JP6504586B1 - How to measure fructosyl lysine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring fructosamine-6-kinase (FN6K) having heat and pH stability, and fructosyl lysine using the same. A method for measuring fructosyl lysine, which comprises reacting FN6K having the following properties (a) to (f) with a test sample; (a) Phosphorus fructosyl lysine in the presence of ATP (B) specific activity: 10 U / mg or more, (c) substrate specificity: fructosyl valine based on 100% reactivity to fructosyl lysine 5% or less, (d) molecular weight: 25 kDa to 35 kDa, (e) pH stability: stable at 6.5 to 8.0, (f) temperature stability: stable at 30 ° C. [Selected figure] Figure 1

Description

  The present invention relates to a method for measuring fructosyl lysine and the like.

  Foods are known to cause browning due to nonenzymatic reaction of proteins and amino acids with sugars (Maillard reaction) during processing and storage. Browning not only plays an important role in quality formation, such as coloring of food, aromatization and imparting of antioxidant ability, but also correlates with heating time and storage time, and therefore it is also an indicator of freshness and quality deterioration .

  In the early stage of the Maillard reaction, a stable Amadori compound is formed by Amadori rearrangement after the amino group of the amino acid and the carbonyl group of the sugar form a Schiff base by the aminocarbonyl reaction. For example, lysine, which is one of the essential amino acids contained in many foods, reacts with sugars contained in foods to produce fructosyl lysine. In addition, since lysine has an amino group not only at the α position but also at the ε position, the amino group is often present in free form in the protein structure, and it is considered that the aminocarbonyl reaction is likely to occur. Therefore, the amount of fructosyl lysine contained in food is correlated with the degree of browning, and the measurement value can be an important indicator of the quality control of food, so establishment of a measurement method is useful.

  A method using fructosamine 6-kinase (hereinafter referred to as FN6K) is known as a method for measuring fructosyl lysine. FN6K is known to phosphorylate fructosyl lysine (ε-FK) glycated at ε position to produce fructosyl lysine-6-phosphate. For example, FN6K derived from Escherichia (Escherichia), Bacillus (Bacillus), and Arthrobacter (Arthrobacter) has been reported (Non-patent Document 1). Among these, Bacillus-derived FN6K has low substrate specificity because it uses both ε-FK and α-fructosyl valine (α-FV) as a substrate. Although FN6K derived from Escherichia coli or Arthrobacter is specific to ε-FK, the former is poor in thermal stability and the latter is low in specific activity (non-patent document 2).

Biotechnology Journal 11 (2016), 797-804 Appl. Biochem. Biotechnol. 170 (2013) 710-717

  An object of the present invention is to provide FN6K excellent in thermal stability, pH stability and substrate specificity, and a method for measuring fructosyl lysine using the same.

  In order to solve the above problems, the present inventors conducted intensive studies on FN6K screening in nature, and found an enzyme having FN6K activity having high thermostability and pH stability from several strains. Furthermore, the inventors have found a method for measuring fructosyl lysine which is excellent in thermostability, pH stability and substrate specificity by using such an enzyme, and completed the present invention.

The present invention relates to the following [1] to [12].
[1] A method for measuring fructosyl lysine, which comprises reacting fructosamine-6-kinase having the following properties (a) to (f) with a test sample.
(A) phosphorylation of fructosyl lysine in the presence of ATP to form fructosyl lysine-6-phosphate (b) specific activity: 10 U / mg or more (c) substrate specificity: ε-fructosyl The reactivity to fructosyl valine is 5% or less when the reactivity to lysine is 100%. (D) Molecular weight: 25 kDa to 35 kDa (e) pH stability: stable at 6.5 to 8.0 (F) temperature stability: stable at 30 ° C. [2] The method for measuring fructosyl lysine according to [1], wherein fructosamine-6-kinase further has the following properties (g) and (h): .
(G) optimum activity pH: 8.0 to 8.5
(H) Optimal activity temperature: 35 to 40 ° C
[3] The method for measuring fructosyl lysine as described in [1] or [2], which is characterized by measuring at 35 ° C. or higher.
[4] The method for measuring fructosyl lysine according to any one of [1] to [3], which is measured in the range of pH 5.0 to 9.0.
[5] A reagent for measuring fructosyl lysine, which comprises fructosamine 6-kinase having the following properties (a) to (f):
(A) phosphorylation of fructosyl lysine in the presence of ATP to form fructosyl lysine-6-phosphate (b) specific activity: 10 U / mg or more (c) substrate specificity: ε-fructosyl The reactivity to fructosyl valine is 5% or less when the reactivity to lysine is 100%. (D) Molecular weight: 25 kDa to 35 kDa (e) pH stability: stable at 6.5 to 8.0 (F) temperature stability: stable at 30 ° C. [6] The reagent for measuring fructosyl lysine according to [5], wherein fructosamine-6-kinase further has the following properties (g) and (h): .
(G) optimum activity pH: 8.0 to 8.5
(H) Optimal activity temperature: 35 to 40 ° C
[7] A kit for measuring fructosyl lysine, which comprises fructosamine 6-kinase having the following properties (a) to (f):
(A) phosphorylation of fructosyl lysine in the presence of ATP to form fructosyl lysine-6-phosphate (b) specific activity: 10 U / mg or more (c) substrate specificity: ε-fructosyl The reactivity to fructosyl valine is 5% or less when the reactivity to lysine is 100%. (D) Molecular weight: 25 kDa to 35 kDa (e) pH stability: stable at 6.5 to 8.0 (F) Temperature stability: [8] The kit for measuring fructosyl lysine according to [7], wherein fructosamine-6-kinase is further stable at 30 ° C., further having the following properties (g) and (h): .
(G) optimum activity pH: 8.0 to 8.5
(H) Optimal activity temperature: 35 to 40 ° C
[9] Fructosamine 6-kinase having the following characteristics (a) to (f):
(A) phosphorylation of fructosyl lysine in the presence of ATP to form fructosyl lysine-6-phosphate (b) specific activity: 10 U / mg or more (c) substrate specificity: ε-fructosyl The reactivity to fructosyl valine is 5% or less when the reactivity to lysine is 100%. (D) Molecular weight: 25 kDa to 35 kDa (e) pH stability: stable at 6.5 to 8.0 (F) Temperature stability: [10] Fructosamine-6-kinase according to [9], which is stable at 30 ° C. [10] Furthermore, having the following properties (g) and (h):
(G) optimum activity pH: 8.0 to 8.5
(H) Optimal activity temperature: 35 to 40 ° C
[11] culturing a microorganism belonging to the genus Chronobacter, Atlanticobacter, Serratia, Bacillus or Escherichia, and collecting the fructosamine 6-kinase described in [9] or [10] from the culture A method for producing fructosamine 6-kinase, which is characterized by the above.
[12] The method for stabilizing fructosamine 6-kinase according to [9] or [10], which comprises adjusting pH to 5.0 to 9.0.

  According to the present invention, FN6K having high stability and excellent substrate specificity was obtained. The FN6K of the present invention has stability in a wide pH range and has high substrate specificity to fructosyl lysine, so it is useful as an enzyme used for measuring fructosyl lysine. Furthermore, because this FN6K has high thermal stability, it has become possible to measure fructosyl lysine at high temperatures.

It is a figure which shows the measurement result of the fructosyl lysine by the enzyme of this invention.

The fructosamine 6-kinase of the present invention has the following properties (a) to (f).
(A) Fructosamine-6-kinase (FN6K) activity: in the presence of ATP, the fructosyl lysine is phosphorylated to form fructosyl lysine-6-phosphate (b) specific activity: 10 U / mg or more ( c) Substrate specificity: The reactivity to fructosyl valine is 5% or less when the reactivity to fructosyl lysine is 100%. (d) Molecular weight: 25 kDa to 35 kDa (e) pH stability: 6 Stable at 0.5 to 8.0 (f) temperature stability: stable at 30 ° C.

Each item will be described in detail below.
1. Physicochemical properties of fructosamine 6-kinase (a) Fructosamine 6-kinase (FN6K) activity Fructosamine 6-kinase phosphorylates fructosyl lysine in the presence of ATP to form fructosyl lysine 6-phosphate Have physicochemical properties to catalyze the reaction. In the present specification, this physicochemical property is referred to as FN6K activity.
FN6K activity can be measured by a known method or a modified method thereof. For example, using NAD (P) H (generic name for NADH and NADPH), ATP, etc., the activity can be measured by using the change in absorbance of the sample at a wavelength of 340 nm before and after the reaction as an indicator. More specifically, the activity can be measured using the following reagents and measurement conditions.

Method of measuring activity of fructosamine 6-kinase (FN6K) <Principle of measurement>
In the present invention, the measurement of FN6K activity can be performed by a method known in the literature or a method obtained by appropriately modifying it. For example, the method described in J. Biol. Chem. 277 (2002) 42523-42529 can be modified and carried out. The method is based on the following measurement principle.

<Reagent and final concentration>
25 mM HEPES buffer (pH 7.1)
25 mM aqueous solution of KCl 1 mM aqueous solution of magnesium chloride 1 mM dithiothreitol 0.25 mM phosphoenolpyruvate 0.15 mM nicotinamide adenine dinucleotide (NADH)
1 mM adenosine triphosphate (ATP)
10 μg / mL pyruvate kinase 5 μg / mL lactate dehydrogenase 0.2 mM ε-fructosyl lysine <measurement conditions>
After incubating 0.9 mL of each reagent solution and water mixed for 10 minutes at 30 ° C. so that the reagent concentration in the reaction becomes the above final concentration, 0.1 mL of the enzyme solution is added to start the reaction. Measure the decrease per minute (ΔA340) of absorbance at 340 nm (NADH has a maximum absorption peak) with progress of the enzyme reaction for 5 minutes from the start of the reaction, and calculate the enzyme activity from the linear part according to the following formula . Under the present circumstances, the enzyme activity defines the amount of enzymes which oxidize 1 micromol NADH in 1 minute as 1 U.

Formula 1

In this formula, 6.3 is the molar absorption coefficient of NADH, 1.0 in the denominator is the optical path length of the cell (cm), 0.1 is the liquid volume of the enzyme solution (mL), 1.0 in the molecule is The liquid volume (mL) of the reaction reagent + enzyme solution, ΔA340 _blank represents the decrease in absorbance per minute at 340 nm when ultra pure water is added instead of the substrate solution to start the reaction.

  The FN6K of the present invention is preferably isolated or purified FN6K. In addition, FN6K of the present invention can be present in a solution (for example, a buffer etc.) suitable for the storage, or in a freeze-dried state (for example, a powder). "Isolated" when used about FN6K of this invention means the state which does not contain components other than the said enzyme substantially. Specifically, for example, in the isolated enzyme of the present invention, the content of contaminating proteins is less than 20% by weight of the whole, preferably less than about 10% by weight.

(B) Specific Activity The specific activity of FN6K of the present invention is 10 U / mg or more, more preferably 20 U / mg or more, and still more preferably 25 U / mg or more. The specific activity is an indicator of the degree of purification or inactivation of the enzyme. The specific activity is calculated by dividing the FN6K activity value by the protein concentration. The FN6K activity value is measured by the above-mentioned activity measurement method, and the protein concentration is measured by the Bradford method. Specifically, bovine serum albumin (BSA, manufactured by Wako Pure Chemical Industries, Ltd., for biochemistry) was prepared as a standard substance using Bio-Rad Protein Assay (manufactured by Japan Bio-Rad Co., Ltd.) according to the instruction manual. It can be determined by converting it from the calibration curve. The enzyme is preferably used after appropriately diluting it to a final concentration of 0.2 to 0.9 mg / mL.

(C) Substrate Specificity The FN6K of the present invention has high substrate specificity for ε-fructosyl lysine which is generated by aminocarbonyl reaction of lysine and sugar. Specifically, the FN 6 K of the present invention is at least less reactive to fructosyl valine than that to ε-fructosyl lysine, and is 100% reactive to 0.2 mM ε-fructosyl lysine When the reaction is performed, the reactivity to 0.2 mM fructosyl valine is 5% or less, preferably 3% or less, more preferably 2% or less, and still more preferably 1% or less.

(D) Molecular weight The molecular weight of FN6K of the present invention is 25 to 35 kDa, preferably 30 to 35 kDa of the polypeptide moiety constituting FN 6 K as measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is there.

  By "polypeptide moiety" is meant FN6K of the protein moiety that is substantially free of carbohydrates. If it is a prokaryote origin FN6K, the molecular weight of a polypeptide part can be calculated | required by measuring FN6K by SDS-PAGE, and may be computed from the amino acid sequence of FN6K. Moreover, if it is FN6K containing sugar_chain | carbohydrate, it can obtain | require by measuring the protein part which removed sugar_chain | carbohydrate by SDS-PAGE.

(E) Thermal Stability The FN 6 K of the present invention is stable at 30 ° C., preferably 35 ° C., more preferably 40 ° C., still more preferably 45 ° C., particularly preferably 50 ° C. In detail, when the remaining activity after standing for 30 minutes is 90% or more in a buffer solution of pH 7.0 at each temperature, it is judged to have heat stability. For example, FN 6 K of the present invention is 30 minutes at each temperature, assuming that 100 U of enzyme activity is allowed to stand for 30 minutes at 0 ° C. in 100 mM potassium phosphate buffer of pH 7.0 at 5.0 U / mL. The residual activity after standing is 90% or more.

(F) pH stability FN6K of the present invention is stable at pH 6.5-8.0. In detail, when the remaining activity after standing for 60 minutes at 30 ° C. in a buffer solution of each pH is 80% or more, it is judged to have pH stability. For example, FN 6 K of the present invention is 5.0 U / mL of enzyme in 100 mM sodium phosphate buffer (pH 6.5), potassium phosphate buffer (pH 7.0) and Tris-HCl buffer (pH 8.0) The residual activity after standing at 30 ° C. for 60 minutes is 80% or more when the enzyme activity before treatment is 100%.

  The FN 6 K of the present invention has the above-mentioned characteristics (a) to (f), preferably further has the characteristics (g) and (h), and more preferably has the characteristics (i).

(G) optimum activity pH
The optimum active pH of FN6K of the present invention is preferably in the range of pH 7.0 to 9.0, more preferably in the range of pH 8.0 to 8.5. In the present specification, the optimum activity pH is an acetic acid-sodium acetate buffer solution (pH 4.0 to 5.0), a sodium citrate buffer solution with an enzyme concentration of 0.05 U / mL as shown in the examples described later. (PH 5.0-6.0), potassium phosphate buffer (pH 6.0-7.5), Tris-HCl buffer (pH 7.5-9.0) or glycine-NaOH buffer (pH 9.0- It can be determined by measuring and comparing the enzyme activity at each pH at 25 ° C. using 9.5). When the highest activity value measured in each buffer is taken as 100%, the pH range showing an activity value of 80% or more is taken as the optimum activity pH.

(H) Optimal Activity Temperature The optimal activity temperature of FN 6 K of the present invention is preferably in the range of 25 to 40 ° C., more preferably in the range of 35 to 40 ° C. In the present specification, the optimum activity temperature is to measure and compare the enzyme activity at each temperature in HEPES buffer (pH 7.1) at an enzyme concentration of 0.05 U / mL, as described in the examples below. It can be determined by When the highest activity value measured at each temperature is taken as 100%, the temperature range showing an activity value of 80% or more is taken as the optimum activity temperature.

(I) Origin The FN6K of the present invention is not particularly limited, but is preferably derived from a microorganism belonging to Chronobacter, Atlantibactor, Serratia or Bacillus. More preferably, it is FN6K derived from Cronobacter sakazakii, Atlantibacter hermannii, or Bacillus vireti.

The fructosamine 6-kinase of the present invention is preferably a protein having the following amino acid sequence (a), (b) or (c) and having fructosamine 6-kinase activity.
(A) The amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8.
(B) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8.
(C) An amino acid sequence having identity with the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8. Identity is preferably an amino acid sequence of at least 90%, 92%, 95%, 97% or 99% identity.
The enzyme is preferably a protein consisting of the amino acid sequence of (a), (b) or (c) and having fructosamine-6-kinase activity.

The polynucleotide of the present invention preferably comprises the following (i), (ii), (iii) or (iv).
(I) A polynucleotide encoding the amino acid sequence described in (a), (b) or (c) above.
(Ii) A polynucleotide having the base sequence shown in SEQ ID NO: 1, 2, 3 or 4.
(Iii) A protein having a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1, 2, 3 or 4 and having fructosamine 6-kinase activity Polynucleotide to encode.
(Iv) a protein having at least 90%, preferably at least 92%, 95% or 97% identity to the base sequence shown in SEQ ID NO: 1, 2, 3 or 4 and having fructosamine-6-kinase activity Polynucleotide encoding

  The identity in this document is the value of identity calculated by BLAST analysis of NCBI.

2. Acquisition of FN6K production stock
According to a known method, the above polynucleotide may be used for prokaryotic cells such as E. coli and Bacillus subtilis, and for eukaryotic cells such as fungi (such as yeast, ascocci, basidiomycetes, etc. of Aspergillus), insect cells, mammalian cells, etc. By introducing, it is possible to acquire a production stock. The production strain is not particularly limited, but can be expressed, for example, in a host / vector system of E. coli.

  Examples of E. coli host / vector systems include known pUC systems, pBluescript II, pET expression systems, pGEX expression systems, pCold expression systems and the like. Moreover, it is also possible to co-express chaperone or lysozyme as needed. In the present invention, as a host, E. coli. coli JM109, E.I. It is preferable to use E. coli BL21 (DE3), a pUC-based or pET-based plasmid as a vector.

  As other host / vector systems, when the host is yeast, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Pichia pastoris etc. are mentioned as examples, and as vectors, pAUR101, pAUR224, pYES2 etc. can be mentioned. When the host is a fungus, for example, Aspergillus oryzae, Aspergillus niger and the like can be exemplified.

  The means for introducing the DNA of the present invention into the host is not particularly limited. For example, FN6K can be obtained by introducing a vector containing the DNA of the present invention into a host cell to obtain a transformant into which the DNA of the present invention has been introduced. Production strains can be obtained. The host cell is not particularly limited as long as it is possible to express the DNA of the present invention to produce FN6K. Specifically, prokaryotic cells such as E. coli and Bacillus subtilis, and eukaryotic cells such as yeast, mold, insect cells, and mammalian cells can be used. In the case of E. coli, transformation is easily possible using commercially available competent cells.

  Since the transformant of the present invention has the ability to produce the FN6K of the present invention, it can be used to efficiently produce the FN6K of the present invention.

3. Method of producing FN6K FN6K of the present invention can be produced by culturing a microorganism and collecting fructosamine 6-kinase having the above-mentioned properties from the culture. Microorganisms and cells to be subjected to culture are not particularly limited as long as they have the ability to produce FN6K of the present invention. For example, a microorganism belonging to the genus Chronobacter, Atlantica, Serratia, Bacillus or Escherichia is preferably exemplified, preferably Cronobacter sakazakii, Atlantica hermanii, Serratia. It is Fontycola (Serratia fonticola) or Bacillus vireti (Bacillus vireti), and more preferably is Kronobacterium sakazakii, Atlantica harmanii or Bacillus vireti. Alternatively, it can be produced by culturing the above-mentioned transformant and collecting FN6K having the above-mentioned characteristics from the culture. The culture method and culture conditions are not particularly limited as long as the FN6K of the present invention is produced. That is, under the condition that FN6K is produced, methods and conditions adapted to the growth of the microorganism or cell to be used can be appropriately set.

  The culture medium, culture temperature, and culture time are exemplified below as culture conditions for the microorganism.

  As the culture medium, a general culture medium for culturing a microorganism can be used, and if it contains a carbon source, a nitrogen source, vitamins, minerals, and other trace nutrients required by the microorganism to be used moderately, a synthetic culture medium or a natural culture medium may be used. Both are usable. As a carbon source, glucose, sucrose, lactose, dextrin, starch, glycerin, molasses etc. can be used. Nitrogen sources include inorganic salts such as ammonium chloride, ammonium nitrate, ammonium sulfate, ammonium phosphate, amino acids such as DL-alanine, L-glutamic acid, etc., and nitrogen containing such as peptone, meat extract, yeast extract, malt extract, corn steep liquor Natural products can be used. As vitamins, riboflavin, pyridoxine, niacin (nicotinic acid), thiamine and the like can be used. As the inorganic substance, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, ferric chloride and the like can be used. Furthermore, compounds necessary for the regulation of protein expression, such as isopropyl-β-thiogalactopyranoside (IPTG) and lactose may be added as appropriate.

  The culture conditions are not particularly limited as long as the culture conditions are suitable for the production of FN6K, but the culture temperature is preferably in the range of 10 to 45 ° C. and pH 5 to 10. If the culture period is batch culture, the range within 1 to 8 days is preferable, but if the production amount is increased by fed-batch culture or continuous culture, the period is extended as long as productivity is judged to be optimal. Can. In addition, as a culture method, for example, a shake culture method or an aerobic submerged culture method using a jar fermenter can be used.

  As a method of obtaining FN6K from the culture, a conventional protein purification method can be used. When FN6K is in the cell body, after culturing the production bacteria, the culture solution is centrifuged to obtain a culture microorganism, and then the culture microorganism is disrupted and / or lysed by an appropriate method and centrifuged from the treatment solution When FN6K is secreted in the medium, the crude enzyme solution is obtained by centrifuging the culture solution to obtain the supernatant fluid by culturing the production bacteria by obtaining the supernatant fluid by using a method such as It can be acquired.

  FN6K contained in these crude enzyme solutions is obtained by combining appropriate purification operations such as ultrafiltration, salting out, solvent precipitation, heat treatment, dialysis, ion exchange chromatography, hydrophobic chromatography, gel filtration, affinity chromatography and the like. It can be refined. The purified enzyme preparation is preferably purified to such an extent as to show a single band by electrophoresis (SDS-PAGE).

  When the FN6K of the present invention is obtained as a recombinant protein, various modifications are possible. For example, if the DNA encoding the enzyme and other appropriate DNAs are inserted into the same vector, and the recombinant protein is produced using the vector, any peptide or protein may be linked to the recombinant protein. An enzyme can be obtained. In addition, modifications may be made to cause addition of sugar chains and / or lipids, and processing of the N-terminus and C-terminus. By the above modifications, extraction of recombinant protein, simplification of purification, and / or addition of biological function, etc. are possible.

4. Applications of FN6K Measurement of Fructosyllysine Next, applications of FN6K obtained by the present invention will be described. Measurement of fructosyl lysine can be performed using FN6K of the present invention. The measurement of fructosyl lysine can be carried out by reacting FN6K of the present invention with a test sample suspected of containing fructosyl lysine. The test sample containing fructosyl lysine is not particularly limited, and examples thereof include beverages, foods, biological samples and the like.

  The fructosyl lysine measurement conditions can be specifically performed under the same conditions as the above-mentioned FN6K measurement activity test, but since ATP is transferred to ε-fructosyl lysine, the generated ADP is colorimetrically determined. It can be quantified.

  Although the measurement conditions of fructosyl lysine can be set appropriately, the measurement temperature is preferably 20 ° C. or more, 25 ° C. or more, or 30 ° C. or more, more preferably 35 ° C. or more, and the upper limit is preferably 50 ° C. or less or 45 ° C. or less, 40 ° C. or less is more preferable, and measurement at high temperature is possible by using highly stable FN 6K. In addition, the pH at the time of measurement is preferably in the range of pH 5.0 to 9.0, more preferably in the range of pH 6.0 to 9.0, still more preferably in the range of pH 6.5 to pH 8.7, and pH7. The range of 2 to 8.5 is more preferable, and the range of pH 7.4 to 8.0 is more preferable. The use of FN6K, which is stable even under alkaline conditions, enables measurement in a high pH range, and it has become possible to measure unstable NADH under acidic conditions under alkaline conditions. Although measurement time can be set suitably, for example, 0.5 minutes-1 hour can be illustrated, and it can carry out by preferably giving to reaction for 1 minute-30 minutes.

  The FN 6 K of the present invention is not necessarily required because of its high substrate selectivity to ε-fructosyl lysine, but the effect of contaminants in the sample may optionally be substantially eliminated for more accurate measurement. .

  The present invention can provide a reagent for measuring fructosyl lysine using the FN6K of the present invention. Since FN6K of the present invention has high substrate specificity and high stability, it is possible to practically measure fructosyl lysine at normal temperature. The reagent may be a reagent containing FN6K of the present invention as an enzyme. The reagent preferably contains a buffer, and suitably contains other optional components known to those skilled in the art such as a stabilizer, and more preferably improves the thermal stability and storage stability of the enzyme or reagent component. .

5. Method of Stabilizing FN6K The present invention can provide a method of stabilizing FN6K. FN6K can be stabilized by adjusting the pH of the composition containing FN6K to the stable range of FN6K. When the composition is allowed to stand at 30 ° C. for 60 minutes, preferably within the range of pH 5.0 to pH 9.0, the residual activity is 80% or more when the enzyme activity before treatment is 100%. The pH is more preferably in the range of 5.5 to 8.7, still more preferably in the range of 6.5 to 8.0.

6. The present invention also relates to a kit containing FN6K. Typically, the composition of the kit comprises, in addition to FN6K of the present invention, buffer, NAD (P) H and ATP. Preferably, a sufficient amount of FN6K for at least one measurement may be included, but about 0.001 to 200 U is preferable per sample, and about 0.05 to 50 U is more preferable. In addition, the composition of the FN6K-containing kit of the present invention is in the form of a composition dissolved in a solution (eg, buffer) suitable for storage or measurement, or in a freeze-dried state (eg, in the form of powder). Is desirable. The kit containing the FN6K can be used as a reagent for measuring fructosyl lysine concentration.

  Also, for example, bovine serum albumin (BSA) or ovalbumin, saccharides (eg, trehalose etc.), sugar alcohols (eg maltitol, sorbitol, glycerol etc.), carboxyl group-containing compounds, alkaline earth metal compounds, ammonium salts The thermal stability of FN6K and reagent components can be obtained by appropriately containing a stabilizing component known to those skilled in the art, such as a stabilizer selected from the group consisting of sulfates, amino acids which do not react with enzymes, proteins, etc. Can improve the stability and storage stability.

  The invention will be illustrated by the following examples, but the invention is not limited to the following examples.

Example 1 Acquisition of FN6K and Measurement of Activity The gene of a known strain was subjected to screening using primers for obtaining FN6K gene designed based on known literatures. As a result, the following four sets of primers were finally obtained In addition, in Clonobacter sakazakii (Cronobacter sakazakii NBRC 102416), Eserichia hermanii (Escherichia hermannii NBRC 105704) (note that Microbiol Immunol. Because they are proposed to be classified, they are identical, and appear to be fructosamine 6-kinase (FN6K) from Serratia fonticola (Serratia fonticola NBRC 102597) and Bacillus vireti (Bacillus vireti NBRC 102452). 4 genes (DNA sequences of SEQ ID NOs: 1, 2, 3 and 4) were obtained.

1) Primer 5'-AAGGAGATATACATAATGAAAACACTGGCGACGG-3 '(SEQ ID NO: 9) for obtaining a gene derived from Chronobacter sakazakii
5'-GGTGGTGGTGCTCGATTACCAGGCACCGTGATAG-3 '(SEQ ID NO: 10)
2) Primer 5 'for obtaining a gene derived from Escherichia coli Harmany-AAGGAGATATACATAATGAACAGCGCTGGCAACG-3' (SEQ ID NO: 11)
5'-GGTGGTGGTGCTCGATTACCAGGCACCATGGTAG-3 '(SEQ ID NO: 12)
3) Primer for gene acquisition from Serratia fonticola 5'-AAGGAGATATACATAATGAAAACATTGATTACGCTTG-3 '(SEQ ID NO: 13)
5'-GGTGGTGGTGCTCGATTACCATGCTCCGTGGTAT-3 '(SEQ ID NO: 14)
4) Primer 5'-AAGGAGATATACATAATGAGAATCGGGGCAGTC-3 '(SEQ ID NO: 15) for obtaining a gene from Bacillus vileti
5'-GGTGGTGGTGCTCGACTACCATGCACCCTTATATT-3 '(SEQ ID NO: 16)

  The four types of obtained genes were respectively inserted into pET-21a (+) vector and introduced into E. coli BL21 (DE3) competent cells to obtain each recombinant E. coli. Each recombinant E. coli was cultured in LB medium, and the resulting cells were sonicated to obtain cell-free extract (CFE). Each CFE was purified by anion exchange chromatography to obtain each purified FN6K enzyme.

  Let FN6K derived from Kronobac sakazakii be FN6K derived from Cs, FN6K derived from Eshersia harmani as FN6K derived from Eh, FN6K derived from Serratia fonticola SN derived from Ff6K and FV6K derived from Bacillus vireti Bv derived FN6K. The Cs-derived FN6K has the amino acid sequence shown in SEQ ID NO: 5, the Eh-derived FN6K has the amino acid sequence shown in SEQ ID NO: 6, the Sf-derived FN6K has the amino acid sequence shown in SEQ ID NO: 7, and the Bv-derived FN6K has the amino acid sequence shown in SEQ ID NO: 8.

  As a comparative example, FN6K (hereinafter referred to as Ec-derived FN6K) derived from Escherichia coli JM109 conventionally known was also obtained according to J. Biol. Chem. 277 (2002) 42523-42529. .

  As a result of measuring the FN6K activity of each of the obtained enzymes by the method for measuring the activity of FN6K, it was confirmed that fructosyl lysine is phosphorylated in the presence of ATP to produce fructosyl lysine-6-phosphate.

  In addition, the protein concentration was measured by the Bradford method, and the specific activity (U / mg) of each FN6K was calculated. The results are shown in Table 2 below.

  The obtained four types of FN6K and Ec-derived FN6K were subjected to SDS-PAGE, and the molecular weight was determined. As a result, all were about 30 kDa.

Example 2 Evaluation of pH Stability of Obtained FN 6 K The four new FN 6 K and Ec-derived FN 6 K were prepared by dissolving them in a buffer solution with different pH, and left at 30 ° C. for 60 minutes of remaining activity. It was measured. As the buffer, 1 M buffer (sodium phosphate buffer (pH 5.0 and 6.0), potassium phosphate buffer (pH 7.0) and Tris HCl buffer (pH 8.0 and 9.0)) The final concentration was adjusted to 100 mM and used. In addition, the pH of the prepared buffer solution was measured with a pH meter, and the value of pH stability was represented by the measured value. The method of measuring FN6K activity followed the method of measuring FN6K activity. The pH range where the remaining activity is 80% or more when the activity before treatment is 100% is shown in Table 3.

  While the Ec-derived FN6K was stable at pH 5.6 to 7.1, all four types of FN6K obtained this time retained an activity of 80% or more at pH 8.0. Therefore, the FN6K of the present invention is easier to use and store than the known FN6K.

Example 3 Evaluation of Thermal Stability of Obtained FN 6 K The four novel FN 6 K and Ec-derived FN 6 K were allowed to stand at 0, 30, 40, 45, 50 ° C. for 30 minutes in the most stable buffer, and residual activity Was evaluated. Cs-derived FN6K, Sf-derived FN6K, Bv-derived FN6K and Ec-derived FN6K used 100 mM potassium phosphate buffer of pH 7.0, and Eh-derived FN6K used 100 mM sodium phosphate buffer of pH 6.0. The residual activity was measured with the activity of the enzyme treated at 0 ° C. for 30 minutes as 100%. The method of measuring FN6K activity followed the method of measuring FN6K activity. The temperature range which showed 90% or more of residual activity is shown in Table 4.

  All four types of FN6K obtained this time showed thermal stability equal to or more than the known Ec-derived FN6K.

Example 4 Evaluation of Substrate Specificity of Obtained FN 6 K With respect to the activity against fructosyl valine when the activity against ε-fructosyllysine is taken as 100%, each substrate concentration is measured as 0.2 mM, and the substrate specificity is measured. The sex was evaluated. The results are shown in Table 5.

  All four types of FN6K obtained this time had comparable substrate specificity compared with known Ec origin FN6K.

Example 5 Evaluation of Optimal Activity pH of FN6K Obtained For the four novel FN6K and Ec-derived FN6K, the buffer solution of the method for measuring the activity of FN6K was changed to a buffer solution having different pH, and FN6K activity was measured. The buffer solution is 200 mM buffer (acetate-sodium acetate buffer (pH 4.0 to 5.0), sodium citrate buffer (pH 5.0 to 6.0), potassium phosphate buffer (pH 6.0 to 7.5), Tris-HCl buffer (pH 7.5-9.0) or glycine-NaOH buffer (pH 9.0-9.5)) was used. The pH range which shows an activity value of 80% or more is shown in Table 6, when the highest activity value measured in each buffer solution is made into 100%.

Example 6 Evaluation of Optimal Activity Temperature of Obtained FN6K For the four novel FN6K and Ec-derived FN6K, the FN6K activity measuring method temperature was 25, 30, 35, and 40 ° C., and FN6K activity was measured. When the highest activity value measured at each temperature is taken as 100%, a temperature range showing an activity value of 80% or more is shown in Table 7.

Example 7 Measurement of ε -Fructosyl Lysine Using the four novel FN6Ks (Cs-FN6K, Eh-FN6K, Sf-FN6K and Bv-FN6K) obtained in Example 1, the method for measuring the activity of FN6K was used. Similarly, the change in absorbance was measured in a buffer having the following composition containing ε-fructosyllysine at a concentration of 0.01, 0.05, 0.1 or 0.2 mM.
<Reagent and final concentration>
25 mM HEPES buffer (pH 7.1)
25 mM aqueous solution of KCl 1 mM aqueous solution of magnesium chloride 1 mM dithiothreitol 0.25 mM phosphoenolpyruvate 0.15 mM nicotinamide adenine dinucleotide (NADH)
1 mM adenosine triphosphate (ATP)
10 μg / mL pyruvate kinase 5 μg / mL lactate dehydrogenase 0.01, 0.05, 0.1 or 0.2 mM ε-fructosyl lysine The relative activity value at each ε-fructosyl lysine concentration is shown in FIG. As a result, it was shown that? -Fructosyl lysine can be quantified using FN6K of the present invention in measurement using an absorbance meter.

Claims (14)

A test sample is characterized by reacting Fructosamine 6-kinase from a microorganism belonging to the genus Chronobacter, Atlantibacter, Serratia or Bacillus having the following properties (a) to (f): How to measure fructosyl lysine.
(A) phosphorylated fructosyl lysine in the presence of ATP to produce fructosyl lysine-6-phosphate (b) specific activity: 10 U / mg or more (c) substrate specificity: for fructosyl lysine reactivity to fructosyl valine when reactivity as 100% is not more than 5% (d) molecular weight: a 25kDa~35kDa (e) pH stability: 6.5 to 8.0 in a stable met Have a stability of 80% or more at pH 8.0 (f) temperature stability: stable at 30 ° C. The method for measuring fructosyl lysine according to claim 1, wherein the fructosamine-6-kinase further has the following properties (g) and (h).
(G) optimum activity pH: 8.0 to 8.5
(H) Optimal activity temperature: 35 to 40 ° C   The method for measuring fructosyl lysine according to claim 1 or 2, wherein the measurement temperature is 35 ° C or higher.   The method for measuring fructosyl lysine according to any one of claims 1 to 3, wherein the measurement is carried out in the range of pH 5.0 to 9.0. A reagent for measuring fructosyl lysine, which comprises fructosamine-6-kinase derived from a microorganism belonging to the genus Chronobacter, Atlantibacter, Serratia or Bacillus having the following properties (a) to (f):
(A) phosphorylated fructosyl lysine in the presence of ATP to produce fructosyl lysine-6-phosphate (b) specific activity: 10 U / mg or more (c) substrate specificity: for fructosyl lysine reactivity to fructosyl valine when reactivity as 100% is not more than 5% (d) molecular weight: a 25kDa~35kDa (e) pH stability: 6.5 to 8.0 in a stable met Have a stability of 80% or more at pH 8.0 (f) temperature stability: stable at 30 ° C. The reagent for fructosyl lysine measurement according to claim 5, wherein the fructosamine-6-kinase further has the following properties (g) and (h).
(G) optimum activity pH: 8.0 to 8.5
(H) Optimal activity temperature: 35 to 40 ° C A kit for measuring fructosyl lysine, which comprises fructosamine-6-kinase from a microorganism belonging to Chronobacter, Atlanticobacter, Serratia or Bacillus having the following properties (a) to (f):
(A) phosphorylated fructosyl lysine in the presence of ATP to produce fructosyl lysine-6-phosphate (b) specific activity: 10 U / mg or more (c) substrate specificity: for fructosyl lysine reactivity to fructosyl valine when reactivity as 100% is not more than 5% (d) molecular weight: a 25kDa~35kDa (e) pH stability: 6.5 to 8.0 in a stable met Have a stability of 80% or more at pH 8.0 (f) temperature stability: stable at 30 ° C. The kit for fructosyl lysine measurement according to claim 7, wherein the fructosamine 6-kinase further has the following properties (g) and (h).
(G) optimum activity pH: 8.0 to 8.5
(H) Optimal activity temperature: 35 to 40 ° C Fructosamine- derived from a microorganism belonging to the genus Chronobacter, Atlantibacter , Serratia or Bacillus, having the characteristics (a) to (f), characterized in that the pH is adjusted to 5.0 to 9.0 6- Stabilization method of kinase.
(A) In the presence of ATP, phosphorylate fructosyl lysine to form fructosyl lysine-6-phosphate
(B) Specific activity: 10 U / mg or more
(C) Substrate specificity: The reactivity to fructosyl valine is 5% or less when the reactivity to fructosyl lysine is 100%.
(D) Molecular weight: 25 kDa to 35 kDa
(E) pH stability: stable at 6.5 to 8.0, having a stability of 80% or more at pH 8.0
(F) Temperature stability: stable at 30 ° C. The method for stabilizing fructosamine 6-kinase according to claim 9, wherein the fructosamine 6-kinase further has the following properties (g) and (h).
(G) optimum activity pH: 8.0 to 8.5
(H) Optimal activity temperature: 35 to 40 ° C A protein having an amino acid sequence of the following (A), (B) or (C) in a test sample, and having fructosamine 6-kinase activity,
(A) the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8 or
(C) an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8
  A method for measuring fructosyl lysine, which comprises reacting fructosamine-6-kinase having the following properties (a), (c), (d) and (e).
(A) In the presence of ATP, phosphorylate fructosyl lysine to form fructosyl lysine-6-phosphate
(C) Substrate specificity: The reactivity to fructosyl valine is 5% or less when the reactivity to fructosyl lysine is 100%.
(D) Molecular weight: 25 kDa to 35 kDa
(E) pH stability: stable at 6.5 to 8.0, having a stability of 80% or more at pH 8.0 A protein having the following amino acid sequence (A), (B) or (C) and having fructosamine 6-kinase activity,
(A) the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8 or
(C) an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8
  A reagent for fructosyl lysine measurement, comprising fructosamine-6-kinase having the following properties (a), (c), (d) and (e):
(A) In the presence of ATP, phosphorylate fructosyl lysine to form fructosyl lysine-6-phosphate
(C) Substrate specificity: The reactivity to fructosyl valine is 5% or less when the reactivity to fructosyl lysine is 100%.
(D) Molecular weight: 25 kDa to 35 kDa
(E) pH stability: stable at 6.5 to 8.0, having a stability of 80% or more at pH 8.0 A protein having the following amino acid sequence (A), (B) or (C) and having fructosamine 6-kinase activity,
(A) the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8 or
(C) an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8
  A kit for fructosyl lysine measurement, comprising fructosamine-6-kinase having the following properties (a), (c), (d) and (e):
(A) In the presence of ATP, phosphorylate fructosyl lysine to form fructosyl lysine-6-phosphate
(C) Substrate specificity: The reactivity to fructosyl valine is 5% or less when the reactivity to fructosyl lysine is 100%.
(D) Molecular weight: 25 kDa to 35 kDa
(E) pH stability: stable at 6.5 to 8.0, having a stability of 80% or more at pH 8.0 The pH is adjusted to 5.0 to 9.0,
  A protein having the following amino acid sequence (A), (B) or (C) and having fructosamine 6-kinase activity,
(A) the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8;
(B) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8 or
(C) an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 5, 6, 7 or 8
  A method for the stabilization of fructosamine-6-kinase having the following characteristics (a), (c), (d) and (e):
(A) In the presence of ATP, phosphorylate fructosyl lysine to form fructosyl lysine-6-phosphate
(C) Substrate specificity: The reactivity to fructosyl valine is 5% or less when the reactivity to fructosyl lysine is 100%.
(D) Molecular weight: 25 kDa to 35 kDa
(E) pH stability: stable at 6.5 to 8.0, having a stability of 80% or more at pH 8.0

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