2.4 mL of distilled water, 0.3 mL of Tris-HCl buffer, and 0.1 mL of NADH aqueous solution and pre-warming at 25 ° C. for 5 minutes.

<Measurement conditions>
To the reaction mixture 2.8 mL, add 0.1 mL of the enzyme solution and 0.1 mL of the DCPIP solution in order, and mix gently. Then, with a spectrophotometer (optical path length 1.0 cm) controlled at 25 ° C. with water as a control, The absorbance change at 600 nm is recorded for 2 to 3 minutes, and then the absorbance change per minute (ΔOD _TEST ) is measured from the linear portion (ie, after the reaction rate becomes constant). In the blind test, an enzyme dilution solution that dissolves diaphorase and a DCPIP solution are added to the reaction mixture instead of the enzyme solution, and the change in absorbance per minute (ΔOD _BLANK ) is similarly measured. From these values, diaphorase activity is determined according to the following formula. Here, 1 unit (U) in the diaphorase activity is the amount of enzyme that reduces the absorbance at 600 nm by 1.0 under the above measurement conditions for 1 minute.

Activity (U / mL) =
{-(ΔOD _TEST -ΔOD _BLANK ) × dilution ratio} / (1.0 × 0.1)

In the formula, 1.0 represents the unit absorbance at 600 nm determined based on the activity definition, and 0.1 represents the volume (mL) of the enzyme solution. In this document, unless otherwise indicated, enzyme activity is measured according to the measurement method described above.

The diaphorase applied to the present invention is preferably an isolated diaphorase or a purified diaphorase. Moreover, the diaphorase applied to the present invention may exist in a state dissolved in a solution suitable for storage or in a lyophilized state (for example, in a powder form). “Isolated” when used in relation to the enzyme (diaphorase) applied to the present invention means that components other than the enzyme (for example, contaminating proteins derived from host cells, other components, culture fluid, etc.) Not included). Specifically, for example, the isolated enzyme applied to the present invention has a contaminating protein content of less than about 20% by weight, preferably less than about 10%, more preferably less than about 5%. Even more preferably, it is less than about 1%. On the other hand, the diaphorase applied to the present invention may be present in a solution (for example, a buffer) suitable for storage or measurement of enzyme activity.

1-2. Polypeptide The diaphorase applied to the present invention is preferably composed of any of the following polypeptides (a) to (c).
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2;
(B) a polyamino acid sequence consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted, added and / or inverted in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and having diaphorase activity peptide;
(C) A polypeptide comprising an amino acid sequence having an identity of 80% or more with the amino acid sequence shown in SEQ ID NO: 1 or 2 and having diaphorase activity.

The amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 is an amino acid sequence of diaphorase derived from the genus Geobacillus as shown in Example 5.
The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of wild-type diaphorase derived from Geobacillus thermodenitificans NG80-2.
The amino acid sequence set forth in SEQ ID NO: 2 is Geobacillus sp. It is the amino acid sequence of wild type diaphorase derived from Y4.1MC1.

In the polypeptide (b) above, one or several amino acid residues are substituted, deleted, inserted and / or added (hereinafter referred to as these) in the amino acid shown in SEQ ID NO: 1 as long as the diaphorase activity is retained. Are collectively referred to as “mutations.”) A polypeptide comprising an amino acid sequence. Here, “several” is not limited as long as the diaphorase activity is maintained, for example, a number corresponding to less than about 20% of all amino acids, preferably a number corresponding to less than about 15%, The number is preferably less than about 10%, more preferably less than about 5%, and most preferably less than about 1%. More specifically, the number of amino acid residues to be mutated is, for example, 2 to 127, preferably 2 to 96, more preferably 2 to 64, still more preferably 2 to 32, and even more. The number is preferably 2 to 20, more preferably 2 to 15, even more preferably 2 to 10, and particularly preferably 2 to 5.

One or several mutations may be introduced into the DNA encoding the diaphorase of the present invention using a known method such as restriction enzyme treatment, treatment with exonuclease or DNA ligase, position-directed mutagenesis or random mutagenesis. It can be carried out by introducing mutations. Variant diaphorase can also be obtained by other methods such as ultraviolet irradiation. Variant diaphorase includes naturally occurring variants (for example, single nucleotide polymorphisms are also included) based on individual differences in microorganisms that retain diaphorase, differences in species or genera, and the like.

In addition, from the viewpoint of maintaining the activity of diaphorase, it is preferable that the mutation is present at a site that does not affect the active site or substrate binding site of diaphorase.

The polypeptide (c) is a polypeptide comprising an amino acid sequence having an identity of 80% or more compared to the amino acid sequence shown in SEQ ID NO: 1 as long as it retains diaphorase activity. Preferably, the identity between the amino acid sequence of the diaphorase of the present invention and the amino acid sequence shown in SEQ ID NO: 1 is 85% or more, more preferably 88% or more, still more preferably 90% or more, and still more preferably 93% or more, more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more. Such a polypeptide comprising an amino acid sequence having a certain identity or more can be prepared based on the known genetic engineering techniques as described above.

The identity of amino acid sequences can be calculated using analysis tools that are commercially available or available through telecommunication lines (Internet). For example, the homology algorithm BLAST (Basic local alignment) of the National Center for Biotechnology Information (NCBI) search tool) http: // www. ncbi. nlm. nih. It can be calculated by using default (initial setting) parameters in gov / BLAST /. In this application, this method is used to calculate the identity of amino acid sequences.

As another origin of diaphorase applied to the present invention, for example, microorganisms present in water systems such as soil, rivers, lakes and marshes or in the ocean, microorganisms resident on the surface or inside various animals and plants, and the like can be mentioned. Isolation sources may be those derived from microorganisms that grow in a low temperature environment, a high temperature environment such as a volcano, an oxygen-free / high-pressure / light-free environment such as the deep sea, and a special environment such as an oil field.

The diaphorase applied to the present invention includes not only a diaphorase directly isolated from a microorganism, but also an isolated diaphorase whose amino acid sequence has been modified by a protein engineering method, or a genetic engineering method. Is also included. For example, a modified enzyme obtained from a microorganism classified into the genus Geobacillus as described above may be used.

1-3. Method for Producing Diaphorase The method for producing diaphorase applied to the present invention is not particularly limited. For example, it can be produced by culturing a microorganism expressing diaphorase and purifying the obtained culture solution.
In general, a method for producing a target protein by culturing a microorganism that expresses the protein and purifying the obtained culture solution has already been established in the art. Therefore, those skilled in the art can apply the knowledge to produce diaphorase, and the embodiment is not particularly limited.

In the method for producing diaphorase applied to the present invention, the method for constructing the diaphorase expression system is not particularly limited. For example, a microorganism having an ability to produce diaphorase may be used as an expression system as it is. Alternatively, a gene recombinant (also referred to as a transformant) prepared by introducing DNA encoding diaphorase into an appropriate host vector system may be used as the expression system. From an industrial viewpoint, it is preferable to use a genetic recombinant for reasons such as easy control and higher safety.

Examples of microorganisms capable of producing diaphorase include those derived from the hyperthermophilic archaeon such as the aforementioned Geobacillus genus.

In the case of producing a recombinant, diaphorase-encoding DNA can be easily prepared using standard genetic engineering techniques (Molecular Cloning 2d Ed, Cold Spring Harbor Lab. Press (1989); Chemistry experiment course "gene research method I, II, III", Japan Biochemical Society edition (1986) etc.). Specifically, a cDNA library is prepared according to a conventional method from the above-mentioned appropriate source microorganism in which the diaphorase applied to the present invention is expressed, and an appropriate sequence peculiar to the DNA sequence of the diaphorase is prepared from the library. This can be performed by selecting a desired clone using a probe or an antibody.

Isolation of total RNA from the above microorganisms, isolation and purification of mRNA, acquisition and cloning of cDNA, determination of base sequence, etc. can all be carried out according to conventional methods. The method for screening the DNA of the present invention from a cDNA library is not particularly limited, and can be performed according to a usual method. For example, for a polypeptide produced by cDNA, a method for selecting a corresponding cDNA clone by immunoscreening using the polypeptide-specific antibody, a plaque high using a probe that selectively binds to a target nucleotide sequence Hybridization, colony hybridization, etc., and combinations thereof can be selected as appropriate.

In obtaining DNA, a PCR method or a modified DNA or RNA amplification method thereof can be suitably used. Primers used in the PCR method can also be appropriately designed and synthesized based on the base sequence determined above. In addition, isolation and purification of the amplified DNA or RNA fragment can be carried out according to a conventional method as described above, for example, by gel electrophoresis, hybridization or the like.

The DNA encoding diaphorase can be incorporated into an appropriate expression vector. The type and structure of the expression vector are not particularly limited as long as the DNA can be replicated in an appropriate host cell and can be expressed. The type of vector is appropriately selected in consideration of the type of host cell. Specific examples of the vector include a plasmid vector, a cosmid vector, a phage vector, a virus vector (an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a herpes virus vector, etc.) and the like. It is also possible to use a vector suitable for self-cloning.

An expression vector in which DNA encoding diaphorase is incorporated can be introduced into an appropriate host cell to be a transformant having the ability to produce the diaphorase. The host cell is not particularly limited as long as it can express the DNA and produce diaphorase. Specifically, prokaryotic cells such as Escherichia coli and Bacillus subtilis, molds such as yeast and filamentous fungi, eukaryotic cells such as insect cells, cultured plant cells, and mammalian cells can be used. Of these, Escherichia coli, Bacillus subtilis and filamentous fungi are preferable. E. coli is more preferred.

In the transformant, exogenous DNA is usually present in the host cell, but a transformant obtained by so-called self-cloning using the microorganism from which the DNA is derived as a host is also a preferred embodiment.

The transformant is preferably prepared by transfection or transformation using the expression vector shown above. Transformation may be transient or stable. Transfection and transformation can be performed using calcium phosphate coprecipitation method, electroporation, lipofection, microinjection, Hanahan method, lithium acetate method, protoplast-polyethylene glycol method, and the like.

The diaphorase applied to the present invention can be produced by culturing a gene recombinant that expresses diaphorase and purifying the obtained culture solution. The culture method and culture conditions are not particularly limited as long as diaphorase is produced. That is, on the condition that diaphorase is produced, a method and conditions suitable for the growth of the microorganism to be used can be appropriately set.

As a method for recovering the obtained culture solution, in the case of using a microorganism that secretes diaphorase to the outside of the cell body, for example, the culture supernatant is filtered and centrifuged to remove insoluble matters, and then an ultrafiltration membrane is used. The diaphorase can be obtained by separating and purifying by appropriately combining the concentration by the above, salting out such as ammonium sulfate precipitation, dialysis, and various chromatography.

On the other hand, when recovering from the microbial cells, for example, the microbial cells are crushed by pressure treatment, ultrasonic treatment, mechanical method, or a method using an enzyme such as lysozyme, and if necessary, such as EDTA. The enzyme can be obtained by solubilizing diaphorase by adding a chelating agent and a surfactant, separating and collecting it as an aqueous solution, separating and purifying it. After the cells are collected from the culture solution in advance by filtration, centrifugation, or the like, the above series of steps (crushing, separating, and purifying the cells) may be performed.

Purification includes, for example, concentration under reduced pressure, membrane concentration, salting-out treatment such as ammonium sulfate and sodium sulfate, or precipitation treatment by a fractional precipitation method using a hydrophilic organic solvent such as methanol, ethanol, acetone, etc., heat treatment or isoelectric point treatment. In addition, gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like can be combined as appropriate.

The purified enzyme preparation is preferably purified to such an extent that it shows a single band on electrophoresis (SDS-PAGE).

In collecting (extracting, purifying, etc.) a protein having diaphorase activity from the culture solution, any one or more of diaphorase activity, thermal stability, etc. may be used as an index.

If this enzyme is obtained as a recombinant protein, various modifications are possible. For example, if a DNA encoding this enzyme and other appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, the peptide consists of a recombinant protein linked to any peptide or protein. This enzyme can be obtained. In addition, modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur. By the modification as described above, extraction of recombinant protein, simplification of purification, addition of biological function, and the like are possible.

The above 1-3. The above-mentioned 1-2. If there is one that satisfies any of the polypeptides (a) to (c) described in (1), it may be applied to the present invention.

2. Any one or more compounds selected from the group consisting of saccharides, sugar alcohols and amino acids Any one or more compounds selected from the group consisting of saccharides, sugar alcohols and amino acids applied to the present invention are particularly It is not limited. In the present application, “amino acids” includes oligopeptides and proteins.
As sugars, sucrose, galactose, arabinose, ribose, melibiose, melezitose, dextrin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, sugar alcohols include inositol, sorbitol, arabitol, xylitol, glucitol, Ribitol, D-mannitol, amino acids alanine, serine, threonine, asparagine, glutamine, valine, leucine, isoleucine, oligopeptides are glycylglycine, alanylglutamine, glycylglutamine, glutathione, and proteins are bovine serum albumin ( BSA) and sericin. More preferably, any one or more selected from the group consisting of mannitol, inositol, xylitol, trehalose, sorbitol and sodium L-glutamate can be mentioned.

These compounds are used as stabilizers to protect the enzyme protein in the process of making the enzyme dry product, to improve the recovery rate in the process, and to prevent inactivation during the storage period of the dry product. The addition amount can be appropriately set within a range in which the object can be achieved. Therefore, the concentration of each compound to be coexisted is not particularly limited, but the preferable lower limit is 2% by weight, more preferably 20% by weight, and further preferably 30% by weight. The upper limit is preferably 300% by weight, more preferably 100% by weight, and still more preferably 70% by weight because of the risk of bringing in foreign substances. In addition, these addition concentrations are represented by weight% with respect to the diaphorase enzyme protein. For example, if 50 wt% stabilizer is added to 40 mg / ml diaphorase, 20 mg stabilizer is dissolved per ml. In addition, the added compound has a protective action even when the enzyme is in a solution state, and contributes to stable retention of the enzyme activity in the solution.

The composition of the buffer solution used for the diaphorase extraction / purification / drying and stability test described above is not particularly limited, but any buffer solution having a buffer capacity in the range of pH 4-9 is preferable. Buffers such as hydrochloric acid and potassium phosphate, and good buffers such as ACES, BES, Bicine, Bis-Tris, CHES, EPPS, HEPES, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TES, Tricine Is mentioned. Mention may also be made of buffers based on dicarboxylic acids, such as phthalic acid, maleic acid, glutaric acid and the like. Of these, only one type may be applied, or two or more types may be used. Furthermore, one or more composite compositions including those other than the above may be used. Further, a chelating agent such as EDTA and / or a surfactant may be contained in the buffer as necessary.
In addition, the concentration of these additives is not particularly limited as long as it has a buffer capacity, but the preferable upper limit is 100 mM or less, more preferably 50 mM or less. A preferred lower limit is 5 mM or more.
The content of the buffer in the dry powder or lyophilized product is not particularly limited, but is preferably 0.1% (weight ratio) or more, particularly preferably 0.1-80% (weight). Ratio).
For these, various commercially available reagents can be used.

The concentration of the enzyme solution used in the drying step is adjusted so that the protein concentration is preferably 5 g / L or more, more preferably 10 g / L or more, and still more preferably 20 g / L or more. If the enzyme used in the drying process is too dilute, the recovery rate often decreases in the drying process, and the resulting dried product often has a shape that is difficult to handle. In addition, when the concentration is excessively high, drying may take time.

3. Composition The composition of the present invention comprises the above-mentioned 1. The diaphorase described in 1., and 2. A composition comprising any one or more compounds selected from the group consisting of saccharides, sugar alcohols, and amino acids described in 1. above.
The form of the composition of the present invention is not particularly limited. Either a dry state such as freeze-drying or powder, or a liquid state may be used. Methods for producing such compositions have already been established in the art. Therefore, those skilled in the art can apply the knowledge to produce the composition of the present invention, and the embodiment is not particularly limited.

4). Stabilization method The diaphorase stabilization method of the present invention is characterized in that any one or more compounds selected from the group consisting of (a) diaphorase, (b) saccharides, sugar alcohols and amino acids coexist. .
Other components may coexist other than the above (a) and (b), and the composition is not particularly limited. Depending on the use of diaphorase (for example, the use exemplified in “5. Product” described later), what components are required in addition to (a) and (b) have already been established in the art. . Therefore, those skilled in the art can apply the knowledge without being limited by the embodiment to construct the stabilization method of the present invention, and can produce a composition for realizing the method.

In the present invention, the improvement in stability means that after the diaphorase is stored at 37 ° C. for 4 weeks, the residual ratio (%) of the diaphorase maintained is higher than when no stabilizer is added. Or at least maintained.

Specifically, whether or not the stability is improved was determined as follows.
In the activity measurement method described in the measurement method of diaphorase enzyme activity described below, the diaphorase activity value per dry product weight after drying (a) and the dry product weight after storage at a constant temperature for a certain period of time The diaphorase activity value (b) was measured, and the relative value ((b) / (a) × 100) with respect to the measured value (a) as 100 was determined. This relative value was defined as the residual rate. And the presence or absence of the addition of the compound was compared, and when the residual ratio increased by the addition, it was judged that the stability was improved.

In addition, it is estimated that the stability is improved only by satisfying the composition of the composition of the present invention.

5. Product containing diaphorase Another aspect of the present invention is the above-mentioned 3. A product containing the diaphorase composition described in 1.
As used herein, “product” means a product that constitutes part or all of a set used by a user for the purpose of carrying out a certain application, and that includes the diaphorase composition of the present invention. .

The product of the present invention can be applied to various uses and is not particularly limited, but typically, one using one of the following two principles can be exemplified.
(A) Measuring a substrate such as NADH by diaphorase.
(B) generating an electric current by an enzymatic reaction with diaphorase;

Examples of using the principle (a) include in vitro diagnostic applications (for example, measurement of various biological components). These biological component measurement methods have already been established in the art. Therefore, according to a known method, the amount or concentration of the biological component in various samples can be measured using the diaphorase of the present invention.
The form is not particularly limited as long as the concentration or amount of the biological component is measured using the diaphorase of the present invention. For example, glucose, lactate dehydrogenase (LDH), creatine kinase (CK), neutral fat (TG), bile Various forms such as reagents, kits, and sensors for measuring biological components such as acids and total branched chain amino acids (BCAA) can be exemplified.
Hereinafter, the case of measuring glucose will be described as an example.

In the case of the composition for measuring glucose, NADH generated by the GDH reaction reduces the electron acceptor such as DCPIP via diaphorase and returns to NAD itself, and the difference in absorbance caused by the change in the structure of DCPIP. Can be determined colorimetrically to determine the glucose concentration. The sample containing glucose is not particularly limited, and examples thereof include blood, beverages, and foods.

The composition for measuring glucose may be in the form of a kit. The kit contains, for example, diaphorase in an amount sufficient for at least one assay, and typically includes buffers, mediators, glucose standard solutions for generating calibration curves, and usage guidelines necessary for the assay. Including. The diaphorase of the invention can be provided in various forms, for example, as a lyophilized reagent or as a solution in a suitable storage solution.

The measurement of the glucose concentration in the form of a sensor can be performed, for example, as follows. Put buffer in constant temperature cell and maintain at constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate, or the like can be used. An electrode on which the diaphorase of the present invention is immobilized is used as a working electrode, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCl electrode) are used. After a constant voltage is applied to the carbon electrode and the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve prepared with a standard concentration glucose solution.

As the electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used, and the enzyme of the present invention is immobilized on this electrode. Immobilization methods include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc., or ferrocene or a derivative thereof. It may be fixed in a polymer or adsorbed and fixed on an electrode together with a representative electron mediator, or a combination of these may be used. Typically, diaphorase is immobilized on a carbon electrode using glutaraldehyde and then treated with a reagent having an amine group to block glutaraldehyde.

Examples of using the principle of (b) include various forms such as an enzyme electrode (may be an immobilized electrode), an enzyme sensor, a fuel cell, and an electronic device having one or more fuel cells. It can be illustrated. *

A fuel cell that uses glucose dehydrogenase and diaphorase to extract electrons by the oxidation reaction of glucose has already been established in the art. Therefore, according to a known method, a fuel cell can be produced and operated using the diaphorase of the present invention.
As long as the fuel cell is produced and operated using the diaphorase of the present invention, its mode is not particularly limited. For example, it can be operated as a battery by the following means. First, the diaphorase of the present invention is immobilized with an electron mediator such as GDH or an osmium complex in a negative electrode of a biofuel cell, and on the other hand, an oxidation-reduction selected from bilirubin oxidase (BOD), laccase, ascorbate oxidase and the like at the positive electrode Immobilize enzyme and mediator such as hexacyanoferrate ion. Further, a structure is constructed in which the negative electrode and the positive electrode are opposed to each other via an electrolyte layer that does not have electron conductivity and conducts only protons. In the negative electrode, glucose supplied as fuel is decomposed by an enzyme to extract electrons and protons (H + In the positive electrode, water is generated by protons transported from the negative electrode through the electrolyte layer, electrons sent from the negative electrode through an external circuit, and oxygen in the air, for example.
The fuel containing glucose is not particularly limited, and examples thereof include blood, beverages, and foods.

The fuel cell of the present invention can be used for anything that requires electric power, and can be of any size. Specifically, this fuel cell is used for, for example, an electronic device, a moving body (automobile, motorcycle, aircraft, rocket, spacecraft, etc.), power unit, construction machine, machine tool, power generation system, cogeneration system, etc. Can do.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to a following example.

Example 1 Acquisition of Transformant An alignment of the polypeptide of SEQ ID NO: 1 and the polypeptide of SEQ ID NO: 2 used in the present invention is shown in FIG.
A structural gene encoding the polypeptide of SEQ ID NO: 1 was synthesized by GenScript. The synthetic gene was inserted downstream of the LacZ promoter of the plasmid pUC57. Therefore, the plasmid into which the synthetic gene was inserted was used as an expression vector as it was, and this was designated as a recombinant expression plasmid pUC-DI-1. The retained expression plasmid was transformed into Escherichia coli DH5α strain competent cell (manufactured by Toyobo), pre-cultured at 37 ° C. for 1 hr in SOC medium, and then developed on LB-amp agar medium. A certain transformant was obtained. The obtained transformant was named Escherichia coli DH5α (pUC-DI-4). In the same manner as for SEQ ID NO: 2, Escherichia coli DH5α (pUC-DI-1) was obtained.

Example 2 Preparation of Enzyme A colony of the transformant Escherichia coli DH5α (pUC-DI-4) obtained in Example 1 was inoculated into a 5 ml LB-amp liquid medium in one platinum ear test tube, and 30 ° C. For 16 hours. This was used as seed culture.
Next, TB liquid medium (tryptone 1.2%, yeast extract 2.4%, glycerol 0.4%, KH ₂ PO ₄ 0.23%, K ₂ HPO ₄ 1.25%, pH 7.0) was added. It put into the test tube and sterilized by the autoclave, and it was set as the culture medium of the main culture medium.
500 mL of TB medium was placed in a 2 L Sakaguchi flask and sterilized by autoclaving to obtain a main culture medium. 5 mL of the seed culture solution was inoculated into the main culture medium, and cultured with shaking at a culture temperature of 30 ° C. and 180 rpm for 24 hours. Thereafter, the cells were collected by centrifugation, and the cells were collected. The obtained bacterial cells were suspended in 20 mM potassium phosphate buffer (pH 7.5).
The same operation was performed for Escherichia coli DH5α (pUC-DI-1).

The suspension was fed to a French press (manufactured by Niro Soavi) at a flow rate of 160 mL / min and crushed at 700 to 1000 bar. Subsequently, a 5% polyethyleneimine solution (pH 7.5) prepared by adjusting ethyleneimine (polymer) (Nacalai Tesque Co., Ltd.) so as to have a polyethyleneimine content of 5% was prepared. %, And the mixture was stirred at room temperature for 30 minutes, and then excess precipitate was removed using a filter aid. Next, ammonium sulfate (manufactured by Sumitomo Chemical Co., Ltd.) is gradually added to reach 0.5 saturation, ammonium sulfate fractionation is performed, and a protein having NAD-dependent glucose dehydrogenase activity is precipitated and recovered. The precipitate was suspended in 20 mM potassium phosphate buffer (pH 7.5). The suspension was then desalted using a Sephadex G-25 gel. Thereafter, it is applied to a 400 mL DEAE Sepharose FastFlow (manufactured by GE Healthcare) column previously equilibrated with 20 mM potassium phosphate buffer (pH 7.5), and 20 mM potassium phosphate buffer (pH 7.5) containing 0.5 M NaCl is added. Elute with a linear gradient. Then, the eluted NAD-dependent glucose dehydrogenase fraction was concentrated with a hollow fiber membrane (manufactured by Spectrum Laboratories) having a fractional molecular weight of 10,000. The concentrated solution was desalted using Sephadex G-25 gel to obtain a purified enzyme.
The same operation was performed for Escherichia coli DH5α (pUC-DI-1).

Example 3 Screening of compounds having a stabilizing effect on diaphorase powder Using purified enzyme derived from Escherichia coli DH5α (pUC-DI-4), mannitol, inositol, xylitol, trehalose, sorbitol, sodium L-glutamate, L- It was examined whether serine and L-threonine have a stabilizing effect. At the same time, the stabilizer concentrations of 30%, 50% and 70% were examined in order to see the concentration at which the stabilizer exerts its effect. Table 1 shows the effects of Geobacillus thermodenitificans NG80-2 derived wild-type diaphorase, stabilizer and concentration.

The preparation obtained in Example 2 contains about 70 mg / ml diaphorase protein in 20 mM potassium phosphate buffer (pH 7.5). Examination of the 30% concentration stabilizer was carried out by dissolving 21 mg of the stabilizer in an enzyme solution containing 70 mg of diaphorase to obtain a 30% concentration, and measuring the activity. For the study of the 50% concentration stabilizer, 35 mg of the stabilizer was dissolved in an enzyme solution containing 70 mg of diaphorase to obtain a 50% concentration, and the activity was measured. In the examination of the 70% concentration stabilizer, 49 mg of the stabilizer was dissolved in an enzyme solution containing 70 mg of diaphorase to obtain a 70% concentration, and the activity was measured. From the enzyme solution to which various stabilizers were added, exactly 2 ml each of the tare weight was dispensed into a measured vial. Moreover, what did not add a stabilizer was prepared for control. This was freeze-dried (FDR) to completely evaporate the water, and the weight of the vial was measured. The weight of the powder obtained by subtracting the tare weight was calculated. Thereafter, about 10 mg of powder was accurately weighed into a spitz roll, (1) immediately measured for activity, and (2) stored for 4 weeks at 37 ° C. and then measured for activity, and the activity per weight of powder was calculated. The activity remaining ratio was calculated by calculating the ratio of activity per powder weight of each sample after 37 ° C. treatment, assuming that the activity per weight of powder immediately after FDR was 100%. As a result, an improvement in stability was observed compared to the case where nothing was added in many sugars, sugar alcohols, amino acids and proteins (Table 1).
Specifically, when no stabilizer was added, the residual activity was 74.6% when stored at 37 ° C. for 4 weeks. When mannitol, inositol, trehalose or sorbitol was added as a stabilizer, the residual activity exceeded 74.6% when stored at 37 ° C. for 4 weeks, and the stabilizing effect could be confirmed. Mannitol, inositol, trehalose and sorbitol were found to have a stabilizing effect in all concentration ranges. When 70% concentration of xylitol was added, the residual activity was 80.8%, indicating a stabilizing effect. Sodium L-glutamate showed a stabilizing effect with a residual activity of 77.0% at a concentration of 30%. When L-serine and L-threonine were added, the residual activity was less than 74.6% at all concentrations, indicating that there was no stabilizing effect.

Example 4
Next, the stabilization effect of the compound that showed a stabilization effect in Example 3 was verified using a purified enzyme derived from Escherichia coli DH5α (pUC-DI-1). Table 2 shows that Geobacillus sp. The influence of Y4.1MC1-derived wild type diaphorase, stabilizer and concentration is shown.

Specifically, when no stabilizer was added, the residual activity was 88.9% when stored at 37 ° C. for 4 weeks. When mannitol, inositol and trehalose were added as stabilizers, the residual activity exceeded 88.9% when stored at 37 ° C. for 4 weeks, and the stabilizing effect could be confirmed. Mannitol, inositol and trehalose were found to have a stabilizing effect in all concentration ranges.
As shown in FIG. 1, it can be seen that SEQ ID NO: 1 and SEQ ID NO: 2 have a remarkable stabilizing effect in common with mannitol, inositol, and trehalose even though the amino acid sequences are different. That is, it can be said that mannitol, inositol, and trehalose are useful stabilizers against diaphorase derived from the genus Geobacillus.

The composition produced according to the present invention can be supplied as a raw material for a reagent for measuring blood glucose level, a blood glucose sensor, and a glucose concentration determination kit.

Claims

(A) A composition containing any one or more compounds selected from the group consisting of diaphorase, (b) saccharides, sugar alcohols and amino acids. *
The composition according to claim 1, wherein the compound (b) is at least one selected from the group consisting of mannitol, inositol, xylitol, trehalose, sorbitol and sodium L-glutamate. *
The composition according to claim 1 or 2, wherein the compound (b) is contained in an amount of 30 to 70% (w / w) based on the amount of diaphorase protein. *
The composition according to any one of claims 1 to 3, wherein the diaphorase is one of the following proteins.
(A) a protein having the amino acid sequence shown in SEQ ID NO: 1 (b) a protein having an amino acid sequence in which one or several amino acids are deleted, inserted, added or substituted in the amino acid sequence shown in SEQ ID NO: 1 A protein having diaphorase activity
(C) a protein having the amino acid sequence of SEQ ID NO: 2
(D) a protein having an amino acid sequence in which one or several amino acids are deleted, inserted, added or substituted in the amino acid sequence shown in SEQ ID NO: 2 and having diaphorase activity
A product comprising the composition according to any one of claims 1-4.
(A) A method for stabilizing diaphorase in a diaphorase composition, wherein any one or more compounds selected from the group consisting of diaphorase, (b) saccharides, sugar alcohols and amino acids are allowed to coexist.