JP2003274976A - Fructosylamine oxidase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new fructosylamine oxidase reactive with fructosylvaline, and to provide a method for producing the oxidase and a method for assaying the oxidase. <P>SOLUTION: The fructosylamine oxidase is produced by culturing a microorganism belonging to Arthrobacter sp., or a transformant obtained by transforming a microorganism by a recombinant vector obtained by incorporating a DNA fragment containing a gene encoding the fructosylamine oxidase. The produced fructosylamine oxidase is specific to the fructosylvaline, and the kit sensor using the oxidase is useful for measuring glycosylated hemoglobin. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel fructosylamine oxidase (FAO). More specifically,
The present invention relates to a fructosylamine oxidase produced by a novel microorganism, and its production. The present invention also relates to a gene encoding a fructosylamine oxidase, said FAO
The present invention relates to a recombinant vector incorporating a gene fragment coding for, a transformant transformed with the recombinant vector, and a method for producing FAO by culturing the transformant. The present invention also relates to HbA1c using fructosylamine oxidase produced by a novel microorganism.
And fructosyl valine measurement kit and sensor

[0002]

2. Description of the Related Art Amino groups of a protein main chain and side chains are non-enzymatically bonded to a reducing end of a reducing sugar such as glucose,
It produces Amadori compounds, or glycated proteins. It is known that hemoglobin is glycated in blood to produce glycated hemoglobin (glycated hemoglobin; HbA1c). The presence ratio of HbA1c to hemoglobin is higher in diabetic patients than in healthy subjects, and
Since the blood level of bA1c reflects the blood glucose level in the past few weeks, the blood level of HbA1c is extremely important in clinical trials as an index for the diagnosis of diabetes and the blood glucose control of diabetic patients.

In HbA1c, since glucose is bound to valine at the N-terminal of the hemoglobin β chain, fructosyl valine can be used as a low molecular weight model compound of HbA1c. That is, using an enzyme having specificity for fructosyl valine, HbA
It is possible to assay 1c.

To date, enzymes that act on Amadori compounds have been isolated from various strains, and it has been suggested that these enzymes can be used to analyze glycoproteins such as glycoalbumin, HbA1c and fructosamine. (JP-A 61-268178, JP-A 61-280)
297, JP-A-3-155780, JP-A-5-1921
93, JP-A-7-289253, and JP-A-8-15467.
2, Agric. Biol. Chem. , 53 (1),
103-110, 1989, Agric. Biol. C
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J. Biol. Chem. , 269 (44), 2729
7-27302, 1994, Appl. Enviro
n. Microbiol. , 61 (12), 4487-
4489, 1995, Biosci. Biotech.
Biochem. , 59 (3), 487-491, 19
95, J. Biol. Chem. , 270 (1), 21
8-224, 1995, J. Biol. Chem. , 2
71 (51), 32803-32809, 1996,
J. Biol. Chem. , 272 (6), 3437-.
3443, 1997).

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel fructosylamine oxidase capable of reacting with fructosyl valine, a method for producing the same, and an analytical method using the same.

[0006]

Means for Solving the Problems As a result of screening a microorganism capable of assimilating fructosyl valine, the present inventors found that Arthrr producing fructosyl amine oxidase.
objecter sp. Was successfully isolated to complete the present invention. As a result of various studies to achieve the above-mentioned object, the present invention also shows that fructosylamine oxidase (F
FAO can be mass-produced by culturing the transformant obtained by transforming a microorganism with a recombinant vector incorporating a DNA fragment containing a gene encoding AO) and collecting FAO from the culture. The inventors have found the present invention and have reached the present invention.

That is, the present invention is based on Arthrobacter
Microorganisms belonging to the genus er and having the ability to produce fructosylamine oxidase, particularly Arthrobacter sp.
Provided is a strain FV1-1 (FERMP-18754), and a method for producing fructosylamine oxidase, which comprises culturing the microorganism and collecting the enzyme from the culture.

The present invention is also a fructosyl amine oxidase produced by the genus Arthrobacter.

The present invention further provides Arthrobacter
sp. Is a fructosylamine oxidase produced by.

The present invention also relates to Arthrobacter
sp. It is a fructosyl amine oxidase produced by FV1-1 strain (FERM P-18754).

The present invention also provides the following protein (a) or (b) having fructosylamine oxidase activity. (A) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 (c) the amino acid sequence (a), in which one or several amino acid sequences are deleted, substituted or added, and A protein having fructosylamine oxidase activity.

Further, the present invention is a gene encoding a fructosylamine oxidase which is the following protein (a) or (b). (A) a protein consisting of the amino acid sequence described in SEQ ID NO: 1 (b) the amino acid sequence (a), consisting of an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added, and Protein with fructosylamine oxidase activity

The present invention is the following sequence (c) or (d), which is a gene encoding a fructosylamine oxidase. (C) DNA consisting of the base sequence described in Sequence Listing / SEQ ID NO: 2 (d) In the sequence of (c) above, one or several bases have been deleted, substituted or added, and fructosylamine D encoding a protein having oxidase activity
NA.

The present invention also relates to the sequence ValProLys
ArgHisPheTyrProGlyGlu Ser
TrpValSerLeu Pro GluLeuGl
It is a fructosyl amine oxidase containing y Asp.

The present invention is also described above.

[0007]

At Arthrobacter s
p. It is a gene encoding a fructosylamine oxidase derived from FV1-1 strain or Arthrobacter genus.

The present invention is also described above.

[0007]

A recombinant vector containing a gene encoding a fructosylamine oxidase.

The present invention also provides the above.

[0016] A transformant or transductant transformed with the recombinant vector according to [1].

The present invention also provides the above.

A method for producing a fructosylamine oxidase, which comprises culturing the transformant according to item 1 and collecting fructosylamine oxidase from the culture, is provided.

The present invention also provides the above.

A fructosylamine oxidase produced by the method described in 1.

The present invention is also described above.

[0007]

A method for spectroscopic analysis of fructosylamine compounds containing fructosylamine oxidase is provided.

The present invention is also described above.

[0007]

An electrochemical analysis method for fructosylamine compounds containing fructosylamine oxidase is provided.

The present invention also provides a method for assaying HbA1c, which comprises degrading HbA1c in a sample to produce fructosyl valine, wherein
Provided is a spectroscopic analysis method comprising quantification using the described fructosylamine oxidase.

The present invention is also described above.

[0007]

A fructosyl valine spectroscopic assay kit containing fructosyl amine oxidase is provided.

The present invention is also described above.

[0007]

In HbA1c spectroscopic analysis method assay kit containing fructosylamine oxidase is provided.

The present invention is also described above.

[0007]

An electrochemical analysis method of fructosyl valine is provided, which comprises using fructosyl amine oxidase in.

The present invention is also described above.

[0007]

[0018] In H., an electrochemical analysis method of HbA1c characterized by using fructosylamine oxidase, and an enzyme electrode characterized by the electrochemical analysis method, and the enzyme electrode are characterized by using. Provide an enzyme sensor.

[0027]

BEST MODE FOR CARRYING OUT THE INVENTION A novel microorganism, Arthrobacter sp. FV1-1 strain is
It is a strain newly isolated from a soil sample by the present inventor, and its mycological characteristics are as follows.

[Mycological properties] Gram stain Positive cell shape Short rod-coccus (diameter 0.3-0.
8 micrometer, length 0.3-0.8 micrometer) No spore formation Optimal growth temperature 30 ℃ Reduction of aerobic bacterial nitrate Negative pyrazine amidase Positive pyrrolidonyl allyl amidase Positive alkaline phosphatase negative β-glucuronidase negative β -Galactosidase positive α-glucosidase positive N-acetyl-β-glucosaminidase negative β-glucosidase positive catalase positive urease negative gelatin hydrolytic degradation positive [fermentability] glucose negative D-ribose negative D-xylose negative D-mannitol negative maltose negative lactose negative saccharose Negative Glycogen Negative [Assimilation] Glycerol Positive Erythritol Negative D-Arabinose Negative L-Arabinose Positive Ribose Negative D-Xylose Positive L-Xylose Negative Adonitol negative β-methyl-xyloside positive galactose positive D-glucose positive D-fucose positive D-mannitol positive L-sorbose negative rhamnose negative doursitol negative inositol positive mannitol positive sorbitol negative α-methyl-D-mannoside negative α-methyl-D glucoside negative N-acetylglucosamine-negative amygdalin-negative arbutin-negative esculin-positive salicin-positive cellobiose-positive maltose-positive lactose-positive melibiose-negative saccharose-positive trehalose-positive inulin-positive melezitose-positive D-raffinose-positive amidone-positive glycogen-positive xylitol-negative β-dentibiose

The glycoassimilation of this strain is similar to that of the genus Bacillus, but it does not form spores.
It is determined that it is not of the s genus. In addition, the cells are not curved and are in the form of bacilli or spheres and have the ability to hydrolyze gelatin, so that they are clearly distinguished from the genus Corynebacterium. From the above properties, Bergey's M
annual of Determinative Ba
When cteriology 9 ^th edition) with reference to the study, belong to the genus Arthrobacter, Arthorobacter sp. The strain was identified as This strain was deposited at the Patent Biological Depositary Center, National Institute of Advanced Industrial Science and Technology, under the deposit number FERMP-1.
Deposited as 8754.

Fructosylamine oxidase The novel fructosylamine oxidase of the present invention has the following properties: it oxidizes fructosyl valine in the presence of oxygen to give the corresponding α-ketoaldehyde and amino acid, and peroxidation. Produces hydrogen. This enzyme specifically oxidizes fructosyl valine as a substrate, and its reaction rate is about 50 times that in the case of using ε-fructosyl lysine as a substrate.

Up to now, there has been no report of fructosylamine oxidase from the genus Arthrobacter. Therefore, this enzyme is a novel enzyme.

Measurement of Enzyme Activity The fructosylamine oxidase activity can be measured by quantifying the amount of oxygen consumed or the amount of hydrogen peroxide generated by the enzymatic reaction. Various methods of quantifying oxygen and hydrogen peroxide are known in the art. For example, a constant temperature cell is charged with a buffer solution containing the fructosylamine oxidase of the present invention and a mediator and maintained at a constant temperature, a sample containing fructosyl valine is added thereto, and the color reaction of the redox dye is monitored. Thus, the activity of the fructosylamine oxidase of the present invention can be measured. As the mediator, potassium ferricyanide, ferrocene, osmium derivative, phenazine methosulfate and the like can be used. Alternatively, peroxidase can be used to quantify the hydrogen peroxide generated. As such a measurement system, peroxidase to 4-aminoantipyrine system is known, and is described in various experimental books (for example, Biotechnology Experiment Book, Biotechnology Society, edited by Baifukan 1992).

Method for Producing Enzyme The enzyme is produced by a bacterium of the genus Arthrobacter that produces fructosylamine oxidase, for example, Arthr.
objecter sp. , And more preferably Arthro
bacteria sp. By culturing the FV1-1 strain in a nutrient medium normally used for culturing microorganisms, more preferably in a nutrient medium containing fructosyl valine, the FV1-1 strain is produced and accumulated in the medium or in the microbial cells. be able to.

As a specific example of the method for producing the present enzyme, Arth
rober sp. FV1-1 strain (FERM P
-18754) as an example. The microorganism is cultured in an appropriate natural or synthetic medium containing fructosyl valine. It is preferable to increase the yield of the enzyme by inducing fructosylamine oxidase using a medium containing fructosyl valine as the sole nitrogen source. Examples of the medium containing fructosyl valine as the only nitrogen source include a minimal medium (for example, M9) supplemented with fructosyl valine. After collecting the bacterial cells from the culture solution by centrifugation or the like, the bacterial cells are crushed with a French press or the like. By subjecting this to ultracentrifugation, a water-soluble fraction containing fructosylamine oxidase can be obtained. The fructosylamine oxidase of the present invention is prepared by purifying the obtained water-soluble fraction by ion exchange chromatography, affinity chromatography, HPLC and the like.

Method for Preparing Gene The DNA fragment containing the gene encoding FAO of the present invention can be obtained from a FAO-producing bacterium. Specific examples of the FAO-producing bacterium include Arthrobacter sp. F
V1-1 strain or Arthrobacter histi
dinovorans, Arthrobacter
nicotinovorans, Arthrobac
ter keyser, Arthrobacter
aurescens, Arthrobacter li
cis, Arthrobacter simplex, A
Bacteria such as rthrobacterium globiformis can be mentioned. Above all, Arthroba
cter sp. Water-soluble FAO derived from FV1-1 strain is preferred.

The gene encoding the FAO may be extracted from these strains or may be chemically synthesized. Furthermore, a DNA fragment containing the FAO gene can be obtained by using the PCR method.

The gene encoding FAO is
For example, (a) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or (b)
FAO, which is a protein consisting of an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence (a) and having FAO activity
The gene encoding is.

Furthermore, (c) a DNA comprising the base sequence described in SEQ ID NO: 2 in the sequence listing, or (d) a deletion of one or several bases in the sequence of (c) above,
There is DNA that is substituted or added and that encodes a protein having FAO activity.

In the present invention, the method for obtaining the FAO-encoding gene includes the following methods.
For example, after separating and purifying a chromosome, DNA is cleaved using ultrasonic treatment, restriction enzyme treatment, etc., and a linear expression vector and both DNAs are ligated and closed at the blunt ends or cohesive ends with DNA ligase or the like. Construct a recombinant vector. After transferring the recombinant vector into a replicable host microorganism, screening is performed using the marker of the vector and expression of enzyme activity as indicators to obtain a microorganism carrying the recombinant vector containing the gene encoding FAO.

Then, the microorganism carrying the above recombinant vector is cultured, the recombinant vector is separated and purified from the cells of the cultured microorganism, and the gene encoding FAO can be collected from the expression vector. . For example, chromosomal DNA that is a gene donor is specifically collected as follows.

A lysate containing the FAO gene can be prepared by collecting a culture solution obtained by culturing the gene-donating microorganism with stirring for 1 to 3 days by centrifugation and then lysing it. . As a lysis method,
For example, it is treated with a lytic enzyme such as lysozyme, and if necessary, a protease or another enzyme and a surfactant such as sodium lauryl sulfate (SDS) are used in combination. Further, it may be combined with a physical disruption method such as freeze-thawing or French press treatment.

From the lysate obtained as described above, DN
Separation and purification of A can be performed according to a conventional method, for example, by appropriately combining deproteinization treatment such as phenol treatment or protease treatment, ribonuclease treatment, and alcohol precipitation treatment.

The method of cleaving the DNA separated and purified from the microorganism can be carried out by, for example, ultrasonic treatment or restriction enzyme treatment. Type II restriction enzymes that act on specific nucleotide sequences are preferred.

As a vector for cloning,
Suitable are those constructed for genetic recombination from phages or plasmids capable of autonomous growth in the host microorganism. Examples of the phage include Escherich
Lambd when ia coli is used as the host microorganism
a gt10, Lambda gt11, etc. are illustrated. Moreover, as a plasmid, for example, Escher
When ichia coli is used as the host microorganism, p
BR322, pUC18, pUC118, pUC19,
pUC119, pTrc99A, pBluescript
t, pET28 or Cosmid SuperCo
sI etc. are illustrated.

At the time of cloning, the above vector can be cleaved with the restriction enzyme used for cleaving the microorganism DNA which is the gene donor encoding FAO to obtain a vector fragment. DNA
It is not necessary to use the same restriction enzyme as that used for the cleavage of The method for ligating the microbial DNA fragment and the vector DNA fragment may be a method using a known DNA ligase. For example, after the cohesive ends of the microbial DNA fragment and the vector fragment are annealed, a suitable D
A recombinant vector of a microbial DNA fragment and a vector DNA fragment is prepared by using NA ligase. If necessary, after annealing, it can be transferred to a host microorganism to utilize a DNA ligase in vivo to prepare a recombinant vector.

The host microorganism used for cloning is not particularly limited as long as the recombinant vector is stable, capable of autonomous growth, and capable of expressing foreign gene. Generally, Escherichia coli
DH5 α, XL-1 BlueMR, Escheri
chia coli BL21 or the like can be used.

As a method for transferring the recombinant vector into the host microorganism, for example, the host microorganism is Escherichi
In the case of a coli, a competent cell method by calcium treatment or an electroporation method can be used.

The transformant microorganism thus obtained was cultured in a nutrient medium to give a large amount of FA.
O can be stably produced. Whether or not the target recombinant vector is transferred into the host microorganism is selected by selecting the target DNA.
It is only necessary to search for a microorganism that simultaneously expresses the drug resistance marker of the vector that retains and FAO activity. For example, a microorganism that grows in a selection medium based on a drug resistance marker and that produces FAO may be selected.

In another aspect, the isolated and purified F
It is also possible to determine the amino acid sequence of AO and clone the FAO structural gene from the genomic DNA prepared from the strain based on the information.

For example, after culturing the FV1-1 cells in an LB liquid medium, the obtained cells were treated with 10 mM NaCl and 20 mM.
It was suspended in Tris-HCl (pH 8.0), a buffer solution (hereinafter, TEbeffer) containing 1 m EDTA, and lyso was added.
Add zyme to 2.5 mg / ml, 37 ° C,
Incubated for 1 h. Further 10% SDS solution, 1
Proteinase k adjusted to 0 mg / ml was added, stirred, and incubated at 37 ° C. for 1 h. 5MNa to this
After adding Cl and CTAB and incubating at 65 ° C. for 10 minutes, an equal amount of phenol-chloroform was added and the mixture was centrifuged to collect the supernatant. To this supernatant was added 0.6 times the amount of isopropanol and the mixture was left at -20 ° C for 2 hours. After centrifugation, the supernatant was discarded, washed with 70% ethanol, and vacuum dried. This is dissolved in a mixed solution of Rnase and ultrapure water, and the mixture is kept at 37 ° C for 1 hour.
By incubating, the genomic DNA of the strain can be prepared.

FA purified by the method described above
After freeze-drying the O sample, ultrapure water was added to the 145 μg sample so that the protein concentration was 2 mg / ml, and SDS-PAGE (10% polyacrylamide gel) was performed using an electrophoresis apparatus. After SDS-PAGE 5 of the gel
Cut out to a size of × 5 cm and perform blotting on a PVDF membrane for 2 h using Fast System ^™ . Further, the PVDF membrane is stained with Coomassie blue, the band of interest is cut out, and the N-terminal amino acid sequence is analyzed by a conventional method to determine the N-terminal sequence of the FAO.

Genomic DN of FV1-1 strain prepared above
5 μg of A, EcoRI, HindIII, Pst
A template DNA for PCR amplification can be prepared by digesting with I and Kpnl at 37 ° C. for overnight, respectively. Dilute this sample 50 times with ultrapure water and then add 4μ from the diluted solution.
l is mixed with 4 μl of solution I and self-ligated at 16 ° C.

Using the primers shown below from the amino acid sequences obtained by the N-terminal amino acid sequence analysis, inverse PCR was performed using the FV1-1 strain genome, which had been self-ligated previously, as a template to obtain the FAO structural gene. Can be prepared. That is, a forward primer (NHNF11) was designed from amino acid residues 11 to 20, and a reverse primer (NHNR10) was designed from amino acid residues 3 to 10. NHNF11 (29mer) 5'-GGCGGGCATCCTGGGCGTCCKCCA
CCGCNGT-3'NHNR10 (24mer) 5'-GCCRATGACGGCGACRTGYTTN
GT-3 ′ However, * K = T + GS = C + G Y = C + TR =
A + G N = A + C + G + T This was subjected to PCR amplification under the following reaction conditions. 94 ° C. 1 minute 60 ° C. 30 seconds 72 ° C. 30 seconds or more in 1 cycle 98 ° C. 30 seconds 60 ° C. 30 seconds 72 ° C. 7 minutes 72 ° C. 8 minutes or more in 25 cycles Taq polymerase used TaKaRaLATaq ^™ .

The target gene can be obtained by analyzing the base sequence of the PCR-amplified fragment thus obtained.
That is, the nucleotide sequence of the amplified fragment obtained by PCR is analyzed by TA cloning and the primer walking method. The analysis of the nucleotide sequence is performed by sequentially repeating the operation of using the already analyzed nucleotide sequence information as a starting point (sequencing primer) for the next sequence analysis. For example, the primers shown below can be used. Forward primer 5'-GGAATCGAGCGTTTCGGCAA-3 'from NFUP69 (20mer) 132base Forward primer 5'-GATGAGATCGAGTCGGTCAC- from NF111 (20mer) 696base.
Reverse primer 5'-CGGACCAGCAGTTTGGAGTC- from 3'NR110 (21mer) 695base.
Reverse primer 5'-GCCGATGACGGGCGACGTGCTTG from 3'NHNR10 (24mer) 367base.
Forward primer 5'-CTGGATCACGACTGGTACG-3 'from GT-3' NF261 (20mer) 1107base.

Further, based on the nucleotide sequence obtained here, FV1-1 was used by using the forward primer (NF110) and reverse primer (NFUPPF69) shown below.
PCR is performed on the genome of the strain to analyze the structural gene of the enzyme. NFUP69 (20mer) 5'-GATGAGATCGAGTCGGTCAC-
3'NR110 (21mer) 5'-CGGAGCCAGCAGTTTGGAGTC-
The 3'reaction conditions were as follows.

The base sequence of the FAO gene obtained by the above method was decoded by a fully automatic base sequence analyzer by a conventional method. Further, the amino acid sequence of FAO was estimated from the nucleotide sequence determined as described above.

As described above, FAO once selected
Transfer of a recombinant vector carrying a gene to a recombinant vector capable of replicating in a microorganism is carried out from the recombinant vector carrying the FAO gene by FA or restriction enzyme or PCR method.
This can be easily carried out by recovering the O gene DNA and ligating it with another vector fragment. For transformation of microorganisms with these vectors, the competent cell method by calcium treatment, the electroporation method, or the like can be used.

For example, for the FAOD structural gene, a forward primer (N
NF1, NNF2), reverse primer (NCR1)
Is designed and PCR amplification of FAOD structural gene is performed. The obtained PCR amplified fragment was digested with AflIII and XhoI, and the expression vector pET28a (+) was digested with Nco.
After digestion with restriction enzymes I and XhoI, each sample was gel-purified and ligated for 2 hours at 16 ° C.
Subcloning into 5α. After confirming the plasmid having the insert, the expression host BL21 (DE3) is transformed to obtain transformed E. coli that recombinantly produces FAO.

Regarding the culture form of the transformant host microorganism, the culture conditions may be selected in consideration of the nutritional physiological properties of the host, and in many cases liquid culture is carried out. Industrially, it is advantageous to carry out aeration stirring culture.

As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, and examples thereof include glucose, sucrose, lactose, maltose, lactose, molasses, and pyruvic acid. The nitrogen source may be any available nitrogen compound, and examples thereof include peptone, meat extract, yeast extract, casein hydrolyzate, and soybean meal alkali extract. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese and zinc, specific amino acids, specific vitamins and the like are used as necessary.

The culture temperature can be appropriately changed within the range in which the bacteria grow and FAO is produced, but it is preferably about 20 to 42 ° C. The culture time varies slightly depending on the conditions, but FAO
The culture may be completed at an appropriate time in consideration of the time when the maximum yield is reached, and it is usually about 12 to 72 hours. The pH of the medium may be appropriately changed within the range in which the bacteria grow and FAO is produced, but the pH is preferably in the range of about 6.0 to 9.0.

The culture solution containing the FAO-producing cells in the culture can be directly collected and used, but in general, when FAO is present in the culture solution, filtration and centrifugation are performed. It is used after separating the FAO-containing solution and the microbial cells by, for example, When FAO is present in the microbial cells, the microbial cells are collected from the obtained culture by means such as filtration or centrifugation, and then the microbial cells are disrupted by a mechanical method or an enzymatic method such as lysozyme. ,
If necessary, a chelating agent such as EDTA and a surfactant are added to solubilize FAO, and FAO is separated and collected as an aqueous solution.

The FAO-containing solution obtained as described above is concentrated under reduced pressure, membrane concentrated, salted out with ammonium sulfate, sodium sulfate or the like, or fractionally precipitated with a hydrophilic organic solvent such as methanol, ethanol or acetone. It may be precipitated by the method. Further, heat treatment and isoelectric point treatment are also effective refining means. afterwards,
Purified FAO can be obtained by performing gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography.

Separation by column chromitography,
It can be purified to obtain a purified enzyme preparation. The purified enzyme preparation is preferably purified so as to show a single band by electrophoresis (SDS-PAGE).

The purified enzyme obtained as described above can be pulverized and distributed by, for example, freeze-drying, vacuum drying or spray drying.

An example of FAO of the present invention has the following physicochemical properties. Thermal stability: about 50 ° C or less (pH 7.5, treatment for 30 minutes) Optimum temperature: about 30 to 50 ° C Optimum pH: 6.0 to 8.5 Molecular weight: 40 kDa (molecular weight by SDS-denaturing polyacrylamide gel electrophoresis) It is water-soluble (as measured) and 60 kDa (by gel filtration chromatography).

In the present invention, the FAO activity was measured by 10
POD / Ph in 0 mM phosphate buffer pH 7.0
enol / 4-a. a. System, hydrogen peroxide produced by the reaction of FAO is reduced by POD, in which case hydrogen peroxide and 4-a. a. Oxidatively condenses, resulting in 500n of quinoneimine dye
tracking the change in absorbance at m (ε = 12.88 × 10 ³ ),
The rate of increase in the absorbance was defined as the reaction rate of the enzyme. At this time, 0.5 μmol of quinoneimine per minute
The enzyme activity produced by e dye was 1 U.

A fructosyl valine measurement kit can be constructed using this enzyme. In addition to the fructosylamine oxidase according to the present invention, a buffer necessary for measurement, a suitable mediator, and an enzyme such as peroxidase if necessary, a standard of fructosyl valine or a derivative thereof for preparing a calibration curve. Includes solutions, as well as directions for use. The fructosylamine oxidase according to the invention can be provided in various forms, for example as a lyophilized reagent or as a solution in a suitable storage solution.

Furthermore, using the enzyme of the present invention, HbA1c
An assay kit can be constructed. HbA1c can be assayed by enzymatically or chemically decomposing HbA1c to produce fructosyl valine, which can be quantified using the fructosylamine oxidase of the present invention. Therefore, the HbA1c assay kit of the present invention further comprises a hydrolytic reagent or a proteolytic enzyme in addition to the fructosyl valine measuring kit described above.

Further, using the fructosylamine oxidase of the present invention, a sensor for measuring fructosyl valine and a sensor for measuring HbA1c can be constructed. That is, the concentration of the substrate fructosyl valine can be determined by measuring the oxygen consumed or the hydrogen peroxide generated by the action of the fructosylamine oxidase of the present invention. Various sensor systems for measuring oxygen or hydrogen peroxide are known in the art. An oxygen electrode, a carbon electrode, a gold electrode, a platinum electrode or the like is used as an electrode, and the enzyme of the present invention is immobilized on this electrode. Examples of the immobilization method include a method using a crosslinking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer, and the like, which are used in combination. Good.

When an oxygen electrode is used, the enzyme of the present invention is immobilized on the electrode surface, inserted into a buffer solution and kept at a constant temperature. A sample is added here and the decrease value of the current is measured. When measuring with an amperometric system using a carbon electrode, a gold electrode, a platinum electrode, etc., these electrodes on which an enzyme is immobilized are used as a working electrode, and a counter electrode (eg platinum electrode) and a reference electrode (eg Ag / (AgCl electrode)
At the same time, it is inserted into a buffer containing a mediator and kept at a constant temperature. A constant voltage is applied to the working electrode, a sample is added, and the increase value of the current is measured. As the mediator, potassium ferricyanide, ferrocene, osmium derivative, phenazine methosulfate and the like can be used.

Further, as a method of measuring with an amperometric system using a carbon electrode, a gold electrode, a platinum electrode or the like, there is a system using an immobilized electron mediator. That is, as the working electrode, an enzyme and an electron mediator such as potassium ferricyanide, ferrocene, an osmium derivative, or phenazine methosulfate immobilized on a polymer matrix by adsorption or covalent bonding are used as a counter electrode (for example, a platinum electrode) and a reference electrode. Along with the electrode (eg Ag / AgCl electrode), it is inserted into a buffer solution and kept at a constant temperature. A constant voltage is applied to the working electrode, a sample is added, and the increase value of the current is measured.

When any of the electrodes is used, the fructosyl valine concentration in the sample can be determined according to the calibration curve prepared by using the standard fructosyl valine solution.

When used as a sensor for measuring HbA1c, the sensor for measuring fructosyl valine described above is further combined with a membrane on which a proteolytic enzyme (eg, protease) is immobilized to construct a composite sensor. Such a structure of a composite sensor using a continuous reaction of a combination of a plurality of enzymes is well known in the art, and for example, Biosensors-Fun.
DAMENTAL AND APPLICATIONS
-Anthony P. F. Tuner, Isao K
arube and Georg S. Wilso
n, OxfordUniversity Press1
987.

[0075]

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1 Production of fructosylamine oxidase Arthrobacter sp. FV1-1 strain is M
9 medium (Na ₂ HPO ₄ 0.6%, KH ₂ PO ₄ 0.
3%, NaCl 0.5%, MgSO ₄ 0.01%,
CaCl ₂ 0.01%, D-glucose 1%) 150
The cells were cultured in a medium containing fructosyl valine at a final concentration of 0.52% as a nitrogen source. Fructosyl valine is described in Keil. P (Acta Chemical Scan)
dinavica. B, 39, 191-193, 198
It was synthesized according to the method described in 5). Approximately 20 g of the bacterial cells obtained here were treated with 100 mM phosphate buffer (pH 7.
0), crushed 6 times with a French press, diluted with the same buffer, and centrifuged (10000 xg, 4 ° C,
15 min) to precipitate unbroken cells and collect the supernatant. The resulting supernatant is ultracentrifuged (69800 xg, 4
C, 90 min). Ammonium sulfate was added to the supernatant obtained here so as to be 30% saturated, and the mixture was stirred at 4 ° C for 1 hour, and then subjected to ultracentrifugation (26000 xg, 4 ° C, 3
0 min) and the supernatant was collected. Add 70% to this supernatant
Ammonium sulfate was added to achieve saturation, and the mixture was stirred for 1 hour as above, followed by ultracentrifugation to recover the precipitate. After dissolving this precipitate with 10 mM phosphate buffer (pH 7.5),
It dialyzed with the same buffer overnight. After dialysis, it was filtered using a nitrocellulose filter, and this was used as a water-soluble fraction in the following purification step.

(1) Anion exchange chromatography a. Packed column DEAE- for anion chromatography equilibrated with 10 mM phosphate buffer (pH 7.5)
The sample obtained above was adsorbed on 5PW (5.0 mm ID × 5 cm, Tosoh Corporation). After equilibration with 10 mM phosphate buffer (pH 7.5) having a column volume of 3 times, FAO was eluted with 10 mM phosphate buffer (pH 7.5) containing 0.75 M NaCl. The flow rate was 1 ml / min, and the eluate was collected every minute. An absorption wavelength of 280 nm was used for detection of the eluate.
The obtained active fraction was treated with 10 mM phosphate buffer (pH 7.
It was desalted by dialysis with 5) overnight at 4 ° C.

B. a. The sample obtained in step 1 was adsorbed on a packed column for anion chromatography Resource Q (1 ml, Amersham Biotech Co., Ltd.) equilibrated with 10 mM phosphate buffer (pH 7.5), and the column volume was 3
After equilibration with a double amount of 10 mM phosphate buffer (pH 7.5), FAO was eluted with 10 mM phosphate buffer (PH 7.5) containing 0.75 M NaCl. The flow rate and the detection of the eluate were carried out under the same conditions as in a).

(2) Hydrophobic Chromatography Ammonium sulfate was added to the active fraction obtained in (1) -b so as to be 40%, and the mixture was equilibrated by stirring at 4 ° C. for 1 hour to obtain a nitrocellulose filter. It was filtered with. This active fraction was equilibrated with 10 mM phosphate buffer (pH 6.6) containing 1.5 M ammonium sulfate, and packed column for the hydrophobic column chromatography Resource.
ePhe (1 ml, Amersham Biotech Co., Ltd.) was allowed to adsorb. After equilibration with 10 mM phosphate buffer (pH 6.6) containing 1.5 M ammonium sulfate in a column volume three times, FAO was eluted with 10 mM phosphate buffer (pH 6.6). Flow rate is 2ml / min, 1
The eluate was collected every minute. The eluate was detected under the same conditions. It is dialyzed against 10 mM phosphate buffer (pH 7.0) overnight at 4 ° C. to obtain a purified enzyme of FAO.

Example 2 Measurement of enzyme activity The fructosylamine oxidase activity was measured by a measurement system using a peroxidase to 4-aminoantipyrine system. In the presence of 2 mM phenol, 1.5 mM 4-aminoantipyrine, 2 U / ml peroxidase, referring to the description given in the biotechnology experiment manual (Bifukan, edited by Japan Society for Biotechnology, p.22, 1992). , 505
The fructosyl amine oxidase activity was measured by monitoring the change in absorbance at nm.

Example 3 Evaluation of pH characteristics of fructosylamine oxidase The pH characteristics of the activity of this enzyme are shown in FIG. The activity was measured by the method described in Example 2. The enzyme has an optimum pH around pH8.

Example 4 Substrate specificity of fructosylamine oxidase The substrate specificity for fructosyl amino acids, glucose, valine and sarcosine was examined using purified FAO. 100% reactivity to fructosyl valine
2 and the results of comparing the reactivity with other substrates are shown in FIG. This enzyme shows the highest activity against fructosylalanine, and is about 1.1 times the activity against fructosyl valine.
It was double. Epsilon-fructosyl lysine, glucose, valine and sarcosine were not used as substrates.

Example 5 Measurement of fructosyl valine Arthrobacter sp. Fructosylamine oxidase derived from FV1-1 strain 0.2 U and 2 mM phenol, 1.5 mM 4-aminoantipyrine, 2 U / ml
The fructosyl valine concentration was measured by monitoring the change in absorbance at 505 nm in the presence of peroxidase (Fig. 3). As a result, using this method, 0.25
It was possible to measure fructosyl valine in the range of up to 5.0 mM.

Example 6 Arthrobacter sp. Preparation of chromosomal DNA from FV1-1 strain After culturing the cells in LB liquid medium, the obtained cells were 10 mM
NaCl, 20 mM Tris-HCl (pH 8.
0) 1m EDTA buffer (hereinafter TE beff
er) and lysozyme 2.5 mg / ml
And incubated at 37 ° C. for 1 h. Furthermore, 10% SDS solution, proteinase k adjusted to 10 mg / ml were added, and the mixture was stirred and incubated at 37 ° C. for 1 h. Add 5M NaCl and CTAB to this,
After incubating at 5 ° C. for 10 minutes, an equal amount of phenol-chloroform was added and the mixture was centrifuged to collect the supernatant. To this supernatant was added 0.6 times the amount of isopropanol, at -20 ° C, 2
It was left for h. After centrifugation, the supernatant was discarded, washed with 70% ethanol, and vacuum dried. This was dissolved in a mixed solution of Rnase and ultrapure water and incubated at 37 ° C. for 1 hour to prepare a chromosomal DNA.

Example 7 Determination of N-terminal amino acid sequence of FAO According to Example 1, the strain was cultured. In the same manner as in Example 1, Arthrobacter sp. A purified enzyme of FAO derived from the FV1-1 strain was obtained. After freeze-drying a sample of this purified enzyme, a protein concentration of 2
Ultrapure water was added to give mg / ml. This sample was subjected to SDS-P using a Rapidus Slab electrophoresis system.
AGE (10% polyacrylamide gel) was performed. S
The gel after DS-PAGE was cut into a size of 5 × 5 cm, and blotted onto a PVDF membrane using Fast System ^™ for 2 hours. This PVDF membrane was stained with Coomassie blue, and the band of interest was cut out. The N-terminal amino acid sequence of the thus obtained enzyme was determined using an amino acid sequencer (PPSQ-10 manufactured by Shimadzu Corp.). As a result, this enzyme was found to have SerSerThrL
ysHisValAlaValIleGlyGlyGl
yIIeLeuGlyValSerThrAlaVal
It was revealed to contain a peptide sequence composed of 20 residues consisting of

Example 8 Cloning of a gene encoding FAO 5 μg of the FV1-1 strain genome extracted according to Example 6 was
EcoRI. Restriction enzyme digestion was performed with HindIII, PstI, and Kpnl at 37 ° C. overnight. After diluting this sample 50 times with ultrapure water, 4 μl of the diluted solution was
It was mixed with 4 μl of solution I and self-ligated at 16 ° C. This sample was used in the following experiment.

(1) PCR amplification of FAOD structural gene From the amino acid sequence obtained by N-terminal amino acid sequence analysis, the following primers were used to reverse the self-ligation of the FV1-1 strain genome as a template. PCR was performed. Amino acid residue 1
From the 1st to the 20th forward primer (N
For HNF11), a reverse primer (NHNR10) was designed from amino acid residues 3 to 10. NHNF11 (29mer) 5'-GGCGGGCATCCTGGGCGTCCKCCA
CCGCNGT-3 'NHNR10 (24mer) 5'-GCCRATGACGGCGACRTGYTTN
GT-3 '* K = T + G S = C + G Y = C + T R = A + G
N = A + C + G + T The reaction conditions were as follows. 94 ° C. 1 minute 60 ° C. 30 seconds 72 ° C. 30 seconds or more in 1 cycle 98 ° C. 30 seconds 60 ° C. 30 seconds 72 ° C. 7 minutes or more in 25 cycles 72 ° C. 8 minutes TaKaRaLATaq ^™ was used as Taq polymerase.

(2) Nucleotide Sequence Analysis of PCR Amplified Fragment The nucleotide sequence of the amplified fragment obtained by PCR was analyzed by TA cloning and primer walking method. The analysis of the base sequence was performed by sequentially repeating the operation of using the already analyzed base sequence information as the starting point (sequencing primer) for the next sequence analysis. NFUP69, NF111, NR110, N
HNR10 and NF261 were used as primers. Forward primer 5'-GGAATCGAGCGTTTCGGCAA-3 'from NFUP69 (20mer) 132base Forward primer 5'-GATGAGATCGAGTCGGTCAC- from NF111 (20mer) 696base.
Reverse primer 5'-CGGACCAGCAGTTTGGAGTC- from 3'NR110 (21mer) 695base.
Reverse primer 5'-GCCGATGACGGGCGACGTGCTTG from 3'NHNR10 (24mer) 367base.
Forward primer 5'-CTGGATCACGACTGGTACG-3 'from GT-3' NF261 (20mer) 1107base.

(3) Determination of nucleotide sequence encoding N-terminal amino acid sequence Based on the nucleotide sequence obtained in (2), forward primer (NF110) and reverse primer (NFUPPF6)
9) was used to perform PCR on the genome of the FV1-1 strain. NFUP69 (20mer) 5'-GATGAGATCGAGTCGGTCAC-
3'NR110 (21mer) 5'-CGGAGCCAGCAGTTTGGAGTC-
The 3'reaction conditions were as follows. 94 ° C 5 minutes 94 ° C 30 seconds 55 ° C 30 seconds 72 ° C 1 minute or more 25 cycles 72 ° C 5 minutes

Example 9 Subcloning Based on the nucleotide sequence of the PCR-amplified fragment obtained in Example 8 (3), a forward primer (NNF1, NNF2) containing the following restriction enzyme sites and a reverse primer (N
CR1) was designed and PCR amplification of FAOD structural gene was performed. The resulting PCR amplified fragment was digested with AflIII and XhoI. In addition, the expression vector pET28a (+)
Was digested with NcoI and XhoI. After gel purification, each sample was ligated at 16 ° C. for 2 hours.
The resulting plasmid was named pFAO01 and was subcloned into the non-expression host DH5α. After confirming the plasmid having the insert, the expression host BL21 (DE
3).

Example 10 Determination of nucleotide sequence The nucleotide sequence of the inserted DNA fragment of pFAO1 was determined by restriction enzyme analysis and a conventional method. As a result, the amino acid N-terminal sequence of FAO revealed in Example 2 was confirmed in this inserted DNA fragment, and an open reading frame containing this sequence was found. The determined nucleotide sequence and amino acid sequence are shown in SEQ ID NOS: 1 and 2. The molecular weight of the protein determined from the amino acid sequence is 39.
754 Da and Arthrobacter sp.
The molecular weight of 40 kDa determined by SDS-PAGE of FV1-1 strain FAO was almost the same.

Example 11 Production of FAO by Recombinant Escherichia coli Escherichia co., Which has been transformed with the plasmid pFAO1 containing the structural gene of FAO.
FAO was produced using li BL21 / pFAO1. The transformant was inoculated into 3 ml of LB medium containing 50 μg / ml of ampicillin, cultured at 37 ° C. for 12 hours, and the cells were collected by a centrifuge. The cells were disrupted with a French press (1500 kgf) and then subjected to ultracentrifugation (4 ° C., 160,400 × g, 90 minutes) to obtain a supernatant water-soluble fraction (10 mM potassium phosphate buffer pH 6.0).
Separated. The transformed Escherichia coli thus prepared expresses FAO intracellularly, and the amount thereof is wild type Artho.
rober sp. It was 30 times or more per unit protein as compared with the FV1-1 strain.

Example 12 Assay of fructosylamines Fructosylvaline was assayed using the fructosylamine oxidase of the present invention. The fructosyl valine oxidative activity of fructosyl valine oxidase (FAO) of the present invention was measured for its fructosyl valine concentration dependency using a measurement system using a peroxidase to 4-aminoantipyrine system. 10 mM potassium phosphate buffer (pH 7.0) containing 1.5 mM 4-aminoantipyrine, 2.0 mM phenol, 2 U / ml peroxidase.
The change in the absorbance at 505 nm when the reaction was carried out at room temperature for 1 minute in the presence of was measured using a spectrophotometer, and the rate of decrease in the absorbance was defined as the enzyme reaction rate. 0.1 mM to 5 mM
Linearity was observed between the two, and it was possible to quantify fructosyl valine within this concentration range.

Example 13 Fructosylvaline oxygen electrode type enzyme sensor Arthrobacter sp. Kimwipes were impregnated with 100 microliters of crude extract enzyme (5.7 mg protein / ml) derived from FV1-1 strain, which was attached to an oxygen electrode manufactured by DKK and covered with a dialysis membrane. The prepared enzyme electrode was inserted into 10 ml PBS (pH 7.4) kept at 30 ° C. 1M fructosyl valine aqueous solution 5
0 microliter was added at any time, and the current decrease value at that time was observed. That is, with an enzyme sensor using the novel fructosylamine oxidase of the present invention, 5 mM-2
It was possible to quantify fructosyl valine in the range of 0 mM.

Example 14 Mediator-type enzyme sensor Arthrobacter sp. A 0.34 unit portion of crude extract enzyme derived from FV1-1 strain was freeze-dried, mixed with 20 mg of carbon paste, filled in a carbon paste electrode, and the surface was flattened on a filter paper. After cross-linking the surface with 1% glutaraldehyde for 30 minutes, the unreacted aldehyde groups were destroyed by treating with 10 mM lysine for 20 minutes. Using this as an enzyme electrode, it was equilibrated by stirring it in a 20 mM potassium phosphate buffer (pH 7.0) for 1 hour or more at room temperature. Methoxy P with a final concentration of 1 mM as a mediator
20 mM potassium phosphate buffer (pH 7.
0) was used as a reaction solution, and the total amount was 10 ml. The prepared carbon paste electrode was used as the working electrode, a platinum electrode was used as the counter electrode, and an Ag / AgCl electrode was used as the reference electrode, and a potential of +0.15 V was applied. The measurement was performed at 25 ° C. When the current value became steady (about 1 hour), 100 μl of the substrate (fructosyl valine) solution was injected. The height of the peak current obtained with the injection of the substrate from the steady current value was used as the response current value. The response current value was measured while setting the current value when fructosyl valine was not added to 0A. The response current value for the fructosyl valine concentration at this time was dependent on the concentration. 0.1 mM to 0.6 mM for the injected fructosyl valine concentration
In the range of, the linearity with the response current value was shown, and it was shown that measurement was possible in this range. It took about 4 minutes after the sample was injected until the steady current value was shown.

Example 15 Sensor for detecting hydrogen peroxide First, an enzyme-immobilized membrane was prepared. Arthrobacter
r sp. 50 μl of crude extract enzyme derived from FV1-1 strain
(0.05 unit) and 0.23 g of a PVA-SbQ aqueous solution as a photo-crosslinkable resin prepolymer were mixed and thinly spread on a plate to have an area of 21 cm ² . After air-drying for 5 hours in the dark, both sides were exposed to the light of a fluorescent lamp for 5 minutes each. This membrane was used as a photo-immobilized enzyme membrane and stored at 4 ° C. The produced film was used as a platinum electrode (Model No. 11-101 manufactured by BAS).
2) After covering the surface, a nylon net was covered and fixed with an O-ring to form an enzyme electrode and a working electrode. Ag / AgCl was used as the reference electrode, and a platinum electrode was used as the counter electrode. 500 mM potassium phosphate buffer (pH) in a constant temperature cell (25 ° C)
10 ml of 7.0) was added and the photocrosslinkable resin-immobilized enzyme electrode was shaken. Potential + using potentiostat
0.6V vs. Ag / AgCl was applied, and when the current value became steady (about 1 hour), 100 μl of the substrate (fructosyl valine) solution was injected. The height of the peak current obtained with the injection of the substrate from the steady current value was used as the response current value. Using the constructed batch type system, a response curve depending on fructosyl valine concentration was obtained. For the injected fructosyl valine concentration,
It showed linearity in the range of 0.05 mM to 2 mM, and it was shown to be measurable in this range.

Example 16 Prussian blue type sensor glassy carbon electrode (Model No. 11 by BAS)
-2012) in a 0.1 M potassium chloride buffer to a final concentration of 2 mM potassium hexacyanoferrate (III) and 2 mM
Of iron (III) chloride was added, and the mixture was added at 25 ° C. for 60 seconds at +0.
A potential of 4V vs Ag / AgCl was applied to deposit the film. After that, a film was formed by applying a potential of -0.05 V to 0.35 V (10 times) to prepare a Prussian blue film-immobilized electrode. The PrOcian blue film-immobilized electrode was coated with the PAO-immobilized film prepared by the method described in Example 15 (0.136 unit). Ag / AgCl was used as the reference electrode, and a platinum electrode was used as the counter electrode. 500m in a constant temperature cell (25 ℃)
10 ml of M potassium phosphate buffer (pH 7.0) was added and used. Using a potentiostat, a Prussian blue film-immobilized enzyme electrode with 0.05 V vs. Ag
/ AgCl was applied, and when the current value became steady (about 1 hour), the substrate (fructosyl valine) solution was added to 100
Each μl was injected. The height of the peak current obtained with the injection of the substrate from the steady current value was used as the response current value. Using the constructed batch type system, reproducible results depending on fructosyl valine concentration were obtained.
It showed linearity with the response current value in the range of 0.1 mM to 1.6 mM with respect to the injected fructosyl valine concentration, and it was shown that measurement was possible in this range.
It took about 6 minutes after the sample was injected until the steady current value was shown.

[0098]

According to the present invention, glycated hemoglobin (HbA)
lc) It has become possible to newly produce an enzyme for measurement, fructosylamine oxidase, from the genus Arthrobacter. The genus Arthrobacter is easily cultivated, and since the culture density is high, fructosylamine oxidase can be efficiently prepared. Furthermore, we succeeded in cloning the structural gene of the enzyme and producing it recombinantly in E. coli. By this, a large amount of F
You can now produce AO. Since this enzyme is selective for fructosyl valine, it can be applied to the analysis of clinical samples.

[Brief description of drawings]

FIG. 1 shows pH characteristics of FV1-1 strain-derived fructosylamine oxidase.

FIG. 2 shows the substrate specificity of FV1-1 strain-derived fructosylamine oxidase.

FIG. 3 shows a calibration curve for quantifying fructosyl valine using fructosylamine oxidase derived from FV1-1 strain.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 5/10 C12Q 1/26 9/06 C12R 1:06 C12Q 1/26 C12N 15/00 ZNAA G01N 27 / 327 5/00 A 27/416 G01N 27/30 353C // (C12N 1/20 353J C12R 1:06) 353R 27/46 336G 27/30 353F F term (reference) 4B024 AA11 BA08 CA04 DA06 EA04 GA11 GA19 HA03 4B050 CC01 CC03 DD02 FF03E FF05E FF09E FF11E LL03 4B063 QA01 QA19 QQ61 QR02 QR41 QR57 QR66 QR84 QS12 QX01 QX04 4B065 AA13X AA13Y AA26X AB01 AC14 BA01 BA22 CA28 CA46

Claims (23)

[Claims] 1. A fructosylamine which belongs to the genus Arthrobacter and which is capable of producing fructosylamine oxidase, is cultured, and the fructosylamine oxidase is collected from the same culture solution or cultured bacterial cells. Method for producing oxidase 2. A fructosylamine oxidase produced by a microorganism belonging to the genus Arthrobacter. 3. The microorganism is Arthrobacter
sp. The fructosylamine oxidase according to claim 3, which is 4. The microorganism is Arthrobacter
sp. The fructosylamine oxidase according to claim 3, which is FV1-1 strain (FERM P-18754). 5. A protein having the following fructosylamine oxidase activity (a) or (b): (A) a protein consisting of the amino acid sequence described in SEQ ID NO: 1 (b) the amino acid sequence (a), consisting of an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added, and A protein having fructosylamine oxidase activity. 6. A gene encoding a fructosylamine oxidase which is the following protein (a) or (b). (A) a protein consisting of the amino acid sequence described in SEQ ID NO: 1 (b) the amino acid sequence (a), consisting of an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added, and Protein with fructosylamine oxidase activity 7. The following sequence (c) or (d):
A gene encoding fructosylamine oxidase. (C) DNA consisting of the base sequence described in Sequence Listing / SEQ ID NO: 2 (d) In the sequence of (c) above, one or several bases have been deleted, substituted or added, and fructosylamine D encoding a protein having oxidase activity
NA. 8. Sequence ValProLysArgHis
PheTyrProGlyGlu SerTrpVal
A fructosylamine oxidase containing SerLeu ProGluLeuGlyAsp. 9. The method according to any one of claims 1 to 8,
ter sp. FV1-1 strain or Arthrobacter
A gene encoding a fructosylamine oxidase derived from the ter genus. 10. A recombinant vector containing a gene encoding a fructosylamine oxidase according to any one of claims 1 to 9. 11. A transformant or transductant transformed with the recombinant vector according to claim 10. 12. A method for producing a fructosylamine oxidase, which comprises culturing the transformant according to claim 11 and collecting fructosylamine oxidase from the culture. 13. A fructosylamine oxidase produced by the method according to claim 12. 14. A method for spectroscopic analysis of fructosylamine compounds containing the fructosylamine oxidase according to any one of claims 1 to 13. 15. A method for electrochemically analyzing fructosylamine compounds containing the fructosylamine oxidase according to any one of claims 1 to 13. 16. A method for assaying HbA1c, which comprises degrading HbA1c in a sample to produce fructosyl valine, the fructosyl valine being used with the fructosyl amine oxidase according to claim 1. Spectroscopic analysis method including quantification. 17. A fructosyl valine spectroscopic assay kit containing the fructosyl amine oxidase according to claim 1. 18. An assay kit for HbA1c spectroscopic analysis method, which comprises the fructosylamine oxidase according to claim 1. 19. A method of electrochemically analyzing fructosyl valine, which comprises using the fructosyl amine oxidase according to any one of claims 1 to 13. 20. An electrochemical analysis method for HbA1c, which comprises using the fructosylamine oxidase according to any one of claims 1 to 13. 21. An enzyme electrode characterized by the electrochemical analysis method according to claim 19 or 20. 22. An enzyme sensor using the enzyme electrode according to claim 21. 23. Arthrobacter sp. FV
1-1 strain (FERMP-18754).