JP3040228B2 - L-fucose dehydrogenase, method for producing the same, and method for quantifying L-fucose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an L-fucose dehydrogenase particularly useful for quantifying L-fucose, a method for producing the same, and a method for enzymatic quantification of L-fucose.

[0002]

2. Description of the Related Art L-fucose dehydrogenases which have been found so far include those described in Journal of Biological Chemistry (J. Biol. Che.).
m. , 244, 4785 (1969), L-fucose dehydrogenase derived from Buda liver, Archives of Biochemistry and Biophysics (Arch. Biochem. Biophys).
s. L-fucose dehydrogenase derived from sheep liver described in Journal of Biochemistry (J. Bioche), vol. 186, p. 184 (1978).
m. 86, p. 1559 (1979), L-fucose dehydrogenase derived from rabbit liver, L-fucose dehydrogenase derived from a bacterium belonging to the genus Corynebacterium described in JP-A-62-155085, and JP-A No. -84177, L-fucose dehydrogenase derived from a bacterium of the genus Pseudomonas is known.

[0003]

The conventionally known L-fucose dehydrogenase has the following drawbacks when used as an enzyme for quantitative analysis of L-fucose. That is, L-fucose dehydrogenase described in Journal of Biological Chemistry, vol. 244, p. 4785 (1969), Archives of Biochemistry and Biophysics,
Both L-fucose dehydrogenase described in 186, 184 (1978) and L-fucose dehydrogenase described in Journal of Biochemistry, 86, 1559 (1979) are animal-derived enzymes. Therefore, it has been difficult to industrially provide the enzyme. Further, L-fucose dehydrogenase described in JP-A-62-155085 and JP-A-2-84
No. 177, the L-fucose dehydrogenase-producing bacterium has the productivity of industrially supplying these enzymes. However, the reduced nicotianamide coenzyme produced by the action of these enzymes is used as the bacterium. When performing quantitative analysis, the Michaelis constant (hereinafter referred to as Km value) for L-fucose was large, and thus there was a problem in measurement sensitivity. Furthermore, these enzymes have low temperature stability, and have a practical problem.

[0004]

Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the conventional drawbacks, and as a result, actinomycetes belonging to the genus Streptomyces act on L-fucose, and
-Converting oxidized nicotinamide adenine dinucleotide (hereinafter referred to as NAD ⁺ ) to reduced nicotinamide adenine dinucleotide (hereinafter NADH)
L-fucose dehydrogenase, which is reduced from L.-fucose dehydrogenase obtained from a culture of this microorganism, has a low Km value for L-fucose and a high temperature stability. The present invention has been found to be effective for measuring L-fucose, and the present invention has been completed.

[0005] That is, the present invention relates to a novel L-fucose dehydrogenase having a low Km value for L-fucose and high stability to temperature, and another invention belongs to the genus Streptomyces and produces L-fucose dehydrogenase. Cultivating a microorganism having the ability to produce L-
L-consisting of collecting fucose dehydrogenase
This is a method for producing fucose dehydrogenase. Furthermore, another invention is directed to a method for producing NADH by reacting a sample containing L-fucose with L-fucose dehydrogenase derived from a microorganism belonging to the genus Streptomyces in the presence of NAD ^+.
Is a method for quantifying L-fucose.

Hitherto, only L-fucose dehydrogenase producing strains such as those of the above-mentioned genus Corynebacterium and Pseudomonas have been reported, and the fact that actinomycetes produced this enzyme was the first time by the present inventors. It is the obtained knowledge.

Hereinafter, the present invention will be described specifically.

The L-fucose dehydrogenase obtained according to the present invention has the following physicochemical properties.

(1) Action As shown in the following formula, the present enzyme oxidizes L-fucose to produce L-fucose.
Fuconolactone and reducing NAD ⁺ to NADH.

L-fucose + NAD ⁺ → L-fuconolactone + NADH + H ⁺ (2) Substrate specificity This enzyme has the highest specificity for L-fucose, but also acts on L-galactose, D-arabinose and the like. . 10m
The relative activity of the enzyme for each saccharide at a concentration of M is
Table 1 shows the activity for L-fucose as 100.

[0011]

[Table 1]

The present enzyme requires NAD ⁺ as a coenzyme and has no effect on NADP ⁺ .

(3) Optimum pH The optimum pH of the present enzyme is in the range of 7.5 to 10.0. (Shown in FIG. 1) (4) pH stability When this enzyme is treated at 30 ° C. for 60 minutes at each pH, it is stable up to pH 6.0 to 10.5. (Shown in FIG. 2) (5) Molecular weight 65,000 ± 5,000 [Bio-Sil TSK-
250 (by BIO-RAD) by gel filtration] (6) Molecular weight of subunit 37,000 ± 5,000 (by SDS-polyacrylamide gel electrophoresis) (7) Number of subunits: 2 Unless otherwise specified, the value of molecular weight in the present invention is measured by gel filtration, and the value of molecular weight of a subunit is measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Further, other physicochemical properties of the L-fucose dehydrogenase of the present invention are as follows.

(1) Km value Lineweaver-Bu
rk) The Km value of this enzyme relative to L-fucose was determined to be 0.24 mM by plotting. (2) Optimal temperature 5% in 0.1 M Tris-HCl buffer (pH 9.0)
0 ° C. (Shown in FIG. 3) (3) Temperature stability In 0.1 M Tris-HCl buffer (pH 9.0),
When treated at each temperature for 10 minutes, it is stable up to 50 ° C and is completely deactivated at 65 ° C. (Shown in FIG. 4) (4) Effect and stabilization of metal ions Table 2 shows the effects of various metal ions and reagents on the enzyme of the present invention at a concentration of 1 mM. Ag ⁺ , Hg ²⁺ and C
Strongly inhibited by d ²⁺ .

[0016]

[Table 2]

(5) Isoelectric point The isoelectric point of the present enzyme determined by isoelectric focusing using Celvalite (pH 3.0-10.0, manufactured by Selva) is 4.0 to 4.4. is there.

As a result of comparing the physicochemical properties of the L-fucose dehydrogenase of the present invention having the above-mentioned physicochemical properties with the conventionally known L-fucose dehydrogenase derived from Corynebacterium and Pseudomonas,
The enzyme of the present invention was found to be an enzyme having properties different from those of conventional L-fucose dehydrogenase. Table 3 shows a comparison of various properties of the L-fucose dehydrogenase according to the present invention and the conventionally known L-fucose dehydrogenase.

[0019]

[Table 3]

The conventional L-fucose dehydrogenase in Table 3 includes L-fucose dehydrogenase derived from the genus Corynebacterium described in JP-A-62-155085 and Pseudomonas sp. Described in JP-A-2-84177. L-fucose dehydrogenase from E. coli.

From Table 3, it is clear that the L-fucose dehydrogenase of the present invention and the conventional L-fucose dehydrogenase are:
Substrate specificity, Km value for L-fucose, stable temperature,
It can be seen that there is a clear difference in the molecular weight and the molecular weight of the subunit. Particularly, the Km value of the present enzyme with respect to L-fucose is 0.24 mM, and the stable temperature is up to 50 ° C.

The method for measuring the enzyme activity of L-fucose dehydrogenase and the method for displaying the enzyme activity value in the present invention are as follows.

0.5 ml of a 0.2 M phosphate buffer (pH 7.5) and 0.2 ml of a 50 mM L-fucose solution
1, 0.2 ml of 1 mM NAD ⁺ solution is placed in a standard cuvette having an optical path length of 1 cm, and after keeping for 5 minutes, 0.1 ml of an appropriately diluted enzyme solution is added and mixed to start the reaction. 2 minutes at 340 nm wavelength or if necessary
Measure the absorbance over a longer period of time. From the amount of increase in absorbance per minute (A), the amount of generated NADH was determined based on the following conversion formula. The enzyme activity value is defined as one unit of the amount of the enzyme that produces 1 μmole of NADH per minute.

[0024]

(Equation 1)

Next, a method for producing L-fucose dehydrogenase according to the present invention will be described. The microorganism used in the present invention may be any strain as long as it belongs to the genus Streptomyces and has an ability to produce L-fucose dehydrogenase, or may be a mutant of these strains. Specific examples of strains belonging to the genus Streptomyces and capable of producing L-fucose dehydrogenase include, for example, Streptomyces olivochromenes.
geneses) IFO 3178, Streptomyces neara
gawaensis) R-19 bacteria. In particular, the present inventors have isolated Streptomyces nailagawaengis from the soil.
awaensis) R-19 bacteria are preferably used.

The bacteriological properties of the Streptomyces neigawaengis R-19 bacterium are as follows.

(A) Morphology This strain elongates aerial hyphae from a base mycelium branched under a microscope, and its tip has a spiral shape. It does not show ring branching, and no formation of special organs such as sclerotia is observed. The mature spore chain shows a chain of 10 or more spores, the size of the spore is 0.8 × 1.1 micron cylindrical, and the surface of the spore is smooth.

(B) Growth conditions and physiological properties in various media Culture was performed at 28 ° C. unless otherwise specified. Unless otherwise described, production of a soluble dye in the medium was not observed.

(1) Sucrose / nitrate agar The growth is colorless, and the aerial hyphae is light gray.

(2) Glucose-asparagine agar The growth is light-colored, aerial mycelia is white to gray-white. Gold brown, aerial hyphae appear light gray. About 2 days after the cultivation, hydrolysis of starch is observed.

(5) Tyrosine agar The growth is dark brown, and the aerial hyphae is white to light gray. Production of a black soluble dye is observed.

(6) Nutrient agar It grows ocher and no aerial hyphae are formed.

(7) Yeast / malt agar The growth develops carkey, gray aerial hyphae.

(8) Oatmeal agar The growth is carkey and the aerial mycelia are light gray.

(9) Peptone / East / Iron agar It grows dark brown and no aerial hyphae are formed. Production of a black soluble dye is observed.

(10) Glucose / Peptone / Gelatin Puncture Culture The growth is golden brown, and the formation of aerial hypha is not observed. After culture 2
Up to one day, no liquefaction of the gelatin is observed.

(11) Skim milk powder (cultured at 37 ° C.) No formation of aerial hyphae is observed. The color changed to brown from about the 12th day after the cultivation, and changed to the color of Kirky on the 21st day after the cultivation, and showed partial coagulation of skim milk powder.

(12) Formation of melanin-like pigment The formation of melanin-like pigment is observed in both tyrosine agar and peptone / yeast / iron agar medium.

(13) D-glucose, L-arabinose, D-xylose, D-fructose, L-rhamnose, raffinose, inositol, D-mannitol,
D-galactose and L-fucose are often used, and sucrose is also used.

(14) Reduction of nitrate As a result of a test using peptone water containing 10% potassium nitrate, reduction of nitrate was observed.

(15) Salt resistance test Salt resistance is up to 4%.

(16) Optimal growth temperature 10% using maltose yeast agar (pH 7.0)
° C, 22 ° C, 25 ° C, 28 ° C, 30 ° C, 37 ° C, 50 ° C
As a result of the test at each temperature, it is possible to grow at 22 ° C. to 37 ° C., but around 28 ° C. seems to be the optimum temperature.

To summarize the above results, this strain belongs to the genus Streptomyces, the tip of the hypha shows a spiral shape, and the surface of the spores is smooth. On various mediums, white-grey-white to light gray aerial mycelia grow on light yellow to dark brown growth, and melanin-like pigments are observed, but other soluble pigments are not observed. Starch has a strong water dissolving power. Proteolytic activity is weak, and coagulation of skim milk is slightly observed, but liquefaction of gelatin is not observed. D-glucose, L
-Arabinose, D-xylose, D-fructose,
L-rhamnose, raffinose, inositol, D-mannitol, D-galactose, L-fucose are often used, and sucrose is also used.

Based on these properties, a similar known bacterial species was identified as I
SP (International Streptomy)
ces Project), Streptomyces neyagawaengis (Strep)
tomyces nailawaensis) and Streptomyces regismitifikas (Strepto)
myces resistomyticus). In addition, the description of Bergeys (Bergeys
Manual of Determinative
Bacteriology, 8th Ed. ), The strain is up to 4% in salt tolerance, and thus seems to be most closely related to Streptomyces neyagawaengis, unlike Streptomyces resistmysisficus.

Next, the present strain and a known ISP strain 558
Table 4 summarizes the results of comparison of the bacteriological properties obtained by culturing S.8 (Streptomyces neyagawaengis) under the same conditions.

[0046]

[Table 4]

As described above, although a slight difference is observed in the coagulation of milk and the reduction reaction of nitrate, in other respects, the present strain satisfies all the characteristics of Streptomyces neyagawaengis. Therefore, this strain was identified as a strain belonging to Streptomyces neigawaengis, and Streptomyces neigawaengis R-19 (Strep
tomyces nailawaensis R-1
9). This strain was sent to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology
-12620). As a medium used for culturing the L-fucose dehydrogenase producing bacterium, any of a synthetic medium and a natural medium to which a carbon source, a nitrogen source, an inorganic salt and other nutrients are added can be used. Commonly used carbon sources such as glucose, sucrose, fructose, glycerol, and starch may be used as the carbon source. As a nitrogen source, ammonium sulfate,
An inorganic nitrogen compound such as ammonium nitrate or an organic nitrogen compound such as peptone, meat extract, yeast extract or asparagine is used. Potassium phosphate, inorganic salt,
Potassium phosphate, magnesium sulfate, sodium chloride, etc. are used.
A surfactant such as 94 is added as needed. Since the L-fucose dehydrogenase of the present invention is an inducing enzyme, the addition of L-fucose to the medium significantly increases the enzyme production. When L-fucose is added in an amount of 0.05% or more, the enzyme production-inducing effect is exhibited, but preferably 0.1 to 0.3
%. Culture is possible in the pH range of 6-8,
It is more desirable to carry out near H7. When culturing with a jar fermenter, the initial pH is adjusted to 7.0.
The culture temperature can be in the range of 22 to 37 ° C, but is preferably around 28 ° C. The culture requires oxygen supply and requires aeration and agitation. The culture time in a jar fermenter is usually 48 to 72 hours, but in order to obtain a highly active strain, the growth of the cells is in a stationary phase (Stationar
y phase).

After culturing the L-fucose dehydrogenase-producing bacterium, when recovering the enzyme, Streptomyces
In the case of Neigawa Engis R-19, the enzyme is mainly present in the cells, so it is preferable to first isolate the cells only by a method such as centrifugation or filtration of the culture.

The L-fucose dehydrogenase of the present invention can be separated and purified as follows. As a method of extracting the enzyme accumulated in the cells from the cells,
Conventional methods include disruption of cells by ultrasonic waves, disruption of cells by a dynomill that rotates together with glass beads, and disruption of cell membranes by enzymes such as lysozyme and organic solvents such as toluene.
By selecting an appropriate method from these and extracting the enzyme from the cells, the enzyme can be collected.

From the crude enzyme solution extracted by these methods, L
-If it is necessary to further purify the fucose dehydrogenase, a commonly used means for purifying common enzymes such as ammonium sulfate precipitation, ion exchange column chromatography, gel filtration, hydrophobic binding column chromatography, etc. Purification can be performed by appropriately combining or repeating the methods.

The specific activity of the completely purified enzyme of L-fucose dehydrogenase according to the present invention is about 130 units / mg-protein. Also, a single protein band is observed in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

Next, the method for quantifying L-fucose according to the present invention will be specifically described.

The measuring principle of the present invention is as follows.

L-fucose dehydrogenase L-fucose + NAD ⁺ → L-fuconolactone + NA
L-fucose in the DH + H ⁺ sample produces L-fuconolactone and NADH by the action of L-fucose dehydrogenase in the presence of NAD ⁺ . Known methods can be used for quantification of the generated NADH. For example, NADH itself can be measured by a method of measuring absorbance at 340 nm.

The buffer used for the reaction is not particularly limited, and a phosphate buffer, a Tris buffer, a glycine buffer, and the like are suitable.
Five to ten buffers are used. Since the Lm-fucose dehydrogenase of the present invention has a small Km value with respect to L-fucose, L-fucose dehydrogenase in the reaction solution is usually used in 0.05 to 20 units.
~ 5 units are preferred. The concentration of NAD ⁺ varies depending on reaction conditions and the like, but usually 0.5 mM to 5 mM is used. The reaction temperature is 30-50 ° C, and the reaction time is 2-20.
Minutes are desirable.

[0056]

Industrial Applicability According to the present invention, there is provided an L-fucose dehydrogenase useful for quantifying L-fucose, a production method suitable for industrial production thereof, and a method for quantifying L-fucose which is simple and highly sensitive.

[0057]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 From L-fucose 0.3%, peptone 0.4%, yeast extract 0.2%, meat extract 0.2% and defoamer (Adecanol LG294) 0.02%, pH 7.0 Medium 1
Dispensed and sterilized (120 ° C, 20 minutes) 5
A 00 ml Sakaguchi flask was inoculated with Streptomyces neigawaengis R-19 and cultured with shaking at 28 ° C for 72 hours. The L-fucose dehumanodenase activity in this culture was 0.58 units / ml. Example 2 Medium 1 comprising 0.3% L-fucose, 0.4% peptone, 0.2% yeast extract, 0.2% meat extract and 0.02% antifoam (ADEKANOL LG294), pH 7.0 , 500 ml into a 5 l Erlenmeyer flask,
After sterilizing for 20 minutes, the medium is inoculated with Streptomyces negawanengis R-19 at 28 ° C. 28 ° C
After shaking culture for 72 hours, this culture solution was added to a sterilized jar fermenter previously charged with 20 l of a medium having the same composition as above, and main culture was performed. After culturing for 70 hours at 28 ° C., agitation frequency of 150 rpm and aeration at 20 l / min, the culture was centrifuged to collect the cells. 70 g of the obtained cells
(Wet cell weight) in 10 mM phosphate buffer (pH 7.5)
The suspension was suspended in 1.5 l, and the suspension was disrupted by an ultrasonic disrupter. The crushed liquid was centrifuged using a centrifuge to obtain a supernatant. The total activity of L-fucose dehydrogenase in this supernatant was 2,470 units, and the specific activity was 0.09 units / mg-protein.

A column (φ9) packed with DEAE-cellulose (trade name: manufactured by Whatman), which was obtained by preliminarily equilibrating the supernatant with a 20 mM phosphate buffer (pH 7.5).
× 40 cm). After washing the column with 10 l of a phosphate buffer (pH 7.5) containing 0.1 M salt, the column was adsorbed with a linear concentration gradient (total volume: 10 l) having a salt concentration of 0.1 M to 0.4 M. The protein is eluted and L
-The fucose dehydrogenase active fraction was collected. The total activity of L-fucose dehydrogenase in this active fraction was 1,840 units, and the specific activity was 1.53 units / mg.
-It was a protein.

After the active fraction was desalted and concentrated using an ultrafiltration apparatus, ammonium sulfate was added to a concentration of 25%, and the resulting precipitate was removed by centrifugation. The total activity of L-fucose dehydrogenase in the obtained supernatant was 1,430 units, and the specific activity was 2.52 units / mg.
-It was a protein.

A column (φ3.5 ×) packed with butyl Toyopearl 650M (trade name, manufactured by Tosoh Corporation), which was obtained by preliminarily equilibrating the supernatant with a 20 mM phosphate buffer (pH 7.5) containing 25% ammonium sulfate. 22.5 cm). After washing the column with the same buffer, the adsorbed L-fucose dehydrogenase was eluted with a linear concentration gradient (total volume: 2 l) having an ammonium sulfate concentration of 25% (about 0.8 M) to 0%. The total activity of L-fucose dehydrogenase in this solution was 1,250 units, and the specific activity was 22.73 units / mg-protein.

The eluate was desalted and concentrated by ultrafiltration, and then Biosil TSK-250 (trade name: Bio Biosil) previously equilibrated with 20 mM phosphate buffer (pH 7.0) containing 0.05 M sodium sulfate. Rat company)
The mixture was passed through a column (φ2.15 × 60 cm) and subjected to gel filtration to collect an active fraction. The total activity of L-fucose dehydrogenase in this active fraction was 840 units, and the specific activity was 1
00 units / mg-protein.

To this solution, ammonium sulfate was added to a concentration of 25%, and then butyltoyopearl 650M (trade name, manufactured by Tosoh Corporation) preliminarily equilibrated with a 20 mM phosphate buffer (pH 7.5) containing 25% ammonium sulfate. ) Through a column (φ1.6 × 5.2 cm). After washing the column with the same buffer, the adsorbed L-fucose dehydrogenase was eluted with a linear concentration gradient (total volume: 100 ml) having an ammonium sulfate concentration of 25% (about 0.8 M) to 0%. A purified enzyme solution was obtained by desalting the L-fucose dehydrogenase active fraction by dialysis. The total activity of L-fucose dehydrogenase in this purified enzyme solution was 560 units, and the specific activity was 130 units / mg-protein.

The purity of the enzyme thus obtained was examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. As a result, only one band was observed, confirming that the enzyme was pure L-fucose dehydrogenase. Was.

Example 3 A 10 mM phosphate buffer (pH 7.
Using the enzyme preparation appropriately diluted and adjusted according to 5), the substrate specificity of the present enzyme, optimal pH, pH stability, optimal temperature,
The temperature stability was investigated.

[Substrate specificity] 0.2 M phosphate buffer (p
H7.5) 0.5 ml, 50 mM solutions of various sugars 0.2 m
l to a reaction solution consisting of 0.2 ml of 1 mM NAD ⁺ solution,
0.1 ml (0.01 unit) of the enzyme preparation was added, and 3
The reaction was performed at 7 ° C. for 2 minutes, and the enzyme activity was determined based on the increase in the absorption of NADH at 340 nm. Table 1 shows the relative activity values of the action on these sugars, with the action on L-fucose being 100.

[Optimal pH] 0.2 M various buffers (pH
5.0-6.5: citrate buffer; pH 6.0-8.
0: phosphate buffer; pH 7.5 to 8.5: Tris-hydrochloric acid buffer; pH 8.5 to 11.5: glycine-caustic soda buffer) 0.5 ml, 50 mM L-fucose solution 0.2
0.1 ml (0.01 units) of the enzyme preparation to a reaction solution consisting of 0.2 ml of 1 mM NAD ⁺ solution,
The reaction was performed at 7 ° C. for 2 minutes, and the enzyme activity was determined based on the increase in the absorption of NADH at 340 nm. After the above operation, the relative activity (Re
latency activity) was calculated and graphed to obtain FIG. From FIG. 1, the optimum pH of this enzyme is 7.5.
It can be seen that it is in the range of ０．０10.0.

[PH stability] 0.1 M Tris-imidazole-sodium acetate buffer (pH 4.5, 5.0,
5.5, 6.0, 6.5, 7.0, 7.5, 8.0,
8.5, 9.0, 9.5, 10.0, 10.5, 10.
7, 11.2, 11.7, 12.2) 0.95 ml was mixed with 0.05 ml of the enzyme preparation (0.4 units),
After standing at 30 ° C. for 60 minutes, 0.025 ml of each solution was added to 0.5 ml of 0.2 M Tris-imidazole-sodium acetate buffer (pH 9.5), 0.2 ml of 50 mM L-fucose solution, 0.2 ml of 1 mM NAD ⁺ solution, The mixture was mixed with 0.075 ml of ion-exchanged water, reacted at 37 ° C. for 2 minutes, and the enzyme activity was determined based on the increase in NADH absorption at 340 nm. After the above operation, the relative activity was calculated with the highest enzyme activity value taken as 100%, and graphed to obtain FIG. As is clear from FIG.
It is stable in the range of 6.0 to 10.5.

[Optimal temperature] 0.1 ml of an enzyme preparation was added to a reaction solution consisting of 0.5 ml of 0.2 M Tris-HCl buffer (pH 9.0), 0.2 ml of 50 mM L-fucose solution, and 0.2 ml of 1 mM NAD ⁺ solution. (0.01 units), 25, 30, 35, 37, 40, 45, 50, 5
The reaction was carried out at each temperature of 5, 60, 65 and 70 ° C. for 2 minutes, and the enzyme activity was determined based on the increase in NADH absorption at 340 nm. After the above operation, the relative activity was calculated with the highest enzyme activity value taken as 100%, and graphed to obtain FIG. According to FIG. 3, the optimal temperature of this enzyme is 50 ° C.
It can be seen that it is.

[Temperature stability] 10 mM phosphate buffer (p
H7.5) was diluted with 0.2 ml (0.04 unit) of the enzyme preparation to 25, 30, 35, 40, 45, 50, 5
The treatment was performed at each temperature of 5,60 ° C. for 10 minutes. 0.05 ml of this enzyme solution was added to a 0.2 M Tris-HCl buffer (pH 9.
0) 0.5 ml, 0.2 mM of 50 mM L-fucose solution
1, 0.2 ml of 1 mM NAD ⁺ solution, 0.1 ml of ion-exchanged water.
Of the mixture, and reacted at 37 ° C. for 2 minutes.
The enzyme activity was determined based on the increase in the absorption of NADH at m. After the above operation, the highest enzyme activity value was 100
% Was calculated and graphed to obtain FIG. As is evident from FIG. 4, the enzyme is stable at temperatures up to 50 ° C.

Example 4 The concentration of L-fucose in a sample solution was determined by the following method using a reagent having the following composition.

A) Reagent 0.2 M phosphate buffer (pH 7.5) 0.6 ml 5 mM NAD ⁺ 0.2 ml 0.7 unit / ml L-fucose dehydrogenase 0.1 ml b) Measurement method Mixed solution of the above reagents 0 0.1 ml of the sample solution was added to 0.9 ml, and reacted at 37 ° C. for 5 minutes, and the absorbance at 340 nm was measured. In the same manner, instead of the sample solution, the same amount of water was added and reacted, and the absorbance was measured. The absorbance of the sample solution was calculated by subtracting the absorbance of the sample solution from the absorbance of the sample solution.

Separately, instead of the sample solution, a known concentration of L-
The fucose solution was added to the mixture of the above reagents and reacted in the same manner to prepare a calibration curve (shown in FIG. 5). When the concentration of L-fucose in the sample solution was determined using this calibration curve, it could be accurately quantified.

[Brief description of the drawings]

FIG. 1: pH of L-fucose dehydrogenase of the present invention
It is a figure which shows an activity curve.

FIG. 2 is a view showing the pH stability of the enzyme.

FIG. 3 is a view showing a temperature activity curve of the enzyme.

FIG. 4 shows the temperature stability of the enzyme.

FIG. 5 is a diagram showing a calibration curve used when measuring the concentration of L-fucose.

Claims (3)

(57) [Claims] An L-fucose dehydrogenase having the following physicochemical properties: Action: Acts on L-fucose in the presence of oxidized nicotinamide adenine dinucleotide to produce L-fuconolactone and reduced nicotinamide adenine dinucleotide. Substrate specificity: L-fucose, L-galactose, D
-Acts on arabinose and has the lowest specificity for D-arabinose. Optimum pH: pH 7.5 to 10.0 pH stability: 60 at each pH at 30 ° C
When treated for minutes, it is stable up to pH 6.0-10.5. Molecular weight: 65,000 ± 5,000 Molecular weight and number of subunits: Molecular weight 37,0
It consists of two 00 ± 5,000 subunits. 2. The method for producing L-fucose dehydrogenase according to claim 1, wherein a microorganism belonging to the genus Streptomyces and capable of producing L-fucose dehydrogenase is cultured, and L-fucose dehydrogenase is collected from the culture. . 3. A reduced nicotine produced by allowing L-fucose dehydrogenase derived from the microorganism belonging to the genus Streptomyces to act on a sample containing L-fucose in the presence of oxidized nicotinamide adenine dinucleotide. A method for quantifying L-fucose, characterized by quantifying amide adenine dinucleotide.