WO1997021818A1 - Novel fructosyl amino acid oxidase originating in fungi of the genus penicillium - Google Patents

Abstract

An expression vector that contains a gene coding for a fructosyl amino acid oxidase originating in the genus Penicillium (FAOD-P) and that is functional in host cells; host cells transformed by said vector; a process for preparing a novel FAOD-P by culturing the obtained transformant; the FAOD-P thus prepared; a method for analyzing Amadori compounds with said FAOD-P; and reagents and kits useful therefor.

Description

 Specification

 Fructosylamino acid oxidase derived from a new fungus of the genus Penicillium

 The present invention relates to the production of a novel fructosylamino acid oxidase by DNA recombinant technology. More specifically, the present invention relates to a DNA encoding fructosyl amino acid oxidase (hereinafter referred to as FAOD-P) derived from Penicillium genus (Penicil_liuffi), containing the DNA and being functional in host cells. Expression vector, host cell transformed by the expression vector, production of novel FA0DP by culturing the obtained transformant, FAOD-P thus obtained, FAOD-P The present invention relates to an Amadori compound analysis method using P and a reagent or kit useful for the analysis method.

Background art

 Amadori compounds are non-enzymatic when an amino group such as protein, peptide and amino acid and a reducing sugar such as aldose coexist. It is a substance produced by irreversibly binding and translocating Amadori, and is contained in foods such as soy sauce and body fluids such as blood. Since the rate of formation is a function of the concentration of the reactive substance, the contact time, the temperature, and the like, measuring the amount of the generated substance can provide various information on the substance containing the reactive substance.

For example, in a living body, a fructosylamine derivative, which is an amadori compound composed of glucose and amino acids, is produced.Fructosylamine derivatives in which hemoglobin in blood is glycated are derivatives in which glycated hemoglobin and albumin are glycated. Glycoalbumin is called glycated albumin, and the derivative of glycated protein in blood is called fructosamine. These blood levels reflect the average blood glucose level over a period of time in the past, and the measured values are important indicators of diabetes symptoms. Establishing a measurement tool is extremely clinically useful because it can serve as an indicator. In addition, by quantifying Amadori compounds in foods, it is possible to know the storage status and period of the foods after production, which will be useful for quality control.

 Thus, quantitative analysis of Amadori compounds is useful in a wide range of fields including medicine and food.

 Conventionally, an analysis method has been proposed in which a redox enzyme is allowed to act on a sample containing an Amadori compound, and the amount of oxygen consumed or the amount of hydrogen peroxide generated is measured to determine the amount (for example, Japanese Patent Publication No. 5-33997). JP-A-61-268178, JP-A-2-195900, JP-A-3-155780). Furthermore, a method for quantifying saccharified tanbugs for diagnosing diabetes has also been disclosed (JP-A-2-195899, JP-A-2-195900).

 The reaction of an amadori compound with an oxidoreductase can be represented by the following general formula.

R ¹ -CO-CH ₂ -NH-R ² + 0 ₂ + H ₂₀ →

^{R'-CO-CHO + R 2} -NH 2 + H 2 0 2

(Where R is an aldose residue and R ² is an amino acid, protein or peptide residue)

 The present applicant has purified fructosylamino acid oxidase (FAOD-P) derived from Penicillium as an enzyme suitable for the above purpose, and has clarified its usefulness (Japanese Patent Application No. 7-146575; EP-A-0737744, published: October 16, 1996)

However, a method of culturing a microorganism and extracting the enzyme from the culture medium to produce the enzyme requires a lot of labor and time and is inefficient. In addition, the enzyme obtained by the purification method contains impurities such as proteins specific to strains of the genus Penicillium. It is highly probable that such impurities may contain substances that have an adverse effect on FAOD-P activity, and the reliability of the measurement may not be sufficiently ensured.

 Therefore, there has been a demand for the development of a method for efficiently producing FAOD-P without impurities derived from bacteria of the genus Penicillium. Such an enzyme is obtained by cloning DNA encoding FAOD-P derived from the genus Niscilium, constructing an appropriate expression vector containing the DNA, and transforming a host cell with the expression vector. The transformant can be obtained by culturing the transformant in an appropriate medium to produce a recombinant FA0D-P. However, there has been no cloning of DNA encoding FAOD-P from P. genus, and it was necessary to clone such a DNA first.

Disclosure of the invention

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above problems, cloned a DNA encoding FA0D-P derived from Penicilliura, and prepared an expression vector containing the DNA. It was constructed. When a host cell was transformed with the obtained expression vector and the obtained transformant was cultured, the transformant produced an expression product having fructosyl amino acid oxidase activity. That is, the transformant produced a recombinant FA0D-P having the same enzymatic activity as fructosylamino acid okinidase (FAOD-P) naturally produced by bacteria of the genus Penicillium. The nucleotide sequence of the DNA encoding FA0D-P of the present invention and the deduced amino acid sequence are shown in SEQ ID NO: 1. According to the present invention, DNA encoding FA OD-P exhibiting fructosylamino acid oxidase activity has been cloned and its nucleotide sequence has been determined. Therefore, the DNA sequence can be easily prepared by a method known in the art. 1. Insertion, substitution or substitution of one or more amino acids from the amino acid sequence described in 1. Has an amino acid sequence derived from the deletion, and it is easy for those skilled in the art to obtain a variant having substantially the same activity or function as FAOD-P having the amino acid sequence of SEQ ID NO: 1. It is. Therefore, the mutant thus obtained is also included in the scope of the present invention.

 Accordingly, the present invention has an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence derived by insertion, deletion or substitution of one or more amino acids with respect to the amino acid sequence, and in the presence of oxygen An object of the present invention is to provide fructosylamino acid oxidase having an enzymatic activity to catalyze a reaction of oxidizing an Amadori compound to produce an α-keto aldehyde, an amine derivative and hydrogen peroxide. The FAOD of the present invention is also characterized in that it does not substantially contain other proteins derived from bacteria of the genus Penicillium (Eenic_liy5).

 The present invention also provides a DNA encoding the above FA〇D-P. The DNA may be either a complementary DNA or a synthetic DNA. In one embodiment, the DNA of the present invention can be represented by the nucleotide sequence of SEQ ID NO: 1 or a partial sequence thereof.

Further, the present invention provides an expression vector containing a gene encoding FAOD-P and being functional in a host cell. The gene preferably encodes a FAOD-P derived from a bacterium of the genus Penicillium, more preferably a Penicillium ′ Jansinerum strain S-341 (Penicillium iant inellum S-3413; FERM BP-5475). bacteria of the preferred _c above it is a gene, have been deposited with the Ministry of International Trade and industry Agency of raw life of industrial Science and technology Research Institute of Tsukuba City, Ibaraki Prefecture Higashi 1-chome 1-chome No. 3 (original date of deposit: 1995 March 28 Date; transfer date to international deposit: March 14, 1996).

In the present specification, the expression vector being "functional" in a host cell means that when the vector is introduced into the host cell, the obtained transformant is transformed into an appropriate medium. Means that it can produce FA〇D-P contained in the vector. The term FAOD-P is used as a term representing both fructosylaminoacid okinidase obtained by the DNA recombination technique of the present invention and natural fructosylaminoacid okinidase derived from bacteria belonging to the genus Penicillium. However, it is clear from the context which enzyme FAOD-P refers to.

 The terms “gene” and “DNA” are used interchangeably, irrespective of their origin, provided that they encode a peptide having the desired FAODP activity.

 The present invention also provides a host cell transformed by the above expression vector.

 Furthermore, the present invention provides a method for producing FAOD-P, which comprises culturing the thus obtained transformant in a medium and recovering fructosylamino acid oxidase from the culture. It is.

 Furthermore, the present invention is characterized in that a sample containing an Amadori compound is brought into contact with a culture obtained by the above culture or a processed product thereof, and the amount of consumed oxygen or the amount of generated hydrogen peroxide is measured. The present invention provides a method for analyzing Amadori compounds in a sample.

 The analysis method of the present invention is preferably a mosquito or a biological component applicable to all of the samples containing the Amadori compound. In this case, it is preferable to measure the amount and / or saccharification rate of the glycated protein in the biological component, or to determine the amount of fructosamine.

The present invention also provides a reagent or a kit for analyzing an Amadori compound containing a culture of the above transformant or a processed product thereof. The reagent and the kit preferably contain the amount of glycated protein in the biological component and Z Alternatively, it is used for measuring the saccharification rate or for quantifying fructosamine.

 The present invention also provides an analytical reagent or kit for an amadori compound containing the culture obtained by the above culture or a processed product thereof.

 In the present specification, the “treated product” of the culture is a substance obtained from the culture, which enhances the enzyme activity for catalyzing the reaction represented by the above formula (I), and uses Z or the enzyme activity. Refers to materials that have been processed by methods common in the art to make them easier.

BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is an explanatory diagram showing the relationship between a primer used for PCR and a partial amino acid sequence of FAOD-P.

 FIG. 2 is a restriction map of plasmid pFAP1 containing DNA encoding FAOD-P.

 FIG. 3 is a mimetic diagram showing the results of agarose electrophoresis in RT-PCR, in which lane 1 is 0X174 / HincII (marker); lane 2 is a PCR using primers 1 and 2. The product electrophoresis pattern is shown. FIG. 4 is a mimetic diagram showing the results of electrophoresis in the subcloning of the PCR fragment of about 690 bp in FIG. 3. In the figure, lane 1 is labeled with S / EcoTl 41 (marker) and lane 2 Represents the migration pattern of the BamHI digest of plasmid PFPP.

 FIG. 5 is a graph showing the time course of FAOD-P production by Escherichia coli transformants transformed with plasmid pFAP1. The horizontal axis shows the time after induction by IPTG, the vertical axis shows the degree of proliferation (ODeon measurement), the black circles show the total activity (U / 1 culture), and the white circles show the specific activity (U / nig).

Figure 6 shows Penicillium ′ Jansinerum strain S-3413 (PenicUlium janthin 11 is a photograph showing a migration pattern obtained by applying purified FAOD-P derived from ellum S-3413; FERM BP-5475) to SDS-PAGE (sodium dodecyl sulfate 'polyacrylamide gel electrophoresis).

 FIG. 7 is a graph showing the results of molecular weight measurement of F AOD-P by gel filtration using Superdex 20 Opg as in FIG.

 FIG. 8 is a graph showing the relationship between the amount of saccharified hemoglobin and the amount of hydrogen peroxide (absorbance at 727 nm) generated by the F AOD action.

 FIG. 9 is a graph showing the relationship between the hemoglobin A 1 c value and the amount of hydrogen peroxide generated by the FAOD action (absorbance at 727ππι).

 FIG. 10 is a restriction map of the vector pNFP1 for expression of FAOD-P in yeast.

BEST MODE FOR CARRYING OUT THE INVENTION

 Cloning of DNA encoding FAOD-P can be performed according to methods known in the art.

 First, FAOD-1P was purified from a culture of P. janthinellum S-3413CFERM BP-5475), and the N-terminal amino acid was determined therefrom. Next, the amino acid sequence of the intermediate portion was determined from the peptide fragment obtained by subjecting FAOD-P to limited digestion, and an oligonucleotide primer was designed based on the amino acid sequence. The determined N-terminal amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the intermediate portion is shown in SEQ ID NO: 3. The nucleotide sequences of oligonucleotide primers 1 and 2 are shown in SEQ ID NOs: 4 and 5, respectively. FIG. 1 shows the relationship between these peptide fragments of SEQ ID NOs: 2 and 3 and primers 1 and 2.

On the other hand, did cultivation in a browning medium (Japanese Patent Application No. 7-85261; EP-A-0737744) induce FA 0DP? , janthinellum S-3413 totalR N From mRNA A, mRNA was obtained using mRNA Purification Kit (Falman), and the mRNA was reverse-transcribed to obtain cDNA, and a cDNA library was constructed using the cDNA ZAPII vector.

 In addition, RT-PCR (reverse transcription polymerase chain reaction) was performed using the total RNA and the above primer to obtain a PCR product of about 690 bp. This PCR product was subcloned, and the above cDNA library was screened using the fragment as a probe. As a result, five positive plaques were obtained. The cDNA fragment contained therein was subcloned into the plasmid pBluescriptllSK- to construct an expression vector. Next, an E. coli host (Escherichia coli S0LR) is transformed using the expression vector, and the resulting transformant is cultured. Among the culture, the N-terminal amino acid sequence of FAOD-P One clone having the plasmid pFAP1 having a nucleotide sequence corresponding to the above was obtained. A restriction map of the expression plasmid pFAPl is shown in FIG. Escherichia coli, coli SOLRZ FAP1, into which plasmid pFAP1 has been introduced, is under contract number FEKM BP-5762 at Tsukuba-Higashi 1-chome 3-chome, Ibaraki Pref. (Hara Deposit: October 4, 1995: Transfer to International Deposit: December 2, 1996).

 Next, the nucleotide sequence of the obtained clone was determined, and the amino acid sequence of FAOD-P was estimated. These are shown in SEQ ID NO: 1.

 As described above, for the purpose of the present invention, not only FAOD-P having the entire amino acid sequence shown in SEQ ID NO: 1 but also amino acids having one or more amino acids inserted, deleted or substituted in the sequence. Mutants having a sequence and similar enzymatic activity are also useful.

The FAOD-P expression vector pFAP1 capable of replicating in an E. coli host of the present invention confers lac promoter, SD sequence, and ampicillin resistance. The DNA encoding FAOD-P contained in this plasmid pFAPl is inserted into the downstream of the promoter of another appropriate expression vector to obtain various hosts. A FAOD-P expression vector that expresses FAOD-P can be constructed.

 The above-described expression vector and host cell are merely examples of many vectors and host cells suitable for expressing the DNA encoding FAOD-P of the present invention. FAOD-P expression vectors that are functional in any host cell can be constructed using methods that are routine in the art. The promoter that can be used for such a vector may be any of those that are appropriately selected from known ones, or those that are newly prepared.

 As described above, the expression vector of the present invention is not limited to the plasmids described in the present specification, but can be modified (for example, by exchanging a promoter) by using ordinary techniques to obtain different types of microorganisms. In addition, an expression vector that is functional in other cells and can produce Z or FAOD-P at a high level can be constructed.

 The host cell used for transformation with the expression vector carrying the DNA encoding FAOD-P of the present invention may be any of prokaryotic cells such as Escherichia coli and eukaryotic cells such as yeast. Commonly used cells of higher organisms are also suitable.

Examples of host cells include microorganisms [prokaryotes (eg, bacteria such as Escherichia coli and Bacillus subtilis), eukaryotes (eg, yeast)], animal cells, and cultured plant cells. Preferred examples of microorganisms include prokaryotes, particularly strains belonging to the genus Escherichia (eg, E. coli), yeasts, particularly strains belonging to Saccharomyces 厲 (eg, S—. Cerevisiae) and strains belonging to Candida 厲 ( For example, C. boidinii), and preferably, a methanol yeast (a methylotrophic yeast or a methanol-utilizing yeast) It is. Preferred animal cell lines include, for example, mouse L929 cells, Chinese hamster ovary (CHO) cells, and the like.

 Expression vectors suitable for using bacteria, particularly Escherichia coli, as host cells are known, and examples thereof include those having a conventional promoter such as a lac promoter or a TAC promoter.

 As an expression vector for the expression of FAOD-P in yeast, a vector containing a promoter such as a GAL motor or an AOD promoter is preferable.

 Examples of expression vectors for expressing FAOD-P in mammalian cells include those having a promoter such as the SV40 promoter.

 In order to enhance the expression efficiency, a multicopy expression vector can be constructed using a multicopy type plasmid known in the art.

 Prokaryotic hosts are preferred in terms of ease of operation and availability, and E. coli is particularly preferred. There are many books on prokaryotic host-vector systems (eg, Molecular Cloning: A LABOLATORY MANUAL Cold Single Harbor Laboratory Press), which are known in the art and are briefly described below.

For example, to express DNA encoding FAOD-P in E. coli, the DNA is inserted downstream of the promoter of an expression plasmid suitable for transforming E. coli. Usually, there are two types of expression: intracellular expression and secretory expression, and there are appropriate expression systems for each. Expression products are usually accumulated in the E. coli host. If it is necessary to secrete extracellularly, the gene for the signal sequence of the secretory protein derived from E. coli is linked to the N-terminus of the DNA encoding FAOD-P. Construct an expression system so that the expression product is secreted into the periplasm. On the other hand, in the case of a eukaryotic host, the F AOD-P of the present invention is originally a eukaryotic microorganism of the genus Penicillium (eg, ^ Penicillium j anthinellum S-3413; FERM BP -5475)) has the advantage that problems that can occur with prokaryotic hosts can be avoided. In other words, it is known that when prokaryotes produce eukaryotic substances, an inclusion body (inclusion body) is formed and efficient enzyme production may not be ensured (Laboratory Genetic Engineering Enhancement Edition, Maruzen (See page 187). However, it is thought that the use of a eukaryotic host can prevent the formation of such inclusion bodies.

 When yeast is used as a host cell, if it is necessary to secrete the expression product extracellularly, as described above, the gene for the signal sequence of the secretory protein derived from the host yeast is located at the N-terminus of the DNA encoding FAOD-P. And an expression system can be constructed so that the expression product is secreted into the periplasm. A method for constructing an expression vector suitable for expression in yeast is shown below, but this is only an example, and the present invention is not limited to the following expression vectors. For expression of the FAOD-P gene in yeast, an FAOD-P expression vector is constructed using plasmid pNOTel (Japanese Patent Application Laid-Open No. 5-344895;), which is a chromosome insertion type expression vector of id nii. The plasmid pNOTe1 contains the A0D promoter and the URA3 gene, and can provide a means for selecting a transformant transformed with the plasmid using Ura requirement as an index.

First, an Escherichia coli expression vector pFAP1 containing the cloned F AOD-Pc DNA was obtained from SOLR / FAP1 (FERM P-15227), which was then digested with the restriction enzymes Ec0RI and Xh0I. By digestion, a FAOD-Pc DNA fragment of about 1.3 kb was obtained and purified. On the other hand, plus After digestion of mid pNOTel with the restriction enzyme Notl, it is dephosphorylated with cyst intestinal phosphatase and used together with the F AOD-Pc 0 Ryohachi fragment described above. 1) Using NA Blunting Kit (Takara Shuzo) And smoothed. These were ligated using DNA Ligation Kit (Takara Shuzo Co., Ltd.) to obtain plasmid pNFP. This was transformed into E. coli by the Hanahan method (Hanahan, D, Techniques for Transformation of E. coli. In: DNA Cloning, vol I, Glover, DM (ed), ppl09-136, IRL Press, 1985). Plasmid was prepared by arbitrarily selecting 84 strains from the obtained transformants. The plasmid was treated with the restriction enzyme Hindlll 2 to confirm the orientation of the insert, and a plasmid pNFFP in which the FA0D—PcDNA fragment was inserted downstream of the AOD promoter was obtained. . Figure 10 shows the restriction map of plasmid pNFP1. The plasmid pNFP1 described above was used to transform the Ura-requiring boidiniiTK62 strain. Transformants are cultured in UNB-free YNB medium, strains arbitrarily selected from URA + -type transformants are cultured in a basal medium containing 1.5% methanol, and strains that produce FAOD-P are selected. did.

 In addition, DNA encoding FAOD-P inserted into E. coli S OLR / FAP1 (FERM P-15227) can be ligated to a suitable enzyme (eg, restriction enzyme, alkaline phosphatase, polynucleotide kinase, DNA ligase). , DNA polymerase, etc.) to obtain a DNA fragment encoding the enzymatic activity of FAOD-P, which is then inserted into an appropriate vector to produce a peptide having FAOD-P activity in various hosts. It can be expressed, and these FA OD-Ps are also included in the scope of the present invention.

Transformation of a host cell with an expression vector is known, and can be performed by the method described in Molecular Cloning: A Laboratories Manual, Cold Sling Harbor Laboratory Press. For example, for prokaryotic hosts, The method can be carried out by a method of producing a pitent cell, in the case of a eukaryotic host, by a method of producing a cell, by a lithium modification method, and in the case of a mammalian cell, by a transfusion method.

 Next, the obtained transformant is cultured in an appropriate medium.

 The medium contains a carbon source (eg, glucose, methanol, galactose, fructose, etc.) and an inorganic or organic nitrogen source (eg, ammonium sulfate, ammonium chloride, sodium nitrate, leptone, casamino acid, etc.). May be. If desired, other nutrients (eg, inorganic salts (sodium chloride, potassium chloride)) and vitamins (eg, vitamins, antibiotics (eg, ampicillin, tetracycline, kanamycin, etc.)) may be added to the medium. Eagle's medium is suitable for culturing animal cells.

When the host cell is a eukaryotic cell, FAOD- medium suitable for production of P of the present invention is 0.1 to 5.0%, preferably 0.5 to 2.0% of the NH ₄ C 1 and / or 0.1 to 5.0%, preferably A basal medium containing 0.1% to 5.0%, preferably 1.5%, of methanol containing 1% of yeast extract.

The culture of the transformant may be carried out usually at pH 5.0 to 8.0, preferably at pH 5.5 to 6.0, at 25 to 40 ° C (preferably 28 ° C) for 16 to 96 hours. If the produced FA0D-P is present in the culture solution or culture filtrate (supernatant), filter or centrifuge the culture. From the culture filtrate, FAOD-P can be used to purify and isolate natural or synthetic proteins by conventional methods (eg, dialysis, gel filtration, affinity using anti-FAOD-P monoclonal antibodies). It can be purified by column chromatography, column chromatography using an appropriate adsorbent, high-performance liquid chromatography, etc.). When the produced FAOD-P is present in the periplasm and cytoplasm of the cultured transformant, the cells are collected by filtration or centrifugation, and their cell walls and And / or disrupting the cell membrane, eg, by ultrasound and lysozyme treatment, to obtain debris. Dissolve the debris in an appropriate aqueous solution (eg, Tris-HCl buffer). From this solution, FAOD-P can be purified by a conventional method.

 When it is necessary to regenerate (refold) F AOD-P produced in the transformant, this can be performed by a conventional method.

 A culture obtained by culturing the transformant obtained by the method of the present invention in an appropriate medium exhibits FAOD-P activity, which is further treated by a usual method known to those skilled in the art as described above to obtain the enzyme. A processed product such as a solution can be prepared. Moreover, you may refine | purify as needed. For example, the culture is centrifuged to collect a FADPD-producing transformant, suspended in a phosphate buffer, and disrupted by sonication or the like. Then, the enzyme sample is obtained by centrifuging the supernatant. Furthermore, the supernatant is filtered and further purified by chromatography or the like to obtain a purified enzyme. Subsequently, a fragment having an enzymatic activity can be obtained by restriction enzyme treatment or exonuclease treatment.

 The above cultures and their processed products (including purified enzyme preparations and fragments having enzyme activity) have FAOD-P enzyme activity and are suitable for quantitative analysis of Amadori compounds. Useful for The DNA encoding FAOD-P has been cloned according to the present invention. Based on the cloned DNA, FAOD-P activity is retained by adding, deleting, inserting, or substituting amino acids using ordinary genetic recombination techniques. It is easy for those skilled in the art to obtain such a derivative. Therefore, active derivatives of FAOD-P obtained by such conventional means are also included in the scope of the present invention.

As described above, a culture obtained by culturing the transformant of the present invention and a processed product thereof are represented by the following reaction formula: R ¹ -C〇1 CH ₂ -NH— R ² + 0 ₂ + H ₂ 0 →

R ¹ — C〇 CHO + R ² -NH ₂ + H ₂ 0 ₂

(Wherein, R ¹ represents an aldose residue, and R ² represents an amino acid, protein or peptide residue)

Catalyzes the oxidation-reduction reaction of the Amadori compound represented by In the above formula, R ¹ gar OH, one (CH _{2) n} -, or one [CH (OH)] (wherein, n an integer from 0- 6) _n -CH ₂ OH is, R ² is - Amadori compounds represented by CHR ³ — [CONHR ³ ] _ra COO H (where R ³ is a residue of one amino acid side chain and m is an integer of 1 to 480) are preferred as 15 as a substrate. Among them, a compound in which R ³ is a side chain residue of an amino acid selected from lysine, polylysine, palin, asparagine and the like, and n is 5 to 6 and m is 55 or less is preferable.

 To carry out the analysis method of the present invention, a sample suspected of containing an Amadori compound is brought into contact with a culture of a transformant expressing FAOD-P of the present invention or a processed product thereof in water or a buffer. The Amadori compound in the sample is analyzed by measuring the amount of oxygen consumed or the amount of hydrogen peroxide generated. The analysis method of the present invention is carried out based on measurement of the amount of glycated protein and Z or glycation rate in a biological component, or quantification of fructosamine.

 For example, to a sample in a buffer containing an Amadori compound, a suspension (solution) of a culture or a processed product thereof in water or a buffer is added. The reaction conditions such as pH, temperature and reaction time are not particularly limited, and may be appropriately selected from the conditions usually used for similar enzyme reactions. However, it is reacted at pH 4.0 to 12.0, preferably pH 7.0 to 8.5, more preferably at a pH of about 7.5, at a temperature of 25 to 50 ° C, preferably at 25 to 40 ° C, more preferably at 25 ° C. .

The test solution used in the method of the present invention may be any solution containing an Amadori compound. A sample solution can be used, and examples thereof include foods such as blood (whole blood, plasma or serum), urine and the like, as well as soy sauce.

 As a buffer, a Tris-monohydrochloride buffer or the like is used. The amount of culture of a transformant expressing FAOD-P or FAOD-P or a processed product thereof is generally 0.1 units or more, preferably 1 to 100 units, in end-point analysis. It is.

 In the analysis method of the present invention, any of the following methods for quantifying an Amadori compound is used.

 (1) Method based on the amount of hydrogen peroxide generated

 Measured by a method known in the art for determining hydrogen peroxide, for example, a coloring method, a method using a hydrogen peroxide electrode, etc., and compared with a standard curve prepared for the amounts of hydrogen peroxide and Amadori compounds. As a result, the Amadori compound in the sample is quantified. Specifically, it is based on the titer measurement method described later. However, the amount of FAOD-1P is set to 1 kN, and an appropriately diluted sample is added, and the amount of generated hydrogen peroxide is measured. As the hydrogen peroxide coloring system, 4-aminoaminoantipyrine ZN-ethyl-1 N-1 (2-hydroxy-13-sulfopropyl) 1 m-toluidine, 4-1-1 instead of 4-aminoantipyrine / funol system Aminoantipyrine ZN, N-Dimethylaniline, 4-Aminoantipyrine / N, N-Jetylaniline, MBTH / N, N-Dimethylaniline, 4-Aminoantipyrine / 2, 4-Dichlorophenol, etc. It is possible.

 (2) Method based on oxygen consumption

A value obtained by subtracting the amount of oxygen at the end of the reaction from the amount of oxygen at the start of the reaction (oxygen consumption) is measured, and is compared with the standard curve created for the amount of oxygen consumption and the amount of the Amadori compound to determine the amount of Amadori in the sample. Quantify compound. Specifically Is performed according to the titer measurement method described later. However, the amount of FA〇D-P used should be 1 unit / ^ 1, and the amount of oxygen absorbed by adding an appropriately diluted sample should be determined.

 Although the method of the present invention can be carried out using a sample solution as it is, depending on the target, it is preferable to release the valine residue to which a sugar is bound beforehand.

 For such purposes, there are cases where a protease is used (enzymatic method) and cases where a chemical substance such as hydrochloric acid is used (chemical method), the former being preferred. Proteolytic enzymes that can be used in the method of the present invention are known to those skilled in the art, and include trypsin, carboxypeptidase B, papain, aminopeptidase, chymotribsine, thermolysin, subsiricin, proteinase, and proteinase. And the like. Enzyme treatment methods are also known. For example, the protease treatment can be performed by the method described in the following Examples.

 As described above, the culture of the transformant expressing the FAOD-P of the present invention or the processed product thereof has a high substrate specificity for fructosyl valin contained in the glycated protein. It is useful for the diagnosis of diabetes, including the measurement of glycated hemoglobin. In addition, since fructosyl lysine also has specificity, it is useful for measuring glycated proteins. When a blood sample (whole blood, plasma or serum) is used as the specimen, the collected blood sample is used as it is or after being subjected to a treatment such as folding.

Furthermore, the culture of the transformant expressing FAOD-P used in the method of the present invention or a processed product thereof, or an enzyme such as peroxidase may be used in the form of a solution, but may be immobilized on a suitable solid support. You may. For example, by packing an enzyme immobilized on beads into a column and incorporating it into an automated device, routine analysis of a large number of samples, such as clinical tests, can be performed efficiently. I Alternatively, the immobilized enzyme can be reused, which is preferable in terms of economic efficiency.

 Furthermore, by appropriately combining enzymes and coloring pigments, it is possible to obtain a kit for the analysis of Amadori compounds useful not only for clinical analysis but also for food analysis.

 Immobilization of the enzyme can be performed by a method known in the art. For example, it is carried out by a carrier binding method, a cross-linking method, an inclusive method, a complex method, or the like. Carriers include polymer gels, microcapsules, agarose, alginic acid, and carrageenan. Coupling is performed by a method known to those skilled in the art, utilizing covalent bonding, ionic bonding, physical adsorption, and biochemical affinity.

 If immobilized enzymes are used, the analysis can be either column or batch. As noted above, immobilized enzymes are particularly useful for routine analysis (glycos) of glycated proteins in blood samples. When the laboratory test is aimed at diagnosing diabetes, the criteria for diagnosis are to express the result as glycated protein concentration or the ratio of glycated protein concentration to total protein concentration in the sample. The total protein concentration can be measured by a conventional method (eg, absorbance at 280 nm, Lowry method, or natural fluorescence of albumin).

 The reagent for quantifying the Amadori compound of the present invention is a culture of a transformant expressing FAOD-P of the present invention or a processed product thereof, and preferably has a pH of 7.5-8.5. Consists of H7.5 buffer. When the culture or its processed product is immobilized, the solid support is selected from a polymer gel or the like, and is preferably alginic acid.

When performing end-point analysis, the amount of culture or processed material in the reagent is usually per sample; ~ 100 units Zml, and the buffer is preferably a Tris-monohydrochloride buffer (pH 7.δ). When quantifying the Amadori compound based on the amount of hydrogen peroxide generated, various combinations of the above can be used as the color developing system.

 A kit can also be prepared by combining the Amadori compound analysis reagent of the present invention with an appropriate color former and a color standard or standard substance for comparison. Such a kit would be useful for preliminary diagnosis and testing. Example

 Hereinafter, the present invention will be described in detail with reference to examples, but these examples do not limit the present invention. Plasmids, various restriction enzymes, T4 DNA ligase, and other enzymes used in the following examples were obtained from commercial products and used according to the supplier's instructions. In addition, DNA cloning, construction of each plasmid, transformation of host, culture of the transformant, and recovery of the enzyme from the culture were performed according to methods known to those skilled in the art or according to methods described in the literature. The enzyme activity was measured according to the following titer measurement method.

Titration method

 (1) A method of measuring the generated hydrogen peroxide by a colorimetric method.

 A. Speed method

A 10 OmM FV solution was prepared by dissolving a previously obtained FV with distilled water. 45 mM 4-aminoantipyrine, 60 units Zm1 peroxidase solution, 100 mM each of 6 OmM phenol solution, 0.1 M Tris-monohydrochloride buffer (pH 7.5) lm], and 50% enzyme solution Mix and make up to 3. Om 1 with distilled water. After equilibration at 25 ° C, 501 of a 10 OmM FV solution was added, and the absorbance at 505 nm was measured over time. From the molecular extinction coefficient (5.161 O'M-'cm- ¹ ) of the generated quinone dye, calculate the micromol of hydrogen peroxide generated in one minute, and use this number as the enzyme activity unit. B. terminal law

 After treating in the same manner as in Method A above, adding the substrate, incubating at 25 ° C for 20 minutes, measuring the absorbance at 505 nm, and separately generating a standard curve prepared using a standard hydrogen peroxide solution in advance. Enzyme activity is measured by calculating the amount of hydrogen peroxide.

 (2) Method for measuring oxygen absorption by enzyme reaction

 0.1 lm 1 of Tris-HCl buffer and 50 u1 of the enzyme solution are mixed, and the total volume is adjusted to 3.0 ml with distilled water. The mixture is placed in the oxygen electrode cell of Rank Brothers. After stirring at 25 ° C to equilibrate the dissolved oxygen with the temperature, 50 mM FV1001 is added, and the oxygen absorption is continuously measured with a recorder to obtain the initial velocity. Calculate the amount of oxygen absorbed per minute from the standard curve, and use this as the enzyme unit. Example 1 Cloning of DNA encoding FAOD-P

1. Determination of the partial amino acid sequence of F A 0D-P of Penicillium janthinellum S-3413 (FERM BP-5475)

 1) Culture of P. janthinellum S-3413 (FERM BP-5475) and purification of F A 0 D— P

 P. Jansinerum strain S-3413 (FERM BP-5475) was transformed from FZL (fructosyl-N.-Z-lysine) 0.5%, glucose 1.0%, dipotassium phosphate 0.1%, sodium phosphate monobasic 0.1%, Inoculate 10 L of a medium (pH 6.0) containing 0.05% of magnesium sulfate, 0.01% of calcium chloride, and 0.2% of yeast extract, and use a jar fermenter to aerate at a rate of 2 L / min and a stirring speed of 40 Orpn. At 28 ° C for 24 hours. Cultures were collected by filtration.

Mycelium 270 g (wet weight), 0.1 M Tris The suspension was suspended in 80 Oml of a monohydrochloric acid buffer (pH 7.5), and the mycelium was disrupted with a Dino mill. The crushed liquid was centrifuged at 9,50 Orpm for 20 minutes, and the obtained liquid was used as a crude enzyme solution and purified by the following method.

 Ammonium sulfate (hereinafter abbreviated as ammonium sulfate) was added to the crude enzyme solution so as to be 40% saturated, stirred, and centrifuged at 12.00 Orpm for 10 minutes. Ammonium sulfate was added to the obtained supernatant to 75% saturation, stirred, and centrifuged at 12,000 で m for 10 minutes. The precipitate was dissolved in 5 OmM Tris monohydrochloride buffer (pH 7.5) containing 2 mM DTT (hereinafter abbreviated as buffer A). An equal volume of buffer A containing 40% ammonium sulfate was added to the solution, and then about 20 Oral of butyl-TOYO PEARL resin was added, followed by gentle stirring. After washing with the same buffer, elution was carried out with the buffer A using the batch method. The eluate was concentrated with ammonium sulfate, and adsorbed on a FUJI TORTO YOP EARL column equilibrated with buffer A containing 25% saturated ammonium sulfate. After washing with the same buffer, elution was performed with a linear gradient of ammonium sulfate concentration of 25-0% saturation. The active fractions were collected, concentrated with ammonium sulfate, and adsorbed on a butyl toe pearl column equilibrated with buffer A with 40% saturated ammonium sulfate. After washing with the same buffer, elution was carried out with a linear concentration gradient of ammonium sulfate concentration of 40-0% saturation. The active fractions were combined and applied to a DE AE-Toyopearl column equilibrated with buffer A. The active fraction was found in the fraction washed with the same buffer, which was collected and concentrated with ammonium sulfate. Subsequently, the obtained enzyme solution was subjected to gel filtration using a Sephacryl S-300 column equilibrated with a 0.1 M Tris-HCl buffer solution (pH 7.5) containing 0.1 M NaCl and 2 mM 0-chome. 100 units of purified enzyme were obtained.

The obtained purified enzyme preparation was subjected to SDS-PAGE (sodium dodecyl sulfate 'polyacrylamide gel electrophoresis) to use phosphorylase B, bovine serum albumin, ovalbumin, and carbonic anhydride as standard proteins. The molecular weight was measured using the enzyme and soybean tribune inhibitor according to the method of Davis. That is, using a 10% gel, electrophoresis was performed at 40 mA for 3 hours, and protein staining was performed with Coomassie Prilian Rebel G-250. The electrophoresis of several proteins of known molecular weight was carried out in the same manner, and the molecular weight was determined from the calibration curve. The result showed that the molecular weight of the subnet was about 48,700 daltons (Fig. 6).

 In addition, the molecular weight measurement by gel filtration using 200 pg of Superdex showed a value of about 38,700 daltons, as is clear from the calibration curve shown in FIG.

 2) Determination of partial amino acid sequence

 The enzyme preparation purified by the above method was fragmented with V8 protease (manufactured by Sigma) and fragmented by the Cleveland method [D. Cleaveland, SGFisher, MW Kirschner and UK Laemnili, J, Biol, Chem., 252 , 1102 (1977) 3. Next, the PVDF (polyvinylidene fluoride, manufactured by Millipore, trade name, Imopiron-P SQ) is transferred to the membrane (at 14 V— 晚 (12 hours)), and the protein sequencer 476A (Applied Biosystems) Was used to determine the amino acid sequence by the Edman degradation method. As a result, the amino acid sequences of 14 and 20 residues shown in SEQ ID NOs: 2 and 3 were determined from the two fragments of the N-terminal and internal peptides, respectively.

 2. Width of partial cDNA fragment by RT-PCR

 1) Preparation of oligonucleotide primer

Based on the nucleotide sequence deduced from the amino acid sequence obtained in 1.2) above, primers for use in PCR (polymerase chain reaction) were designed as shown in FIG. In designing this primer, the codon usage of Penicillium was taken into consideration, and in order to facilitate subcloning, A BamHI recognition sequence was added to the end of the primer. The nucleotide sequences of these primers 1 and 2 are shown in SEQ ID NOs: 4 and 5, respectively. Primer 2 is synthesized from the C-terminal side based on the sequence shown in FIG. 1 so as to attach to DNA complementary to DNA to which primer 1 attaches.

 2) Preparation of total RNA

 From 1 g of the mycelium of P. janthinelluro S-3413 cultured by the method described in 1.1) above, the guanidine / fuyunol / cloguchi-form method (Chomczynski, P. and Sacc hi, N. (1987) Single-step method of According to RNA isolation by acid guanidini um thiocyanate-PhOi chloroform extraction, Anal. _Biochem. 162, 156-159), 5 mg of total RNA was obtained.

 3) RT-PCR

 Using the primer designed in 1.2) above and the total RNA prepared in 2.2), a reverse transcription polymerase tune reaction (RT-PCR) was performed according to the following procedure. a) Add sterile water 361 to total RNA (5 ^ g / ^ l) 21, heat at 65 ° C for 5 minutes, and quench on ice.

 b) Add the following solution to the solution of a).

 5 X buffer 20 ul

 d NT Pmix (2 OmM each) 5 j l

 RNase inhibitor (1 1 5 U / ml) 2 ju l

 Oligo dt (0.4.2 fig / fil 2 A nl

 R Tase (L V) (2 0 0 UZ / 1) 1 l

 D TT (0.1 M) 1 0〃1

c) Leave the solution of a) + b) at 25 ° C for 10 minutes, and react at 42 ° C overnight. Then, after heating at 95 for 5 minutes, quench on ice to obtain cDNA. d) Add the following solution to cDNA2n synthesized in c).

 10 P C R buffer 2.5 / 1

 d NTP mi x 1.8 / 1

 Primer 1 1 1

 Primer 2 1 i 1

 Sterile water 16.575 1 e) Heat the solution of d) at 95 ° C for 5 minutes, cool rapidly on ice, and add 0.125〃 1 of Taq DNA Polymerase (5 U / m).

 f) Overlay mineral oil on e) and perform PCR under the following conditions.

 (95. C, 1 minute; 62 ° C, 1 minute; 72 ° C, 2 minutes) After a series of 30 cycles, treat at 72 ° C for 3 minutes.

 g) Then, run on agarose gel electrophoresis.

 As a result, when primers 1 and 2 were used, a fragment width of about 690 bp was confirmed as shown in FIG. FIG. 3 is a mimetic diagram showing the results of agarose electrophoresis. In the figure, lane 1 shows 0X174ZHincII (marker), and lane 2 shows the electrophoresis pattern of the PCR product when primers 1 and 2 were used. Electrophoresis was performed using a marker to determine the size of the fragment amplified by PCR.

 3. Sub-cloning of PCR fragment

 The PCR fragment of about 690 bp obtained in step 2 above was cut out from the agarose gel, and the DNA was collected at 10,000 rpm.4 ° using a centrifuge tube with a filter for DNA recovery (pore size 0.22 μπι, Takara Shuzo Co., Code No. 9040). C. After centrifugation for 1 hour, purification was performed by ethanol precipitation.

Next, the PCR fragment (11), K buffer (11), BaniHI (1D and sterilized water (7 // 1)) were mixed and digested at 37 ° C for 4 hours. The BamHI digested fragment was ligated (16 ° C for 30 minutes) to pBluescreipt II SK ⁺ (manufactured by STRATAGENE: an expression vector for Escherichia coli having a 1 ac promoter) also digested with BamBI. The resulting ligation mixture was used to transform E. coli JMl09 strain. Transformation was performed according to the Hanahan method (supra) using TaKaRa Ligation Kit Ver. 2.0 (Takara Shuzo).

 As a result, as shown in FIG. 4, a plasmid pFPP in which a PCR fragment of about 69 Obp was inserted into the BaniHI site of pBluescript II SK + was obtained. In FIG. 4, lane 1 represents: I ZEcoT 141 (marker 1) and lane 2 represents a BamHI digest of plasmid pFPP. When its nucleotide sequence was determined by the dideoxy method, it was confirmed to be a partial sequence of FAOD-P cDNA.

4. Preparation of cDNA Library and Plaque Hybridization mRNA was obtained from total RNA obtained by the method of 2.2) above using mRNA Purification Kit (Falman). From 5 g of the mRNA, a cDNA library was prepared using a ZAP-cDNA Synthesis Kit (manufactured by STRATAGENE). That is, cDNA is synthesized from 5 g of mRNA using reverse transcriptase, ligated to λII vector (manufactured by STRATAGENE), packaged in vitro using Gigapack III Gold (manufactured by STEATAGENE), and in vitro cloned into a cDNA library. (The conditions and the like were in accordance with the manual.) Then, the titer of cDNA was measured, and as a result, it was 1.8 × 10 ⁵ piu / 〃g vector.

This phage library was infected with E. coli XLI-Blue MRF strain, and cultured at 37 ° C for 12 hours to form plaque. Next, the PCR fragment subcloned in step 3 was labeled with ³² P and used as a probe. And screened by plaque hybridization. That is, the obtained plaque was transferred to a nitrocellulose filter, and after denaturation, hybridized with a probe labeled with ³² P at 42 ° C. for 12 hours. After washing, X-ray film was exposed for 12 hours. As a result, five positive plaques were obtained from about 184,000 plaques.

5. Subcloning of DNA encoding F A〇D—P

 Subcloning of DNA encoding FAOD-P into plasmid was performed by in vivo excision. Escherichia coli S0LR was transformed from five positive plaques using ExAssist helper phage (manufactured by STRATAGENE) according to the attached manual. Plasmid was extracted from the obtained transformant and the nucleotide sequence was determined. As a result, one clone containing the plasmid pFAP1 into which an approximately 1.5 kb DNA fragment having a nucleotide sequence corresponding to the N-terminal amino acid sequence of FAOD-P was inserted (E. coli transformant) was obtained. (And. ^ I SOL RZFAP 1)). FIG. 2 shows the restriction map of pFAP1. The nucleotide sequence and deduced amino acid sequence of this clone are shown in SEQ ID NO: 1.

Example 2 Activity of FAOD-1P of Escherichia coli transformant (E. coli SOLR-NO FAP 1)

 Escherichia coli (. ^ L SOLR / FAP 1) transformed in Example 1 was transformed into LB medium (1% Bacto-Trypton, 0.5% Bacto -Yeast extract, 1% NaCI, pH7.2) 5 Oml. IPTG was added 2 hours after inoculation with E. coli.

After the culture, the cells were collected by centrifugation (10,000 rpm, 4 ° C, 1 minute), and the pellet was washed with 0.85% KC1 and suspended in 0.1 M Tris-HCl buffer (pH 7.5). 3.800 rpm with MINI-BEAT BEATER (Japan Lambda) The beads were crushed 6 times in 30 seconds with ice cooling, and centrifuged (1,400 卬 π 4 ° C, 5 minutes) to prepare a cell-free extract. Subsequently, FAOD-P activity was measured by the A. rate method of the above titration method. As a control, a cell-free extract obtained by similarly culturing Escherichia coli transformed with the plasmid pBluescreipt II SK was used. The results are shown in Table 1 together with the activity detected from the culture of P. janthinellum S-3413.

 Expression of FAOD-P by E. coli SOL R transformed with 1_ plasmid p FAP1

 Strain specific activity (UZmg)

 U / nig • Protein

 SOLR / p F AP 1 0.0953

 S 0 L R pBluescript II SK .D.

 P. janthinellum S-3413 0. 0971 It is clear from the table that pFAPl contains a cDNA encoding FAOD-P, and the E. coli transformant transformed with the plasmid is FAOD-P Is produced. Escherichia coli transformed with this plasmid p F AP I. ^ ii SOLR / FAP 1 was contracted with Tsukuba-Higashi 1-chome 3-chome, Ibaraki Pref. Deposited under BP-57 62 (Original deposit: October 4, 1995; transfer to international deposit: December 2, 1996) 0

The production of FAOD-P by E. coli transformants reached a maximum 8 hours after the addition of IPTG, and showed the same productivity as the parent strain, Penici ^ ium-janthinellum S-3413 (see Fig. 5). In FIG. 5, the horizontal axis is the time after induction with IPTG, the vertical axis is the degree of proliferation (OD ₆ Qo measurement value), and the solid circle is the total activity (ϋ Zl culture) and open circles indicate specific activity (UZmg).

Example 3 Measurement of glycated hemoglobin amount

 1) Sample processing

 0 to 15 mg glycohemoglobin control E (Sigma) was dissolved in 100% distilled water. 1 ml of acetone hydrochloride (11/1 hydrochloric acid 7acetone: 1Z100) was added to these samples, and centrifuged at 12,000 rpm for 10 minutes. The precipitate was washed with getyl ether 5001, and dried under reduced pressure. Further, 8 M urea 1001 was added, heated in boiling water for 20 minutes, cooled, mixed with 5.4 unit Zml trypsin 3001, and incubated at 37 ° C for 3 hours. Thereafter, the sample was heated in boiling water for 5 minutes to prepare a sample.

 2) Activity measurement

 The FA〇D reaction solution was prepared as follows.

 3 mM N— (carboxymethylaminocarbonyl)

 4.4—Bis (dimethylamino) biphenylamine solution 30 1

60 units Zml peroxidase solution 30〃 1

0.1 M Tris-HCl buffer (pH 8.0) 300 1

25 units 1111 8 00-solution 51 The total volume was adjusted to 1 ml with distilled water.

 The 25 u / ml FA〇D-P solution was prepared by diluting the FA0D-P obtained in the method of Example 2 with 0.1 M potassium phosphate buffer (pH 7.5) to 25 u / p. Prepared.

150 1 of each of the above treated substrates was added to the FAOD reaction solution, the mixture was incubated at 30 ° C, and the absorbance at 727 nm was measured 30 minutes later. FIG. 8 shows the relationship between the amount of saccharified hemoglobin obtained by this method and the absorbance. The vertical axis in the figure is the absorbance of 727ηπι (corresponding to the amount of hydrogen peroxide), and the horizontal axis is saccharified Represents the amount of The figure shows that there is a correlation between the amount of glycated hemoglobin and the amount of hydrogen peroxide generated.

Example 4 Measurement of hemoglobin A 1 c value

 Hemoglobin A0 reagent (Sigma) was dissolved in distilled water to make 2.3. This solution was fractionated using an automatic glycohemoglobin measuring device (Kyoto Daiichi Kagaku), and the hemoglobin A1c fraction and the hemoglobin A0 fraction were fractionated and purified. By mixing the two fractions in proportion, a base sample having an A1c value of 0% to 52.0% was obtained.

 2 9

 1) Sample processing

 Substrate sample 250】

500 units / ^ 1 aminopeptidase solution 5 1 0.1M Tris-HCl buffer (pH 8.0) 15〃] These were mixed and made up to a total volume of 200] with distilled water.

 This mixture was incubated at 30 ° C for 30 minutes. Thereafter, 10% trichloroacetic acid (200α1) was added and the mixture was stirred, allowed to stand at 0 ° C for 20 minutes, and centrifuged at 12,000 rpm for 10 minutes. About 40] of 5N NaOH was added to the obtained supernatant to make a neutral solution.

 2) Activity measurement

 The F A 0 D reaction solution was prepared as follows.

 3mM N— (carboxymethylaminocarbonyl)

 4.4-Bis (dimethylamino) biphenylamine solution 100〃]

60 units / ml peroxidase solution 100 0.1 M Tris-HCl buffer (pH 8.0) 1000 116 units Zml FAOD-P solution 51 The total volume was adjusted to 2.6 ml with distilled water. The 16 units Zml FAOD-P solution was prepared by diluting the FA〇D-P obtained by the method of Example 2 with 0.1 M potassium phosphate buffer (pH 7.5) to 16 units Zml. Prepared.

 After incubating the FAOD reaction solution at 30 ° C. for 2 minutes, 400 1 of each of the above treated substrates was added, and the absorbance at 727 nm was measured after further incubation for 30 minutes. FIG. 9 shows the relationship between the hemoglobin Ale value and the absorbance of the substrate obtained by this method. The vertical axis in the figure represents the absorbance at 727 nm (corresponding to the amount of hydrogen peroxide), and the horizontal axis represents the hemoglobin Ale value. The figure shows that there is a correlation between the hemoglobin A1c value and the amount of generated hydrogen peroxide. Example 5 Expression of FAOD-P in yeast

 (1) Construction of FAOD-P expression vector

 From the E. coli strain obtained in Example 2 and £ 2 cS OLR / FAP1 (FERM BP-5762), an Escherichia coli expression vector pFAP1 containing a cloned cDNA derived from Penicillium. Obtained.

 The obtained pFAP1 was digested with restriction enzymes EcoRI and Xhol to obtain a FAOD-Pc DNA fragment of about 1.3 kb.

 Plasmid pNOTel (Japanese Patent Laid-Open No. 5-344895) is digested with restriction enzyme Not I, dephosphorylated using cyst intestinal phosphatase (Boehringer Mannheim), and DNA together with the FAOD-P cDNA fragment described above. Smoothing was performed using Blunting Kit (Takara Shuzo). These were ligated using a DN ligation Kit (Takara Shuzo Co., Ltd.) to obtain plasmid pNFP.

Next, E. coli J109 strain was transformed by the Hanahan method (supra) using plasmid pNFP. Plasmid was prepared by arbitrarily selecting 84 strains from the obtained transformants. Plasmid is treated with restriction enzyme Hindlll and inserted. After confirming the orientation, plasmid pNFFP, in which the FAOD-Pc DNA fragment was inserted downstream of the AOD promoter, was obtained. The restriction map of the plasmid pNFP1 is shown in FIG.

 (2) Transformation

 The above plasmid pNFP1 was linearized with the restriction enzyme BamHI, and then transformed into the .boidiniiTK62 strain using a lithium modification method. This TK62 strain is required for lira, and since the plasmid KpNOTel contains the URA3 gene, transformants can be selected based on the Ura requirement. Eight transformants were arbitrarily selected from URA + -type transformants obtained by spreading the transformants on YNB medium without Ura, inoculated into a 1.5% methanol-containing basic medium, and incubated at 28 ° C for 3 days. The cells were cultured with shaking. After collection, the F AOD-P activity in the cells was measured by the A. rate method in the above titration method, and the results in Table 2 were obtained.

Table 2 From £ .boidiniiTK62 strain transformed with plasmid pNF P1

FAOD-P expression

 Share ratio activity

 U / mg 'protein

 TK62 / pNFP l-l 0.0816

TK62 / pNFP 1-2 0.071 1

TK62 / pNFP 1-3 0.10 70

TK62 / pNFP 1-4 0.0268

TK62 / pNF P 1-5 0.0312

TK62 / pNF P 1-6 0.0394

TK 62 / pNF P 1-7 0.0409

TK62 / pNFP l-8 0.0249

TK 62 / pNOTel ND (control) It is clear from Table 2 that eight strains had FAOD-P activity, of which ς. BoidiniiTK62 / pNFP1-3 showed the highest activity.

Arrangement table

 SEQ ID NO: 1

 Array length: 1 3 1 4

 Sequence type: nucleic acid

 Number of chains: double strand

 Topology: linear

 Sequence type: c D N A to m R N A

 Origin

 Organism name: Penicillium j anthinellum S-3413CFERM BP-5475) Sequence

 AT

 Me

GG

 Gl

GG

Two

 Two

Gly Tyr Thr Pro Ser Asn He Thr Val Leu Asp Val Tyr Pro lie

 35 40 45

CCA TCC TTG CAA TCC GCA GGA TAT GAT CTT AAC AAG ATC ATG AGC 180

Pro Ser Leu Gin Ser Ala Gly Tyr Asp Leu Asn Lys lie Met Ser

 50 55 60

ATC CGA TTA CGC AAC GGG CCT GAC TTG CAA CTT TCC CTG GAG GCT

lie Arg Leu Arg Asn Gly Pro Asp Leu Gin Leu Ser Leu Glu Ala

65 70 75 CTC GAT ATG TGG Leu Asp Met Trp

AAC GTT GGC ATG

Asn Val Gly Met

AGC CTT CGA CGG

Ser Leu Arg Arg

CTA GAG AAG ACG

Leu Glu Lys Thr

GCA AAA GCC CCG

Ala then ys Ala Pro

GGC TTG TTT TGC

Gly leu Phe Cys

ATC AAT GCG ATC

lie Asn Ala lie

GGA TTT GGA AGT

Gly Phe Gly Ser

GAT GGG GCG ACA

Asp Gly Ala Thr Cys Ser Gly Val Glu Thr Val Asp Gly Thr Lys

200 205 210

TAC TTC GCC GAC AAG GTG GTT TTG GCC GCT GGT GCT TGG AGT TCG

Tyr Phe Ala Asp Lys Val Val then eu Ala Ala Gly Ala Trp Ser Ser

 215 220 225

ACG TTA GTA GAT TTG GAG GAC CAA TGT GTT TCG AAG GCC TGG GTC 720 Thr Leu Val Asp Leu Glu Asp Gin Cys Val Ser Lys Ala Trp Val

 230 235 240

TTC GCT CAT ATC CAA CTC ACG CCC CAA GAA TCG CCC CAG TAC AAG 765 Phe Ala His lie Gin Leu Thr Pro Gin Glu Ser Ala Gin Tyr Lys

 245 250 255

GAC GTG CCC GTA GTA TAC GAC GGT GAT TAT GGC TTT TTC TTC GAG 810 Asp Val Pro Val Val Tyr Asp Gly Asp Tyr Gly Phe Phe Phe Glu

 260 265 270

CCC AAC GAA CAC GGA GTA ATC AAA GTC TGC GAT GAG TTC CCC GGG 855 Pro Asn Glu His Gly Val lie Lys Val Cys Asp Glu Phe Pro Gly

 275 280 285 6

 7 δ

TTC TCC CGC TTC AAG CTG CAT CAA CCT TAC GGT GCC ACC TCT CCT 900 Phe Ser Arg Phe lys Leu His Gin Pro Tyr Gly Ala Thr Ser Pro

 290 295 300

AAG CTT ATA TCC GTT CCT CGA TCA CAC GCC AAG CAT CCC ACC GAT 945

Lys leu lie Ser Val Pro Arg Ser His Ala Lys His Pro Thr Asp

 305 310 315

ACC TAC CCA GAT TCT TCT GAA GAG ACC ATT CGA AAA GCG ATT GCG 990 Thr Tyr Pro Asp Ser Ser Glu Glu Thr lie Arg Lys Ala lie Ala 320 325 330

AGG TTT ATG CCA CGC TTC AAG GAT AAG GAG CTT TTT AAT AGG AGC 1035 Arg Phe Met Pro Arg Phe lys Asp Lys Glu Leu Phe Asn Arg Ser

 335 340 345

ATG TGC TGG TGC ACC GAT ACT GCT GAT GCC AAC TTG TTG ATC TGC 1080 Met Cys Trp Cys Thr Asp Thr Al a Asp Ala Asn Leu Leu He Cys

 350 355 360

GAG CAC CCC AAG TGG AAG AAC TTT ATC TTG GCC ACA GGA GAC AGC 1125 Glu His Pro Lys Trp Lys Asn Phe lie Leu Ala Thr Gly Asp Ser

 365 370 375

GGC CAT AGT TTC AAG GTT TTG CCC AAT ATA GGA AAA CAT GTC GTT 1170 Gly His Ser Phe Lys Val leu Pro Asn lie Gly Lys His Val Val

 380 385 390

GAG TTG ATA GAA GGA CGC CTA CCA CAA GAC CTG GCT GGT GCG TGG 1215 Glu Leu lie Glu Gly Arg leu Pro Gin Asp Leu Ala Gly Ala Trp

 395 400 405

AGA TGG AGA CCA GGG GGA GAT GCC CTT AAG TCC AAA CGC AGT GCT 1260 Arg Trp Arg Pro Gly Gly Asp Ala Leu Lys Ser lys Arg Ser Ala

 410 415 420

CCG GCA AAG GAC CTT GCT GAA ATG CCG GGC TGG AAG CAT GAT GCG 1305 Pro Ala Lys Asp Leu Ala Glu Met Pro Gly Trp lys His Asp Ala

 425 430 435

AAG CTC TGA 1314 Lys Leu

437 SEQ ID NO: 2

 Array length: 1 4

 Sequence type: amino acid

 Topology: linear

 Sequence type: Peptide

 Origin

 Organism name: Penicillium janthinellum S-3413 (FERM BP-5475) sequence

 Ser Thr Lys lie Val lie Val Gly Gly Gly Gly Thr Met Gly

 1 5 10 14 SEQ ID NO: 3

 Array length: 20

 Sequence type: amino acid

 Topology: linear

 Sequence type: peptide

 Origin

 Organism name: Penicillium janthinellum S-3413 (FERM BP-5475) Sequence

 Gly Val Glu Thr Val Asp Gly Thr Lys Tyr Phe Ala Asp Lys Val Val 1 5 10 16

Leu Ala Ala Gly

 20

 SEQ ID NO: 4

 Array length: 2 3

Sequence type: nucleic acid Number of chains: single strand

 Topology: linear

 Sequence type: synthetic DNA

Array

 ACNAARATHG TNATHGTNGG NGG 23 SEQ ID NO: 5

 Array length: 35

 Sequence type: nucleic acid

 Number of chains: single strand

 Topology: Linear

 Sequence type: synthetic DNA

Array

 ACNGTNGAYG GNACNAARTA YTTYGCNGAY AARGT 35

Claims

The scope of the claims

 1. Oxidizing an Amadori compound in the presence of oxygen, which has an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence derived by inserting, deleting or substituting one or more amino acids with the amino acid sequence And a fructosyl amino acid oxidase having an enzymatic activity to catalyze a reaction for producing a ketoaldehyde, an amide derivative and hydrogen peroxide.

 2. P. janthinellum S-3413 (FERM BP-5475) is obtained by cloning DNA encoding fructosylamino acid oxidase induced by culturing in browning medium. Item 7. The fructosylamino acid oxidase according to Item 1.

 3. A DNA encoding the fructosylamino acid oxidase according to claim 1 or 2.

 4. A DNA described in SEQ ID NO: 1 or a DNA capable of hybridizing with the DNA under stringent conditions, wherein the Amadori compound is oxidized in the presence of oxygen to produce a peak-aldehyde, an amine derivative and DNA encoding fructosylamino acid oxidase which has enzymatic activity to catalyze the reaction to produce hydrogen peroxide.

 5. An expression vector containing the DNA according to claim 3 or 4.

 6. A host cell transformed by the expression vector according to claim 5.

7. The host cell according to claim 6, which is a prokaryotic or eukaryotic host.

 8. The host cell according to claim 7, which is Escherichia coli or yeast.

 9. The host cell according to claim 8, which is a methanol yeast.

 10. A method for producing a recombinant fructosylamino acid oxidase, which comprises culturing the host cell according to claim 5 in a medium.

11. medium, methanol from 0.1 to 5.0%, and 0.1 to 5.0% of NH ₄ C1 and / Or the production method according to claim 10, which comprises 0.1 to 5.0% of yeast extract.

12. Contacting a sample containing an Amadori compound with the culture of the host cell according to claim 10 or 11 or a processed product thereof, and measuring the amount of consumed oxygen or the amount of generated hydrogen peroxide. wherein the analysis method _c Amadori compound in a sample

13. The sample is a biological component, and the analysis of the Amadori compound is performed by measuring the amount and / or saccharification rate of the glycated protein in the biological component or by determining the amount of fructosamine. 2. The analytical method according to 2.

 14. A reagent or kit for analyzing an Amadori compound comprising the culture of the host cell according to any one of claims 6 to 9 or a processed product thereof.

 15. The reagent or kit according to claim 14, wherein the reagent or kit is used for measuring the amount and / or saccharification rate of a glycated protein in a biological component or for quantifying fructosamine.