JP6521238B2 - Modified diaphorase - Google Patents

Description

The present invention relates to modified diaphorase.

Diaphorase is an enzyme having an activity (diaphorase activity) that catalyzes the reaction of oxidizing NADH or NADPH with a pigment such as potassium ferricyanide, methylene blue, 2,6-dichloroindophenol, tetrazolium salt, etc. For example, Clostridium (Clostridium) genus (Non-patent document 2) or those isolated and purified from a microorganism belonging to the genus Bacillus (patent document 1) are commercially available. Bacillus stearothermophilus which produces diaphorase described in Patent Document 1 was reclassified in 2001 as Geobacillus stearothermophilus (Non-patent document 3) ). As to Geobacillus stearothermophilus, those further modified are also known, and their gene sequences, amino acid sequences and physicochemical properties are examined (Patent Document 1, Patent Document 2, Non-patent document 1)

Diaphorase [EC. 1.6.99. -] Plays an important role in the electron transfer system in vivo. By this diaphorase, NADH or NADPH generated by the dehydrogenation reaction from the substrate by NAD or NADP-dependent dehydrogenases is oxidized at the electron acceptor, and the electron acceptor is in a reduced form. In various technical fields, diaphorase has been considered for its in vitro use, and part of it has been put to practical use. Such technical fields include production of useful substances, production of energy-related substances, measurement or analysis, environmental protection, medical treatment and the like. For example, in the field of clinical diagnostics, diaphorase has a variety of extracorporeal activities utilizing reactions in which it oxidizes reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) with a dye. It is used to improve the sensitivity of diagnostic reagents.

The industrial use of diaphorase has various required characteristics depending on the purpose. For example, properties required for use in in vitro diagnostic reagents include reactivity with a substrate NADH, stability in a diagnostic reagent, and reactivity (affinity) with a dye.

Patent 3953578 Japanese Patent Application Laid-Open No. 2007-143493

Sugiyama et al. Biosens Bioelectron. 2010 Oct 15; 26 (2): 452-7. Epub 2010 Aug 3. Kaplan, N. O. , Et al. , Arch. Biochem. Biophys, vol 132, p91-98, 1969 T. N. Nazina. , Int. Jour. Syst. Evol. Micro. 51: 433-446. 2001

An object of the present invention is to provide a more stable diaphorase in an in vitro diagnostic reagent.

The present inventors examined the stability of the Geophorus genus diaphorase, and found that the stability of the diaphorase is affected by various components mixed in, and among them, affected by various preservatives. I found that. Then, as a result of further investigation, the coexistence of the isothiazoline-based preservative, which is a widely used preservative, by replacing the 119th cysteine residue of the Geophorus genus diaphorase with another amino acid residue. It succeeded in producing diaphorase whose activity is stable even after long-term storage under, and came to complete this invention.

That is, the summary of the present invention is as follows.
Item 1.
The diaphorase shown by either of following (1) to (3).
(1) A polypeptide comprising an amino acid sequence in which the 119th amino acid of the amino acid sequence set forth in SEQ ID NO: 2 is substituted with another amino acid other than methionine.
(2) In the polypeptide shown in (1), one or several amino acids other than the 119th amino acid in the amino acid sequence of SEQ ID NO: 2 are further substituted, deleted, inserted and / or added A polypeptide consisting of an amino acid sequence and stable to an isothiazoline preservative.
(3) In the polypeptide shown in (1), it is a polypeptide in which a portion other than the 119th amino acid in the amino acid sequence of SEQ ID NO: 2 is further modified, wherein the amino acid sequence of the polypeptide and SEQ ID NO: 2. A polypeptide having an identity of 80% or more with the amino acid sequence shown in 2, and being stable to an isothiazoline preservative.
Item 2.
Diaphorase shown by the following (4).
(4) In diaphorase derived from genus Geobacillus (except for those having the amino acid sequence described in SEQ ID NO: 2), substitution of the amino acid corresponding to the 119th amino acid in the amino acid sequence described in SEQ ID NO: 2 with another amino acid other than methionine And a polypeptide which is stable to an isothiazoline preservative.
Item 3.
The diaphorase according to item 1 or 2, wherein the amino acid corresponding to 119th or 119th of the amino acid sequence set forth in SEQ ID NO: 2 is substituted with alanine.
Item 4.
The DNA in any one of the following (A)-(C).
(A) DNA encoding the amino acid sequence of diaphorase shown in item 1
(B) In the base sequence shown in SEQ ID NO: 1, the base sequence corresponding to the amino acid change site shown in (1) of item 1 is modified to encode a different amino acid other than cysteine or methionine A DNA comprising a nucleotide sequence having a mutation at a site other than the altered site, wherein the identity of the sequence and the nucleotide sequence shown in SEQ ID NO: 1 is 80% or more, and a polypeptide having diaphorase activity is encoded DNA to be
(C) In the base sequence shown in SEQ ID NO: 1, the base sequence corresponding to the amino acid change site shown in (1) of item 1 is modified to encode a different amino acid other than cysteine or methionine A DNA comprising a nucleotide sequence having a mutation at a site other than the altered site, wherein said sequence hybridizes to the nucleotide sequence complementary to SEQ ID NO: 1 under stringent conditions and has diaphorase activity DNA encoding a polypeptide
Item 5.
5. A vector into which the DNA according to item 4 has been incorporated.
Item 6.
Item 6. A transformant comprising the vector of Item 5 or having the DNA of Item 4 inserted into genomic DNA.
Item 7.
A method for producing diaphorase, comprising the steps of culturing the transformant according to item 6 in a medium, accumulating diaphorase in or in the transformant, and recovering the accumulated diaphorase.
Item 8.
Item 4. A product comprising the diaphorase according to any one of Items 1 to 3.

The modified diaphorase of the present invention is improved in preservative resistance as compared to the wild-type, and therefore, it is possible to stably retain the activity even in the in vitro diagnostic reagent containing it.

Hereinafter, the present invention will be described in detail.
1. Modified diaphorase 1-1. Method of measuring diaphorase activity The diaphorase activity can be measured by a known method. For example, DCPIP can be used as an electron acceptor, and the activity can be measured by using the change in absorbance of the sample at a wavelength of 600 nm before and after the reaction as an indicator. More specifically, the activity can be measured using the following reagents and measurement conditions, and in the present specification, the value measured by this method is used.
<Reagent>
Distilled water 200 mM Tris-HCl buffer (pH 8.0)
36.0 mM aqueous solution of NADH 2.4 mM 2,6-dichlorophenolindophenol (DCPIP) solution Enzyme dilution solution: 200 mM Tris-HCl buffer (pH 8.0) containing 0.5% Tween 20
<Step 1>
The diaphorase solution is diluted to 0.1 to 0.3 U / mL with the above-mentioned enzyme dilution solution which has been ice-cooled in advance, and the one kept on ice-cold is used as the enzyme solution.
<Step 2>
After mixing 2.4 mL of the above distilled water, 0.3 mL of Tris-HCl buffer, and 0.1 mL of an aqueous solution of NADH and prewarming at 37 ° C. for 4 minutes, 0.1 mL of the enzyme solution is added and another 1 min. The prewarmed solution is used as the reaction mixture.
<Measurement conditions>
After adding 0.1 mL of DCPIP solution to 2.9 mL of the reaction mixture and mixing gently, water was used as a control at 37 ° C. with a spectrophotometer (1.0 cm optical path length) to change absorbance at 600 nm by 2 to 3 Record for a minute and then measure the change in absorbance per minute (ΔOD _TEST ) from the linear portion (ie, after the reaction rate becomes constant). In the blind test, instead of the enzyme solution, an enzyme dilution solution in which diaphorase is dissolved and a DCPIP solution are added to the reaction mixture, and the absorbance change per minute (ΔOD _BLANK ) is similarly measured. From these values, the diaphorase activity is determined according to the following equation. Here, 1 unit (U) in the diaphorase activity is an amount of enzyme that reduces 1 μM DCPIP in 1 minute under the above-mentioned measurement conditions.

Activity (U / mL)
= {(ΔOD _TEST −ΔOD _BLANK ) × 3.0 × dilution factor} ÷ {20.1 × 1.0 × 0.1}
= (ΔOD _TEST -ΔOD _BLANK ) × dilution ratio × 1.49
In the formula, 3.0 is the total amount (mL) of the reaction solution, 20.1 is the millimolar molecular extinction coefficient of DCPIP, 1.0 is the optical path length (cm), 0.1 is the volume of the enzyme solution (mL) Indicates In Examples 1 and 2 described later, measurement of diaphorase activity was performed by this measurement method.

1-2. Polypeptide The diaphorase of the present invention may be represented by any of the following (1) to (3).
(1) A polypeptide comprising an amino acid sequence in which the 119th amino acid of the amino acid sequence set forth in SEQ ID NO: 2 is substituted with another amino acid other than methionine.
(2) In the polypeptide shown in (1), one or several amino acids other than the 119th amino acid in the amino acid sequence of SEQ ID NO: 2 are further substituted, deleted, inserted and / or added A polypeptide consisting of an amino acid sequence and stable to an isothiazoline preservative.
(3) In the polypeptide shown in (1), it is a polypeptide in which a portion other than the 119th amino acid in the amino acid sequence of SEQ ID NO: 2 is further modified, wherein the amino acid sequence of the polypeptide and SEQ ID NO: 2. A polypeptide having an identity of 80% or more with the amino acid sequence shown in 2, and being stable to an isothiazoline preservative.

The amino acid sequence shown by SEQ ID NO: 2 is the amino acid sequence of diaphorase derived from Geobacillus sp. Y4.1 MC1. The microorganism is a strain stored in ATCC (American Type Culture Collection), and can be distributed by subjecting it to a predetermined procedure.

One or several mutations may be treated with restriction enzymes, treatment with exonuclease or DNA ligase, position-directed mutagenesis or random mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New It is possible to implement by introduce | transducing a mutation into DNA which codes the diaphorase of this invention mentioned later using well-known methods, such as York | yoke). Also, variant diaphorase can be obtained by other methods such as ultraviolet irradiation. Variant diaphorases also include naturally occurring variants (e.g., single nucleotide polymorphisms) such as those based on individual differences of microorganisms that retain PCNA, differences in species and genera, and the like.

In the polypeptide of (1), the amino acid residue at position 119 in SEQ ID NO: 2 is cysteine. The amino acid substitution at position 119 is substitution with other amino acids except for cysteine or methionine on which an isothiazoline preservative acts, and is not particularly limited as long as it retains diaphorase activity, but substitution of alanine is preferable .

The substitution of the codon on the diaphorase gene to the codon encoding another amino acid creates a gene in which the codon encoding the above-mentioned cysteine residue on the diaphorase gene sequence is replaced with another codon, and this is used as a host cell. It can be carried out by transforming, culturing the transformant to express the gene, and purifying the expression product, the modified diaphorase. Such mutant diaphorase gene can also be produced by chemical synthesis of the entire designed engineered diaphorase gene sequence by DNA synthesis using a wild-type diaphorase gene as a template and a mismatch primer. Is also possible.

The lower limit of “several” in the above (2) is two. The upper limit is not limited as long as the diaphorase activity is maintained, but is, for example, a number corresponding to less than 20% of the total amino acids, preferably a number corresponding to less than 15%, more preferably a number corresponding to less than 10%. More preferably, it is a number corresponding to less than 5%, more preferably a number corresponding to less than 1%. In another aspect, the number of mutated amino acid residues is, for example, 40 or less, preferably 30 or less, more preferably 20 or less, more preferably 15 or less, more preferably 10 or less, more preferably Is 5 or less, more preferably 4 or less, and further preferably 3 or less.

The identity of the polypeptide of (3) and the amino acid sequence shown in SEQ ID NO: 2 is 80% or more, and is not particularly limited as long as the diaphorase activity is maintained. It is preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, and more preferably 99% or more.

The identity of the amino acid sequences can be calculated using analytical tools available commercially or through telecommunication links (Internet). As used herein, the National Center for Biotechnology Information (NCBI) homology algorithm BLAST. (Basic local alignment search tool) http: // www. ncbi. nlm. nih. Calculated by using default (default) parameters in gov / BLAST /.

In the above (2) and (3), the isothiazoline-based preservative is a general term for sterilization / antifungal / algal / preservative of isothiazolinone derivatives, and is not particularly limited, but methylisothiazolinone (2-Methyl-4 -Isothiazolin-3-one; MIT), chloromethylisothiazolinone (5-chloro-2-methyl-4-isothiazolin-3-one; CMIT), octylisothiazolinone (2-n-octyl-4-isothiazolin- 3-one; OIT) dichlorooctylisothiazolinone (4,5-Dichloro-2-octyl-4-isothiazolin-3-one; DCOIT) benzisothiazolinone (1,2-benzisothiazolin-3-one; BI) ), And the like.

These isothiazoline preservatives are thought to act on the cysteine or methionine of diaphorase to destabilize diaphorase. The present invention is intended to improve this problem, and can be applied to various isothiazoline-based preservatives. Among them, it is MIT that the stabilization effect of the present invention is preferably obtained.

[Evaluation method of stability]
As used herein, “stable to isothiazoline preservative (eg, MIT)” refers to a 50 mM HEPES buffer containing 0.05% isothiazoline preservative and 0.1% Tween 20 at 37 ° C. The residual enzyme activity after sealing storage of diaphorase at a final concentration of 5 U / mL for 1 week in pH 7.5 does not show a substantial reduction compared with the enzyme activity before treatment (ie, 90% or more) And the diaphorase is judged to be stable in isothiazoline based preservatives.

Preferably, the modified diaphorase of the present invention has improved stability as compared to wild-type diaphorase. In the present specification, “the stability is improved as compared to wild-type diaphorase” is defined by 50 mM HEPES buffer containing 0.05% isothiazoline preservative and 0.1% Tween 20 at 37 ° C. After keeping sealed for 5 weeks at a final concentration of 5 U / mL diaphorase in pH 7.5, the enzyme activity retention rate is compared, and if it is modified type> wild type, the modified diaphorase is wild type and It is judged that the stability is improved in comparison. (If there is no difference in the enzyme activity retention rate for one week of storage, the storage period is appropriately extended and judged until the two are different.)

The diaphorase of the present invention can also be shown in the following (4).
(4) In diaphorase derived from genus Geobacillus (except for those having the amino acid sequence described in SEQ ID NO: 2), substitution of the amino acid corresponding to the 119th amino acid in the amino acid sequence described in SEQ ID NO: 2 with another amino acid other than methionine And a polypeptide which is stable to an isothiazoline preservative.

The type of Geobacillus microorganism listed as the source of diaphorase before modification in (4) above is not particularly limited except for Geobacillus sp. Y4.1 MC1 having the amino acid sequence set forth in SEQ ID NO: 2, Geobacillus stearothermophilus, Geobacillus kaustophilus HTA 426, Geobacillus thermoleovorans, Geobacillus thermoglucosidasius, Geobacillus caldoxylosiliticus, Geobacillus tepidamans, Geobacillus toebii subsp. Decanicus ga, Lactosidasius, Geobacillus sp. Y412MC61, Geobacillus sp. Y412 MC52, Geobacillus sp. G11 MC16, Geobacillus zalihae, Geobacillus thermodonitrificans can be shown. In addition to the diaphorase directly isolated from the microorganism, the diaphorase before the modification described above is not only the diaphorase isolated directly from the microorganism, but also the isolated diaphorase modified with the amino acid sequence etc. by the protein engineering method, or modified by the genetic engineering method Things are also included.

The diaphorase of the present invention is preferably an isolated diaphorase or a purified diaphorase. In addition, the diaphorase of the present invention may be present in a solution or in a freeze-dried state (for example, in the form of powder) in a solution suitable for the storage method described later. The term "isolated" when used in the context of the enzyme (diaphorase) of the present invention substantially includes components other than the enzyme (eg, contaminating proteins derived from host cells, other components, culture fluid, etc.) Not) state. Specifically, for example, the isolated enzyme of the present invention has a content of contaminating proteins of less than about 20%, preferably less than about 10%, more preferably less than about 5%, and even more preferably less than about 10% of the total. Is less than about 1%. On the other hand, the diaphorase of the present invention may be present in a solution (eg, buffer) suitable for storage or measurement of enzyme activity.

2. Production of Modified Diaphorase As another aspect of the present invention, the following may be shown.
(5) A DNA encoding an amino acid sequence of the diaphorase of the present invention, which is any of the following (A) to (C).
(A) DNA encoding the amino acid sequence of diaphorase shown in claim 1
(B) In the base sequence shown in SEQ ID NO: 1, the base sequence corresponding to the amino acid change site shown in (1) of claim 1 is modified to encode a different amino acid other than cysteine or methionine A DNA comprising a nucleotide sequence having a mutation at a site other than the altered site, wherein the identity of the sequence and the nucleotide sequence shown in SEQ ID NO: 1 is 80% or more, and a polypeptide having diaphorase activity Coding DNA
(C) In the base sequence shown in SEQ ID NO: 1, the base sequence corresponding to the amino acid change site shown in (1) of claim 1 is modified to encode a different amino acid other than cysteine or methionine. A DNA comprising a nucleotide sequence having a mutation at a site other than the altered site, wherein the sequence hybridizes to the nucleotide sequence complementary to SEQ ID NO: 1 under stringent conditions and has a diaphorase activity Encoding the polypeptide
(6) a vector into which the above DNA has been incorporated,
(7) A transformant comprising the above vector or having the above DNA inserted into genomic DNA, and
(8) A method for producing diaphorase, comprising the steps of culturing the above-mentioned transformant in a medium, accumulating diaphorase in or in the transformant, and recovering the accumulated diaphorase.

The diaphorase of the present invention is preferably produced by isolation and purification from a culture of transformants containing an expression vector carrying a DNA encoding the protein or the DNA inserted into genomic DNA. can do.

In the above, the term "DNA encoding a protein" refers to DNA from which the protein is obtained when it is expressed, ie, DNA having a base sequence corresponding to the amino acid sequence of the protein. Therefore, DNAs that differ due to the degeneracy of codons are also included.

The identity of the DNA of (B) and the base sequence shown in SEQ ID NO: 1 is 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 98 % Or more, more preferably 99% or more.

Various methods are known as methods for calculating the identity of base sequences. For example, it can be calculated using a commercially available or analysis tool available through a telecommunication line (Internet).
In this specification, nucleotide sequence homology is obtained by performing a search using blastn as a program and setting various parameters as default values in the homology algorithm Advanced BLAST 2.1 of the National Center for Biotechnology Information (NCBI). Calculate the value (%) of

In the above (C), "stringent conditions" generally means conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such stringent conditions are known to those skilled in the art and can be set, for example, with reference to Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York).
As used herein, "stringent conditions" refer to the conditions shown below.
50% formamide, 5 × SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) as a hybridization solution, 1 × Denhardt's solution, 1% SDS, 10% dextran sulfate, 10 μg / mL denatured salmon sperm DNA, Use 50 mM phosphate buffer (pH 7.5)).
Although some hybridizing DNA under such conditions may have a stop codon generated along the way, or may have lost activity due to mutation of the active center, these can be incorporated into a commercially available active expression vector. It can be expressed in a suitable host and easily removed by measuring the enzyme activity by a known method.

The host cell into which the DNA encoding the diaphorase of the present invention is introduced is not particularly limited as long as a recombinant expression system is established as described later, but preferably bacteria such as E. coli and Bacillus subtilis, actinomycetes And microbial hosts such as filamentous fungi, bacilli and yeast, as well as insect cells, animal cells, higher plants and the like.

Examples of vectors include E. coli-derived plasmids such as pBR322, pBR325, pUC18, pUC19, pBluescript SK (-), pBluescript KS (+), and yeast-derived plasmids such as pSH19 and pSH15, such as Bacillus subtilis-derived plasmids. pUB110, pTP5, pC194 and the like can be mentioned. Also, as viruses, bacteriophages such as λ phage, papovaviruses such as SV40, bovine papilloma virus (BPV), retroviruses such as Moloney murine leukemia virus (MoMuLV), adenovirus (AdV), adeno-associated virus (AAV), Animal and insect viruses such as the vaccinia virus, baculovirus and the like are exemplified.

In particular, it is preferable to use a diaphorase expression vector in which a DNA encoding a diaphorase is disposed under the control of a functional promoter in a host cell of interest, and the vector used is a prokaryotic and / or eukaryotic cell. A promoter region that can function in various host cells to control transcription of a gene located downstream thereof (eg, trp promoter, lac promoter, lecA promoter, etc. when host is E. coli, SPO1 when host is Bacillus subtilis) Promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, G diaphorase promoter, ADH promoter, etc. When the host is mammalian cell, SV40 derived early promoter, MoMuLV derived long terminal Peat, a viral promoter such as an adenovirus-derived early promoter, and a transcription termination signal of the gene, ie, a terminator region, wherein the promoter region and the terminator region are at least one restriction enzyme recognition site, preferably the vector There is no particular limitation as long as it is linked via a sequence containing a unique restriction site that cuts at only the site, but a selectable marker gene (tetracycline, ampicillin, kanamycin, hygromycin, etc.) for selection of transformants. It is preferable to further contain a gene that imparts resistance to a drug such as phosphinothricin, a gene that complements the auxotrophic mutation, and the like. Furthermore, when the DNA encoding the inserted diaphorase does not contain the initiation codon and the termination codon, the initiation codon (ATG or GTG) and the termination codon (TAG, TGA, TAA) are respectively downstream of the promoter region and the terminator region The vector contained upstream of is preferably used.

When using bacteria as the host cell, in general, the expression vector should contain, in addition to the above promoter and terminator regions, a replicable unit capable of autonomous replication in the host cell. Also, the promoter region includes operator and Shine-Dalgarno (SD) sequences in the vicinity of the promoter.

When yeast, an animal cell or an insect cell is used as a host, the expression vector preferably further includes an enhancer sequence, 5 'and 3' untranslated regions of diaphorase mRNA, a polyadenylation site, and the like.

Examples of host organisms into which the recombinant vector has been prepared include bacteria such as E. coli and Bacillus subtilis for which recombinant expression systems have been established, microbial hosts such as actinomycetes, bacilli and yeast, insect cells, animal cells, higher plants, etc. Among them, it is preferable to use E. coli which is excellent in protein expression ability. As a method for introducing a recombinant plasmid, in addition to the introduction by electroporation, introduction by heat shock is also possible if the host is made competent by treatment with a chemical such as calcium chloride. Selection for the presence or absence of transfer of the target recombinant plasmid into the host vector may be carried out by searching for a microorganism that simultaneously expresses the marker represented by various drug resistance genes of the vector holding the target DNA and the diaphorase activity, For example, a microorganism that grows in a selective medium based on a drug resistance marker and expresses diaphorase may be selected.

Preferred host-vector systems for producing the diaphorase of the present invention include combinations of E. coli and pUC19, combinations of yeast and pSH19, and the like. Among them, a preferred combination is E. coli and pUC19.

The diaphorase of the present invention can be produced by culturing, in a medium, a transformant containing the diaphorase expression vector prepared as described above, and recovering diaphorase from the resulting culture.

The medium to be used preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of host cells (transformants). As carbon sources, for example, glucose, dextran, soluble starch, sucrose etc., as inorganic nitrogen sources or organic nitrogen sources, for example, ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large Bean meal, potato extract and the like are exemplified. Also, if desired, other nutrients (eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.), etc.) may be contained. .

Culturing is performed by methods known in the art. Specific media and culture conditions to be used depending on the host cell are exemplified below, but the culture conditions in the present invention are not limited to these.

When the host is bacteria, actinomycetes, yeast, filamentous fungi, etc., for example, a liquid medium containing the above-mentioned nutrient source is suitable. Preferably, it is a medium having a pH of 5-9. When the host is E. coli, LB medium, M9 medium [Miller. J. , Exp. Mol. Genet, p. 431, Cold Spring Harbor Laboratory, New York (1972)] and the like. The cultivation can be carried out usually at 14 to 43 ° C. for about 3 to 72 hours, while aeration and stirring as necessary. When the host is Bacillus subtilis, the reaction can be carried out usually at 30 to 40 ° C. for about 16 to 96 hours, while aeration and stirring as necessary. When the host is yeast, for example, Burkholder minimal medium [Bostian. K. L. et al, Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)], and the pH is preferably 5-8. The culture is usually carried out at about 20 to 35 ° C. for about 14 to 144 hours, and if necessary, aeration and agitation can be carried out.

When the host is an animal cell, the medium is, for example, minimal essential medium (MEM) [Science, 122, 501 (1952)] containing about 5 to 20% fetal bovine serum, Dulbecco's modified Eagle's medium (DMEM) [Virology, 8 , 396 (1959)], RPMI 1640 medium [J. Am. Med. Assoc. , 199, 519 (1967)], 199 medium [Proc. Soc. Exp. Biol. Med. , 73, 1 (1950)] can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually performed at about 30 to 40 ° C. for about 15 to 72 hours, and may be aerated or stirred as needed.

When the host is an insect cell, for example, Grace's medium containing fetal bovine serum [Proc. Natl. Acad. Sci. USA, 82, 8404 (1985)] and the like, and the pH thereof is preferably about 5-8. Culturing is usually carried out at about 20 to 40 ° C. for 15 to 100 hours, and if necessary, aeration and / or stirring can be carried out.

Purification of diaphorase can be carried out by combining various separation techniques usually used depending on the fraction in which the diaphorase activity is present.
The diaphorase present in the culture medium is obtained by centrifuging or filtering the culture to obtain a culture supernatant (filtrate), and from the culture supernatant, for example, salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration By appropriately selecting and performing known separation methods such as non-denaturing PAGE, SDS-PAGE, ion exchange chromatography, hydroxylapatite chromatography, affinity chromatography, reverse phase high performance liquid chromatography, isoelectric focusing, etc. You can get it.

On the other hand, the diaphorase present in the cytoplasm is collected by centrifuging or filtering the culture to collect the cells, and suspending it in a suitable buffer, for example, sonication, lysozyme treatment, freeze-thawing, osmotic shock, and / or triton After disrupting (dissolving) the cells and organelle membrane by surfactant treatment such as X100, etc., the debris is removed by centrifugation, filtration or the like to obtain a soluble fraction, and the soluble fraction is the same as above. It can be isolated and purified by treatment with a method.

In the above purification step, treatments such as membrane concentration, concentration under reduced pressure, and addition of an activator and a stabilizer can be performed as necessary. The solvent used in these steps is not particularly limited, but various buffers typified by K-phosphate buffer having buffer capacity in the range of about pH 6 to 9, Tris-HCl buffer, GOOD buffer and the like are preferable. .

When the diaphorase thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein is obtained as a salt, a method known per se Alternatively, the salt can be converted to an educt or other salt by a method analogous thereto.

The purified enzyme may be in liquid form for industrial use, but may be powdered or further granulated. The liquid enzyme is powdered by freeze-drying according to a conventional method. When provided in liquid form, it is preferable to contain a buffer, metal salt and preservative, and it is preferable to contain an antifreezing agent, surfactant and the like as required. As the buffer, phosphate, tris (hydroxymethyl) aminomethane hydrochloride, triethanolamine hydrochloride, a buffer of GOOD, etc. are suitably selected, but not limited thereto. The concentration of the buffer is not particularly limited as long as it can keep the pH of the solution constant, but is preferably in the range of 1 mM to 500 mM, and more preferably in the range of 2 mM to 200 mM. In addition, the pH of the solution is preferably in the range that can stably maintain the activity of diaphorase, and is preferably in the range of pH 6-9. Examples of antiseptics include, but are not limited to, azides, antibiotics, procrine 150, procrine 300 and the like. Further, as the antifreezing agent, non-proteins such as glycerin and dimethylsulfoxide are preferable. Furthermore, when a surfactant is added, Triton X-100, Tween 20, Tween 80, Emulgen A60, Emulgen 430, Brij 35, etc. are preferably selected.

3. Use of Diaphorase As another aspect of the present invention, the following may be shown.
(9) A product containing the diaphorase of the present invention described above

The diaphorase of the present invention can be applied to various products.
In the present specification, the term "product" means a product that constitutes a part or all of a set used by a user for the purpose of carrying out a certain application and that includes the diaphorase of the present invention.

The product of the present invention can be applied to various applications, and its application is not particularly limited, but typically utilizing the principle of measuring a substrate such as NADH by diaphorase, for example, extracorporeal The reagents and kits for diagnosis can be exemplified.

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

When measuring LDH, NADH generated by the LDH reaction reduces nitrotetrazolium blue etc via the diaphorase and returns to NAD itself to form formazan dye, so the activity of LDH is determined by colorimetry. You can determine the value.
Even in the case of measuring bile acid, the reaction proceeds with 3-α-hydroxysteroid dehydrogenase using bile acid as a substrate to generate NADH. Therefore, the bile acid can be determined by colorimetrically determining it in the same manner as described above. The concentration can be determined.
Even in the case of measuring BCAA, the reaction proceeds with leucine dehydrogenase using BCAA as a substrate to generate NADH. Therefore, the concentration of BCAA can be determined by performing colorimetric determination of this according to the same method as described above.

When CK is measured, since NADH is not directly generated in the CK reaction, ATP generated in the CK reaction is reacted with glucokinase together with glucose previously added to the reagent to form glucose 6 phosphate, and further glucose By designing a so-called conjugation reaction in which hexaphosphate is reacted with glucose hexaphosphate dehydrogenase together with NAD previously added to a reagent to produce NADH, it becomes possible to quantify the value of CK activity by diaphorase.
In the case of measuring TG, it is possible to quantify TG concentration by diaphorase by producing NADH using lipoprotein lipase using TG as a substrate and glycerol dehydrogenase as a coupling enzyme.
By appropriately designing such a conjugation reaction, it is also possible to measure the concentration or amount of biological components other than the above.

When glucose is measured, NADH generated by the glucose dehydrogenase reaction reduces an electron acceptor such as DCPIP via diaphorase to return itself to NAD, and the difference in absorbance caused by the change in the structure of DCPIP is The concentration of glucose can be determined by colorimetric determination. The sample containing glucose is not particularly limited, and examples thereof include blood, beverages, food and the like.

The form of the kit in the product of the present invention will be described in the case of a glucose assay kit applied to the above glucose measurement. The glucose assay kit of the present invention comprises the diaphorase of the present invention in an amount sufficient for at least one assay. Typically, the kit comprises, in addition to the diaphorase of the present invention, NAD-dependent glucose dehydrogenase, a buffer necessary for the assay, a mediator, a glucose standard solution for calibration curve generation, and guidelines for use. The diaphorase of the invention can be provided in various forms, for example as a lyophilised reagent or as a solution in a suitable storage solution.

The above-mentioned diaphorase of the present invention exhibits good stability even in the coexistence of an isothiazoline-based preservative. Therefore, in the above-mentioned reagent or kit of the present invention, an isothiazoline based preservative may coexist.

Example 1 Introduction of Mutation and Obtaining Transformant (1) Introduction of Mutation In order to replace the 119th cysteine in the amino acid sequence shown by SEQ ID NO: 2 with alanine, Geobacillus sp. Using pUC57 (patent document 1) into which 211 amino acid residues of Y4.1 MC1 were inserted as a template, a replication reaction was performed using mismatched primers shown in SEQ ID NOS: 5 and 6 and a PCR kit (KOD plus made by Toyobo Co., Ltd.). The reaction solution composition and reaction conditions were in accordance with the recommended conditions for ordinary PCR described in the manual attached to the kit. The template pUC57 is digested by adding 1 μL of the restriction enzyme DpnI to 50 μL of the reaction solution containing the replication product for 2 hours at 37 ° C., and the digested product is heat shocked into E. coli strain JM109 (competent high JM109 manufactured by TOYOBO) Transformation was carried out, SOC medium was added, shaken at 37 ° C. for 1 hour, and then coated on LB agar medium containing 100 μg / mL of ampicillin, and cultured at 30 ° C. overnight to form transformed colonies. Transformed colonies were inoculated in 5 mL of LB medium containing 100 μg / mL of ampicillin with a toothpick and cultured with shaking overnight at 30 ° C. From this culture solution, a plasmid was extracted and purified using a plasmid extraction kit (manufactured by TOYOBO, NPK-3). As a result of analyzing the sequence of the diaphorase gene in the plasmid, the codon TGC encoding the 119th cysteine was converted to GCG encoding alanine (ie, 355th T in the nucleotide sequence shown in SEQ ID NO: 1 to G, 356th G was converted to C, and 357th C was converted to G), respectively, and this plasmid was named as pUC-DI-1 C119A.
(2) Preparation of transformant Escherichia coli DH5α strain competent cell (Toyobo Co., Ltd.) is transformed with pUC-DI-1 C119A, and precultured at 37 ° C. for 1 hr in SOC medium. Then, it was developed on LB-amp agar medium to obtain the transformant, which is a colony. The resulting transformant was named Escherichia coli DH5α (pUC-DI-1 C119A).

Example 2 Preparation of culture supernatant Colonies of the transformant Escherichia coli DH5α (pUC-DI-1 C119A) obtained in Example 1 were treated with 5 mL of LB liquid medium (polypeptone 1.0%, one platinum ear test tube) The strain was inoculated into 0.5% of yeast extract, 1.0% of NaCl, pH 7.0) plus 100 μg / mL of ampicillin and cultured at 30 ° C. for 16 hours. At the end of culture, the cells are collected by centrifugation, suspended in 50 mM phosphate buffer (pH 7.5), sonicated with a sonicator, and further centrifuged to obtain a supernatant as a crude enzyme solution Got as.

Example 3 Confirmation of diaphorase activity The diaphorase activity in the crude enzyme solution obtained in Example 2 was compared with the above 1-1. It measured using the measuring method of the diaphorase activity shown to.
As a result, it was confirmed that diaphorase activity is present in a crude enzyme solution derived from the transformant Escherichia coli DH5α (pUC-DI-1 C119A).

Example 4 Preparation of mutant diaphorase in E. coli as a host (culture and purification)
The transformant E. coli DH5α (pUC-DI-1 C119A) obtained in Example 1 was inoculated in 5 mL of pre-preculture LB liquid medium (containing 100 μg / mL of ampicillin), The shaking culture was carried out at 30 ° C. and 180 rpm for 16 hours. After completion of the pre-pre-incubation, 600 μL of the culture solution was inoculated into 60 mL of B liquid medium (containing 100 μg / mL of ampicillin) in a 500 mL Sakaguchi flask, and shake cultured overnight at 30 ° C. and 180 rpm. 6 L production medium (Mist P1G 4.2%, glycerol 0.4%, KH2PO4 0.23%, K2 HPO4 1.25%, adecanol 0.2%, ampicillin sodium 100 μg / 100 μg / ml) in a 10 L volume jar fermenter. The whole amount was added to mL, pH 7.0), and culture was carried out with stirring and aeration at a temperature of 30 ° C. for 66 hours with aeration amount of 2 L / min, stirring at 380 rpm. This produced 1100 U / mL diaphorase.
Next, the obtained culture solution was dispensed into a 500 mL centrifuge tube, centrifuged at 8000 rpm for 30 minutes with a high-speed cooling centrifuge, and the supernatant was decanted to obtain bacterial cells. The cells were suspended in 1.0 L of 20 mM potassium phosphate buffer (pH 7.5), and crushed with a French press crusher at a pressure of 80 MPa. Subsequently, a 5% polyethyleneimine solution (pH 7.5) prepared by adjusting ethyleneimine (polymer) (Nacalai Tesque, Inc.) to a polyethyleneimine content of 5% is prepared, and 5 to the crushed liquid amount The mixture was gradually added to 100%, stirred at room temperature for 30 minutes, and then filter aid was used to remove excess precipitate. Next, ammonium sulfate (manufactured by Sumitomo Chemical Co., Ltd.) was gradually added so as to be 0.2 saturated, ammonium sulfate fractionation was performed, and a solution of a protein having diaphorase activity was recovered. Furthermore, ammonium sulfate is gradually added to a final concentration of 0.6, and ammonium sulfate fractionation is performed again to precipitate diaphorase, which is recovered using a filter aid, and the aid containing diaphorase is 20 mM potassium phosphate buffer It suspended in liquid (pH 7.5). Next, the mixture was heated at 60 ° C. for 16 hours, and the precipitate formed by the aggregated protein was removed using a filter aid to obtain a supernatant containing diaphorase. The supernatant is concentrated using a hollow fiber membrane (Spectrum Laboratories, Inc.) with a molecular weight cut off of 10,000, and then desalted using a gel of Sephadex G-25, and at the same time, 50 mM Tris-HCl from 20 mM potassium phosphate buffer The buffer was replaced with buffer (pH 8.5). Then, gradient elution was performed by applying a 400 mL DEAE Sepharose FastFlow (manufactured by GE Healthcare) column equilibrated with 50 mM Tris-HCl buffer (pH 8.5) in advance and raising the NaCl concentration to 0.3 M with the same buffer. The Then, after eluting the eluted diaphorase fraction with a hollow fiber membrane with a molecular weight cut off of 10,000, desalt the concentrate using a gel of Sephadex G-25 and replace the buffer solution with 20 mM potassium phosphate buffer , 835 U / mg of purified enzyme was obtained.
In this example, the diaphorase “mutated diaphorase” obtained from the transformant Escherichia coli DH5α (pUC-DI-1 C119A) is described.

Next, the obtained purified enzyme was subjected to SDS-polyacrylamide gel electrophoresis (Phast System and PhastGelTM Gradient 10-15, manufactured by GE Healthcare Biosciences). Under the present circumstances, as a protein molecular weight marker, phosphorylase b (97,000 daltons), albumin (66,000 daltons), ovalbumin (45,000 daltons), carbonic anhydrase (30,000 daltons), trypsin inhibitor (20, 100 daltons) and alpha-lactalbumin (14,400 daltons) were used.
As a result, a single band was obtained for each of the enzymes, indicating that wild-type diaphorase and mutant diaphorase were sufficiently purified.

Example 5 Evaluation of stability for preservative MIT Using the obtained diaphorase enzyme solution, evaluation of stability for preservative MIT was performed. Evaluation is based on activity before and after storage in 50 mM HEPES buffer (pH 7.5) containing 5 U / mL diaphorase, 0.1% Tween 20, 0.05% MIT at final concentration for 1 to 2 weeks at 37 ° C. Based on the calculated residual activity of diaphorase. Combined with the conditions in which MIT is not added as a control, 1-1. The results obtained by the diaphorase activity measurement method described in Table 1 are shown in Table 1, and the results obtained by calculating the residual activity after 1 to 2 weeks with the activity immediately before storage as 100% are shown in Table 2.

As a result, Geobacillus sp. The mutant diaphorase derived from Y4.1 MC1 is Geobacillus sp. The stability against MIT was found to be twice as high as that of wild-type diaphorase derived from Y4.1 MC1.

The present invention is not limited to the description of the embodiments and examples of the above-mentioned invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive of the claims without departing from the scope of the claims.
The contents of articles, published patent publications, patent publications, etc. specified in the present specification are incorporated by reference in their entirety.

The diaphorase of the present invention is excellent in stability to MIT, and is not limited to the type of preservative, and can be generally used for in vitro diagnostic reagents and the like. Therefore, the diaphorase of the present invention is suitable for various applications.

Claims (8)

A polypeptide represented by any one of the following (1) to (3), and at 37 ° C., 50 mM HEPES buffer (pH 7) containing 0.05% isothiazoline preservative and 0.1% Tween 20: .5) Diaphorase, in which residual enzyme activity after sealed storage of a final concentration of 5 U / mL diaphorase for 1 week maintains 90% or more compared to the enzyme activity before treatment.
(1) In the polypeptide represented by the polypeptide (2) (1) consisting of an amino acid sequence in which the 119th amino acid of the amino acid sequence of SEQ ID NO: 2 is substituted with alanine, the amino acid of SEQ ID NO: 2 Furthermore, in the polypeptide represented by polypeptide (3) (1), which comprises an amino acid sequence in which 1 to 20 amino acids other than amino acid 119 in the sequence have been substituted, deleted, inserted and / or added. A polypeptide in which the portion other than the 119th amino acid in the amino acid sequence described in No. 2 is modified, and the identity of the amino acid sequence of said polypeptide and the amino acid sequence shown in SEQ ID NO: 2 is 90% or more Polypeptide The diaphorase before modification is Geobacillus sp. Y4.1 MC1, Geobacillus kaustophilus HTA 426, Geobacillus thermoleovorans, Geobacillus thermoglucasidasius, Geobacillus celdoxylos ilybacillusi, which is a Geobacillus topibacillusi, which is a bacillus bacillus bacile bacile ich, which is a bacillus bacillus bacillus bacilli G11 MC16, Geobacillus zalihae and Diaphorase according to claim 1 derived from microorganisms of any Geobacillus a genus selected from the group consisting of Geobacillus thermodenitrificans. Isothiazoline based preservatives are methylisothiazolinone (2-Methyl-4-isothiazolin-3-one; MIT), chloromethylisothiazolinone (5-Chloro-2-methyl-4-isothiazolin-3-one; CMIT) Octylisothiazolinone (2-n-Octyl-4-isothiazolin-3-one; OIT), dichlorooctylisothiazolinone (4,5-Dichloro-2-octyl-4-isothiazolin-3-one; DCOIT) and The diaphorase according to claim 1 or 2, which is any one selected from the group consisting of benzisothiazolinone (1,2-benzisothiazolin-3-one; BIT). The DNA in any one of the following (A)-(C).
(A) DNA encoding the amino acid sequence of diaphorase shown in claim 1
(B) In the base sequence shown in SEQ ID NO: 1, the base sequence corresponding to the amino acid modification site shown in (1) of claim 1 is modified to encode alanine, and further, a site other than the above modification site A DNA consisting of a nucleotide sequence having a mutation in at least 90% identity between the sequence and the nucleotide sequence shown in SEQ ID NO: 1 and encoding a polypeptide having diaphorase activity , and After leaving sealed for one week in a final concentration of 5 U / mL diaphorase in 50 mM HEPES buffer (pH 7.5) containing 0.05% isothiazolin preservative and 0.1% Tween 20 at 37 ° C. DNA encoding diaphorase, wherein the enzyme activity maintains 90% or more compared to the enzyme activity before treatment
(C) In the base sequence shown in SEQ ID NO: 1, the base sequence corresponding to the amino acid modification site shown in (1) of claim 1 is modified to encode alanine, and further, a site other than the above modification site a DNA having a base sequence having a mutation in the sequence, and hybridizes under stringent conditions to a nucleotide sequence complementary to SEQ ID NO: 1, with DNA encoding a polypeptide having diaphorase activity Sealed storage of diaphorase at a final concentration of 5 U / mL for 1 week in 50 mM HEPES buffer (pH 7.5) containing 0.05% isothiazoline preservative and 0.1% Tween 20 at 37 ° C. Enzymatic activity of the remaining enzyme encodes diaphorase, which maintains 90% or more compared to the enzyme activity before treatment DNA A vector into which the DNA according to claim 4 has been incorporated. A transformant comprising the vector according to claim 5 or having the DNA according to claim 4 inserted into genomic DNA. A method for producing diaphorase, comprising the steps of culturing the transformant according to claim 6 in a culture medium, accumulating diaphorase in or in the transformant, and recovering the accumulated diaphorase. A product comprising the diaphorase according to any one of claims 1 to 3.