JP2007143493A - Variant protein having diaphorase activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a variant protein having diaphorase activities and heat-resistant degree of diaphorase at a prescribed level or more. <P>SOLUTION: The variant protein comprises a specific natural amino acid sequence having an amino acid sequence wherein at least one or more acid residues are deleted, substituted, added or inserted residues from, with or to, and has the diaphorase activities having an enzyme activity of ≥245. Preferably, the thermostable variant protein has an enzyme activity of ≥245 and residual ratio of the enzyme activity after heat treatment of ≥41%. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a mutant protein having diaphorase activity. More specifically, the present invention relates to a mutant protein having diaphorase activity having a predetermined level or more of enzyme activity, and further, the degree of heat resistance.

  Enzymes are in-vivo catalysts that smoothly carry out many reactions related to the maintenance of life under mild conditions in the living body. This enzyme turns over in vivo and is produced in vivo as necessary to exert its catalytic function.

  Currently, a technique for utilizing this enzyme in vitro has already been put into practical use, or studies for practical use have been conducted. For example, enzyme utilization technologies are progressing in various technical fields such as production of useful substances, production of energy-related substances, measurement or analysis, environmental conservation, and medical treatment. In recent years, technologies such as an enzyme battery (see, for example, Patent Document 1), an enzyme electrode, and an enzyme sensor (a sensor that measures a chemical substance using an enzyme reaction), which are a type of fuel cell, have been proposed.

  Since the chemical body of this enzyme is a protein, the enzyme has the property of being denatured by the degree of heat and pH. For this reason, the enzyme is less stable in vitro than other chemical catalysts such as metal catalysts. Therefore, when the enzyme is used in vitro, it is important to make the enzyme work more stably against environmental changes so as to maintain its activity.

When the enzyme is used in vitro, approaches such as a method of artificially modifying the properties and functions of the enzyme itself and a method of devising the environment where the enzyme works are adopted. In the former method, an artificially mutated protein is produced by artificially changing the base sequence of a gene encoding a protein, and expressing the changed gene in an organism such as Escherichia coli, and using it. In general, screening (mutation) of mutant proteins having functions and properties suitable for the purpose is performed (see, for example, Patent Document 2).
JP-A-2004-71559. JP 2004-298185A.

  The main object of the present invention is to provide a mutant protein having a diaphorase activity and a degree of heat resistance exceeding a predetermined level in view of wide applicability of diaphorase in vitro.

  In the present invention, in the natural amino acid sequence of SEQ ID NO: 1 (the number of amino acid residues 211), it comprises an amino acid sequence in which at least one amino acid residue is deleted, substituted, added or inserted, and the enzyme activity value A mutant protein having a diaphorase activity of 245 or more, an enzyme activity value of 245 or more, and a residual enzyme activity rate of 27% or more after heat treatment, more preferably remaining after heat treatment Provided is a mutant protein having a diaphorase activity with an enzyme activity rate of 41% or more.

  Further, in the natural amino acid sequence of SEQ ID NO: 1, it comprises an amino acid sequence in which at least one amino acid residue is deleted, substituted, added or inserted, has a diaphorase activity with an enzyme activity value of 170 or more, and Provided is a mutant protein having a residual enzyme activity rate of 41% or more after heat treatment.

  These mutant proteins are, for example, mutant proteins of a protein having a diaphorase activity derived from a thermophilic Bacillus genus bacterium, particularly Bacillus stearothermophilus, and more specifically, SEQ ID NO: 2 Mention may be made of mutant proteins having an amino acid sequence of 7.

  In the amino acid sequence of SEQ ID NO: 2, lysine (Lysine) at position 139 from the N-terminus of the natural amino acid sequence of SEQ ID NO: 1 is replaced with asparagine, and alanine at position 187 from the N-terminus. (Alanine) is replaced by glutamic acid (hereinafter abbreviated symbol K139N / A187E). In the amino acid sequence of SEQ ID NO: 3, phenylalanine at the 105th position from the N-terminal of the natural amino acid sequence of SEQ ID NO: 1 is substituted with leucine (hereinafter abbreviated symbol F105L). In the amino acid sequence of SEQ ID NO: 4, glycine (Glycine) at position 122 from the N-terminal of the natural amino acid sequence of SEQ ID NO: 1 is substituted with aspartic acid (hereinafter abbreviated symbol G122D). In the amino acid sequence of SEQ ID NO: 5, glycine (Glycine) at position 131 from the N-terminal of the natural amino acid sequence of SEQ ID NO: 1 is replaced with glutamic acid (hereinafter abbreviated symbol G131E). In the amino acid sequence of SEQ ID NO: 6, alanine at position 146 from the N-terminal of the natural amino acid sequence of SEQ ID NO: 1 is replaced with glycine (hereinafter abbreviated symbol A146G). In the amino acid sequence of SEQ ID NO: 7, arginine at position 147 from the N-terminal of the natural amino acid sequence of SEQ ID NO: 1 is replaced with histidine (hereinafter abbreviated symbol R147H).

  Here, main technical terms related to the present invention will be described.

“Diaphorase” is an enzyme having an activity that catalyzes a reaction of oxidizing NADH or NADPH with a dye such as potassium ferricyanide, methylene blue, 2,6-dichloroindophenol, tetrazolium salt (ie, diaphorase activity). It is widely distributed 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 diaphorase by the dehydrogenation reaction from the substrate by NAD ⁺ or NADP ⁺ dependent dehydrogenases is an electron. Oxidized at the acceptor, the electron acceptor is reduced.

  A “mutant protein” is a protein expressed from a gene obtained by artificially changing the sequence of DNA bases encoding the amino acid sequence constituting the protein.

  The “enzyme activity value” generally means a catalytic reaction rate of a certain reaction under a certain condition. In the present invention, Nicotinamide dinucleotide, oxidized form (NAD +) and 2-amino-1,4-dihidroxynaphthene are obtained by reduction reaction of 2-amino-1,4-naphthoquinone (ANQ) with Nicotinamide dinucleotide, reduced form (NADH). The reaction rate of the resulting reaction, specifically, 1 mol per unit time in the presence of 0.3 mM ANQ and 40 mM NADH in 0.1 M potassium phosphate buffer at 25 ° C. in an argon or nitrogen atmosphere. The number of moles of product produced by the enzyme-catalyzed reaction (hence the unit is sec-1). The enzyme activity value of 245 or more corresponds to about 1.5 times or more of the natural protein having diaphorase activity derived from Bacillus stearothermophilus.

  “Residual activity after heat treatment (residual activity)” may be referred to as “enzyme activity remaining rate” or “enzyme activity maintenance rate”. When the enzyme is subjected to a predetermined heat treatment, It is a value representing how the activity changes before and after that. That is, the enzyme activity measurement is performed under the same conditions before and after the heat treatment, and the percentage value indicates how much activity value after the heat treatment is present before the heat treatment. In the present invention, the “heat treatment” is a stationary treatment at 80 ° C. for 10 minutes in a buffer solution, and the ratio of the enzyme activity values before and after the heat treatment is expressed as a percentage. The residual enzyme activity rate of 41% or more after the heat treatment corresponds to about 1.5 times or more of the natural protein having diaphorase activity derived from Bacillus stearothermophilus.

  In the mutant protein according to the present invention, the degree of diaphorase activity and / or heat resistance thereof is at a predetermined level or more.

(Example 1).
Cloning, expression and purification of diaphorase from Bacillus stearothermophilus.

(1-1) Isolation and purification of genomic DNA from Bacillus stearothermophilus.
Bacillus stearothermophilus was purchased from RIKEN Microbial System Storage Facility (JCM) (JCM No.2501, NCBI diaphorase gene accession number: AF112858). The lyophilized product of Bacillus stearothermophilus was cultured overnight on agar medium A at 55 ° C.

  The colonies thus obtained were cultured in the same manner on a new agar medium A, and a part thereof was taken out as a clean colony and cultured overnight at 55 ° C. in liquid medium A. Bacteria were collected by centrifugation, and genomic DNA was isolated using Wizard Genomic DNA Purification Kit (Promega). The composition of medium A is as shown in the following “Table 1” (adjusted to pH 7.0-7.2 in 1 L).

(1-2) Cloning of diaphorase gene.
The diaphorase gene was cloned from the genomic DNA obtained in (1-1) by PCR. Pfu DNA polymerase (Stratagene) was used as the DNA polymerase, and primers having the sequences shown in Table 2 below were used.

  Next, the diaphorase gene of the PCR product was purified using PCR Cleanup Kit (Qiagen) and confirmed by agarose electrophoresis. In addition, the base sequence was confirmed with a DNA sequencer.

(1-3) Introduction of a diaphorase gene into a vector.
The cloned fragment of the diaphorase gene was treated with BamH I and Nde I and purified using PCR Cleanup Kit (Qiagen). The vector pET12a (Novagen) was treated with BamH I and Nde I and purified in the same manner. These two types of fragments were ligated with T4 ligase, and XL1-blue electrocompetent cells (Stratagene) were transformed with the product and cultured in LB-amp medium to increase production.

  The obtained plasmid was treated with Bss I, insertion of the diaphorase gene was confirmed by agarose electrophoresis, and the nucleotide sequence was measured and analyzed. Then, there was a slight difference between the nucleotide sequence of the database (NCBI). This seems to be because there was a discrepancy between the gene sequence of the cloned DI and the database DI because there was a slight difference between the strain obtained from the microbial strain storage facility of RIKEN and the database strain. There were 11 genotypes (base sequences), of which 2 residues in the phenotype (amino acid sequence) were inconsistent (see Table 3).

  Therefore, a gene in which these two amino acid residues were corrected in the same manner as in the database was prepared using the Quick Change Site-Directed Mutagenesis Kit (Stratagene). This gene was named “pET12a-BsDI”.

(1-4) Transformation of E. coli.
The pET12a-BsDI was introduced into E. coli BL21 (DE3) (Novagen) by the heat shock method and transformed. After pre-culturing at 37 ° C for 1 hr in SOC, the cells were developed on an LB-amp agar medium, a part of the colonies were liquid-cultured, and the expression of diaphorase was confirmed by SDS-page.

(1-5) A cryopreserved sample of the transformant.
The transformant culture solution (3 mL) obtained in the above (1-4) was centrifuged, and 2XYT medium was added to the Escherichia coli pellet, dispersed, and stored at -80 ° C.

(1-6) Mass culture and protein purification.
From a frozen sample of BL21 (DE3) / pET12a-BsDI, develop it into LB-amp agar medium, pick up the colonies, pre-culture them with 100mL LB-amp until OD600 is around 1, and add 18 L of LB- The amp was developed and cultured at 37 ° C. until the OD600 was saturated at about 2. The cells were collected from this culture by centrifugation (5 kG) (wet yield 20 g). The cell pellet is frozen at -80 ° C, lysed, lysed by sonication in 200 mL of 50 mM Tris-HCl, pH 7.8, 1 mM EDTA, 1 mM DTT, 1 mM PMSF at 0 ° C, and centrifuged. The solution fraction was collected by separation (9.5 kG).

  Next, purification by an ammonium sulfate precipitation method was performed. While stirring until a 35% saturated solution was obtained, powdered ammonium sulfate was gradually added and allowed to stand overnight. The precipitate was removed by centrifugation (9.5 kG) and desalted using a dialysis membrane tube (final solution: 5 mM Tris-HCl, pH 7.8). A 50 mL sample concentrated by ultrafiltration was applied to an anion exchange column (Sepharose Q FastFlow, Amersham Bioscience), and the diaphorase-containing fraction was collected and concentrated by ultrafiltration (solution volume 20 mL, Centriplus centrifugal filter) Unit YM-30, Millipore). The sample was then applied to a gel filtration column (Sephacryl S-200, Amersham Bioscience) and the diaphorase-containing fraction was collected.

(Example 2).
Creation of mutant library by random mutation of diaphorase from Bacillus stearothermophilus and screening of thermostable mutant.

  First, FIG. 1 shows a flow of an experiment performed in the second embodiment. A gene library of diaphorase mutants was prepared by error-prone PCR, and this gene was introduced into vector DNA and expressed in E. coli. This library was subjected to heat resistance screening, and the target heat-resistant diaphorase mutant was extracted.

(2-1) Error-Prone PCR by GeneMorph (registered trademark).
The "Error-prone PCR method" is a method of randomly causing mutations in a replicated DNA fragment by utilizing the fact that DNA polymerase misreads the base sequence during a DNA fragment replication reaction by PCR. . Although various methods have been reported, Stratagene's GeneMorph (registered trademark) was selected from those commercially available. As the template DNA, the above-described pET12a-BsDI into which a diaphorase gene of Bacillus stearothermophilus was incorporated was used. The primer used for the cloning of this gene was also used.

  The primer sequences are as shown in “Table 4” below, and have an Nde I sequence at the 5 ′ end of the coding and a BamH I sequence at the 5 ′ end of the complementary (underlined portion). -prone PCR products can be inserted into the multiple cloning site of pET12a by these restriction enzyme treatments (similar to cloning of natural diaphorase).

  According to the GeneMorph (registered trademark) manual, PCR was performed based on the preparation of the reaction solution and the temperature control cycle as shown in Table 5 below.

(2-2) Introduction of a diaphorase gene into a vector.
The entire amount of Error-prone PCR product was used for restriction enzyme treatment with Nde I and BamH I except for the amount used for agarose gel electrophoresis. After performing the reaction at 37 ° C. for 2 hours, the reaction product was purified by Qiaquick PCR purification Kit (Qiagen). On the other hand, as for the vector, pET12a was treated with Nde I and BamH I for restriction enzyme treatment (at 37 ° C. for 2 hours) like the PCR product.

  These restriction enzyme-treated reaction products were separated by low-melting point agarose gel electrophoresis, and the corresponding circularly open vector DNA was purified using Qiaquick Gel Extraction Kit (Qiagen). Subsequently, the 5 'end was dephosphorylated by treating the restriction enzyme-treated product of the purified vector with alkaline phosphatase. The reaction product was purified by Qiaquick PCR purification Kit (Qiagen). The error-prone PCR product (that is, the diaphorase mutant gene library) thus obtained was ligated to a restriction enzyme / dephosphorylated vector. Ligation Kit Mighty Mix (Takara Bio Inc.) was used for the ligation reaction. The reaction product was purified by ethanol precipitation.

(2-3) Production and transformation of competent cells.
Competent cells used were BL21 (DE3) electrocompetent cells (competency ˜10 ⁸ / ng) prepared in-house. Thaw a frozen sample of 40 μL of competent cell on ice, mix 0.5 μL of a DNA sample with a concentration of about 1 μg / μL, set the entire volume in a 0.1 cm gap eleltroporation cuvette, and apply a voltage of 1800 kV. Was transformed. To this, 960 μL of SOC medium was added, and preculture was performed at 37 ° C. for 1 hour. This culture was inoculated into 5-50 μL LB-amp agar medium, and incubated at 37 ° C. overnight.

(2-4) Screening method.
Single colonies on the agar medium obtained in the above (2-3) were inoculated into a LB-amp liquid medium (150 μL) in a 96-well plate using a toothpick. Two wells were applied to the E. coli strain producing the wild type. The upper surface of the well plate was sealed with a gas-permable adhesive sheet (ABgene), and the attached lid was put on, followed by incubation at 37 ° C. overnight (˜14 hours). 25 μL of this culture solution was mixed in a new well plate with the same amount of 0.2N NaOH aq (previously dispensed) by pipetting, then the plate was covered and incubated for 15 minutes at 37 ° C. (incubator) for lysis .

  Next, 100 μL of 0.1 M K-pi, pH 6.8 was added at room temperature to neutralize the solution. At this time, one of two wild-type samples as an unheated control sample was placed in a microtube and stored at room temperature. The plate was sealed with a commercially available OPP tape, heat-treated at 80 ° C. for 75 minutes (incubator), allowed to cool at room temperature, and the wild-type sample that had been separated was returned to the plate. To each sample, 10 μL of 20 mM anthraquinone sulfonic acid (AQS) 20% DMSO solution and 50 μL of the 80 mM NADH aqueous solution prepared immediately before were sequentially added, the plate was sealed with OPP tape, and stirred with a vortex mixer for 5 seconds. The state of color development was recorded with a camera, and those having strong color development due to the reduction of the AQS compared to the wild type sample were selected as heat resistance candidates.

(2-5) Sample storage.
For the specimens that passed the screening, a part of the culture solution remaining in the 96-well plate was inoculated into 4.5 mL of LB medium, cultured overnight, the plasmid was purified, and stored in a freezer. Separately, inoculate into 4 mL of LB medium and incubate until OD ₆₀₀ reaches about 0.4, collect by centrifugation, suspend in 2 mL of 2xYT medium, freeze with liquid nitrogen, and store at -80 ° C. did.

(2-6) Mass expression and purification of diaphorase mutants.
Large-scale expression and purification of diaphorase mutants was carried out by previously reported methods. However, E. coli in large-scale expression was cultured with 1 L LB-amp, and the volume in each subsequent purification step was adjusted according to the culture scale.

(2-7) Activity evaluation test.
The activity of the diaphorase mutant was evaluated under the following conditions. The reaction solution is 100 mM K-pi, pH 8.0, [ANQ] = 0.3 mM, [NADH] = 40 mM, [diaphorase] = 48 nM. Prior to the measurement, oxygen was sufficiently removed by argon bubbling, and the reaction was performed in an argon atmosphere. The reaction was started by the addition of diaphorase, and the reaction progress was monitored by a decrease in the absorbance of ANQ at 520 nm (molar extinction coefficient 680 M ⁻¹ cm ⁻¹ ) to calculate the reaction rate.

(2-8) Heat resistance test.
A 50 mM Tris-HCl, pH 7.8, 300 mM NaCl solution of the purified diaphorase mutant sample was concentrated / buffer exchanged by an ultrafiltration method to obtain a 0.1 M K-pi, pH 8.0 solution. This solution was appropriately diluted so that the absorbance at 460 nm of diaphorase was 0.1 (the solution having an enzyme concentration of 8.3 μM). This solution was incubated at 80 ° C. for 10 minutes with an aluminum block heater or the like, immediately cooled on ice, and cooled down to measure the activity. In addition, a control experiment was performed using a sample that was not incubated.

(2-9) Results.
A total of about 8000 colonies were screened according to the above method. Results 43 samples were extracted as positive. As a practical example, a part of a photograph of a well plate is shown in FIG. FIG. 2 shows an example of detection of diaphorase that maintains the activity in screening. Arrows (A, A) are samples of thermostable mutant candidates detected on this plate, and arrows are used as control experiments. (B) shows the wild type specimen, and arrow (C) shows the wild type specimen not subjected to the heat treatment.

  In consideration of possible errors such as differences between plates and diaphorase expression levels between strains, the selected specimens were collected and rescreened at once. That is, the cryopreserved Escherichia coli sample was streaked on LB agar medium, and the obtained colonies were inoculated into a 96-well plate, and a heating experiment was similarly performed. However, in order to minimize the error, eight colonies were picked up for each specimen and screened.

  A part of a photograph of the result of this double check experiment is shown in FIG. In FIG. 3, the same mutant sample is placed in the column, and as a reference, the upper 4 wells of the column numbers 11 and 24 are the wild type after the heat treatment, and the lower 4 wells of the column numbers 12 and 24 are not subjected to the heat treatment. It is a wild type sample. In the example of the photograph shown in FIG. 3, column numbers 7, 14, 17, 19, 20, and 21 were selected as positive and selected as heat-resistant mutant candidates.

  Thus, 14 samples that ensured reproducibility were used as extracted thermostable diaphorase mutant candidates. When the gene sequences of these 14 variants were analyzed, the results were as shown in the attached FIG. 4 (drawing substitute table). Three of the 14 samples overlapped with the same mutant G149D, and one sample was wild type. Therefore, the number of thermostable mutant candidates finally extracted was 10.

  FIG. 5 (drawing substitute graph) shows the results of the heat resistance test conducted on the thermostable diaphorase mutant candidates obtained above. In FIG. 5, the activity (reaction rate, left) of the thermostable diaphorase mutant candidate before and after heat treatment, the residual enzyme activity rate by heat treatment (right), and the dotted line in the graph indicate the wild-type value at each value. ing. “Amano” in FIG. 5 is a diaphorase manufactured by Amano Enzyme, which has the same sequence as WT.

  As a result, the enzyme residual activity rate after heat treatment of the mutant protein is particularly superior to that of the wild type (WT) in F105L (47.7%) and K139N / A187E (53.3%), G131E (28.1%), G122D (27.7%) was slightly better than the wild type (WT, 25.3%) (see FIG. 5). Others were less than or equal to the wild type. Therefore, it can be seen that in the screening, not only mutants with improved heat resistance but also mutants with improved normal activity even though the residual enzyme activity rate did not improve so much. However, an improvement in activity was confirmed in many of these mutants, and in particular, K139N / A187E showed an activity twice or more that of the wild type. In any case, this experimental system succeeded in obtaining the target diaphorase mutant with improved thermostability. Therefore, it was confirmed that the method for creating mutant libraries generated by random mutation by error-prone PCR and screening by heat treatment used in this study was actually effective.

(Example 3).
Detailed study on diaphorase thermostable mutants.

  (3-1). Among the mutants obtained in the above examples, further detailed examination was performed on the heat resistance of the mutant protein K139N / A187E, which has particularly improved heat resistance compared to the wild type. FIG. 6 shows a graph obtained by measuring and plotting the residual enzyme activity rate when the temperature of the heat treatment is variously changed.

  As shown in FIG. 6 (drawing substitute graph), the enzyme activity begins to drop at a temperature of 70 ° C., but the mutant protein K139N / A187E has a residual enzyme activity of the natural protein in the temperature range of 75 to 85 ° C. It was found that 1.5 times or more of the rate (heat resistance) can be maintained.

  (3-2). Among the mutants obtained by the above study, G122D and K139N / A187E, which have improved enzyme activity as compared with the wild type, were used as representative examples to further study enzyme kinetics.

  First, FIG. 7 (drawing substitute graph) plots the enzyme reaction rate when the concentration of ANQ was changed variously under the condition of NADH 40 mM. FIG. 7 shows that the mutant proteins G122D and K139N / A187E show ANQ concentration dependency in the same manner as the wild type (natural type), and these mutant proteins are compared with the wild type (natural type). It can be seen that greater affinity for the substrate ANQ has been obtained.

  Further, in FIG. 8 (drawing substitute graph), the enzyme reaction rate when the concentration of NADH was changed under various conditions of ANQ 2.2 mM was plotted. Mutant protein G122D draws the same ANQ concentration-dependent curve as wild type (natural type), and mutant protein K139N / A187E has rather lower affinity for substrate NADH than wild type (natural type). ing.

Enzymatic reactions were in good agreement with the Michaelis-Menten equation, and k _cat, K _M (NADH), and K _M (ANQ) were determined. For comparison, wild type (natural type) diaphorase (DI (DH “Amano” 3) k _cat and K _M (NADH), K _M (ANQ) are also shown. Diaphorase has a so-called ping-pong type reaction format. taking. here, k _cat is the number of turnovers per unit time of the catalyst _{reaction, K M (NADH), K} M (ANQ) is Michaelis constant for each substrate, to the active site for each of the substrate for the enzyme This factor reflects the ease of substrate binding, and the above is summarized as shown in FIG.

  From these results, it can be seen that the binding site of the mediator ANQ of these mutants has a property of binding more easily than the wild type (natural type) (see the ANQ related table in FIG. 9), while the binding site of NADH It is considered that there is no particular change compared to the wild type (natural type), or rather it is reduced (see the NADH-related table in FIG. 9). Therefore, it can be predicted that the higher catalytic ability of these mutants (mutant proteins) is due to the acquisition of affinity for the substrate ANQ.

  This means that it does not show higher catalytic activity at high concentrations, but it shows superiority at low concentrations, leading to the advantage of being able to reduce the mediator ANQ concentration in, for example, enzyme cells. Conceivable.

  Mutant proteins having diaphorase activity according to the present invention include, for example, NADH or NADPH assay kits, NADH or NADPH sensors, NADH or NADPH measurement methods, fuel cells in which enzymes are immobilized and used as electrodes, quinones, etc. It can be used in the field of the reduction reaction catalyst of

It is a schematic diagram which shows the flow of the experiment concerning Example 2 (library creation and screening by random mutation). A part of a well plate photo (drawing substitute photo) when a total of about 8000 colonies were screened. It is a part of well plate photograph of the result of the double check experiment. It is a drawing substitute table | surface which shows the result of having analyzed the gene sequence of 14 variants which are thermostable diaphorase variant candidates. It is a drawing substitute graph which shows the result of having performed the thermostability test about the thermostable diaphorase variant candidate (mutant protein). It is the graph which measured and plotted the residual enzyme activity rate when changing the temperature of heat processing variously regarding the heat resistance regarding the mutant protein K139N / A187E which improved heat resistance compared with the wild type. It is a drawing substitute graph plotting the enzyme reaction rate when the concentration of ANQ is changed variously under the condition of NADH 40 mM. FIG. 5 is a drawing-substituting graph that plots the enzyme reaction rate when the concentration of NADH is variously changed under the condition of ANQ 2.2 mM. It is a drawing substitute table summarizing factors reflecting the ease of binding of the substrate to the active site for each of the diaphorase enzymes of G122D and K139N / A187E, which are representative examples of the wild type and the mutant type.

Claims (14)

In the natural amino acid sequence of SEQ ID NO: 1, consisting of an amino acid sequence in which at least one amino acid residue is deleted, substituted, added or inserted,
A mutant protein having diaphorase activity having an enzyme activity value of 245 or more.   The mutant protein according to claim 1, wherein the residual enzyme activity rate after the heat treatment is 27% or more.   The mutant protein according to claim 2, wherein the residual enzyme activity rate after the heat treatment is 41% or more. In the natural amino acid sequence of SEQ ID NO: 1, consisting of an amino acid sequence in which at least one amino acid residue is deleted, substituted, added or inserted,
A mutant protein having a diaphorase activity with an enzyme activity value of 170 or more and having a residual enzyme activity rate of 41% or more after heat treatment.   The mutant protein according to claim 1, which is a mutant form of a protein having diaphorase activity derived from a thermophilic Bacillus bacterium. The mutant protein according to claim 4, which is a mutant form of a protein having a diaphorase activity derived from a thermophilic Bacillus bacterium.   The mutant protein according to claim 5, which is a mutant protein having a diaphorase activity derived from Bacillus stearothermophilus.   The mutant protein according to claim 6, which is a mutant protein having a diaphorase activity derived from Bacillus stearothermophilus.   The mutant protein according to claim 3, which has the amino acid sequence of SEQ ID NO: 2.   The mutant protein according to claim 3, which has the amino acid sequence of SEQ ID NO: 3.   The mutant protein according to claim 2, which has the amino acid sequence of SEQ ID NO: 4.   The mutant protein according to claim 2, which has the amino acid sequence of SEQ ID NO: 5.   The mutant protein according to claim 1, which has the amino acid sequence of SEQ ID NO: 6.   The mutant protein according to claim 1, which has the amino acid sequence of SEQ ID NO: 7.