JPS5935592B2 - Determination method of D-fructose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for treating D-fructose in a sample in the presence of oxygen.
Increase in 5-keto D-fructose or reduced electron acceptor produced by the action of D-fructose dehydrogenase (D-fructose: ferricytochrome oxidoreductase, hereinafter abbreviated as enzyme in the present invention) and electron acceptor The present invention relates to a method for quantifying D-fructose, which is characterized by quantifying D-fructose by measuring the amount or amount of oxidized electron acceptor or oxygen reduction.

Conventionally, methods for quantifying D-fructose include the cysteine carbazole method, which uses chemical reagents, the Anthrone sulfuric acid method, and the Berndt-Bergmeier method (E, B), which uses enzymes.
emtandU, Bergmeyer method). The cysteine carbazole method and the anthrone sulfuric acid method both use concentrated sulfuric acid, which is dangerous to handle.
It also reacts with saccharides such as mannose and xylose to develop color, resulting in poor accuracy and complicated operations.

On the other hand, the Berndt-Bergmeier method as an enzymatic method uses hexokinase (EC, 2.7.1.1), phosphoglucose isomerase (EC, 5.3.1.9), glucose-6-phosphate dehydrogenase (EC , 1.1.1.49
) and adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADP).
It not only has the disadvantage of being expensive because it requires two types of coenzymes, but also has the disadvantage of poor accuracy because many sugars react with hexokinase and is easily affected by enzyme inhibitors.

The present inventors have demonstrated that D-
As a result of searching for an enzyme capable of quantifying fructose, the inventors discovered that bacteria belonging to the Acetobacter group produce a significant amount of the novel enzyme of the present invention, and established a method for obtaining a protein-specific enzyme.

A separate patent application has been filed for this patent. Conventionally, D-
Fructose dehydrogenase is an enzyme containing nicotinamide adenine dinucleotide (oxidized NAD) or nicotinamide adenine dinucleotide phosphate (oxidized NADP) derived from microorganisms and animal liver as a coenzyme, D-
Fructose: NAD (P-synthesis reductase (E, C, 1.
1.1.123) is known.

The present inventors have discovered for the first time that microorganisms, particularly acetic acid bacteria, produce a novel redox dye-dependent D-fructose dehydrogenase, ie, D-fructose-refructose cytochrome oxidoreductase. This enzyme is localized in the membrane compartment of bacterial cells, solubilized by detergents, and purified to a single preparation by conventional methods. This purified enzyme contains cytochrome as a prosthetic molecule and is an enzyme that oxidizes D-fructose to the corresponding 5-keto-D-fructose only in the presence of a suitable redox dye as an electron acceptor, converting NAD or NADP into electrons. A novel D- that is not a receptor at all
EC. l. l. 2 (conventionally, this enzyme was conveniently classified as ECl.1.99.1O because its physical and chemical properties were unknown). As the above-mentioned microorganisms producing the enzyme of the present invention, any microorganisms such as acetic acid bacteria, bacteria, yeast, and molds that produce the enzyme of the present invention can be used. Examples of bacteria that produce the enzyme of the present invention in large quantities include the genus Gluconobacter belonging to Acetobacteria, such as the well-known GlucOnObactercerinus IF.
O-3268, G. industrius IFO-3260, Acetobacter genus, such as the known Acetobacter Xylinus I
There are FO3288 etc. In carrying out the method for producing the enzyme of the present invention, the enzyme-producing bacteria of the present invention are cultured in a medium,
As the nutrient source for the medium, a wide variety of nutrients commonly used for culturing microorganisms can be used. In other words, the carbon source may be any carbon compound that can be assimilated, such as sugars such as glucose, fructose, sucrose, maltose, pentose, and molasses, organic acids such as gluconic acid and acetic acid, and alcohols such as glycerol and ethanol. sugar alcohols such as mannite, sorbitol, etc. are used. As the nitrogen source, any available nitrogen compound may be used, and for example, natural products such as yeast factory scratches, casein hydrolyzate, corn steep liquor, peptone, meat factory scratches, urea, ammonium salts, etc. are used. As suitable inorganic salts to be added to the medium, for example, sodium salts, potassium salts, calcium salts, magnesium salts, phosphates, etc. can be used as appropriate. Furthermore, various organic substances, inorganic substances, etc. necessary for enzyme production can be added to the culture medium for the growth and growth of bacteria, if necessary. In the method for producing the enzyme of the present invention, liquid culture is usually preferred, and from an industrial perspective, it is advantageous to perform deep aeration agitation culture.

In the method for producing the enzyme of the present invention, the culture temperature can be changed as appropriate within the range in which the enzyme-producing bacteria of the present invention can grow and produce the enzyme of the present invention, but a particularly preferred temperature is 28 to 32°C.

The culture period varies depending on the conditions, but is approximately 1 to 3 days, and the culture may be terminated at an appropriate time by examining the time when the enzyme of the present invention reaches its optimum yield.

Although it is not necessary to particularly adjust the pH during culturing, it is advantageous to adjust the pH to around 6 to 7 when preparing the culture medium. The enzyme of the present invention can be collected from a culture in which the enzyme of the present invention has been sufficiently produced in this manner by a conventional method for isolating and purifying the intracellular enzyme.

That is, the culture solution and the bacterial cells are separated by a known appropriate method such as vacuum filtration method, centrifugation method, etc., and the collected bacterial cells are suspended in an appropriate buffer solution. For example, the bacterial cells are destroyed by ultrasonic destruction, French press, dyno mill, etc. The enzyme of the present invention is localized in the membrane fraction precipitated by ultracentrifugation at 27,000 rpm for 90 minutes. This can also be carried out by suspending a membrane fraction prepared by centrifugation in a buffer containing an appropriate concentration of surfactant. The solubilized enzyme reacts with various ion exchange agents such as carboxymethylcephadex (hereinafter referred to as ``CM-cephadex'') and diethylaminoethylcephadex (hereinafter referred to as ``DEAE-cephadex'') in the presence of a surfactant. Ion-exchange chromatography that applies differences in affinity, adsorption chromatography that uses adsorbents such as hydroxyapatite, or conventional methods such as gel filtration using gel filtration agents such as biogel and Cephadex. The enzyme of the present invention purified as desired can be obtained by applying the enzymes alone or in appropriate combinations.
The enzyme titer of the enzyme of the present invention is at a temperature of 25°C and a pH of 4.5.
The amount of enzyme that acts on D-fructose and produces 1 μmol of 5-keto-D-fructose per minute under the following conditions is 1
Do it as a unit.

The measurement principle of the present invention is that when the enzyme of the present invention is applied to D-fructose, two hydrogen atoms of D-fructose are dehydrogenated to become 5-keto-D-fructose.

At this time, two electrons are transferred to the electron acceptor due to the coexistence of the electron acceptor. This is expressed in the following formula.

That is, D-fructose can be quantified by measuring a decrease in electron acceptors, a decrease in oxygen, an increase in 5-keto-D-fructose, or an increase in substituted electron acceptors.
Either method can be used.

More specifically, to quantify D-fructose, 1: Colorimetric method in which the decrease in absorbance is measured with a spectrophotometer in the presence of potassium ferricyanide, which is an electron acceptor.2: 2.6, which is an electron acceptor. - Spectrometry method to measure the decrease in absorbance in the coexistence of dichlorophenol/indophenol and phenazine methosulfate 3: Pressure method to measure the amount of decrease in oxygen in the reaction solution using a Whirlburg manometer (M
anOmetry) 4: Polarometric method that measures the amount of oxygen reduction using an oxygen electrode 5: Electron acceptor phenazine methosulfate and nitrotetrazolium blue, nicotinamide adenine dinucleotide, enzyme diafluorase (ECl.6.4) .3) A method of measuring the color produced in the presence of formosan using a spectrophotometer.

etc.

A method for quantifying D-fructose using the enzyme of the present invention and potassium ferricyanide as an electron acceptor will be described below.

The reaction in the present invention is shown as follows.

In particular, enzymatic methods that utilize the substrate specificity of enzymes are rapidly growing. Currently, methods for diagnosing kidneys include administering para-aminohippuric acid, creatinine, sorbitol, sodium thiosulfate, and inulin into the blood and measuring the amount or rate of excretion into the urine.

Generally, methods using para-aminohippuric acid or sodium thiosulfate are widely used, but because these compounds have small molecular weights, they easily pass through the kidney membrane, making them difficult to distinguish between normal kidneys and diseased kidneys. The disadvantage is that it is difficult to distinguish between Therefore, there was a need for a molecular species that had a large molecular weight and could not be broken down by the enzymes present in the human body, and inulin was found to be the most suitable and is gradually being used in the United States. However, since there is no simple, inexpensive, and highly accurate method for quantifying inulin, it has not become widely used.

By using the method of the present invention, the amount of inulin can be easily determined.

That is, after inulin is administered into the blood, inulin excreted in the urine is treated with inulase (EC3.2.l.7), particularly preferably Candida Kefyr.
) or Kluyveromyces fragilis (Kluyver).
Inulin is 100% degraded by the action of inulase produced by Omyces-Fragilis, and the amount of inulin is calculated by quantifying the produced D-fructose using the method of the present invention. Both the inulase used in the above method and the enzyme of the present invention have an optimum pH of 4.0 to 4.5;
This can be said to be the optimal method for quantifying inulin.

The method for quantifying D-fructose of the present invention will be described below with reference to Examples.

Example 1 (Quantification of D-fructose) (1) Preparation of enzyme The enzyme of the present invention is Gluconobacter I. Industrius I
FO-3260 was cultured using glycerol-glutamic acid medium, and the bacterial cells were collected and pressed to 1000k using a French press.
g/Cwi, and centrifuge the crushed cell ends (50 g/Cwi).
After removing the cell homogenate using an ultracentrifuge (
Treat with Hitachi 55P-7 Model 6800y for 90 minutes and collect the cell membrane fraction.

This cell membrane fraction was dissolved in pine kilvain buffer (PH6.O
), and after solubilizing FDH by adding 10% Triton Collect the liquid. This supernatant was DA
After adsorption to E-cellulose (PH6.O) and hydroxyapatite (PH6.O), a single protein chemical sample (172U/Wly protein) was obtained by desorption.

B) Preparation of enzyme solution The enzyme preparation of the present invention (172U/W) prepared by the above method
ly-protein) was adjusted to IOU/W9 using pine kilvaine buffer (PH4.5) containing 1% Triton X-100.

3) Preparation of potassium ferricyanide solution 0. Adjusted to IM concentration.

4) Preparation of D-fructose substrate solution A standard solution of D-fructose was prepared in distilled water.

・) Preparation of ferric sulfate/dupanol reagent Ferric sulfate [
Fe2(SO4)3・NH2O] 5t, dupanol (sodium lauryl sulfate) 3V) 80%
Add distilled water to 95 ml of phosphoric acid to make 11.

5) Creating a calibration curve graph - Dilute the fructose stock solution (100 μmol) with distilled water and add 0.01, 0.00 D-fructose in 0.1 ml.
02, 0.04, 0.06, 0.08, 0.10) 0.
Seven solutions containing 5 μmol were prepared.

0.8 ml of enzyme solution was added to 0.1 ml of these solutions to make the total volume 0.9 ml, and the mixture was allowed to stand at 25° C. for 5 minutes, and then 0.1 ml of potassium ferricyanide solution was added to start the reaction. The reaction was carried out at 25°C for 20 minutes, and the reaction stop solution, sulfuric acid 2
The reaction was stopped by adding 0.5 mι of iron/dupanol reagent.

Next, 3.5 g of distilled water was added to this reaction solution, and the mixture was heated to 25°C.
After holding for 20 minutes to stabilize the color development, use a spectrophotometer (
The absorbance at 660 nm was measured using a Hitachi Model 101). The results are shown in FIG. As shown in FIG. 1, the amount of D-fructose became linear from 0.01 to 0.5 μmol, making ultra-trace quantification possible.・) D- in isomerized sugar
Quantification of Fructose Obtain commercially available isomerized high-fructose sugar (two types with an isomerization rate of 42% and 55%), measure it using the currently used cysteine carbazole method and the method of the present invention, and obtain the results shown in Table 1. Obtained.

In addition, the reagents are 0.3 x 1, O-6 Shika^,
The sample was diluted to two levels of 0.6 x 10-6 times (B). From the above results, it can be seen that the results of the method of the present invention and the cysteine carbazole method are in very good agreement.
Example 2 (Quantification of inulin) (1) Preparation of inulase Candida Kefyr IFO-0616 was used as the inulase-producing strain, and cultured and purified by conventional methods to yield 102.5 U/mι.
50ml of purified enzyme was obtained.

This purified enzyme solution was diluted with M/50 acetate buffer and IU/
ml. To display the inulase titer, add 1 ml of the enzyme solution to 1 ml of a 2% solution of inulin in M/50 acetate buffer (PH 4.5) as a substrate solution, and react at 40°C for 30 minutes. and D-fructose is 1
~The enzyme titer produced is defined as 1 unit. (2) Preparation of D-fructose dehydrogenase Prepared by the method of Example 1.

(3) Preparation of inulin solution 334.2 Tny of inulin (manufactured by Hanui Chemical, special grade) was accurately weighed and dissolved in M/50 acetate buffer to give an exact concentration of 11.

One molecule of inulin produces 35 moles of D-fructose through hydrolysis, so 1 ml of this preparation contains 2 μmol of D-fructose.
It will contain fructose. (4) Preparation of potassium ferricyanide solution0. Adjusted to IM concentration.

(5) Preparation of ferric sulfate/dupanol solution Prepared by the method of Example 1.

(6) Determination of inulin Add 1 ml of inulase enzyme solution to 1 inulin solution (substrate) (containing a theoretical amount of 2 μmol of D-fructose),
The reaction was carried out at ℃ for 30 minutes.

0.1ml of this reaction solution! -(theoretical amount of 0.1 μmol of D
- containing fructose) was added with 0.1 ml of the enzyme solution of the present invention and 0.7 ml of pine kirvane buffer, and then heated at 25°C for 5 minutes.
Leave for a minute. Add 0.1ml of potassium ferricyanide solution to this.
was added and reacted for 30 minutes at 25℃, then 0.5ml of ferric sulfate/dupanol solution as a reaction stop solution was added, and further 3.5771ι of distilled water was added, and the mixture was kept at 25℃ for 20 minutes to stabilize the coloration. After converting, use a spectrophotometer (Hitachi model 101)
The absorbance at 660 nm was measured. As a control, distilled water was used instead of inulin solution, and the 660 nm of the test solution was
The amount of D-fructose was determined from the calibration curve determined in the example by subtracting that of the control from the absorbance of , and converted to the amount of inulin. The difference in absorbance between the test solution and the control was 0.401, and D-fructose was 0.1 μmol. This corresponded exactly to the theoretical D-fructose content of the inulin solution used.

[Brief explanation of the drawing]

FIG. 1 shows a calibration curve for D-fructose.

Claims (1)

[Claims] 1. Increase the amount of 5-keto-D-fructose or ring-type electron acceptor or oxidized electron by allowing D-fructose dehydrogenase and electron acceptor to act on D-fructose in the sample in the presence of oxygen. A method for quantifying D-fructose, which comprises quantifying D-fructose by measuring the amount of decrease in receptors or oxygen.