WO1997021818A1 - Nouvelle oxydase d'acide amine fructosyle provenant d'un champignon du genre penicillium - Google Patents

Nouvelle oxydase d'acide amine fructosyle provenant d'un champignon du genre penicillium Download PDF

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Publication number
WO1997021818A1
WO1997021818A1 PCT/JP1996/003651 JP9603651W WO9721818A1 WO 1997021818 A1 WO1997021818 A1 WO 1997021818A1 JP 9603651 W JP9603651 W JP 9603651W WO 9721818 A1 WO9721818 A1 WO 9721818A1
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faod
amino acid
dna
host cell
amount
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PCT/JP1996/003651
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Japanese (ja)
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Nobuo Kato
Yasuyoshi Sakai
Yoshiki Tani
Hiroshi Fukuya
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Kyoto Daiichi Kagaku Co., Ltd.
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Publication of WO1997021818A1 publication Critical patent/WO1997021818A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)

Definitions

  • 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.
  • FAOD-P fructosyl amino acid oxidase
  • 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.
  • a fructosylamine derivative which is an amadori compound composed of glucose and amino acids
  • Fructosylamine derivatives in which hemoglobin in blood is glycated are derivatives in which glycated hemoglobin and albumin are glycated.
  • Glycoalbumin is called glycated albumin
  • fructosamine the derivative of glycated protein in blood is called fructosamine.
  • reaction of an amadori compound with an oxidoreductase can be represented by the following general formula.
  • 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)
  • 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.
  • 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.
  • there has been no cloning of DNA encoding FAOD-P from P. genus and it was necessary to clone such a DNA first.
  • 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.
  • FAOD-P fructosylamino acid okinidase
  • 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.
  • 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.
  • 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 ⁇ -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 ⁇ D-P.
  • the DNA may be either a complementary DNA or a synthetic DNA.
  • the DNA of the present invention can be represented by the nucleotide sequence of SEQ ID NO: 1 or a partial sequence thereof.
  • 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).
  • 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 ⁇ 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 present invention also provides a host cell transformed by the above expression vector.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 727 ⁇ ).
  • FIG. 10 is a restriction map of the vector pNFP1 for expression of FAOD-P in yeast.
  • Cloning of DNA encoding FAOD-P can be performed according to methods known in the art.
  • FAOD-1P was purified from a culture of P. janthinellum S-3413CFERM BP-5475), and the N-terminal amino acid was determined therefrom.
  • 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.
  • RT-PCR reverse transcription polymerase chain reaction
  • 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.
  • 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.
  • 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.
  • host cells include microorganisms [prokaryotes (eg, bacteria such as Escherichia coli and Bacillus subtilis), eukaryotes (eg, yeast)], animal cells, and cultured plant cells.
  • microorganisms include prokaryotes, particularly strains belonging to the genus Escherichia (eg, E. coli), yeasts, particularly strains belonging to Saccharomyces ⁇ (eg, S—. Cerevisiae) and strains belonging to Candida ⁇ ( 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.
  • 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.
  • 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.
  • the DNA is inserted downstream of the promoter of an expression plasmid suitable for transforming E. coli.
  • an expression plasmid suitable for transforming E. coli.
  • intracellular expression and secretory expression 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.
  • 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.
  • Penicillium eg, ⁇ Penicillium j anthinellum S-3413; FERM BP -5475
  • 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.
  • 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.
  • an FAOD-P expression vector is constructed using plasmid pNOTel (Japanese Patent Application Laid-Open No.
  • 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.
  • 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.
  • 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.
  • DNA encoding FAOD-P inserted into E. coli S OLR / FAP1 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.
  • a suitable enzyme eg, restriction enzyme, alkaline phosphatase, polynucleotide kinase, DNA ligase.
  • DNA polymerase DNA polymerase, etc.
  • 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.
  • 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.
  • 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.
  • a carbon source eg, glucose, methanol, galactose, fructose, etc.
  • an inorganic or organic nitrogen source eg, ammonium sulfate, ammonium chloride, sodium nitrate, leptone, casamino acid, etc.
  • other nutrients eg, inorganic salts (sodium chloride, potassium chloride)
  • vitamins eg, vitamins, antibiotics (eg, ampicillin, tetracycline, kanamycin, etc.)
  • Eagle's medium is suitable for culturing animal cells.
  • 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.).
  • 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.
  • an appropriate aqueous solution eg, Tris-HCl buffer
  • 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.
  • the culture is centrifuged to collect a FADPD-producing transformant, suspended in a phosphate buffer, and disrupted by sonication or the like.
  • the enzyme sample is obtained by centrifuging the supernatant.
  • the supernatant is filtered and further purified by chromatography or the like to obtain a purified enzyme.
  • a fragment having an enzymatic activity can be obtained by restriction enzyme treatment or exonuclease treatment.
  • the above cultures and their processed products 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.
  • 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 ⁇ 1 CH 2 -NH— R 2 + 0 2 + H 2 0 ⁇
  • R 1 represents an aldose residue
  • R 2 represents an amino acid, protein or peptide residue
  • 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.
  • 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.
  • 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.
  • 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. .
  • 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.
  • a Tris-monohydrochloride buffer or the like is used as a buffer.
  • 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.
  • any of the following methods for quantifying an Amadori compound is used.
  • 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.
  • 4-aminoaminoantipyrine ZN-ethyl-1 N-1 (2-hydroxy-13-sulfopropyl) 1 m-toluidine, 4-1-1 instead of 4-aminoantipyrine / funol system
  • 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 ⁇ D-P used should be 1 unit / ⁇ 1, and the amount of oxygen absorbed by adding an appropriately diluted sample should be determined.
  • 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.
  • protease 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.
  • the protease treatment can be performed by the method described in the following Examples.
  • 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.
  • a blood sample whole blood, plasma or serum
  • the collected blood sample is used as it is or after being subjected to a treatment such as folding.
  • 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.
  • 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.
  • 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.
  • 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.
  • the solid support is selected from a polymer gel or the like, and is preferably alginic acid.
  • 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. ⁇ ).
  • the buffer is preferably a Tris-monohydrochloride buffer (pH 7. ⁇ ).
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • FZL fructtosyl-N.-Z-lysine
  • 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 ⁇ 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.
  • buffer A OmM Tris monohydrochloride buffer
  • 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).
  • 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.
  • the PVDF polyvinylidene fluoride, manufactured by Millipore, trade name, Imopiron-P SQ
  • the protein sequencer 476A (Applied Biosystems) was used to determine the amino acid sequence by the Edman degradation method.
  • 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.
  • primers for use in PCR were designed as shown in FIG.
  • 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.
  • RT-PCR reverse transcription polymerase tune reaction
  • FIG. 3 is a mimetic diagram showing the results of agarose electrophoresis.
  • lane 1 shows 0X174ZHincII (marker)
  • 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.
  • 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 ⁇ , Takara Shuzo Co., Code No. 9040). C. After centrifugation for 1 hour, purification was performed by ethanol precipitation.
  • 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.
  • STRATAGENE an expression vector for Escherichia coli having a 1 ac promoter
  • 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).
  • a plasmid pFPP in which a PCR fragment of about 69 Obp was inserted into the BaniHI site of pBluescript II SK + was obtained.
  • lane 1 represents: I ZEcoT 141 (marker 1) and lane 2 represents a BamHI digest of plasmid pFPP.
  • its nucleotide sequence was determined by the dideoxy method, it was confirmed to be a partial sequence of FAOD-P cDNA.
  • cDNA Library and Plaque Hybridization 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 ⁇ II 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ ⁇ 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.
  • 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.
  • acetone hydrochloride 11/1 hydrochloric acid 7acetone: 1Z100
  • the FA ⁇ D reaction solution was prepared as follows.
  • the 25 u / ml FA ⁇ 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.
  • 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 727 ⁇ (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.
  • 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.
  • the F A 0 D reaction solution was prepared as follows.
  • 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.
  • 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.
  • 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.
  • 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.
  • Sequence type nucleic acid
  • Organism name Penicillium j anthinellum S-3413CFERM BP-5475) Sequence
  • Lys leu lie Ser Val Pro Arg Ser His Ala Lys His Pro Thr Asp
  • Organism name Penicillium janthinellum S-3413 (FERM BP-5475) sequence
  • Organism name Penicillium janthinellum S-3413 (FERM BP-5475) Sequence
  • Sequence type nucleic acid Number of chains: single strand
  • Sequence type nucleic acid

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Abstract

La présente invention concerne un vecteur d'expression contenant un gène codant pour une oxydase d'acide aminé fructosyle issu du genre Penicillium (FAOD-P) et présentant une activité fonctionnelle au niveau de cellules hôtes. L'invention concerne également des cellules hôtes transformées par ce vecteur. L'invention concerne aussi un procédé de préparation d'une nouvelle FAOD-P selon l'invention par mise en culture du transformant obtenu ainsi que la FAOD-P préparée de cette façon. L'invention concerne enfin un procédé d'analyse des composés Amadori au moyen de ladite FAOD-P, ainsi que les réactifs et matériels utiles à cet effet.
PCT/JP1996/003651 1995-12-14 1996-12-13 Nouvelle oxydase d'acide amine fructosyle provenant d'un champignon du genre penicillium WO1997021818A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1291416A1 (fr) * 2001-09-04 2003-03-12 Kikkoman Corporation Fructosyle peptide oxidase
WO2004104203A1 (fr) * 2003-05-21 2004-12-02 Asahi Kasei Pharma Corporation Methode de mesure d'hemoglobine glycolee a1c, enzyme a utiliser pour cette methode et son procede de production

Citations (5)

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Publication number Priority date Publication date Assignee Title
JPS61268178A (ja) * 1985-05-22 1986-11-27 Noda Sangyo Kagaku Kenkyusho フルクトシルアミノ酸オキシダーゼ
JPH03155781A (ja) * 1989-10-20 1991-07-03 Bristol Myers Squibb Co アリールスルファターゼ
JPH044874A (ja) * 1990-04-20 1992-01-09 Nakano Vinegar Co Ltd フラクトシルアミノ酸分解酵素、その製造法及びその利用
JPH05192193A (ja) * 1991-07-29 1993-08-03 Genzyme Ltd 非酵素的グリコシル化タンパク質の検定法
JPH0646846A (ja) * 1992-06-05 1994-02-22 Nakano Vinegar Co Ltd フルクトシルアミンデグリカーゼ、その製造法及び該酵素を用いたアマドリ化合物の定量方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61268178A (ja) * 1985-05-22 1986-11-27 Noda Sangyo Kagaku Kenkyusho フルクトシルアミノ酸オキシダーゼ
JPH03155781A (ja) * 1989-10-20 1991-07-03 Bristol Myers Squibb Co アリールスルファターゼ
JPH044874A (ja) * 1990-04-20 1992-01-09 Nakano Vinegar Co Ltd フラクトシルアミノ酸分解酵素、その製造法及びその利用
JPH05192193A (ja) * 1991-07-29 1993-08-03 Genzyme Ltd 非酵素的グリコシル化タンパク質の検定法
JPH0646846A (ja) * 1992-06-05 1994-02-22 Nakano Vinegar Co Ltd フルクトシルアミンデグリカーゼ、その製造法及び該酵素を用いたアマドリ化合物の定量方法

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Title
APPL. ENVIRONMENT. MICROBIOL., Vol. 61(12), 1995, YOSHIDA N. et al., "Distribution and Properties of Fructosyl Amino Acid Oxidase in Fungi", pages 4487-4489. *
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Vol. 59(3), 1995, YOSHIDA N. et al., "Purification and Perperties of Fructosyl Lysine Oxidase from Fusarium Oxysporum S-1F4", pages 487-491. *
BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, Vol. 60(1), 1996, YOSHIDA N. et al., "Production of Fructosyl Lysine Oxidase from Fusarium Oxysporum S-1F4 on Autoclave-Browned Medium", pages 150-151. *
J. BIOL. CHEM., Vol. 252(3), 1977, D.W. CLEAVELAND et al., "Peptide Mapping by Limited Proteolysis in Sodium Dodecyl Sulfate and Analysis by Gel Electro-Phoresis", pages 1102-1106. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1291416A1 (fr) * 2001-09-04 2003-03-12 Kikkoman Corporation Fructosyle peptide oxidase
US7018823B2 (en) 2001-09-04 2006-03-28 Kikkoman Corporation Fructosyl peptide oxidase
US7419813B2 (en) 2001-09-04 2008-09-02 Kikkoman Corporation Fructosyl peptide oxidase
WO2004104203A1 (fr) * 2003-05-21 2004-12-02 Asahi Kasei Pharma Corporation Methode de mesure d'hemoglobine glycolee a1c, enzyme a utiliser pour cette methode et son procede de production
US7588910B2 (en) 2003-05-21 2009-09-15 Asahi Kasei Pharma Corporation Hemoglobin A1c determination method, enzyme to be used therefor, and production method thereof
US7943337B2 (en) 2003-05-21 2011-05-17 Asahi Kasei Pharma Corporation Method for screening a protease

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