JP3152855B2 - Novel sorbitol dehydrogenase, method for producing the same, reagent and method for quantifying sorbitol using the enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the use of NAD ^{+ in} the presence of
Novel sorbitol dehydrogenase that oxidizes D-sorbitol to produce D-fructose, and reduces D-fructose to produce D-sorbitol in the presence of NADH by the reverse reaction of this reaction, and a method for producing the same And a reagent and a method for quantifying D-sorbitol using the enzyme.

[0002]

2. Description of the Related Art D-sorbitol is present in trace amounts in human serum and urine, and its content is known to be an important index in the diagnosis of diseases such as diabetes. In research institutions, D-sorbitol in serum is measured using an enzymatic method.

[0003] Powdered or granular D-sorbitol can be obtained by hydrogenating glucose under high temperature and high pressure to obtain a D-sorbitol solution, and purifying this solution with an ion exchange resin or the like. . D-sorbitol has no smell, has an invigorating sweetness, and gives a cool feeling to the tongue, and is excellent in hygroscopicity, so that it can be used as a sweetener for chewing gum, candy, other confectioneries, etc. It is widely used for thickening sake and sweet humectants for tobacco.

[0004] D-fructose can be obtained by a method of hydrolyzing a plant containing a large amount of inulin, for example, a method of inverting sucrose by invertase, etc. This D-fructose is used extensively in the food industry as a sweetener and the like because it has an elegant and strong sweetness and gives wettability.

[0005] From the above, it is difficult to obtain D-sorbitol present in trace amounts in serum or urine and an enzyme usable for measuring D-sorbitol or D-fructose contained in foods. It is extremely important in industry. Conventionally known sorbitol dehydrogenases include those derived from animal liver, for example, those derived from sheep liver sold by SIGMA and the like. Examples of microorganism-derived enzymes include, for example, enzyme microbacteria
Technology (EnzymeMicrob.Techn)
ol. ) Volume 13, April, 332-337, 19
91 years, Pseudomonas sp.
domonas sp. ) And Journal of Biological Chemistry (Journal)
of Biological Chemistry) Vol. 267, No. 35, 24989-24994, 19
1992, Bacillus subtilis (Bacill)
subtilis) are known.

[0006] However, the above-mentioned sorbitol dehydrogenase derived from animal liver is complicated in its production method and has problems in terms of price. In addition, the above-mentioned Pseudom
onas sp. The activity of the derived enzyme is reduced by half at 40 ° C. for 5 minutes under the conditions of 20 mM Tris-HCl buffer pH 7.0, and the reaction is carried out at 37 ° C. for 30 minutes, which is widely used in the measurement by the enzymatic method. The method has drawbacks such as a lack of stability of the enzyme activity.

[0007]

SUMMARY OF THE INVENTION The present invention overcomes the above-mentioned drawbacks of the conventional sorbitol dehydrogenase, and includes, for example, D-sorbitol or D-fructose contained in foods or human serum or urine. A novel enzyme derived from a microorganism, which can be used for the measurement of D-sorbitol present in a microorganism, and has a stable enzyme activity even under temperature conditions often used for measurement by a usual enzymatic method, and this enzyme at a low cost An object of the present invention is to provide a method for easily producing the enzyme, and a reagent and a method for quantifying sorbitol using the enzyme.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above-mentioned object, and as a result of extensively searching for microorganisms from nature, belonged to the genus Pseudomonas isolated from soil. The strain is D
Producing D-fructose by acting on sorbitol, and producing a novel enzyme acting on D-fructose to produce D-sorbitol by a reverse reaction of this reaction; It has been found that the enzyme activity is stable even under the temperature conditions often used in the measurement by the method described above, and that D-sorbitol can be accurately quantified by the enzymatic method using this enzyme. It was completed.

That is, the first aspect of the present invention is the following physicochemical properties: (1) Action: In the presence of NAD ⁺ , D-sorbitol is oxidized to produce D-fructose and NADH. The reverse reaction reduces D-fructose in the presence of NADH to produce D-sorbitol and NAD ⁺ . (2) Substrate specificity: specifically acts on D-sorbitol and galactitol.

(3) Optimal pH and stable pH range: optimal p
H is around 10, and the stable pH range is pH 5.5 to 10.5 after treatment at 30 ° C. for 4 hours. (4) Range of suitable working temperature: The range of suitable working temperature is around 50 ° C. with 100 mM Tris-HCl buffer (pH 9.0).

(5) Deactivation conditions depending on pH, temperature, etc .:
It is stable at pH 5.5 to 10.5 when treated at 30 ° C for 4 hours, and completely deactivated at pH 5.0 or lower and pH 11.0 or higher. It is stable up to around 40 ° C. in 30 minutes of treatment at pH 7.0 and completely deactivated in 24 hours at 40 ° C. at pH 7.0. (6) Inhibition: strongly inhibited by HgCl ₂ .

(7) Molecular weight: about 64,500 ± 5,000
0 (gel filtration method). A second aspect of the present invention is the sorbitol dehydrogenase, which belongs to the genus Pseudomonas, wherein the sorbitol dehydrogenase-producing strain is cultured in a medium, and sorbitol dehydrogenase is collected from the culture. The third aspect of the present invention is a method for producing dehydrogenase, and the third aspect of the present invention is the method for producing sorbitol dehydrogenase and NA.
A fourth aspect of the present invention is a D-sorbitol quantification reagent containing D ⁺ , wherein the sorbitol dehydrogenase and NAD ⁺ are allowed to act on a D-sorbitol-containing sample, and the resulting NADH is quantified. -A method for quantifying sorbitol.

Hereinafter, the present invention will be described in detail. First, the details of the physicochemical properties of the novel enzyme of the present invention, sorbitol dehydrogenase (hereinafter sometimes referred to as “the present enzyme”) are as follows. (1) Action: This enzyme is 1 mol in the presence of NAD ⁺
Oxidized D-sorbitol to produce 1 mol of D-fructose and NADH, and by the reverse reaction of this reaction, reduced 1 mol of D-fructose in the presence of NADH to produce 1 mol of D-sorbitol and NAD ⁺ Generate

From the above results, the present enzyme has the following reaction formula:

[0015]

Embedded image

It has been found that it catalyzes the reaction represented by (2) Substrate specificity: specifically acts on D-sorbitol and galactitol, and also acts on L-iditol.
Table 1 shows an example of the results obtained by examining the relative activities of the present enzyme for various substrates.

[0017]

[Table 1]

(3) Optimum pH and stable pH range: The optimum pH is 100 mM potassium phosphate buffer (pH 6.0 to 7.5), 100 mM Tris-HCl buffer (pH 7.5 to 7.5). 9.0), 100 mM glycine-sodium hydroxide buffer (pH 9.0 to 10.5), 10
0 mM sodium carbonate-sodium bicarbonate buffer (p
H10.5-11.5), using 100 mM cyclohexylaminopropanesulfonic acid (CAPS) -sodium hydroxide buffer solution (pH 10.5-11.0), the activity of the enzyme at each pH was measured at 37 ° C. I asked. Enzyme activity was measured in 2.4 ml of each 100 mM buffer, 1.
0.5 ml of 1 M D-sorbitol aqueous solution and 30 mM
After mixing 0.05 ml of NAD ⁺ aqueous solution and preincubating at 37 ° C., 0.05 ml of the enzyme solution was added, reacted at 37 ° C., and measured for the increase in absorbance at 340 nm for 70 to 80 seconds. . The results are as shown in FIG. 1, and the optimal pH of this enzyme was 1
It is around 0, especially pH 9.5 to 10.5.

The stable pH range is as follows:
00 mM acetic acid-sodium acetate buffer (pH 4.5-
6.0), 100 mM potassium phosphate buffer (pH 6.0).
0-8.0), 100 mM Tris-HCl buffer (pH
7.5-9.0), 100 mM glycine-sodium hydroxide buffer (pH 9.0-10.5), 100 mM sodium carbonate-sodium bicarbonate buffer (pH 11.0
１11.5) at pH 4.5 to 11.5.
After each treatment at 0 ° C for 4 hours, 100 mM Tris-
The enzyme was diluted 10-fold with a hydrochloric acid buffer, and the remaining activity of the enzyme was measured and determined. The results are as shown in FIG. 2, and the stable pH range of the present enzyme is pH 5.5 to 10.5.

(4) Range of suitable temperature for action: Measurement of titer described later
At various temperatures using a substrate / enzyme mixture in the standard method
The activity of this enzyme was measured. That is, 100 mM Tris-
Hydrochloric acid buffer (pH 9.0) 2.4 ml, 1.1M D-
0.5 ml of sorbitol aqueous solution and 30 mM NAD ⁺
Mix 0.05 ml of the aqueous solution and pre-incubate at the specified temperature.
After addition, add 0.05 ml of enzyme solution and
Reaction at 340 nm for 30-90 seconds
By measuring the increase in absorbance, the activity of this enzyme can be measured.
went. The results are as shown in FIG.
The range of suitable temperature for operation is around 50 ° C.

(5) Deactivation conditions depending on pH, temperature, etc .:
The enzyme is treated at 30 ° C. for 4 hours to obtain a pH of 5.5 to 10.
5 and completely inactivated at pH 5.0 or lower and pH 11.0 or higher, as can be seen from FIG.

A 20 mM Tris-HCl buffer (pH
9.0) and 20 mM phosphate buffer (pH 7.0) at room temperature (30 ° C, 37 ° C, 40 ° C).
The thermal stability when treated for 0 minutes to 24 hours was examined. The results are as shown in FIG. 4 and FIG. 5.
The treatment at each temperature for 30 minutes is stable up to around 40 ° C., and the activity half-life of the enzyme at 40 ° C. is about 3.5 hours at pH 9.0 and about 2 hours at pH 7.0. And the enzyme has a pH of 7.0 to pH
It is completely inactivated in 24 hours at 9.0 at 40 ° C.

(6) Inhibition: Various additives (metal salts) were added to the substrate / enzyme mixed solution in the titer measurement method described later, for each one.
The enzyme activity was measured by adding to a concentration of mM.
The results are as shown in Table 2, where the enzyme was HgCl
Strongly inhibited by ₂ .

[0024]

[Table 2]

(7) Molecular weight: TSKgel G3000
According to a gel filtration method using SWXL (manufactured by Tosoh Corporation), the molecular weight is about 64,500 ± 5,000. In addition, since the molecular weight of the subunit by SDS-polyacrylamide gel electrophoresis is about 27,400, this enzyme is considered as a dimer. Also, acrylamide 10-20%
As a result of performing SDS-polyacrylamide gel electrophoresis using a polyacrylamide gel having a concentration gradient of (W / V) according to a conventional method, a single band was observed as shown in FIG. confirmed. (8) Method for measuring titer: The titer of the present enzyme was measured by the following method, and the amount of the enzyme that produced 1 μmol of NADH per minute was defined as 1 U.

(Preparation of Reagent) 1 solution: 20 g of D-sorbitol substrate solution is dissolved in distilled water to make 100 ml. Liquid 2: 86 mg of substrate solution NAD ⁺ is dissolved in distilled water to make 4 ml. Solution 3: Buffer solution After dissolving 6.05 g of Tris-hydrochloric acid in distilled water, adjusting the pH to 9.0 with 4 N hydrochloric acid, and adjusting to 500 ml.

(Measurement procedure) 1) 0.5 ml of 1 solution, 0.05 ml of 2 solutions, 2.4 m of 3 solutions
Mix and preincubate at 37 ° C for 5 minutes. 2) The solution pre-incubated for 5 minutes and 0.1
0.05 ml of the enzyme solution adjusted to ０．８0.8 U / ml is mixed, and the amount of increase in sample absorbance per minute at 340 nm at 37 ° C. is measured. 3) Measurement of blank value is 0.5m of distilled water instead of 1 solution
1 and 0.05 ml of solution 2 and 2.4 ml of solution 3 were mixed and pre-incubated at 37 ° C. for 5 minutes.
0.05 ml of the enzyme solution adjusted to 0.8 U / ml is mixed, and the amount of increase in blank absorbance per minute at 340 nm at 37 ° C. is measured.

(Calculation of titer)

[0029]

(Equation 1)

(9) Purification method: The isolation and purification of the enzyme
It can be carried out according to a conventional method.For example, ammonium sulfate precipitation, organic solvent precipitation, ion exchange chromatography, gel filtration chromatography, adsorption chromatography, affinity chromatography, electrophoresis, etc. can be used alone or appropriately. Used in combination.

Next, examples of other physicochemical properties of the present enzyme will be described. (10) Km value and relative ratio of Vmax / Km (value with D-sorbitol value as 100): line weber
From the Bark plot, the Km value is 1.41 × 10 ⁻² M.
(At pH 9.0 at 37 ° C. using D-sorbitol as a substrate).

The Km value (mM) and Vmax (μmol · mi) of the enzyme at pH 9.0 and 37 ° C. for each substrate.
n ⁻¹ · mg ⁻¹ ), Vmax / Km, and the relative ratio when Vmax / Km of D-sorbitol is 100, as shown in Table 3.

[0033]

[Table 3]

From Table 3, it can be seen that the Km value is the smallest when D-sorbitol is used as a substrate, and the Vm of D-sorbitol is the lowest.
The relative ratio when max / Km (min ^-1 · mg ^-1 ) is set to 100 indicates that this enzyme acts most intimately on D-sorbitol even from the viewpoint that D-sorbitol is the largest. Understand. The main physicochemical properties of the enzyme of the present invention are as described above, and the grounds for the novelty of this enzyme are shown below.

Conventionally, the above-mentioned sorbitol dehydrogenase derived from sheep liver, Pseudomonas sp.
omonas sp. ) Or Bacillus subtilis (B
It is known that sorbitol dehydrogenase produced by A. subtilis oxidizes D-sorbitol in the presence of NAD ⁺ to produce D-fructose.

[0036]

[Table 4]

(Note 1): The molecular weight, the optimum pH, and the stable pH range were measured using the enzyme derived from sheep liver (“sorbitol dehydrogenase S-3376” manufactured by SIGMA) in the same manner as in the enzyme of the present invention. did. (Note 2): Data on enzymes derived from known Pseudomonas sp. Are described in Enz-yme Microb. Tech
nol. (Vol. 13, April, pp. 332-337, 19
1991).

(Note 3): Data of known Bacillus-derived enzymes are based on the Journal of Biological C
hemistry (Vol. 267, No. 35, No. 24989
-24994, 1992). (Note 4): Thermal stability was measured using a 20 mM phosphate buffer (pH
7.0) and 20 mM Tris-HCl buffer (pH 7.0).
0), an enzyme activity of 10 at 37 ° C. and 40 ° C.
Indicates the time remaining at 0%.

The activity was halved at 40 ° C. in the same buffer.
Shows the time at which the activity is reduced by half.

However, from Table 4, it can be seen from Table 4 that the enzyme derived from sheep liver was 129,00
The enzyme derived from Bacillus subtilis is 153,000 (gel filtration method), and both are tetramers and 64,500 ±
Since it is a dimer at 5,000 (gel filtration method), the enzyme of the present invention is completely different from these enzymes.

Particularly, in terms of thermostability, the Pseudomonas sp.-derived enzyme has an activity half-life at pH 7.0 of 40 minutes at 40 ° C., whereas the enzyme has an activity half-life of about 2 at pH 7.0. Because it ’s time,
This enzyme is found to be completely different from the enzyme of the present invention. That is, the enzyme of the present invention acts on D-sorbitol, and is stable to about 40 ° C. and keeps about 100% of the activity at a pH of 7.0 to 9.0 by treating for 30 minutes. A novel sorbitol dehydrogenase having an unprecedented property that the time is about 2 hours at pH 7.0.

Next, a method for producing the novel sorbitol dehydrogenase of the present invention will be described. First, as a bacterium to be used, any bacterium belonging to the genus Pseudomonas and having the present enzyme-producing ability may be used, and a variant or mutant of these bacterium may be used. As a specific example of the microorganism, Pseudomonas (Pseudomon)
onas) sp. KS-E1806, and a variant or mutant of the strain can also be used. This Pseudomonas sp. KS-E1806 is a bacterial strain obtained by the present inventors by separating it from soil in Kyoto Prefecture, and its bacteriological properties are as follows.

Experiments for identification of mycological properties are as follows:
It was mainly carried out by Takeji Hasegawa, "Classification and Identification of Microorganisms", University of Tokyo Press (1975). As a criterion for classification and identification, “Bergies Manual of Deterministic Bacteriology (Bergey's
s Manual of Determinative
Bacteriology), 8th Edition (1974)
Was referenced.

Pseudomonas sp. Mycological properties of KS-E1806 (A) Morphological properties Microscopic observation [meat agar medium (pH 9.0), 30 ° C,
Culture for 24-48 hours] (1) Shape and size of cells: 0.5-1.0 × 2.0-
It is a short rod of 4.0 μm. (2) Presence / absence of cell polymorphism: No (3) Presence / absence of motility: Yes. Has extreme hair. (4) Presence or absence of spores: None (5) Gram stainability: negative (6) Acid resistance: negative

(B) Growth state in each medium (pH 8.0) (1) Broth agar plate culture: A 4 to 5 mm diameter, slightly milky brown circular colony with a stationary culture at 30 ° C. for 48 hours. To form The surface is smooth and glossy and the periphery is smooth. No pigment production is observed. (2) Gravy agar slant culture: culture at 30 ° C. for 48 hours,
It shows filamentous growth. (3) Gravy liquid culture: static culture at 30 ° C. for 48 hours,
Growth (turbidity) is observed throughout the medium, a pellicle is formed, and sediment is also observed. (4) Meat juice gelatin stab culture: A stationary culture at 20 ° C. for 10 days grows to the middle of the medium and slowly liquefies the gelatin. (5) Litmus milk culture: After standing at 30 ° C. for 14 days, litmus milk does not coagulate, but liquefaction is observed. No acid or alkali production is observed.

(C) Physiological properties The test was carried out mainly using a medium adjusted to pH 6.5. (1) Reduction of nitrate: not reduced. (2) Denitrification reaction: None. (3) MR test: negative (4) VP test: negative (5) Indole generation: not generated (6) Generation of hydrogen sulfide: No generation. (7) Hydrolysis of starch: no hydrolysis. (8) Use of citric acid: Use.

(9) Use of inorganic nitrogen source: A nitrate is not used, but an ammonium salt is used. (10) Formation of dye: No formation. (11) Ureaase: positive (12) Oxidase: positive (13) Catalase: positive (14) Growth range: temperature; 25-40 ° C, pH;
0 to 8.0 (15) Attitude to oxygen: aerobic (16) OF test (Hugh-Leifson)
Method): Oxidation (17) Generation of acid and gas from saccharides: As shown in Table 5, generation of acid from D-xylose, D-glucose, D-mannose, D-galactose, and sucrose was observed. Is not observed from any of them.

[0048]

[Table 5]

The present strain having the above-mentioned mycological properties and having a novel sorbitol dehydrogenase-producing ability has a negative Gram stain, has motility, has extreme hair, and has an OF-F test. It is determined to belong to the genus Pseudomonas because it is oxidized. Therefore, this strain was designated as Pseudomonas sp.
It was named KS-E1806. This strain was sent to FERM P-1429 by the National Institute of Bioscience and Human Technology.
9 has been deposited.

The sorbitol dehydrogenase in the present invention may be one having the main physicochemical properties such as the above-mentioned action and substrate specificity, and other physicochemical properties exhibit some differences. Are also included as enzymes of the present invention. The microorganism is an example of a bacterium used for obtaining such sorbitol dehydrogenase. In the present invention, any microorganism that belongs to the genus Pseudomonas and has the ability to produce sorbitol dehydrogenase can be used.

Next, a liquid culture method is used to produce the sorbitol dehydrogenase of the present invention using the microorganism having the ability to produce sorbitol dehydrogenase. As the medium, any of a synthetic medium and a natural medium can be used as long as the medium appropriately contains a carbon source, a nitrogen source, an inorganic substance, and other nutrients.

The carbon source may be any assimilable carbon compound, for example, D-sorbitol, glucose, fructose, maltose, starch hydrolyzate, molasses and the like. As the nitrogen source, any available nitrogen compound may be used, and for example, yeast extract, polypeptone, meat extract, corn steep liquor, soybean powder, amino acid, ammonium sulfate, ammonium nitrate and the like are used. As other inorganic substances, various salts such as common salt, potassium chloride, magnesium sulfate, manganese chloride, ferrous sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, other vitamins, antifoaming agents and the like are used. These nutrient sources are
Each can be used alone or in combination.

In order to produce the present enzyme using the liquid medium prepared as described above, it is preferable to culture aerobically by aeration, agitation culture, shaking culture, or the like. At that time, the initial pH of the medium was adjusted to about 6.5 to 7.5, and
Incubate at a temperature of 7 ° C, preferably around 30 ° C for 10 to 36 hours. By doing so, the present enzyme is produced in the culture. After completion of the culture, a normal enzyme collecting means can be used to collect sorbitol dehydrogenase from the culture.

Since the present enzyme is mainly present in the cells, it is preferable to separate the cells from the culture by, for example, filtration, centrifugation, etc., and to collect the cells from the cells. . In this case, the cells can be used as they are, for example, an ultrasonic crusher, a French press, a method of destroying the cells using various destruction means such as dynamill, using a cell wall lysing enzyme such as lysozyme. A method of lysing the cell wall of the cells, a method of extracting the enzyme from the cells using a surfactant such as Triton X-100, or the like can be used alone or in combination. Then, impurities are removed by filtration or centrifugation to obtain a crude enzyme solution of the present enzyme.

The above-mentioned purification method can be applied to isolate and purify the present enzyme as needed from the crude enzyme solution thus obtained. The present enzyme obtained as described above is extremely useful for the quantification of D-sorbitol, and the use of the present enzyme enables enzymatic quantification of D-sorbitol. For example, it is used for the measurement of the amount of D-sorbitol present in human serum or urine, and is used for diagnosis of various diseases such as diabetes, quantification of sorbitol contained in foods and the like, and further, the reverse reaction of the enzyme. It can also be used for quantification of D-fructose by the method described above.

The reagent for quantifying D-sorbitol of the present invention comprises:
It contains this enzyme and NAD ⁺ . As an advantageous system for quantifying D-sorbitol, for example, as a reaction reagent, a system of pH 6.5 to 10.0 containing 0.5 to 20 U / ml of the present enzyme and 10 to 500 mM of a buffer, a system of 0.5 to 3 mM NAD
⁺ System and the like.

The buffers used in these systems include:
For example, potassium phosphate, tris hydrochloride and the like can be mentioned. In such a system, in addition to the above-mentioned components, various commonly used additive components as necessary within a range not to impair the object of the present invention,
For example, surfactants (such as Triton X-100), reducing agents (such as 2-mercaptoethanol, dithiothreitol), bovine serum albumin, and sugars (such as glycerin and sucrose) as solubilizers and stabilizers. It can also be added. These components may be added at an appropriate stage during the preparation of the above system, and may be used alone or in combination of two or more.

The reagent of the present invention may be used in a dried or dissolved state, or may be used after being impregnated in a thin-film carrier such as a sheet-impregnable paper. The enzyme used may be immobilized by a conventional method and used repeatedly. By using such a reagent of the present invention, D-sorbitol contained in various samples can be accurately quantified by a simple operation.

Next, as described above, the method for quantifying D-sorbitol of the present invention is performed by adding the present enzyme and NAD ⁺ to a sample containing D-sorbitol and quantifying NADH produced by the action. However, any sample containing D-sorbitol may be used as the D-sorbitol-containing sample. For example, D-sorbitol is to be measured, and human serum, urine and other diagnostic samples, and liquid or powdered foods , Juices, chewing gums, candies and other confectionery products, synthetic liquors, tobacco and other favorite items.

The sample may be extracted as it is or with water, a buffer, or the like, and concentrated or diluted so that D-sorbitol has an appropriate concentration, and then subjected to quantification. For quantification, the pH of these samples may be unadjusted,
It is desirable to adjust the pH to 8 to 10 with a pH adjuster such as hydrochloric acid, sulfuric acid, nitric acid, sodium hydroxide, potassium hydroxide and the like.

The amount of the present enzyme to act on the sample containing D-sorbitol is appropriately selected depending on the content of D-sorbitol contained in the sample, the enzyme reaction conditions, and the like. Add so that the concentration is 0.5 to 20 U / ml. Next, the method of allowing the present enzyme and NAD ⁺ to act on a D-sorbitol-containing sample and quantifying the generated NADH is not particularly limited, and any method may be used. As a method for quantifying NADH, for example, (i) 340 nm
Method for measuring the amount of increase in absorbance at (2) excitation wavelength 3
By using a calibration curve prepared in advance by a method of measuring fluorescence using 66 nm and a fluorescence wave of 452 nm, the D-
A method for calculating sorbitol is exemplified.

Next, a preferred example of the method for quantifying D-sorbitol of the present invention will be described. First, the enzyme was added to a D-sorbitol-containing sample in an amount of 0.5 to 20 U / ml, preferably 2 to 6 U / ml.
ml and buffer (pH 7-9) at 50-150 mM, NAD ⁺ at 0.
Enzyme reaction is carried out at pH 7 to 9 and at a temperature of 25 to 40 ° C. The reaction time at this time may be a time sufficient to oxidize D-sorbitol, and is 15 to 60 minutes, preferably 25 to 35 minutes. Then generate N
ADH is quantified by the above-described method, and a quantitative value of D-sorbitol in the sample is calculated using a calibration curve of D-sorbitol prepared in advance by the same method.

[0063]

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples. (Example 1) D-sorbitol 0.5%, polypeptone 2.0%, yeast extract 0.5%, KH ₂ PO ₄ 0.01
%, K ₂ HPO ₄ 0.01%, MgSO ₄ .7H ₂ O
Medium (pH 7.2) 1 consisting of 0.01% and tap water
00 ml was placed in Sakaguchi Kolben and sterilized at 121 ° C. for 15 minutes. Pseudomonas
sp. KS-E1806 (FERM P-14299)
Was inoculated from a stock slant of No. 1 and shake-cultured at 30 ° C. for about 24 hours with a shaker to obtain a seed culture solution.

Next, 100 ml of the seed culture solution (for one Sakaguchi Koruben) was inoculated into a 30-liter jar fermenter containing 20 L of a medium prepared and sterilized in the same manner as described above, and the number of rotations was 300 rpm, and the air flow rate was 20 L / min. About 2 at 30 ° C
The cells were cultured for 0 hours. After completion of the culture, the cells were collected from 20 L of the culture solution using Asahi Kasei Microza (PW-303) and collected.
After washing the cells with 0 mM phosphate buffer (pH 7.5), the cells were suspended in about 5 L of the same buffer. Purification of this enzyme was performed by the following operation.

Step 1 (Preparation of Crude Enzyme Solution): 10 g of lysozyme and 1 g of Triton X-100 were added to the cell suspension.
0 ml and 0.55 M EDTA 2Na (pH 8.0)
Add 500 ml, mix and leave at 20 ° C. overnight,
200 ml of a 5% aqueous protamine solution (pH 8.0) was added dropwise with stirring to remove nucleic acids. The supernatant was filtered using an ultrafiltration membrane containing 1 mM containing 50 mM potassium chloride.
It was dialyzed against 0 mM Tris-HCl buffer (pH 8.0) (hereinafter referred to as "buffer A").

Step 2 (DEAE-cellulose treatment): The dialysate (about 10 L) was added to a wet weight of about 9 kg of DEA.
After adding and mixing E-cellulose to adsorb the present enzyme, DEAE was performed with buffer A containing 50 mM potassium chloride.
-The cellulose was washed and then the enzyme was eluted with buffer A containing 0.5 M potassium chloride and ultra-concentrated. Step 3 (QAE-Sephadex A-50 treatment): 1000 ml of Q was added to the concentrate (1000 ml).
AE-Sephadex A-50 is added and mixed,
After adsorbing this enzyme, QAE-Sephadex A-50 was added to buffer A containing 0.05 M potassium chloride.
The enzyme was then eluted with buffer A containing 0.5 M potassium chloride, ultra-concentrated, and further concentrated at 40 mM
It was dialyzed against 20 mM acetic acid-sodium acetate buffer (pH 6.0) containing potassium chloride (hereinafter referred to as "buffer B").

Step 4 (QAE-Toyopearl)
550c column chromatography): The above concentrated dialysate (100 ml) was subjected to QAE-Toyopearl 5
Adsorbed to a 50c column (2.5 × 33 cm).
Wash with buffer B containing 04M potassium chloride, then elute by linear gradient with buffer B containing 0.05M-0.1M potassium chloride and elute with about 0.6M potassium chloride. The eluted active fraction was ultra-concentrated.

Step 5 (Biogel A 1.5 m
200-400 mesh column chromatography):
The concentrated solution (about 2 ml) was applied to Biogel A 1.5 m 2
The mixture was passed through a 00-400 mesh column (2.5 × 95 cm), subjected to gel filtration with a buffer A containing 0.1 M sodium chloride and 0.02% sodium azide, and the eluted active fraction (46 ml) was collected. Collected.

The fraction obtained by the above-mentioned purification procedure was SD
A purified sample of the enzyme determined to be homogeneous by S-polyacrylamide gel electrophoresis (FIG. 6), with a total protein amount of 5.3 mg, a total activity of 480 U, and a specific activity of 90 U / m.
g.

(Example 2) (1) Preparation of reagent for quantifying D-sorbitol The following components were dissolved in purified water at the following concentrations to prepare a reagent. For the enzyme solution, 100m
10 mM Tris-HCl buffer containing M potassium chloride (p
H8.0), the concentration was adjusted to a predetermined unit.

Component concentration Tris-HCl buffer (pH 9.0) 250 mM NAD ⁺ (manufactured by Oriental Yeast Co., Ltd.) 30 mM the present enzyme 40 U / ml

(2) Method for quantifying D-sorbitol D-sorbitol standard (first grade, manufactured by Wako Pure Chemical Industries, Ltd.)
The Tris-HCl buffer (pH 9.0) was added to 1.0 ml of each sample adjusted to 25, 50, 75, and 100 μM using
0) 0.8 ml and 0.1 ml of NAD ⁺ were added,
For 5 minutes. Then, 0.1 ml of the present enzyme was added, and a spectrophotometer (U-20, manufactured by Hitachi, Ltd.) was added.
The reaction was carried out at 37 ° C. for 30 minutes in the ００00 type), and the absorbance at 340 nm after 30 minutes was determined.
D) was determined. There was a linear correlation between this value and the D-sorbitol content. The calibration curve is shown in FIG.
The equation of the calibration curve is y = 0.0298X + 0.002 (r = 1.000). This proved to be effective as a calibration curve, and D-sorbitol in the sample could be accurately quantified.

[0073]

Industrial Applicability The present enzyme acts on D-sorbitol,
Moreover, it is a novel enzyme whose enzyme activity is stable even under the conditions often used in the measurement by the usual enzymatic method (reaction at pH 9.0, 37 ° C., 30 minutes). Can accurately measure the amount of D-sorbitol present in serum or urine. Further, for example, D-fructose in food can be measured by the reverse reaction of the present enzyme.

The present enzyme is different from conventional enzymes of animal origin, such as those derived from sheep liver, and is derived from microorganisms. Therefore, it can be easily and inexpensively produced. It is. Further, according to the quantification reagent and quantification method of the present invention using the present enzyme, it is possible to accurately measure the amount of D-sorbitol present in, for example, food or human serum or urine.

[Brief description of the drawings]

FIG. 1 is a graph showing the optimum pH of the present enzyme at 37 ° C.

[Explanation of symbols]

○ 100 mM potassium phosphate buffer ▲ 100 mM Tris-HCl buffer □ 100 mM glycine-sodium hydroxide buffer △ 100 mM CAPS-sodium hydroxide buffer ● 100 mM sodium carbonate-sodium bicarbonate buffer

FIG. 2 is a graph showing a stable pH range at 30 ° C. of the present enzyme.

[Explanation of symbols]

× 100 mM sodium carbonate-sodium bicarbonate buffer ○ 100 mM glycine-sodium hydroxide buffer ● 100 mM tris-hydrochloride buffer △ 100 mM potassium phosphate buffer ▲ 100 mM acetic acid-sodium acetate buffer

FIG. 3 is a graph showing a suitable temperature range of the present enzyme at pH 9.0.

FIG. 4 is a graph showing the thermostability of the present enzyme at pH 9.0.

[Explanation of symbols]

 ○ 30 ℃ ● 37 ℃ △ 40 ℃

FIG. 5 is a graph showing the thermal stability of the present enzyme at pH 7.0.

[Explanation of symbols]

 ○ 30 ℃ ● 37 ℃ △ 40 ℃

FIG. 6 is an electrophoretogram of the present enzyme.

FIG. 7 is a diagram showing a calibration curve of D-sorbitol.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 9/04 C12Q 1/32 BIOSIS (DIALOG) JICST file (JOIS) MEDLINE (STN) WPI (DIALOG)

Claims (4)

(57) [Claims] 1. A sorbitol dehydrogenase having the following physicochemical properties. (1) Action: D-sorbitol is oxidized in the presence of NAD ⁺ to produce D-fructose and NADH, and D-fructose is reduced by the reverse reaction of this reaction to reduce D-fructose in the presence of NADH. -Generates sorbitol and NAD ⁺ . (2) Substrate specificity: specifically acts on D-sorbitol and galactitol. (3) Optimum pH and stable pH range: The optimum pH is around 10, and the stable pH range is 30 ° C. for 4 hours,
5.5 to 10.5. (4) Range of suitable working temperature: The range of suitable working temperature is around 50 ° C. with 100 mM Tris-HCl buffer (pH 9.0). (5) Deactivation conditions by pH, temperature, etc .: stable at pH 5.5 to 10.5 by treatment at 30 ° C. for 4 hours;
It is completely inactivated at 0 or less and at pH 11.0 or more. pH
At 7.0, it is stable up to around 40 ° C. after 30 minutes treatment, and completely inactivated at pH 7.0 at 40 ° C. in 24 hours. (6) Inhibition: strongly inhibited by HgCl ₂ . (7) Molecular weight: about 64,500 ± 5,000 (gel filtration method). 2. The sorbitol dehydrogenase according to claim 1, wherein the strain belonging to the genus Pseudomonas and having the ability to produce sorbitol dehydrogenase according to claim 1 is cultured in a medium, and sorbitol dehydrogenase is collected from the culture. Production method. 3. A reagent for quantifying D-sorbitol, comprising the sorbitol dehydrogenase according to claim 1 and NAD ⁺ . 4. The method according to claim 1, wherein the sample containing D-sorbitol is
A method for quantifying D-sorbitol, comprising reacting the described sorbitol dehydrogenase and NAD ⁺ to quantify the generated NADH.