JPH05168474A - L-fucose dehydrogenase, method for producing the same and method for quantifying L-fucose - Google Patents

Abstract

(57) [Summary] (Modified) [Constitution] Has the following physicochemical properties (1) Action: N
Acts on L-fucose in the presence of AD ⁺ to produce L-fuconolactone and NADH (2) Acts on L-fucose, L-galactose and D-arabinose, and has the lowest specificity for D-arabinose ( 3) Optimum pH 7.5
~ 10.0 (4) pH stability: pH 6.0 to 10.5 by treatment at 30 ° C for 60 minutes (5) Molecular weight: 65,000
± 5,000 (6) Molecular weight 37,000 ± 5,000
It consists of 2 subunits. A method for producing L-fucose dehydrogenase from a culture of a microorganism belonging to the genus Streptomyces. NA for samples containing L-fucose
A method for quantifying L-fucose in which L-fucose dehydrogenase derived from a microorganism belonging to the genus Streptomyces is allowed to act in the presence of D ⁺ to measure NADH produced. [Effects] An L-fucose dehydrogenase useful for quantifying L-fucose, a production method suitable for industrial production thereof, and a method for quantifying L-fucose with simple operation and high sensitivity are provided.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art As L-fucose dehydrogenase which has been found so far, Journal of Biological Chemistry (J. Biol. Che.
m. ), 244, p. 4785 (1969), L-fucose dehydrogenase derived from Buda liver, Archives of Biochemistry and Biophysics (Arch. Biochem. Biophys).
s. ), 186, 184 (1978), L-fucose dehydrogenase derived from sheep liver, Journal of Biochemistry (J. Bioche).
m. ), 86, p. 1559 (1979), L-fucose dehydrogenase derived from rabbit liver, L-fucose dehydrogenase derived from corynebacterium bacterium described in JP-A-62-155085, and JP-A-2. An L-fucose dehydrogenase derived from a bacterium belonging to the genus Pseudomonas described in JP-A-84177 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, 244, page 4785 (1969), Archives of Biochemistry and Biophysics,
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 was difficult to industrially provide the enzyme. Further, L-fucose dehydrogenase described in JP-A-62-155085 and JP-A-2-84.
The L-fucose dehydrogenase-producing bacterium described in Japanese Patent Publication No. 177 has productivity to industrially supply these enzymes, but it produces a reduced nicotianamide coenzyme produced by acting these enzymes. In the quantitative analysis, the Michaelis constant for L-fucose (hereinafter referred to as Km value) was large, and thus there was a problem in measurement sensitivity. Furthermore, these enzymes have low stability against temperature and have problems in practical use.

[0004]

Means for Solving the Problems As a result of intensive studies conducted by the present inventors to solve the above-mentioned conventional drawbacks, actinomycetes belonging to the genus Streptomyces act on L-fucose to give L-fucose.
-Fuconolactone is used and oxidized nicotinamide adenine dinucleotide (hereinafter referred to as NAD ⁺ ) is reduced nicotinamide adenine dinucleotide (hereinafter referred to as NADH).
It is found that the L-fucose dehydrogenase produced by the culture of this microorganism has a small Km value for L-fucose and high stability against temperature. The present invention has been completed by discovering that it can be effectively used for the measurement of L-fucose.

That is, the present invention is a novel L-fucose dehydrogenase having a low Km value for L-fucose and high stability against temperature, and another invention belongs to the genus Streptomyces and produces L-fucose dehydrogenase. The microorganism having the ability is cultivated, and L-
L-consisting of collecting fucose dehydrogenase
It is a method for producing fucose dehydrogenase. Further, another invention is that NADH produced by allowing a sample containing L-fucose to act on L-fucose dehydrogenase derived from a microorganism belonging to the genus Streptomyces in the presence of NAD ^+.
Is a method for quantifying L-fucose.

[0006] Conventionally, as the L-fucose dehydrogenase producing strain, only the above-mentioned bacteria of the genus Corynebacterium, Pseudomonas, etc. have been reported. This is the knowledge obtained.

The present invention will be specifically described below.

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

(1) Action As shown by the following formula, this enzyme oxidizes L-fucose to give L-fucose.
It is converted to fuconolactone and NAD ⁺ is reduced 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 this enzyme for each saccharide at the concentration of M is
Table 1 shows the activity against L-fucose as 100.

[0011]

[Table 1]

Further, the present enzyme requires NAD ⁺ as a coenzyme and has no effect on NADP ⁺ .

(3) Optimum pH The optimum pH of this enzyme is 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 (manufactured by BIO-RAD) by gel filtration method] (6) Molecular weight of subunit 37,000 ± 5,000 (by SDS-polyacrylamide gel electrophoresis) (7) Number of subunits: 2 Unless otherwise specified, the molecular weight values in the invention are those measured by gel filtration, and the subunit molecular weights are measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Other physicochemical properties of the L-fucose dehydrogenase of the present invention are as follows.

(1) Km value Lineweaver-Burk
The Km value of this enzyme for L-fucose was determined from the rk) plot and found to be 0.24 mM. (2) Optimum temperature 5 in 0.1M Tris-HCl buffer (pH 9.0)
It is 0 ° C. (Shown in FIG. 3) (3) Temperature stability In 0.1M Tris-hydrochloric acid buffer (pH 9.0),
When it was treated at each temperature for 10 minutes, it was stable up to 50 ° C and completely deactivated at 65 ° C. (Shown in FIG. 4) (4) Effects 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
It is strongly inhibited by d ²⁺ .

[0016]

[Table 2]

(5) Isoelectric point The isoelectric point of the present enzyme determined by the isoelectric focusing method using Cellbarite (pH 3.0-10.0, manufactured by Selva Co.) 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 and the conventionally known L-fucose dehydrogenase derived from Corynebacterium and Pseudomonas,
It was revealed that the enzyme of the present invention is 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 means L-fucose dehydrogenase derived from the genus Corynebacterium described in JP-A-62-155085 and Pseudomonas described in JP-A-2-84177. It is an L-fucose dehydrogenase derived from.

From Table 3, 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 molecular weight and subunit molecular weight. Particularly, the Km value of this enzyme for 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 0.2 M phosphate buffer (pH 7.5) and 0.2 m of 50 mM L-fucose solution
1, 0.2 mM of 1 mM NAD ⁺ solution was placed in each standard cuvette with an optical path length of 1 cm, kept for 5 minutes, and 0.1 ml of an appropriately diluted enzyme solution was added and mixed to start the reaction. 2 minutes at 340 nm wavelength or if needed,
Absorbance is measured over that time. The amount of NADH produced was calculated from the increase (A) in absorbance per minute based on the following conversion formula. The enzyme activity value is defined as 1 unit of the amount of enzyme that produces 1 μmole of NADH per minute.

[0024]

[Equation 1]

Next, the 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 the ability to produce L-fucose dehydrogenase, or a mutant strain of these strains. Then, as a specific example of the strain belonging to the genus Streptomyces and having the ability to produce L-fucose dehydrogenase, for example, Streptomyces olivine chromogenes (Streptomyces oliveovochromo
genes) IFO 3178, Streptomyces neya
Gawaensis) R-19 bacterium. In particular, the present inventors separated from the soil by Streptomyces neyagawa endis (Streptomyces neyag).
awaensis) R-19 bacterium is preferably used.

The mycological properties of this Streptomyces nyagagawadis R-19 bacterium are as shown below.

(A) Morphology This strain extends aerial hyphae from the basal hyphae branched under a microscope, and the tip thereof has a spiral shape. It does not show limbus branching and no formation of special organs such as sclerotium is observed. The matured spore chain has a chain of 10 or more spores, the size of the spores is 0.8 × 1.1 micron, and the surface of the spores is smooth.

(B) Growth state and physiological properties in various media Unless otherwise specified, the culture was carried out at 28 ° C. In addition, no production of soluble dyes was observed in the medium unless otherwise specified.

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

(2) Glucose / asparagine agar Growth is light-colored, aerial mycelia are white to grayish white (3) Glycerin / asparagine agar Growth is carkey color, aerial mycelia is white to light gray (4) Starch / inorganic salt agar Golden brown with a light gray mycelium. Hydrolysis of starch is observed from about the second day after culturing.

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

(6) Nutrient agar It grows in ocher color and no aerial mycelium is observed.

(7) Yeast / malt agar During development, aquatic hyphae of kerky color and gray are settled.

(8) Oatmeal agar Growth is carkey color, and aerial mycelium is light gray.

(9) Peptone / Yeast / Iron Agar It grows dark brown and no aerial hyphae are observed. Production of a black soluble pigment is observed.

(10) Glucose / peptone / gelatin puncture culture The growth was golden brown and no aerial hyphae were observed. After culturing 2
Up to 1 day, no liquefaction of gelatin was observed.

(11) Nonfat dry milk (37 ° C. culture) No aerial hyphae are observed. About 12 days after culturing, the color changed to brown, and on the 21st day after culturing, the color changed to kerky color and partial coagulation of skim milk powder was shown.

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

(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 the test using peptone water containing 10% potassium nitrate, reduction of nitrate was observed.

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

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

In summary of the above results, this strain belongs to the genus Streptomyces, the tips of mycelia show a spiral shape, and the surface of spores is smooth. In various media, white to off-white to light gray aerial hyphae grow on light yellow to dark brown growth, and melanin-like pigments are observed, but other soluble pigments are not observed. The starch is highly water-degradable. The proteolytic activity is weak, and coagulation of skim milk is slightly observed, but liquefaction of gelatin is not observed. As a carbon source, 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.

From these properties, similar known bacterial strains were identified as I
SP (International Streptomy)
ces Project) to search for Streptomyces neyagawaenjis (Strep)
tomyces nyagawaensis) and Streptomyces resist mimicus (Strepto)
myces resistomycicus). In addition, the description of Bergey (Bergeys
Manual of Determinative
Bacteriology, 8th Ed. 2), the salt tolerance of this strain is up to 4%, and unlike Streptomyces registris michificus, it seems to be most closely related to Streptomyces neyagawaenjis.

Next, this strain and known ISP strain 558
Table 4 summarizes the results of the comparison of the mycological properties obtained by culturing 8 (Streptomyces nayagawaenjis) under the same conditions.

[0046]

[Table 4]

As described above, a slight difference is recognized in the coagulation of milk and the reduction reaction of nitrate, but in other respects, this strain satisfies all the characteristics of Streptomyces neyagawa endis. Therefore, this strain was identified as a strain that belongs to Streptomyces nyagawaensis, and Streptomyces nyagawaensis R-19 (Strep
tomyces neyagawaensis R-1
9). In addition, this strain was sent to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Micro Engineering Research Institute No. 12620 (FERM P
-12620). As a medium used for culturing the L-fucose dehydrogenase-producing bacterium, any of a synthetic medium and a natural medium containing a carbon source, a nitrogen source, an inorganic salt and other nutrient sources can be used. As the carbon source, commonly used ones such as glucose, sucrose, fructose, glycerol and starch may be used. Ammonium sulfate as a nitrogen source,
Inorganic nitrogen compounds such as ammonium nitrate or organic nitrogen compounds such as peptone, meat extract, yeast extract and asparagine are used. Inorganic salt potassium phosphate,
Dibasic potassium phosphate, magnesium sulfate, sodium chloride, etc. are used, and ADEKA NOL LG-2 is also used as an antifoaming agent.
A surfactant such as 94 is added if necessary. Since the L-fucose dehydrogenase of the present invention is an inducing enzyme, addition of L-fucose to the medium markedly increases the enzyme production. When the amount of L-fucose added is 0.05% or more, an enzyme production inducing effect is exhibited, but preferably 0.1 to 0.3.
%. Cultivation is possible within a pH range of 6 to 8, but p
It is more desirable to carry out near H7. When culturing with a jar fermenter, the initial pH is 7.0.
The culture temperature can be in the range of 22 to 37 ° C, but is preferably around 28 ° C. Oxygen must be supplied for culturing, and aeration and agitation must be performed. The culture time in the jar fermenter is usually 48 to 72 hours, but in order to obtain a highly active strain, the bacterial growth is stationary phase (Stationar).
y phase) is desirable.

After culturing the L-fucose dehydrogenase-producing bacterium, when recovering the enzyme, Streptomyces
In the case of Neigawa Engendis R-19, the enzyme is mainly present in the microbial cells, so it is preferable to first separate only the microbial cells 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 for extracting the enzyme accumulated in the bacterial cells from the bacterial cells,
Examples of the conventional methods include cell disruption by ultrasonic waves, cell disruption by a Dynomill disruptor rotating with glass beads, or cell membrane disruption by an enzyme such as lysozyme or an organic solvent such as toluene.
The enzyme can be collected by selecting an appropriate method from these and extracting the enzyme from the bacterial cells.

L was obtained from the crude enzyme solution extracted by these methods.
-When it is necessary to further purify the fucose dehydrogenase, a commonly used purification method for an enzyme such as ammonium sulfate precipitation method, ion exchange column chromatography method, gel filtration method and hydrophobic binding column chromatography method is used. Purification can be carried out by appropriately combining or repeating the methods.

The specific activity value 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 measurement 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 ⁺ . A known method can be used for the quantification of the produced NADH. For example, NADH itself can be measured by a method of measuring the absorbance at 340 nm.

The buffer solution used in the reaction is not particularly limited, and a phosphate buffer solution, a Tris buffer solution, a glycine buffer solution and the like are suitable, and a pH of 6 to 10.5, preferably a pH of 7.
5-10 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 an amount of 0.05 to 20 units.
~ 5 units are preferred. The concentration of NAD ⁺ varies depending on the reaction conditions and the like, but is usually 0.5 mM to 5 mM. Reaction temperature is 30 to 50 ° C., reaction time is 2 to 20
Minutes are desirable.

[0056]

INDUSTRIAL APPLICABILITY The present invention provides 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 easy and highly sensitive to operate.

[0057]

EXAMPLES The present invention will now be described in more detail 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 antifoaming agent (Adecan LG294) 0.02%, pH 7.0. Naruto medium 1
00 ml was dispensed and sterilized (120 ° C, 20 minutes) 5
A 00 ml Sakaguchi flask was inoculated with Streptomyces nayagawa Endis R-19 and shake-cultured at 28 ° C. for 72 hours. The L-fucose dehydrogenase activity in this culture was 0.58 units / ml. Example 2 Medium 1 consisting of 0.3% L-fucose, 0.4% peptone, 0.2% yeast extract, 0.2% meat extract, 0.02% defoamer (Adecanol LG294), pH 7.0. , 500ml into a 5L Erlenmeyer flask, 120 ℃
After sterilizing for 20 minutes, this medium is inoculated with Streptomyces nayagawaengdis R-19 at 28 ° C. 28 ° C
After 72 hours of shaking culture, this culture solution was added to a jar fermenter which had been sterilized with 20 l of a medium having the same composition as above to carry out main culture. Culturing conditions were 28 ° C., stirring frequency 150 rpm, aeration 20 l / min, and after culturing for 70 hours, the culture solution was centrifuged to collect bacterial cells. 70 g of the obtained cells
(Wet cell weight) 10 mM phosphate buffer (pH 7.5)
The cells were suspended in 1.5 l and the suspension was disrupted by an ultrasonic disrupter. The disrupted liquid was centrifuged using a centrifuge to obtain a supernatant liquid. 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) in which this supernatant was equilibrated with a 20 mM phosphate buffer (pH 7.5) in advance.
X 40 cm). After washing the column with 10 l of a phosphate buffer (pH 7.5) containing 0.1 M sodium chloride, the column was adsorbed with a linear concentration gradient (total volume: 10 l) of 0.1 M to 0.4 M. Elute the protein, L
-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.

This active fraction was desalted and concentrated using an ultrafiltration device, ammonium sulfate was added to 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.

This supernatant solution was preliminarily equilibrated with a 20 mM phosphate buffer solution (pH 7.5) containing 25% ammonium sulfate, and the column (φ3.5 ×) was packed with Butyl Toyopearl 650M (trade name: manufactured by Tosoh Corporation). 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.

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

Ammonium sulfate was added to this solution at 25% and then equilibrated with 20 mM phosphate buffer (pH 7.5) containing 25% ammonium sulfate in advance. Butyl Toyopearl 650M (trade name: manufactured by Tosoh Corporation) ) Was packed 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 thus obtained enzyme was examined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. As a result, only one band was observed, which confirmed that the enzyme was pure L-fucose dehydrogenase. It was

Example 3 The obtained purified enzyme was treated with 10 mM phosphate buffer (pH 7.
Using the enzyme preparation appropriately diluted by 5), the substrate specificity of this enzyme, optimum pH, pH stability, optimum temperature,
The temperature stability was investigated.

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

[Optimal pH] 0.2 M buffer solutions (pH
5.0-6.5: citrate buffer; pH 6.0-8.
0: phosphate buffer; pH 7.5-8.5: Tris-hydrochloric acid buffer; pH 8.5-11.5: glycine-caustic soda buffer) 0.5 ml, 50 mM L-fucose solution 0.2
0.1 ml (0.01 unit) of the enzyme preparation was added to the reaction mixture consisting of 0.2 ml of 1 mM NAD ⁺ solution, and
The reaction was carried out at 7 ° 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 (Re
1) 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 up to 10.0.

[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 enzyme preparation (0.4 unit),
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 enzyme activity was calculated based on the increase in NADH absorption at 340 nm by mixing with 0.075 ml of ion-exchanged water and reacting at 37 ° C. for 2 minutes. After the above operation, the relative activity was calculated with the maximum enzyme activity value as 100% and graphed to obtain FIG. As is clear from FIG. 2, the pH of this enzyme is
It is stable in the range of 6.0 to 10.5.

[Optimum temperature] 0.5 ml of 0.2 M Tris-hydrochloric acid buffer (pH 9.0), 0.2 ml of 50 mM L-fucose solution, 0.2 ml of 1 mM NAD ⁺ solution, and 0.1 ml of enzyme preparation. (0.01 unit) is added to add 25, 30, 35, 37, 40, 45, 50, 5
At each temperature of 5, 60, 65, and 70 ° C., the reaction was carried out 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 maximum enzyme activity value as 100% and graphed to obtain FIG. From Fig. 3, the optimum temperature of this enzyme is 50 ° C.
It can be seen that it is.

[Temperature stability] 10 mM phosphate buffer (p
0.2 ml (0.04 unit) of enzyme preparation diluted with H7.5) was added to 25, 30, 35, 40, 45, 50, 5
It processed at each temperature of 5,60 degreeC for 10 minutes. 0.05 ml of this enzyme solution was added to 0.2 M Tris-HCl buffer (pH 9.
0) 0.5 ml, 50 mM L-fucose solution 0.2 m
l, 1 mM NAD ⁺ solution 0.2 ml, deionized water 0.
Mix with 05 ml, react at 37 ° C for 2 minutes, 340n
Enzyme activity was determined based on the increased absorption of NADH in m. After the above operation, the maximum enzyme activity value is 100
The relative activity value in% was calculated and graphed to obtain FIG. As is clear from FIG. 4, this enzyme is stable at temperatures up to 50 ° C.

Example 4 The concentration of L-fucose in a sample solution was quantified 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) Assay method Mixture 0 of the above reagents 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. Similarly, instead of the sample solution, the same amount of water was added and reacted to measure the absorbance. The absorbance of the sample solution was calculated by subtracting the absorbance when water was added from the absorbance when the sample solution was added.

Separately, instead of the sample solution, L- of a known concentration was used.
The fucose solution was added to the mixed solution 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 was possible to accurately quantify.

[Brief description of 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 diagram showing pH stability of the enzyme.

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

FIG. 4 is a view showing 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)

[Claims] 1. 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 is least specific 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 to 10.5. Molecular weight: 65,000 ± 5,000 Molecular weight of subunit and its number: Molecular weight 37.0
It consists of two subunits of 00 ± 5,000. 2. A method for producing L-fucose dehydrogenase, which comprises culturing a microorganism belonging to the genus Streptomyces and capable of producing L-fucose dehydrogenase, and collecting L-fucose dehydrogenase from the culture. 3. A reduced nicotinamide adenine dinucleotide produced by reacting a sample containing L-fucose with L-fucose dehydrogenase derived from a microorganism belonging to the genus Streptomyces in the presence of oxidized nicotinamide adenine dinucleotide. A method for quantifying L-fucose, which comprises quantifying