JP6311270B2 - Flavin adenine dinucleotide-dependent glucose dehydrogenase with excellent thermostability - Google Patents

Description

The present invention relates to a modified glucose dehydrogenase (also referred to as GDH) with improved thermostability, and a modified flavin adenine dinucleotide-dependent glucose dehydrogenase (also referred to as FAD) as a coenzyme. It is also referred to as FADGDH.), A method for producing the FADGDH, a glucose measuring method using the FADGDH, a glucose sensor, and a glucose assay kit.

Self-monitoring of Blood Glucose (SMBG) is important for diabetic patients to manage their blood glucose level and utilize it for treatment. In recent years, a simple self-blood glucose meter using an electrochemical biosensor has been widely used for SMBG. A biosensor has an electrode and an enzyme reaction layer formed on an insulating substrate.

Examples of the enzyme used here include glucose dehydrogenase and glucose oxidase (also referred to as GO). It has been pointed out that the method using GO (EC 1.1.3.4) is easily affected by dissolved oxygen in the measurement sample, and the dissolved oxygen affects the measurement result. On the other hand, pyrroloquinoline quinone-dependent glucose dehydrogenase (also referred to as PQQ-GDH) (EC 1.1.5.2 (formerly EC 1.1.9.17)) is also affected by dissolved oxygen. Although it acts on sugars other than glucose such as maltose and lactose, it is not suitable for accurate blood glucose measurement.

FADGDH is not affected by dissolved oxygen and hardly acts on maltose. The inventors have so far discovered FADGDH that is less susceptible to maltose and xylose by screening from ascomycetes. A representative example is FADGDH derived from Mucor Himaris described in Patent Document 1. The enzyme had a thermal stability of less than 50 ° C., leaving room for improvement.

In the manufacturing process of the blood glucose sensor chip, heat drying may be performed, and a step of evaporating and drying the GDH solution on the reaction layer is performed. Here, heating at a temperature of 50 ° C. or higher often increases the efficiency of evaporation to dryness and increases the efficiency of production. While such heating treatment is effective in increasing the efficiency of production, it is generally known that proteins are denatured by heat. Therefore, an enzyme that does not have sufficient heat stability has a risk of causing significant heat inactivation, and it is necessary to improve the heat stability.

Further, the sensor strip after fabrication is usually provided with a warranty period of up to two years at a temperature of about room temperature. However, in general users who use glucose sensors, it is rare to perform strict temperature control when storing the sensor strip, especially in the summer, when the temperature exceeds 35 ° C or sometimes exceeds 40 ° C, the enzyme itself It is not difficult to imagine that high stability is desired. Enzymes with excellent thermal stability generally have a stable steric structure and can be said to be more suitable for long-term storage under harsh conditions.

JP2013-116102A

The objective of this invention is providing the enzyme which can be used for the reagent for blood glucose level measurement provided with high heat resistance compared with the above-mentioned known enzyme for blood glucose sensors.

As a result of diligently searching for a location where the heat stability of FAGDDH derived from the Mucor genus described in Patent Document 8 is described, the inventors have found that the residual activity after treatment at 55 ° C. for 15 minutes is 50% or more, that is, a substantial loss of activity We succeeded in developing an enzyme that does not cause The present invention has been completed as a result of such research and improvement, and typical inventions are as follows.

Item 1
In the flavin adenine dinucleotide-dependent glucose dehydrogenase having 60% or more identity with the sequence shown in SEQ ID NO: 1, positions 60, 94, 182, 189, 228, 230 in the amino acid sequence of SEQ ID NO: 1 Modified flavin adenine having an amino acid substitution at one or more positions selected from the group consisting of positions 317, 399, 411, 419, 439, 466, and 619, or equivalent positions Nucleotide-dependent glucose dehydrogenase.
Item 2
A DNA encoding the modified flavin adenine dinucleotide-dependent glucose dehydrogenase according to Item 1.
Item 3
A vector incorporating the DNA of Item 2.
Item 4
A transformant comprising the vector according to Item 3.
Item 5
Item 5. A method for producing a modified flavin adenine dinucleotide-dependent glucose dehydrogenase according to Item 1, comprising culturing the transformant according to Item 4.
Item 6
A method for measuring a glucose concentration, comprising causing the modified flavin adenine dinucleotide-dependent glucose dehydrogenase according to Item 1 to act on glucose.
Item 7
A glucose assay kit comprising the modified flavin adenine dinucleotide-dependent glucose dehydrogenase according to Item 1.
Item 8
A glucose sensor comprising the modified flavin adenine dinucleotide-dependent glucose dehydrogenase according to Item 1.

The present invention provides FADGDH having high substrate specificity, sufficient thermal stability, and preferably suitable for efficient production using Escherichia coli, yeast, brute and the like as host cells.

It is a figure which shows the temperature stability of modified FADGDH. It is a figure which shows the optimal temperature of modified type FADGDH. It is a figure which shows the optimal pH of modified FADGDH. It is a figure which shows pH stability of modified FADGDH. It is a figure which shows the response with respect to the glucose solution of the glucose sensor using modified FADGDH.

Hereinafter, the present invention will be described in detail.
1. Modified FADGDH with improved thermal stability
The modified FADGDH of the present invention is FADGDH having 60% or more identity with the sequence shown in SEQ ID NO: 1, and in the amino acid sequence of SEQ ID NO: 1, positions 60, 94, 182, 189, 228, 230 Obtained by introducing an amino acid substitution at one or more positions selected from the group consisting of positions 317, 399, 411, 419, 439, 466, and 619, or equivalent positions be able to.

The amino acid sequence represented by SEQ ID NO: 1 refers to Mucor himalis f. It is an amino acid sequence of FADGDH derived from silvaticus NBRC6754 and has various characteristics described in Patent Document 1 filed by the applicant in the past.

The modification source of the modified FADGDH of the present invention is 60% or more (preferably 80% or more) of the sequence shown in SEQ ID NO: 1 as long as the modified FADGDH has improved thermal stability compared to the wild type FADGDH. %, More preferably 90% or more, still more preferably 95% or more, still more preferably 98% or more, more preferably 99% or more), and FADGDH is not particularly limited. The modification source may be a wild type or may have already been modified in some way.

From another point of view, the modified amino acid sequence of the modified FADGDH of the present invention is represented by SEQ ID NO: 1 as long as the modified FADGDH has improved thermal stability compared to the wild-type FADGDH. The amino acid sequence may consist of an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added.

In the present specification, the “position equivalent to the amino acid sequence of SEQ ID NO: 1” refers to the same position in the alignment when the amino acid sequence of SEQ ID NO: 1 and GDH having another amino acid sequence are aligned. means.

The correspondence between amino acid sequences and the identity of amino acid sequences when two amino acid sequences are aligned can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). In this specification, the National Biotechnology Information Center (NCBI) homology algorithm BLAST. (Basic local alignment search tool) http: // www. ncbi. nlm. nih. Calculation is performed by using default (initial setting) parameters in gov / BLAST /.

The modified FADGDH of the present invention has positions 60, 94, 182, 189, 228, 230, 317, 399, 411, 419, 439, 439 and 466 in the amino acid sequence of SEQ ID NO: 1. And FADGDH having at least one amino acid substitution among amino acids belonging to the position of the group consisting of positions 619 and 619.

More preferably, the modified FADGDH of the present invention is one selected from the group consisting of A60E, E94P, S182A, T228V, R 230V, D317A, R411M, W419Y, A439S, D466M, and A619S in the amino acid sequence of SEQ ID NO: 1. A modified FADGDH having the above amino acid substitution.

In the present specification, for amino acid substitution in a certain amino acid sequence, a number representing the order from the N-terminal on the amino acid sequence is sandwiched between a one-letter code indicating the amino acid before substitution and a one-letter code indicating the amino acid after substitution. It expresses by. For example, “A60E” means that A (Ala) at position 60 is replaced with E (Glu).

In addition, the polypeptide which comprises the said FADGDH may have deleted the signal peptide part. Using SignalP 4.1 (http://www.cbs.dtud./default.) Of The Technical University of Denmark's The Center for Biological Sequence Analysis (CBS) According to the estimation, the first to the 17th amino acid sequence shown in SEQ ID NO: 1 is predicted as the signal sequence. Therefore, it is speculated that deleting this part does not have an adverse effect on the enzyme properties.

1-1. Thermal stability The modified FADGDH of the present invention has improved thermal stability compared to wild-type FADGDH.
In this specification, thermal stability is evaluated by the activity maintained even after a 15-minute heating treatment in a state where 2 U / ml GDH is contained in 50 mM potassium phosphate buffer (pH 6.0). .
The FAD-GDH of the present invention preferably has an activity remaining rate of 50% or more when heated at 55 ° C. for 15 minutes, more preferably 60% or more, still more preferably 70% or more, and further preferably Is 80% or more, more preferably 90% or more.
The FAD-GDH of the present invention preferably has an activity remaining rate of 35% or more after heating at 55 ° C. for 30 minutes, more preferably 40% or more, and even more preferably 50% or more. More preferably, it is 60% or more, more preferably 70% or more. Furthermore, the FAD-GDH of the present invention preferably has an activity remaining rate of 25% or more after heating at 60 ° C. for 15 minutes. More preferably, it is 40% or more, more preferably 50% or more

2. Method for producing modified GDH The method for producing the modified FADGDH of the present invention is not particularly limited. For example, the above-described 1-1. It is possible to produce a transformant containing a vector incorporating a DNA encoding the amino acid sequence of the modified FADGDH shown in the above by culturing in a nutrient medium and purifying the obtained culture solution.
A DNA encoding the amino acid sequence of modified FADGDH, a vector incorporating the DNA, and a transformant containing the vector, which are related to the production method, are also embodiments of the present invention.

As used herein, “DNA encoding a protein” refers to DNA from which the protein is obtained when it is expressed, that is, DNA having a base sequence corresponding to the amino acid sequence of the protein.

As a method for introducing a mutation into DNA for producing FADGDH of the present invention, for example, it has a sequence in which a portion corresponding to a codon encoding an amino acid residue to be substituted is replaced with a codon encoding an amino acid after substitution A mismatch primer is prepared, and using this primer and DNA polymerase, a sequence into which a mutation is introduced using a DNA encoding SEQ ID NO: 1 (for example, the DNA represented by SEQ ID NO: 6 described in Patent Document 8) as a template A method for elongating and producing DNA having the same is used. In performing site-specific modification of such a gene, it is also possible to use various commercially available site direct mutagenesis kits such as Transformer Mutagenesis Kit (Clontech) or QuickChange Site Direct Mutagenesis Kit (Stratagene). Is applicable, but is not limited thereto.

In addition, as a method for obtaining DNA used for GDH production of the present invention, a DNA strand is chemically synthesized or a synthesized partially overlapping oligo DNA short chain is connected by using a PCR method. It is also possible to construct a DNA encoding the full length of the GDH of the present invention. The advantage of constructing full-length DNA in combination with chemical synthesis or PCR is that the codons used can be designed over the entire length of the gene in accordance with the host into which the gene is introduced. A plurality of codons encoding the same amino acid are not used uniformly, and the frequency of use varies depending on the species. In general, codons contained in genes that are highly expressed in a given species contain many codons that are frequently used in that species, and conversely, the presence of codons that are used infrequently becomes a bottleneck for genes with low expression levels. There are many examples that prevent high expression. In the expression of a heterologous gene, many examples have been reported so far in which the expression level of the heterologous protein has been increased by replacing the gene sequence with a codon that is frequently used in the host organism. It is expected to be effective in increasing the expression level of heterologous genes.

For the above reasons, it is desirable to modify the DNA encoding the GDH of the present invention into a codon more suitable for the host cell into which it is introduced (ie, a codon that is frequently used in the host). The codon usage frequency of each host is defined by the ratio of each codon used in all genes present on the genome sequence of the host organism, and is expressed, for example, by the number of times used per 1000 codons. In addition, the codon usage frequency can be approximately calculated from the sequence of a plurality of typical genes in an organism whose entire genome sequence has not been elucidated. The data on the frequency of codon usage in the host organism to be recombined uses, for example, the genetic code frequency database published on the website of Kazusa DNA Research Institute (http://www.kazusa.or.jp). Or you can refer to the literature describing the codon usage in each organism, or you can determine the codon usage data for the host organism you are using. By referring to the obtained data and the gene sequence to be introduced and replacing the codon used in the gene sequence that is not frequently used in the host organism with a codon that encodes the same amino acid and is frequently used Good. As such a frequently used codon, for example, when the host is E. coli K12, Gly is GGT or GGC, Glu is GAA, Asp is GAT, Val is GTG, and Ala is GCG and Arg are CGT or CGC, Ser is AGC, Lys is AAA, Ile is ATT or ATC, Thr is ACC, Leu is CTG, Gln is CAG, Pro is CCG, and the like.

The prepared DNA having the genetic information of the modified FADGDH is transferred to a host microorganism in a state of being linked to a vector, and becomes a transformant that produces the modified FADGDH.
As the type of vector, an appropriate vector is 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 using a filamentous fungus as a host or a vector suitable for self-cloning.
When Escherichia coli is used as a host, for example, M13 phage or a variant thereof, λ phage or a variant thereof, pBR322 or a variant thereof (pB325, pAT153, pUC8, etc.) can be used. When yeast is used as a host, pYepSec1, pMFa, pYES2, etc. can be used. When insect cells are used as hosts, for example, pAc and pVL can be used. When mammalian cells are used as hosts, for example, pCDM8 and pMT2PC can be used, but the present invention is not limited thereto. .
Insertion of DNA into a vector, insertion of a selectable marker gene (if necessary), insertion of a promoter (if necessary), etc. can be performed using standard recombinant DNA techniques.

The means for introducing the DNA encoding the enzyme into the host is not particularly limited. For example, it is introduced into the host in a state of being incorporated into the vector. The host cell is not particularly limited as long as it can express the DNA of the present invention and produce FADGDH. Specifically, prokaryotic cells such as Escherichia coli and Bacillus subtilis, eukaryotic cells such as yeast, mold, insect cells, plant culture cells, and mammalian cells can be used.
When the host is a prokaryotic cell, examples include the genus Escherichia, the genus Bacillus, the genus Brevibacillus, the genus Corynebacterium, and the like. Examples include Escherichia coli C600, Escherichia coli HB101, Escherichia coli DH5α, Bacillus subtilis, Examples include Brevibacillus choshinensis and Corynebacterium glutamicum. Examples of the vector include pBR322, pUC19, and pBluescript.
When the host is yeast, examples include Saccharomyces genus, Schizosaccharomyces genus, Candida genus, Pichia genus, Cryptococcus genus, etc., respectively. An example is Cryptococcus sp. Examples of the vector include pAUR101, pAUR224, and pYE32.
When the host is a filamentous fungal cell, Aspergillus genus, Trichoderma genus, Mucor genus and the like can be mentioned as examples, and Aspergillus oryzae, Aspergillus niger, Trichoderma reesei, Mucor Himalis and the like can be exemplified.
In the present invention, it is also preferable to use a microorganism belonging to the genus Mucor from which FADGDH is isolated as a host. That is, in the transformant, exogenous DNA is usually present in the host cell, but a transformant obtained by so-called self-cloning using a 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 above expression vector. 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 microorganism, which is the transformant thus obtained, can stably produce a large amount of modified FADGDH by being cultured in a nutrient medium. The culture form of the host microorganism, which is a transformant, may be selected in consideration of the nutritional physiological properties of the host. Usually, the culture is performed in liquid culture, but industrially, aeration and agitation culture is performed. Is advantageous. The culture method and culture conditions are not particularly limited as long as the FADGDH of the present invention is produced. That is, on the condition that FADGDH is produced, a method and conditions suitable for the growth of the microorganism to be used can be appropriately set. Hereinafter, examples of the culture conditions include a culture medium, a culture temperature, and a culture time.

The medium is not particularly limited as long as the microorganism to be used can grow. For example, carbon sources such as glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein Nitrogen sources such as hydrolysates, bran and meat extracts, and further added with inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts and zinc salts can be used. In order to promote the growth of the microorganisms to be used, vitamins, amino acids and the like may be added to the medium.

The pH of the medium is only required to be suitable for the growth of the microorganism to be cultured. For example, it is adjusted to about 3 to 8, preferably about 5 to 7, and the culture temperature is usually about 10 to 50 ° C., preferably about 25 to 35. Culturing is carried out under aerobic conditions at about 0 ° C. for 1 to 15 days, preferably about 3 to 7 days. As the culture method, for example, a shaking culture method or an aerobic deep culture method using a jar fermenter can be used.

After culturing under the above conditions, FADGDH is preferably recovered from the culture solution or the bacterial cells. When using microorganisms that secrete FADGDH outside the cells, for example, the culture supernatant is filtered, centrifuged to remove insolubles, concentrated by ultrafiltration membrane, salting out such as ammonium sulfate precipitation, dialysis, The present enzyme can be obtained by performing separation and purification by appropriately combining various types of 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. This enzyme can be obtained by adding a chelating agent and a surfactant to solubilize FADGDH, separating and collecting the solution as an aqueous solution, and performing separation and purification. 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.

When column chromatography is used, for example, gel filtration using Sephadex gel (manufactured by GE Healthcare Bioscience), DEAE Sepharose CL-6B (manufactured by GE Healthcare Bioscience), octyl Sepharose CL-6B (GE Healthcare Bioscience, Inc.) can be used. The purified enzyme preparation is preferably purified to the extent that it shows a single band on electrophoresis (SDS-PAGE).

When collecting (extracting, purifying, etc.) a protein having glucose dehydrogenase activity from the culture solution, one or more of glucose dehydrogenase activity, maltose activity, thermal stability, etc. are used as indicators. May be.

In each purification process, in principle, fractionation is performed using GDH activity as an index, and the process proceeds to the next step. However, this does not apply when appropriate conditions can be set in advance by a preliminary test or the like.

3. Method for measuring glucose etc. Another embodiment of the present invention is the use of the FADGDH of the present invention having the above characteristics. As a use, the measuring method of glucose can be illustrated and it can utilize suitably for the measurement of the blood glucose level, the measurement of the glucose level in foodstuffs (a seasoning, a drink, etc.), etc.
Yet another embodiment of the present invention is a variety of products for measuring glucose, including FADGDH of the present invention having the above characteristics, such as a glucose assay kit and a glucose sensor. In this specification, “product” means a product that constitutes a part or all of one set used by a user for the purpose of executing a certain application, and includes the FADGDH of the present invention. .

A method for measuring glucose using FADGDH has already been established in the art. Therefore, according to a known method, the amount or concentration of glucose in various samples can be measured using the FADGDH of the present invention. As long as the concentration or amount of glucose can be measured using the FADGDH of the present invention, the embodiment is not particularly limited.

3-1. Glucose assay kit The glucose assay kit of the present invention comprises a modified FADGDH in an amount sufficient for at least one assay according to the present invention. Typically, it contains reagents necessary for measurement such as GDH, buffer, mediator, etc. of the present invention, a glucose standard solution for preparing a calibration curve, and usage guidelines. The kit of the invention can be provided, for example, as a lyophilized reagent or as a solution in a suitable storage solution. In addition, in the reagent containing GDH, as a stabilizer and / or an activator, a protein such as bovine serum albumin and sericin, a surfactant such as Triton X-100, Tween 20, cholate and deoxycholate, glycine Amino acids such as serine, glutamic acid, glutamine, aspartic acid, asparagine, glycylglycine, sugars such as trehalose, inositol, sorbitol, xylitol, glycerol, sucrose and / or sugar alcohols, inorganic salts such as sodium chloride, potassium chloride, Furthermore, hydrophilic polymers such as pullulan, dextran, polyethylene glycol, polyvinyl pyrrolidone, carboxymethyl cellulose, and polyglutamic acid may be added as appropriate.
In the present invention,

3-2. Glucose sensor The glucose sensor using the modified FADGDH according to the present invention uses a carbon electrode, a gold electrode, a platinum electrode or the like as an electrode, and the enzyme of the present invention is immobilized on the electrode. As immobilization methods, there are a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a method of using a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc. NAD or NADP Such a coenzyme or an electron mediator typified by ferrocene or a derivative thereof may be fixed in a polymer or adsorbed and fixed on an electrode, or may be used in combination. Since FADGDH of the present invention is excellent in thermal stability, it can be immobilized under relatively high temperature conditions (for example, 50 ° C. and 55 ° C.). Typically, after the FADGDH of the present invention is immobilized on a carbon electrode using glutaraldehyde, the glutaraldehyde can be blocked by treatment with a reagent having an amine group. Examples of the electron mediator to be used include those that receive electrons from FAD which is a coenzyme of GDH, and can donate electrons to the color-developing substances and electrodes. N, N, N ′, N′-tetramethylphenylenediamine, 1-methoxy-phenazine methosulfate, 2,6-dichlorophenolindophenol, 2,5-dimethyl-1,4-benzoquinone, 2,6-dimethyl Examples include, but are not limited to, -1,4-benzoquinone, 2,5-dichloro-1,4-benzoquinone, nitrosoaniline, ferrocene derivatives, osmium complexes, ruthenium complexes and the like. Further, in the GDH composition on the electrode, as a stabilizer and / or an activator, a protein such as bovine serum albumin or sericin, a surfactant such as Triton X-100, Tween 20, cholate or deoxycholate , Glycine, serine, glutamic acid, glutamine, aspartic acid, asparagine, glycylglycine and other amino acids, trehalose, inositol, sorbitol, xylitol, glycerol, sucrose and other sugars and / or sugar alcohols, sodium chloride, potassium chloride and other inorganic substances It may contain a salt or a hydrophilic polymer such as pullulan, dextran, polyethylene glycol, polyvinyl pyrrolidone, carboxymethyl cellulose, polyglutamic acid.

The glucose concentration can be measured as follows. A reaction solution containing a buffer solution, GDH and 2,6-dichlorophenolindophenol (DCPIP) as a mediator is placed in a constant temperature cell, and maintained at a constant temperature. A sample containing glucose is added thereto and reacted at a fixed temperature for a fixed time. During this time, glucose can be quantified by monitoring the decrease in absorbance at 600 nm. Further, it is possible to determine the amount of diformazan produced by adding phenazine method (PMS) as a mediator and nitrotetrazorium blue (NTB) as a coloring reagent, and measuring the absorbance at 570 nm, and calculating the glucose concentration. Needless to say, the mediator and coloring reagent used are not limited to these.

The glucose concentration can also be measured as follows. Put the buffer in a constant temperature cell and add GDH and mediator as needed to maintain constant temperature. As the mediator, potassium ferricyanide, phenazine methosulfate, or the like can be used. An electrode on which the GDH 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.

4). Glucose dehydrogenase activity measurement method Glucose dehydrogenase (GDH) is a physicochemical property that catalyzes the reaction of oxidizing the hydroxyl group of glucose in the presence of an electron acceptor to produce glucono-δ-lactone. It has an enzyme. In this document, this physicochemical property is referred to as glucose dehydrogenase activity, and unless otherwise specified, “enzyme activity” or “activity” means the enzyme activity. The electron acceptor is not particularly limited as long as it is capable of transferring electrons in a reaction catalyzed by GDH. For example, 2,6-dichlorophenolindophenol (DCPIP), phenazine methosulfate (PMS), 1-methoxy-5-methylphenadium methyl sulfate, a ferricyan compound, and the like can be used.

Various methods are known as methods for measuring glucose dehydrogenase activity. In this document, DCPIP is used as an electron acceptor, and the activity is measured by using the change in absorbance of the sample at a wavelength of 600 nm before and after the reaction as an index. Apply. The specific reagent composition and measurement conditions are as follows unless otherwise specified.

<Reagent>
150 mM phosphate buffer pH 6.5 containing 0.3 M D-glucose (including 0.1% Triton X-100)
1.64 mM 2,6-dichlorophenolindophenol (DCPIP) solution 20 mL of phosphate buffer containing D-glucose and 10 mL of DCPIP solution are mixed to obtain a reaction reagent.

<Measurement conditions>
Prewarm 3 mL of reaction reagent at 37 ° C. for 5 minutes. After adding 0.1 mL of GDH solution and mixing gently, the absorbance change at 600 nm was recorded for 5 minutes with a spectrophotometer controlled at 37 ° C. with water as a control, and from the linear portion (ie, the reaction rate became constant). Measure the change in absorbance per minute (ΔOD _TEST ). In the blind test, a change in absorbance per minute (ΔOD _BLANK ) is similarly measured by adding a solvent that dissolves GDH to the reagent mixture instead of the GDH solution. From these values, the GDH activity is determined according to the following formula. Here, 1 unit (U) in GDH activity is the amount of enzyme that reduces 1 micromole of DCPIP per minute in the presence of D-glucose at a concentration of 1M.

Activity (U / mL) =
{− (ΔOD _TEST −ΔOD _BLANK ) × 3.1 × dilution ratio} / {16.3 × 0.1 × 1.0}

In the formula, 3.1 is the amount of the reaction reagent + enzyme solution (mL), 16.3 is the molar molecular extinction coefficient (cm ² / micromole) under the conditions for this activity measurement, and 0.1 is the solution of the enzyme solution. Amount (mL), 1.0 indicates the optical path length (cm) of the cell. In this document, unless otherwise indicated, enzyme activity is measured according to the measurement method described above.

Hereinafter, the present invention will be specifically described by way of examples. However, these examples do not limit the present invention.

Example 1 Construction of a FADGDH random mutation library using Saccharomyces as a host In the example of Patent Document 8, there is a description of pYESMh6754. Using this plasmid, random mutation was introduced into the FADGDH gene by error-prone PCR. On pYESMh6754, a GAL1 promoter and a CYC1 terminator are arranged with the FADGDH gene interposed therebetween. Random mutation was introduced using Diversity PCR Random Mutagenesis Kit (Clontech) using universal primers capable of complementary binding to GAL1 promoter and CYC1 terminator according to the protocol attached to this product. As a result, a DNA fragment containing the FADGDH gene into which mutation was introduced at a certain rate was obtained.

Next, the obtained DNA fragment containing the FADGDH gene having a mutation introduced at a certain ratio is treated with the restriction enzymes KpnI and NotI, and mixed with the vector pYES3 (Invitrogen) similarly treated with the restriction enzymes KpnI and NotI. Then, ligation was performed by adding an equal amount of ligation reagent (Toyobo Lagation High) to the mixed solution and incubating. The ligated DNA was transformed into Escherichia coli DH5α competent cells (Toyobo Competent High DH5α) according to the protocol attached to the product, and the transformants were obtained. The transformant was cultured in an LB medium, a plasmid was extracted, and a random mutant plasmid library was obtained in which mutations were introduced at a constant rate into the FADGDH gene inserted into the plasmid.
Subsequently, Saccharomyces cerevisiae INVSc1 (Invitrogen) was transformed with the random mutant plasmid library. About 2000 grown colonies were used as a FADGDH random mutation library using Saccharomyces as a host.

Example 2 Search for FADGDH excellent in heat stability 3% yeast extract in which FADGDH random mutation library using Saccharomyces constructed in Example 2 as a host was dispensed to each culture cell of ScreenMates (Matrix Technology). The cells were inoculated into a medium containing 1% polypeptone and 3% galactose, and cultured with shaking at 25 ° C. for 60 hours. Next, the obtained culture solution was centrifuged at 2000 rpm for 15 minutes to obtain a culture supernatant. The obtained culture supernatant is used as a crude enzyme solution and heated at 55 ° C. for 30 minutes, and the above 1-5. The GDH activity was measured using the FADGDH activity measurement method shown in Fig. 1, and the residual ratio was measured in comparison with the GDH activity before the treatment.

As a result, 13 types of GDH of the present invention with improved heat resistance were obtained compared to FADGDH (hereinafter, wild type) obtained from a transformant containing pYESMh6754 not subjected to mutation treatment. These 13 kinds of plasmids are pYESMh6754-M1, pYESMh6754-M2, pYESMh6754-M3, pYESMh6754-M4, pYESMh6754-M5, pYESMh6754-M6, pYESMh6754-M7, pYESMh6754-M11hYESM67654-M9, YES. PYESMh6754-M12 and pYESMh6754-M13, and FADGDH sequences in the respective plasmids were determined using a multi-cavity DNA analysis system ABI3700 (Applied Biosystems).

As a result, A60E for pYESMh6754-M1, E94P for pYESMh6754-M2, S182A for pYESMh6754-M3, T228V for pYESMh6754-M5, R230V for pYESMh6754-M7, D317A for pYESMh6754-M7, pYESMh6799N, 54 In pYESMh6754-M10, the amino acid was substituted with W419Y, pYESMh6754-M11 with A439S, pYESMh6754-M12 with D466M, and pYESMh6754-M13 with A619S. The results are shown in the table.

Example 3 Further Improvement of Thermal Stability by Accumulation of Mutations Synthetic oligonucleotides of SEQ ID NOs: 2 and 3 designed to replace the 399th Gly with Asn using the pYESMh6754-M3 plasmid introduced with the mutation of S182A as a template Based on the above, PCR reaction was performed using KOD-Plus- (Toyobo Co., Ltd.) according to the protocol attached to this product. Next, the resulting reaction solution was transformed into Escherichia coli DH5α strain competent cells (Toyobo Competent High DH5α) according to the protocol attached to the product, and the transformants were obtained. The transformant was cultured in LB medium, and plasmid pYESMh6754-M14 was extracted. Next, using the obtained plasmid pYESMh6754-M14 as a template, KOD-Plus- (Toyobo Co., Ltd.) was used based on the synthetic oligonucleotides of SEQ ID NOS: 4 and 5 designed to replace the 439th Ala with Ser. The PCR reaction was performed according to the protocol attached to this product. Next, the obtained reaction solution was transformed into Escherichia coli DH5α strain competent cell (competent high DH5α manufactured by Toyobo Co., Ltd.) according to the protocol attached to the product, and the transformant was obtained. The transformant was cultured in LB medium, and the plasmid pYESMh6754-M15 was extracted.

In addition, based on the synthetic oligonucleotides of SEQ ID NOs: 6 and 7 designed to replace the 439th Ala with Ser, using the plasmid of pYESMh6754-M3 introduced with the mutation of S182A as a template, KOD-Plus- (Toyobo) ) Was performed according to the protocol attached to this product. Next, the obtained reaction solution was transformed into Escherichia coli DH5α strain competent cell (competent high DH5α manufactured by Toyobo Co., Ltd.) according to the protocol attached to the product, and the transformant was obtained. The transformant was cultured in LB medium, and the plasmid pYESMh6754-M16 was extracted.

PYESMh6754-M13, pYESMh6754-M14, and pYESMh6754-M15 obtained by the above procedure were transformed into Saccharomyces cerevisiae INVSc1 (Invitrogen). Next, the obtained transformant was cultured in a medium containing 3% yeast extract, 1% polypeptone, and 3% galactose at 25 ° C. for 60 hours, and the culture solution was centrifuged at 12000 rpm for 5 minutes. I got Qing. Next, the step of concentrating the obtained culture supernatant using a hollow fiber membrane (manufactured by Spectrum Laboratories) having a molecular weight cut-off of 10,000, and adding 50 mM phosphate buffer (pH 6.0) to the concentrate. Repeatedly removed low molecules. Thereafter, it was applied to a DEAE Sepharose Fast Flow (manufactured by GE Healthcare) column equilibrated with 50 mM potassium phosphate buffer (pH 6.0) to adsorb only contaminating proteins, and the obtained eluate was used as a crude purified solution. The obtained crude purified solution is heated at 55 ° C. for 15 minutes, 55 ° C. for 30 minutes, and 60 ° C. for 15 minutes, respectively. The GDH activity was measured using the FADGDH activity measurement method shown in Fig. 1, and the residual ratio was measured in comparison with the GDH activity before the treatment.

As a result, improvement in thermal stability due to accumulation of mutations was confirmed. The results are shown in Table 2.

Example 4 Purification of mutant FADGDH with improved thermostability Saccharomyces cerevisiae INVSc1 (Invitrogen) incorporating pYESMh6754-M15, a mutant with the highest heat resistance in Example 3, was used in a tryptophan-requiring medium. The culture was performed at 25 ° C. for 20 hours to obtain a seed culture solution.
Next, 6.0 L of production medium (galactose 3.0%, yeast extract 3.0%, peptone 1.0%, pH 5.5) was placed in a 10 L jar fermenter, sterilized by autoclaving, and main culture. A medium was used. 50 mL of the seed culture solution was inoculated into the main culture medium, and cultured for 3 days under conditions of a culture temperature of 25 ° C., a stirring speed of 380 rpm, an aeration rate of 2.0 L / min, and a tube pressure of 0.2 MPa. Thereafter, the culture solution is centrifuged at 4500 rpm, and the supernatant is concentrated using a UF membrane (Millipore Corp.) having a molecular weight cut off of 30,000, and 50 mM phosphate buffer (pH 6.0) is added to the concentrate. The process of adding was repeated to remove low molecules.
Then, it was applied to a DEAE Sepharose Fast Flow (GE Healthcare) column equilibrated with 50 mM potassium phosphate buffer (pH 6.0) to adsorb only contaminating proteins. Thereafter, the eluate was applied to a CM Sepharose Fast Flow (GE Healthcare) column to adsorb the protein, and then eluted with a linear gradient of 0.5 M sodium chloride 50 mM phosphate buffer (pH 6.0). Further, the eluted GDH fraction was concentrated with a hollow fiber membrane (Spectrum Laboratories) having a molecular weight cut off of 10,000, and then applied to a Superdex S-200 (GE Healthcare) column to obtain a purified enzyme.
The purified enzyme obtained here is called modified FADGDH.

Example 5 Temperature Stability of Modified FADGDH Using the modified FADGDH enzyme solution (2 U / mL) obtained in Example 4, the temperature stability was examined. The modified FADGDH enzyme solution is treated with each temperature (4 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65 ° C) for 15 minutes using 50 mM potassium phosphate buffer (pH 6.0), and then 1-5. The GDH activity was measured using the FADGDH activity measurement method shown in Fig. 1, and the residual ratio was measured in comparison with the GDH activity before the treatment. The results are shown in FIG.

As a result, the modified FADGDH had a residual rate of 95% or more after treatment at a temperature in the range of 4 ° C to 55 ° C. From the above, it was confirmed that the modified FADGDH was not substantially inactivated by the heating treatment up to 55 ° C.

Example 6 Optimal activity temperature of modified FADGDH Using the modified FADGDH enzyme solution (2.5 μg / mL) obtained in Example 4, the optimum activity temperature was examined. The GDH activity at 30 ° C., 37 ° C., 40 ° C., 45 ° C., 52 ° C. and 60 ° C. was determined. The results are shown in FIG.

As a result, the modified FADGDH showed the highest activity value at 60 ° C. From the above, it was shown that the optimum activity temperature of the modified FADGDH has an optimum activity of 60 ° C. or higher.

Example 8 Optimum active pH
Using the modified FADGDH enzyme solution (0.5 U / mL) obtained in Example 5, the optimum pH was examined. 100 mM potassium acetate buffer (pH 5.0-5.5, plotted with ■ in the figure) 100 mM MES-NaOH buffer (pH 5.5-6.5, plotted with □ in FIG. 1), 100 mM potassium phosphate buffer Solution (pH 6.3-7.7, plotted with ▲ in FIG. 1), 100 mM Tris-HCl buffer solution (pH 7.0-8.9, plotted with Δ in the figure), and at each pH, the temperature was 37 ° C. Enzymatic reactions were carried out and the relative activities were compared. The results are shown in FIG.

As a result, the optimum activity pH of the modified FADGDH showed the highest activity value at pH 6.3 when the potassium phosphate buffer was used. Moreover, in the range of pH 6.3-6.5, the relative activity of 90% or more was shown compared with the case where the activity in pH 6.3 was 100%. From the above, it was shown that the optimum active pH of the modified FADGDH is pH 6.3 to 6.5.

Example 9 pH stability Using the modified FADGDH enzyme solution (2 U / mL) obtained in Example 5, pH stability was examined. 100 mM Glycine-NaOH buffer (pH 2.5-3.5, plotted with ■ in the figure), 100 mM potassium acetate buffer (pH 3.5-pH 5.5: plotted with □ in FIG. 3), 100 mM MES-NaOH buffer (PH 5.5-pH 6.5: plotted with ▲ in FIG. 3), 100 mM potassium phosphate buffer (pH 6.0-pH 8.0: plotted with △ in FIG. 3), 100 mM Tris-HCl buffer (pH 7) 5-pH 9.0: plotted with a circle in FIG. 3), 100 mM Glycine-NaOH buffer solution (pH 9.0-pH 10.5: plotted with a circle in FIG. 3), each buffer at 25 ° C. for 16 hours The activity was measured when glucose was used as a substrate after maintaining the enzyme in the liquid. The activity value after the treatment and the activity value before the treatment were compared to determine the residual activity rate. The results are shown in FIG.

As a result, the modified FADGDH showed an activity remaining rate of 90% or more in the range of pH 3.5 to pH 10.3. From the above, it was shown that the stable pH range of the modified FADGDH is pH 3.5 to pH 10.3.

Example 10 Substrate specificity 1-5. According to the method for measuring FADGDH activity shown in Fig. 1, the activity was measured using D-glucose, maltose, D-galactose and D-xylose as substrates. The activity when D-glucose was used as a substrate was defined as 100%, and the activity against other sugars compared with that was determined. The concentration of each sugar was 200 mM. The results are shown in Table 2. Regarding the enzyme concentration, the reaction was carried out at a final concentration of 2.7 μg / mL for glucose and at a final concentration of 0.9 mg / mL for other sugars. The results are shown in Table 3.

From the results of Table 3, the substrate specificity of the FADGDH of the present invention is that the apparent activity against maltose, D-galactose and D-xylose is 1% or less when the activity value against D-glucose is 100%. In other words, FADGDH of the present invention was shown to be excellent in substrate specificity.

Example 11 An electrode sensor in which a working electrode, a counter electrode, and a reference electrode were arranged on a glucose sensor insulating substrate was obtained from Biodevice Technology Co., Ltd. (Nomi City, Ishikawa Prefecture). This electrode sensor has electrodes printed on a 4.0 mm × 17 mm substrate. 3 μL of an aqueous solution serving as a reagent layer was dispensed onto the working electrode (area: about 1.3 mm 2) of this sensor. The aqueous solution to be the reagent layer includes the following composition.
・ FAD-GDH
・ 200 mM potassium ferricyanide ・ 50 mM potassium phosphate buffer (pH 7.0)
Here, the modified FADGDH purified in Example 4 was used as FAD-GDH. This was dried by heating at 50 ° C. for 15 minutes to obtain a glucose sensor chip.
Subsequently, glucose solutions having concentrations of 10 mM, 20 mM, and 35 mM were prepared. 15 μL of these sample solutions were dropped onto the above chip connected to a potentiostat with a micropipette, and a voltage of +300 mV was applied 35 seconds after dropping to measure the current value. FIG. 5 shows the results of current response values at each glucose concentration.

As a result, at a glucose concentration of at least 10 mM to 35 mM, the glucose sensor of the present invention shows a concentration-dependent increase in current response value.

Example 12 Confirmation of Influence of Other Sugars on Glucose Sensor Using the same electrode sensor as used in Example 11, a glucose sensor chip was prepared in the same manner as in Example 11 from 3 μL of an aqueous solution having the following composition. did.
FADGDH (purified in Example 6)
・ 200 mM potassium ferricyanide ・ 50 mM potassium phosphate buffer (pH 7.0)
Each chip prepared as described above was connected to a potentiostat, 15 μL of glucose solution (concentration: 10 mM) was dropped onto the electrode using a micropipette, and a voltage of +300 mV was applied 35 seconds after dropping, and the current value was measured. did. Subsequently, a liquid containing 10 mM glucose and any one sugar of 20 mM maltose, galactose, and xylose was prepared and reacted in the same manner. Table 3 shows the results of comparison of response signals when using a solution containing only glucose and when using a solution obtained by adding another sugar to glucose. In addition, the numerical value in Table 3 is shown as a relative value when the intensity of the electrochemical signal when no other sugar is added is 100.

As a result, in the sensor using the modified FADGDH, an increase in signal value due to the coexistence of maltose, galactose, and xylose was not observed.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.

    The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

    The FADGDH of the present invention is excellent in thermal stability, can suppress enzyme inactivation during the production of a glucose sensor, and can more accurately measure the amount of glucose. Therefore, it can be said that FADGDH of the present invention is suitable for blood glucose measurement and the like.

Claims (9)

In the flavin adenine dinucleotide-dependent glucose dehydrogenase having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, at least 2 selected from the group consisting of positions 182, 399, and 439 in the amino acid sequence of SEQ ID NO: 1. It has amino acid substitution at one position or equivalent position, the amino acid substitution is as shown in the following (a) to (c), and the optimum activity temperature is 60 ° C. or higher A modified flavin adenine dinucleotide-dependent glucose dehydrogenase characterized by
(A) Serine at position 182 is alanine and glycine at position 399 is asparagine (b) Serine at position 182 is alanine and alanine at position 439 is serine (c) Serine at position 182 is alanine and glycine at position 399 is asparagine and 439th alanine is serine In the flavin adenine dinucleotide-dependent glucose dehydrogenase comprising the amino acid sequence set forth in SEQ ID NO: 1, at least two positions selected from the group consisting of positions 182, 399, and 439 in the amino acid sequence of SEQ ID NO: 1, or equivalent thereto A modified flavin characterized by having an amino acid substitution at the position, wherein the amino acid substitution is as shown in the following (a) to (c) and the optimum activity temperature is 60 ° C. or higher: Adenine dinucleotide-dependent glucose dehydrogenase.
(A) Serine at position 182 is alanine and glycine at position 399 is asparagine (b) Serine at position 182 is alanine and alanine at position 439 is serine (c) Serine at position 182 is alanine and glycine at position 399 is asparagine and 439th alanine is serine DNA encoding the modified flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 1 or 2 . A vector incorporating the DNA according to claim 3. A transformant comprising the vector according to claim 4 . A method for producing a modified flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 1 or 2 , comprising culturing the transformant according to claim 5 . A method for measuring a glucose concentration, comprising causing the modified flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 1 or 2 to act on glucose. A glucose assay kit comprising the modified flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 1 or 2 . A glucose sensor comprising the modified flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 1 or 2 .