JP2000270855A - Fructosylvaline oxidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new enzyme, having specific physical properties, capable of oxidizing fructosylvaline thereby and useful for diagnosis of diabetes mellitus and index, etc., for blood glucose control of diabetes mellitus patients. SOLUTION: This new enzyme has properties (A) oxidizing fructosylvaline, (B) having about 57 kDA molecular weight, (C) having nearly 40 deg.C optimal temperature and (D) having <=40 deg.C stable temperature range by heat treatment for 10 min. Furthermore, the new enzyme is preferably obtained by culturing a microorganism (FERM P-17326) having ability to produce fructosylvaline oxidase and fructosylvaline in a sample is preferably determined by using fructosylvaline oxidase and further, a sensor for measuring fructosylvaline and a sensor for measuring HbAlc are preferably prepared by using the fructosylvaline oxidase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel fructosyl valine kinase. More specifically, the present invention relates to a fructosyl valine kinase produced by a novel microorganism, and a sensor for measuring fructosyl valine using the same.

[0002]

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

[0003] In HbA1c, fructosyl valine can be used as a low-molecular model compound of HbA1c because glucose is bonded to valine at the N-terminal of the hemoglobin β chain. That is, using an enzyme having specificity for fructosyl valine, HbA
It is possible to assay 1c.

[0004] Enzymes acting on Amadori compounds have been isolated from various strains, and it has been suggested that glycated proteins such as glycoalbumin, HbA1c and fructosamine can be analyzed using these enzymes. (JP-A-61-268178, JP-A-61-280)
297, JP-A-3-155780, JP-A-5-1921
93, JP-A-7-289253, JP-A-8-15467
2, Agric. Biol. Chem., 53 (1), 103-110, 1989, Agri
c. Biol. Chem., 55 (2), 333-338, 1991, J. Biol. Che.
m., 269 (44), 27297-27302, 1994, Appl.Environ.Mic
robiol., 61 (12), 4487-4489, 1995, Biosci. Biotech.
Biochem., 59 (3), 487-491, 1995, J. Biol. Chem., 27
0 (1), 218-224, 1995, J. Biol. Chem., 271 (51), 3280
3-32809, 1996, J. Biol. Chem., 272 (6), 3437-3443,
1997).

[0005]

An object of the present invention is to provide a novel enzyme capable of reacting with fructosyl valine and a sensor for measuring fructosyl valine using the enzyme of the present invention.

[0006]

The present inventors screened a microorganism capable of assimilating fructosyl valine and, as a result, succeeded in isolating a novel microorganism that produces fructosyl valine kinase and succeeded in isolating the present invention. Was completed.

That is, the present invention provides a novel enzyme having the following properties: (1) oxidizes fructosyl valine; (2) molecular weight of about 57 kDa; (3) optimum temperature around 40 ° C .; (4) stable temperature range Provide 40 ° C or less (heat treatment for 10 minutes). The present invention also relates to a microorganism belonging to the genus Pichia and capable of producing fructosyl valine kinase, and a method of culturing the microorganism and collecting the enzyme from the culture. A method for producing fructosyl valine kinase is provided.

[0008] The present invention further relates to the use of fructosyl valine and HbA using the fructosyl valine kinase of the present invention.
Provided are a method for assaying 1c, an assay kit, and a sensor for measuring fructosyl valine and a sensor for measuring HbA1c.

[0009]

DETAILED DESCRIPTION OF THE INVENTION The novel microorganism Pichia sp. The N1-1 strain is a strain newly isolated from a marine sample by the present inventor, and its bacteriological characteristics are as follows.

[0010] A film is formed on the surface of the YM liquid medium by static culture. According to observation under a microscope, the cell morphology is elliptical. Cells placed in an anaerobic state in glass slide culture form Mycocandida-type pseudohyphae. Since no color was observed in colony staining with DBB, the strain belongs to Ascomycete yeast or Ascomyces incomplete yeast. As a result of staining with Brilliant Green / Safranin, the presence of spores inside and outside the cells was confirmed, but the presence of ascosium was not confirmed. D
-No fermentation is performed on any of the six sugars, glucose, D-galactose, sucrose, maltose, lactose and raffinose. It performs oxidative metabolism on D-glucose, D-galactose, and, with some delay, lactose and raffinose. Has no assimilation of nitrate.

From the above properties, the "classification and identification method of yeast"
(Hiroshi Iizuka, Shoji Goto, Third Edition, University of Tokyo Press, 19
1980), this strain was identified as a strain belonging to the genus Pichia. This strain was deposited with the National Institute of Bioscience and Biotechnology, the Ministry of International Trade and Industry, under the deposit number FERM.
Deposited as P-17326. Fructosyl valine kinase The novel fructosyl valine kinase of the present invention has the following properties: (1) action oxidizes fructosyl valine in the presence of oxygen to form the corresponding α-keto aldehyde and amino acid, And produce hydrogen peroxide. (2) Molecular weight By both gel filtration and SDS-PAGE,
It was shown to have a molecular weight of about 57 kDa. Therefore, it is understood that the present enzyme is a single peptide enzyme. (3) Optimum temperature FIG. 1 shows the temperature characteristics of the present enzyme. The activity was measured in Example 3.
Was carried out according to the method described in the above. This enzyme has an optimum temperature around 40 ° C. (4) Stable temperature range After subjecting this enzyme to heat treatment in 10 mM phosphate buffer (pH 7.0) at each temperature for 10 minutes, the residual activity was measured.
The results are shown in FIG. This enzyme is stable in a temperature range of 40 ° C. or less.

There has been no report of a fructosylamine oxidase from the genus Pichia. Further, the existence of the same type of enzyme composed of a subunit having a molecular weight of 57 kDa is not known. Therefore, this enzyme is a novel enzyme. Measurement of Enzyme Activity Fructosyl phosphatase activity can be measured by quantifying the amount of oxygen consumed by the enzyme reaction or the amount of hydrogen peroxide generated. Various methods for quantifying oxygen and hydrogen peroxide are known in the art. For example, a buffer solution containing the fructosyl valine kinase of the present invention and a mediator is placed in a thermostatic cell and maintained at a constant temperature, and a sample containing fructosyl valine is added thereto, and the color reaction of the redox dye is monitored. By doing so, the activity of the fructosyl phosphatase of the present invention can be measured. As the mediator, potassium ferricyanide, ferrocene, osmium derivative, phenazine methosulfate, and the like can be used. Alternatively, the generated hydrogen peroxide can be quantified using peroxidase. As such a measurement system, a peroxidase to 4-aminoantipyrine system is known and described in various experiment books (for example, Biological Engineering Experiment Book, edited by Biotechnology Society, Baifukan 1992). Enzyme Production Method The microorganism used in the production of the present enzyme may be any microorganism that produces fructosyl valine kinase. As a specific example, Pichia sp. N1
-1 strain (FERM P-17326). The microorganism is cultured in a suitable natural or synthetic medium containing fructosyl valine. It is preferable to increase the enzyme yield by inducing fructosyl valine kinase using a medium containing fructosyl valine as the sole nitrogen source.
As a medium using fructosyl valine as the sole nitrogen source, a medium obtained by adding fructosyl valine to a minimal medium (for example, M9S) can be mentioned. After recovering the cells from the culture solution by centrifugation or the like, the cells are disrupted by a French press or the like. This is subjected to ultracentrifugation to obtain a water-soluble fraction containing fructosyl phosphatase. The obtained water-soluble fraction is purified by ion exchange chromatography, affinity chromatography, HPLC, or the like to prepare the fructosyl valine kinase of the present invention. Assay kits In another aspect, the invention features a fructosyl valine assay kit that includes the novel fructosyl valine kinase of the present invention. The fructosyl valine assay kit of the present invention comprises a fructosyl valine kinase according to the present invention in an amount sufficient for at least one assay. Typically, the kit includes, in addition to the enzyme of the present invention, buffers necessary for the assay, appropriate mediators, and enzymes such as peroxidase, if necessary, fructosyl valine or ca. Includes standard solutions of the derivative, as well as guidelines for use. The fructosyl phosphatase according to the invention can be provided in various forms, for example as a lyophilized reagent or as a solution in a suitable storage solution.

[0013] In yet another aspect, the present invention relates to an Hb
Features an A1c assay kit. HbA1c is enzymatically or chemically degraded to produce fructosyl valine, and HbA1c can be assayed by quantifying this using the fructosyl valine kinase of the present invention. Therefore, the HbA1c assay kit of the present invention further includes a hydrolyzing reagent or a protease in addition to the fructosyl valine assay kit described above. In another aspect of the enzyme sensor , the present invention features a sensor for measuring fructosyl valine and a sensor for measuring HbA1c. Using the sensor of the present invention, the concentration of fructosyl valine as a substrate can be determined by measuring the oxygen consumed by the action of fructosyl phosphatase or the generated hydrogen peroxide. Various sensor systems for measuring oxygen or hydrogen peroxide are known in the art. As an electrode, an oxygen electrode, a carbon electrode, a gold electrode, a platinum electrode, or the like is used, and the enzyme of the present invention is immobilized on the electrode. Examples of the immobilization method include a method using a crosslinking reagent, a method of enclosing in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, and a redox polymer. Is also good.

When an oxygen electrode is used, the enzyme of the present invention is immobilized on the electrode surface, inserted into a buffer, and maintained at a constant temperature. A sample is added thereto and the decrease value of the current is measured. FIG. 3 shows a schematic diagram of a sensor system combining the enzyme of the present invention and an oxygen electrode.

When the measurement is performed in an amperometric system using a carbon electrode, a gold electrode, a platinum electrode, or the like, these electrodes on which an enzyme is immobilized are used as working electrodes, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, a platinum electrode). For example, Ag / Ag
(Cl electrode) together with a buffer containing a mediator to maintain a constant temperature. A constant voltage is applied to the working electrode, a sample is added, and the increase in current is measured. As the mediator, potassium ferricyanide, ferrocene, osmium derivative, phenazine methosulfate, and the like can be used.

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

Regardless of which electrode is used, the concentration of fructosyl valine in a sample can be determined according to a calibration curve prepared using a standard concentration of a fructosyl valine solution.

When used as an HbA1c measurement sensor, a composite sensor is constructed by combining the above-described fructosyl valine measurement sensor with a membrane on which a protease (eg, a protease) is immobilized. Such a structure of a composite sensor using a continuous reaction by a combination of a plurality of enzymes is well known in the art, for example, Biosensors-Fundamental and
Applications-AnthonyP. F. Tuner, Isao Karube and G
eroge S. Wilson, Oxford University Press1987.

[0019]

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 Purification of fructosyl phosphatase Pichia sp. The N1-1 strain is an M9S medium (Na
₂ HPO ₄ 6%, KH ₂ PO ₄ 0.3%, NaCl 3
%, MgSO ₄ 0.01%, CaCl ₂ 0.01%, D
(Glucose 1%) in 150 ml of a medium in which fructosyl valine having a final concentration of 0.52% was added as a nitrogen source. Fructosyl valine is available from Keil. P (Acta
Chemica Scandinavica. B, 3
9, 191-193, 1985). The cells 60 hours after inoculation are collected, crushed by a French press, and centrifuged (9000 g, 20 minutes, 2 minutes).
Times) to remove unbroken cells. Next, ultracentrifugation (6
9,800 g, twice for 90 minutes), and the supernatant was collected and dialyzed overnight against 10 mM phosphate buffer (pH 7.0). The water-soluble fraction thus prepared was subjected to DEAE
Purified by anion exchange chromatography using -5PW. The fraction containing fructosyl phosphatase was eluted near the NaCl concentration of 0.33 M (FIG. 4). Example 2 Assay of molecular weight of fructosyl phosphatase The purified enzyme obtained in Example 1 was subjected to G-30
When the molecular weight was determined by gel filtration chromatography using a 00 column, it was found that the molecular weight was about 57 kDa (FIG. 5). Further analysis of these active fractions by SDS-PAGE revealed a band at about 57 kDa. From these results,
It was revealed that fructosyl phosphatase produced by the N1-1 strain was a single peptide enzyme having a molecular weight of about 57 kDa. Example 3 Measurement of Enzyme Activity The fructosyl phosphatase activity was measured by a measurement system using a peroxidase to 4-aminoantipyrine system. With reference to the description in the Biotechnology Experiment Book (edited by the Society of Biotechnology, Baifukan, p. 22, 1994), in the presence of 2 mM phenol, 1.5 mM 4-aminoantipyrine, 2 U / ml peroxidase. , 505
By monitoring the change in absorbance at nm, the fructosyl valine kinase activity was measured. Example 4 Evaluation of temperature characteristics of fructosyl phosphatase The temperature dependence of the activity of the present enzyme is shown in FIG. The activity was measured by the method described in Example 3. This enzyme has an optimum temperature around 40 ° C. Next, the thermostability of this enzyme was examined. After subjecting this enzyme to a heat treatment at a temperature of 30, 35, 40, 42, 43 and 45 ° C. for 10 minutes in a 10 mM phosphate buffer (pH 7.0), the residual activity was measured. The results are shown in FIG. This enzyme is found to be stable in a temperature range of 40 ° C. or lower. Example 5 Inhibitor of fructosyl phosphatase The effects of various metal ions and inhibitors on the present enzyme were examined. Various metal salts or inhibitors were added to a 10 mM solution of fructosyl valine as a substrate to a concentration of 1 mM, and the fructosyl valine kinase activity was measured using the above-described peroxidase-aminoantipyrine method. The values were expressed as specific activities when the activity of the enzyme in the absence of the inhibitor was defined as 100%. FIG. 6 shows the results. Example 6 Kinetic Evaluation of Fructosyl valine Kinase The fructosyl valine concentration activity of fructosyl valine enzyme activity was measured using a measurement system using a peroxidase to 4-aminoantipyrine system. 1.5
1 mM at room temperature in the presence of mM 4-aminoantipyrine, 2.0 mM phenol, 2 U / ml peroxidase.
Allowed to react for minutes. FIG. 7 shows the results. The Km value of this enzyme for fructosyl valine was about 3 mM. Example 7 Construction of enzyme sensor for measuring fructosyl valine Pichia sp. Crude enzyme 10 derived from N1-1 strain
0 microliter (5.7 mg protein / ml) was impregnated in Kimwipe, which was attached to a DKK oxygen electrode and covered with a dialysis membrane. The prepared enzyme electrode was inserted into 10 ml of PBS (pH 7.4) kept at 30 ° C. 50 μl of a 1 M aqueous solution of fructosyl valine was added thereto at any time, and the current decrease value at that time was observed. FIG. 8 shows the obtained calibration curve. That is, with the enzyme sensor using the novel fructosyl valine kinase of the present invention, fructosyl valine could be quantified in the range of 5 mM to 20 mM.

[Brief description of the drawings]

FIG. 1 shows the temperature characteristics of the fructosyl phosphatase of the present invention.

FIG. 2 shows the thermostability of the fructosyl phosphatase of the present invention.

FIG. 3 shows a schematic diagram of a sensor for measuring fructosyl valine of the present invention.

FIG. 4 shows the purification of the fructosyl phosphatase of the present invention by anion exchange chromatography using DEAE-5PW.

FIG. 5 shows the assay of the molecular weight of the fructosyl phosphatase of the present invention by gel filtration chromatography.

FIG. 6 shows the effects of various metal ions and inhibitors on fructosyl phosphatase of the present invention.

FIG. 7 shows the substrate concentration dependence of the fructosyl phosphatase activity of the present invention.

FIG. 8 shows a calibration curve of the sensor for measuring fructosyl valine of the present invention.

[Explanation of symbols]

 1. Recorder 2. 2. pH-mV meter IV converter Oxygen electrode 5. Dialysis membrane 6. 6. Fructosyl valine kinase 7 Constant temperature bath 8. Stirrer 9.10 mM phosphate buffer (pH 7.0)

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) (C12N 1/14 C12R 1:84)

Claims (9)

[Claims] 1. A novel enzyme having the following properties: (1) oxidizes fructosyl valine; (2) molecular weight of about 57 kDa; (3) optimal temperature around 40 ° C .; (4) stable temperature range of 40 ° C. or less (Heat treatment for 10 minutes). 2. A microorganism belonging to the genus Pichia and capable of producing fructosyl phosphatase. 3. A method for producing a fructosyl phosphatase according to claim 1, wherein a microorganism belonging to the genus Pichia is cultured, and the enzyme is collected from the culture. 4. A method for assaying fructosyl valine, comprising quantifying fructosyl valine in a sample using the fructosyl valine kinase according to claim 1. 5. A method for assaying HbA1c, which comprises decomposing HbA1c in a sample to produce fructosyl valine, and converting the fructosyl valine using the fructosyl valine kinase according to claim 1. A method comprising quantifying. 6. A fructosyl valine assay kit comprising the fructosyl valine kinase according to claim 1. 7. An HbA1c assay kit containing the fructosyl phosphatase according to claim 1. 8. A sensor for measuring fructosyl valine, which uses the fructosyl valine kinase according to claim 1. 9. A sensor for measuring HbA1c, wherein the fructosyl phosphatase according to claim 1 is used.