JPH04325089A - Production of fructkinase - Google Patents

Abstract

PURPOSE:To readily and efficiently obtain the title enzyme having high specificity to fructose, accurately measuring fructose by culturing a bacterium belonging to the genus Pseudomonas, capable of producing fructokinase and collecting a product from the culture mixture. CONSTITUTION:A Pseudomonas sp. No921 (FERM P-3,310), a bacterium belonging to the genus Pseudomonas, capable of producing fructokinase, is cultured in a medium, the culture solution is centrifuged to collect cells, the prepared cells are suspended in 20mM tris hydrochloric acid buffer solution (pH8.0), ground by an ultrasonic grinder, centrifuged, a supernatant liquid is collected, ammonium sulfate is dissolved in the supernatant liquid, formed precipitate is separated by centrifuging, dissolved in 20mM tris hydrochloric acid buffer solution (pH8.0), dialyzed, passed through an anion exchange column, an adsorbed component is eluted and concentrated to give efficiently the objective fructokinase having high specificity to fructose but not acting on other saccharides.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to a method for efficiently producing fructokinase, and more particularly, to a method for efficiently producing fructokinase with excellent properties using bacteria belonging to the genus Pseudomonas. .

0002

BACKGROUND OF THE INVENTION Fructose is a biochemically and physiologically important substance. For example, in the food field, fructose generally coexists with large amounts of glucose and is consumed in large quantities as fructose or invert sugar.In the agricultural field, fructose is used as an indicator of fruit sweetness and is used to evaluate product quality. It is considered an item. Furthermore, normally 0.06mM fructose is present in the serum of a healthy person. Normally, glucose is present in an amount 50 to 100 times that of fructose. Therefore, fructose is considered to be one of the important factors for understanding a person's health condition, for example, monitoring diabetes. Therefore, it is important to quantify fructose accurately.

[0003] Fructose has conventionally been used, for example, as “M
methods of enzymatic analysis
sis [Second Edition], Ver.
lag Chemie Weinheim Acade
micPress, Inc. , 1305-13
This method is based on the following principle:
(1) Through the action of HK (hexokinase), F6 is converted from D-fructose and ATP (adenosine triphosphate).
P (D-fructose 6-phosphate) and ADP (adenosine diphosphate) are produced. (2) The F6P in (1) above is converted to G6P (D-glucose hexaphosphate) by the action of PGI (phosphoglucose isomerase). (3) Due to the action of G6PDH (D-glucose 6-phosphodehydrogenase), the G6P and NAD of (2) above are
P+ (nicotinamide adenine dinucleotide phosphate) and D-glucono-δ-6-phosphate (D-gluc
ono-δ-lactone 6-phosphate)
, NADPH (reduced nicotinamide adenine dinucleotide phosphate) and H+ (hydrogen ion) are generated. (4) The amount of fructose in NAPDH in (3) above is determined by the absorbance at 340 nm, which is the maximum absorption wavelength of NADPH. Furthermore, fructokinase is known as a type of hexokinase. The enzyme that phosphorylates fructose is fructokinase or D-fructose 6-phosphotransferase (D-fructos).
e6-phosphotransferase), is an enzyme with enzyme number EC2.7.1.4, and is an enzyme with enzyme number EC2.7.1.4.
It is an enzyme that catalyzes the reaction that generates D-fructose 6-phosphate and ADP from D-fructose and ATP in the presence of g2+. Examples of fructokinase-producing bacteria include (1) Streptomyces viola
ceruber [Sabater, B. , Sebas
tian, J. & Asensio, C. (19
72) Biochem. Biophys. Acta,
284, 414-420], (2) Leucono
stoc mesenteroides [Anders
on, R. L. & Sapico, V. L. (1
975) Meth. Enzymol. 42, 39-
43], (3) Streptococcus fae
calis [Moore, L. D. &O'Kan
e, D. J. (1963) J. Bacteriol.
86, 766-772], (4) Aerobac
ter aerogenes [Kelker, N.
E. & Hanson, T. E. (1970) J.
Biol. Chem. 8, 2060-2065],
and (5) Echinococcus granu
losus [bean; Chen. M. & Whiste
ler, R. L. (1977) Adv. Carboh
tdr. Chem. & Biochem. 34,
285-343], etc. are known.

0004

[Problems to be solved by the invention] For example, in serum,
When quantifying fructose, a fructose sample generally contains a larger amount of glucose than fructose, as described above. In conventional techniques using commonly available fructokinase, the enzyme acts on glucose in addition to fructose as a substrate in the sample, and glucose is always present in an amount 50 to 100 times that of fructose, resulting in high blank measurements. Not only did this result in large errors, but it was also difficult to measure fructose in many cases. In addition, fructokinase obtained from the above-mentioned producing bacteria has poor performance, for example, Streptomycin
Fructokinase from ess violaceruber has poor stability and is completely inactivated in 2 weeks at 0 to 4°C.
Fructokinase from P. teriodes has low specificity for fructose and acts on mannose as well as fructose, so no one has yet been put to practical use. If it is highly specific for fructose and has no substantial effect on other sugars,
If a fructokinase with high phosphorylation activity and excellent properties such as various stability can be found and produced easily and efficiently, the use of such a fructokinase will enable the production of blanks without errors. This makes it possible to assemble a fructose measurement system that is difficult to use.

[0005]

[Means for Solving the Problems] The present inventors screened a wide range of natural organisms to find microorganisms that produce fructokinase with high substrate specificity for fructose and excellent stability. Pseudomonas sp. No. isolated from field soil. 921
The present invention was completed based on the discovery that the enzyme was producing an enzyme with the desired properties. That is, the present invention provides a method for producing fructokinase, which comprises collecting fructokinase from a culture obtained by culturing a fructokinase-producing bacterium belonging to the genus Pseudomonas. This fructokinase-producing bacterium belongs to the genus Pseudomonas, and includes, for example, No. 1 isolated by the present inventors. The 921 strain is an example of a strain most effectively used in the present invention. For the identification of this bacterial strain, the identification experiment was performed in accordance with the “Guidebook for Medical Bacteria Identification (2nd Edition)” 19.
74”, “Microbiological Meth
ods (Volume 3)”, the DNA GCmol
The percentage was measured by treating DNA purified by the phenol method with nuclease P1 and using HPLC using a DEAE column. For analysis of quinone, quinone was purified according to the "Bacterial Identification Method Accompanying New Taxonomy" and analyzed by HPLC. Except for the growth temperature test, culturing was carried out at 28-30°C. The experimental results are summarized in “Bergey’s Manual”.
of Determinative Bacteri
ology (8th edition), Bergey's Manual
of Systematic Bacteriolo
gy Vol. 1 (1984), same magazine, Vol. 2(1
Pseudomonas sp. No. 921 was identified by comparison with Pseudomonas sp.
No. 310 (FERM BP-3310) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry.

[0006] Pseudomonas sp. no. 92
It shows the mycological properties of 1. That is, 1) Characteristics of growth Grows well in a linear pattern on an ordinary agar slant medium. It is semi-glossy and has a grayish-white to pale ocher color. No soluble pigments are produced. The edges of the plain agar medium form smooth, round, flat colonies. It has a grayish-white to pale ocher color. No soluble pigments are produced. Liquid medium (peptone water) Growth is weak, but it is uniformly cloudy. Litmus milk medium unchanged. 2) DNA GC mol % 64.5 ± 1.0 mol % (HPLC analysis using DEAE column) 3) Main isoprenoid quinone Q8 4) Morphological characteristics A rod-shaped bacterium with rounded ends, straight or slightly curved.
Exercise with polar hair. Size is 0.5 x 1.5~2.0μm
and does not form spores. Sequences are single, sometimes double, and sometimes short chains. 5) Physiological/biochemical properties Gram staining
- KOH reaction
+ Acid-fast staining
−
Capsule formation
- OF test (Hugh
-Leifson) 0
OF test (NH4H2PO4 as N source)
0 Aerobic growth
+
Growth in anaerobic conditions
- Growth temperature 3
7℃ -
30℃
+
20℃
+
10℃
NT salt tolerance 0%
+
1.0%
(+)
3.0%
- Growth pH 4.7
+
9.0
+
10.0
- Gelatin degradation
+
Starch decomposition
＋ Casein decomposition
− (no growth) Aesculin decomposition
- Cellulose degradation
− Tyrosine degradation
−
Deconstructing Tween 80
− Arginine decomposition
+
catalase production
+ Oxidase production −
Lecithinase production
- Urease production (S
SR) -
Urease production (Chris.)
− Indole production
−
Hydrogen sulfide production (lead acetate pa
per) - acetoin production (K
2HPO4) −
Acetoin production (NaCl)
- MR test
−
Nitrate reduction test (gas production)
−
(Detection of No. 2 −) −
(Detection of No3 −) + Usability in Simmons medium Citrate
- malate
− Maleate salt
−
malonate
- propionate
−
Gluconate
- succinate

- Usability in Christensen medium
citrate
- malate

− Maleate salt
- malonate
- propionate
−
Gluconate
- succinate
−
Production of gas from glucose
− Production of acid from sugar Adonitol
+L(+
) − arabinose +
cellobiose
- dulcitol
− Mesoerythritol
− Fructose −
D-galactose
+ D-glucose +
glycerin
- inositol
+ Inulin
−
lactose
− Maltose
−
mannitol
+ Mannose
+ Melezitose
−
melibiose
− Raffinose
−
L(+)-rhamnose
+ D-ribose
+
Salicin
- L-sorbose +
Sorbitol
+ Starch
- saccharose
−
trehalose
+ D-xylose
+

0007
] Pseudomonas sp. No. The identification of 921 will be described. Namely: 1) Main characteristics: Gram-negative, rod-shaped bacteria that move using polar hairs. Oxidatively breaks down glucose to produce acid. No oxidase production. Catalase production. GCmol% of DNA is 64.5±1
．． 0%. The main quinone is Q8. 2) Identification No. 921 is an aerobic Gram-negative rod-shaped bacterium with motile (polar hairs), so it is classified as Pseudomonas genus of Pseudomonas family, Xanthomona
It belongs to either the genus S or the genus Gluconobacter. The identification table for each genus is listed. No.
921 Pseudomonas Xantho
monas Gluconobacter pigment production W W,G
, Y Y W
Oxidase −
+(-) −
− Catalase +
＋ ＋
+ Growth at pH 4.5
− − −
− Quinone
Q8 Q8,9,10
Q8 Q10 DNA
GCmol% of 64.5
58-70 63-71
56-64 W: White, G: Gray, Y: Yellow No. 921 is Pseudomonas due to its growth at pH 4.5, pigment production, and quinone type.
It was identified as belonging to the genus. GCmol% of DNA
Bacterial species with 64±5% were selected and their properties were compared, but there were no descriptions of bacterial species whose properties matched well, so it was not possible to determine the species. Therefore, No. 921, Pseudomonas
sp. No. It was named 921 and deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology.

[0008] A method for producing fructokinase will be explained. That is, in the present invention, first, a fructokinase-producing bacterium belonging to the genus Pseudomonas is cultured in an appropriate medium. A representative example of the fructokinase-producing bacteria is the Pseudomonas sp.
921 is mentioned. As a general property of bacteria, mycological properties can vary naturally or artificially. Therefore, the strain may be mutated naturally or by artificial mutagenesis means such as conventional ultraviolet irradiation, irradiation, or mutagenic agents such as N-methyl-N-nitro-N-nitrosoguanidine or ethyl methanesulfonate. All mutant strains or variants belonging to the genus Pseudomonas, including naturally occurring mutant strains, that have the ability to produce fructokinase in harvestable amounts can be used in the present invention (these can be used in the present invention). (Sometimes collectively referred to as this bacterium).

[0009] The above culture can be carried out under conditions generally used for culturing bacteria. As a medium,
A nutrient medium containing a carbon source that can be assimilated by the bacteria, a nitrogen source that can be digested, and, if necessary, an inorganic salt, is used. As carbon sources that can be assimilated by this bacterium, glucose, fructose, sucrose, mannose, etc. are used alone or in combination. As nitrogen sources that can be assimilated by this bacterium, peptone, meat extract, yeast extract, malt extract, etc. are used alone or in combination. In addition, inorganic salts, metal salts, etc. are used as necessary. It goes without saying that in addition to the above, carbon sources and nitrogen sources that can be assimilated by the bacteria can be used. Cultivation is usually carried out under aerobic conditions such as shaking or aerated agitation culture, and industrially, deep aeration agitation culture is preferred. The culture temperature can be changed as appropriate within the range in which the fructokinase-producing bacteria grow and produce this enzyme, but it is usually 15 to 15 minutes.
32°C, particularly around 28°C is preferred. Although the culture time varies depending on the culture conditions, the culture may be stopped at an appropriate time by determining the time when the enzyme reaches its maximum titer, and is usually about 1 to 2 days. When foaming occurs, an antifoaming agent such as silicone oil is used as appropriate. Fructokinase is
Generally, the bacteria obtained in this way are mainly contained within the bacterial bodies, so the bacteria are collected from the resulting culture solution by conventional methods such as filtration or centrifugation, and these bacteria are subjected to ultrasonic treatment and French A crude fructokinase-containing solution can be obtained by appropriately using various methods alone or in combination, such as mechanical destruction means such as press treatment and glass bead treatment, and enzymatic dissolution of lysozyme.

[0010] Next, purified fructokinase can be obtained from this fructokinase-containing solution by using known protein and enzyme purification means. For example, ammonium sulfate may be added to a crude fructokinase-containing solution to recover the enzyme by ammonium sulfate precipitation, and then, if necessary, the enzyme may be purified using an appropriate combination of molecular sieves and various chromatography methods. The purified enzyme thus obtained can be freeze-dried to obtain a fructokinase powder, with stabilizers such as sugars such as saccharose and mannitol, and proteins such as bovine serum albumin added as necessary.

The properties of the fructokinase thus obtained (hereinafter referred to as the present enzyme) are as follows. That is, (1) Enzyme action As shown in the following formula, in the presence of Mg2+, it catalyzes the reaction of producing D-fructose 6-phosphate and ADP from D-fructose and ATP.

(2) Substrate specificity fructose 100
% N-acetyl-D-glucosamine 0 Glucose 0
lactose
0 Sorbose
0 saccharose
0 galactose
0 maltose
0 xylose
0 Sorbitol 0
Mannose
0.1* 2-deoxy-D-ribose
0 2-deoxy-D-glucose 0*: This is the result of a reaction with a substrate (unidentified) that existed as an impurity in mannose without acting on mannose, or it showed a relative activity of 0.1% against mannose. Although it was not possible to determine whether the result was due to the quantification of fructose, it can be considered that it does not have a substantial effect on the determination of fructose.

(3) Molecular weight 75,000±8000 TSK gel G3000SW (0.75×6
0 cm). Eluent; 0.2M NaCl
Contains 0.1M phosphate buffer (pH 7.0). The following marker manufactured by Oriental Yeast Co., Ltd. was used as a standard protein. Molecular weight 12,400 Cytochrome C32,
000 Adenylate Kinase 67,000
Enolase 142,000 Lactate dehydrogenase 29
0,000 Glutamate dehydrogenase

00
14] (4) Isoelectric point 5.12 ± 0.52 After applying current at a constant voltage of 700 V for 40 hours at 4°C by focusing electrophoresis using carrier ampholite (pH 3.5 to 10), fractionation was carried out. The enzyme activity of each fraction was measured.

(5) Km value (Michaelis constant) 100m
M Tris-HCl buffer (pH 8.0) 30mM
D-Fructose (manufactured by Wako Pure Chemical Industries) 0.2%
Bovine serum albumin (Sigma) 5U/ml Diaphorase (Toyo Jozo) 5U/ml Phosphoglucose isomerase (Boehringer Mannheim Yamanouchi) 20U/ml Glucose 6-phosphate dehydrogenase (Toyo Jozo) 1mM NADP+ 5mM MgCl2 The Km value for ATP was measured by changing the concentration of ATP in a reaction solution containing 0.025% Nitrotetrazolium Blue (manufactured by Wako Pure Chemical Industries, Ltd.), and the result showed a value of 0.71 mM. On the other hand, 10mM ATP was added instead of 30mM fructose in the reaction solution, and the concentration of D-fructose was changed, and the Km value for D-fructose was measured, and the result showed 1.0mM.

(6) Thermostability The present enzyme solution (1.0 U/ml) was prepared with 20 mM Tris-HCl buffer (pH 8.0), and after heat treatment at each temperature for 15 minutes, the residual activity was determined using the enzyme described below. It was measured according to the activity assay method. The results are as shown in FIG. 1, and the enzyme activity was stable up to 40°C.

(7) Optimal temperature 100mM Tris-HCl buffer (pH 8.0), 30mM
D-fructose, 10mM ATP and 5mM
This enzyme (1 U/m
l) Add 10 μl, 30, 35, 40, 45, 50
and 55°C for 15 minutes, and immediately ultrafiltrated at 2°C using Amicon Reflow CF25 (manufactured by Amicon) to obtain a filtrate, and the D-fructose 6- Phosphoric acid was measured using the reaction solution described below. The results were as shown in Figure 2, with the highest activity at 45°. Reaction solution 100mM Tris-HCl buffer (pH
8.0) 0.2% Bovine serum albumin 5U/ml Diaphorase 5U/ml Phosphoglucose isomerase 20
U/ml glucose 6-phosphate dehydrogenase 1mM NADP+ and 0.025% nitrotetrazolium blue Add 0.05ml of filtrate to 1ml of the above reaction solution, react at 37°C for 5 minutes, then add 2ml of 0.1N hydrochloric acid The reaction was stopped, and the absorbance at 550 nm was measured.

(8) pH stability This enzyme solution (1.0 U/ml) was dissolved in 40 mM acetate buffer (
pH5.0-6.0 (represented by an open triangle), phosphate buffer (pH6.0-8.0 represented by an open circle), and Tris-HCl buffer (pH8.0-10.0 represented by a black circle) After each buffer solution was prepared and heat-treated at 40° C. for 15 minutes, the residual activity was measured according to the enzymatic activity measurement method described below. The results are shown in Figure 3, with pH 6.5
The activity was maintained at 95% or more within the range of ~8.0.

(9) Optimum pH Acetate buffer (pH, 5.0, 5.5, 6.0 indicated by open triangle), phosphate buffer (pH, 6.0, 6.5, 7)
．． 0,7.5,8.0 (indicated by open circles) and Tris-HCl buffer (pH, 8.0, 8.5, 9.0, 9.5)
This enzyme was added to a reaction solution prepared with each buffer solution (indicated by a black circle), and the reaction was allowed to proceed at 37°C. The reaction was stopped with 2 ml of 0.1N hydrochloric acid, and the absorbance at 550 nm was measured. The results are shown in Figure 4. As shown in , the activity was highest around pH 7.

(10) Effect of metal ions on the activity When enzyme activity was measured by adding hydrochloride of the divalent metal shown below instead of MgCl2 in the enzymatic activity measurement method described later, it was found that MnCl2 Other metal ions had no effect. Metal salt Relative activity none 0% MgCl2 100 BaCl2 0 CaCl2 0 MnCl2 8.2 ZnCl2 0 NiCl2 0 CuCl2 0 CoCl2 0

(11) Storage stability in aqueous solution 0
．． 40mM Tris-HCl buffer containing 05% NaN3 (
Prepare this enzyme solution (5 U/ml) at pH 8.0) and incubate at 5°C.
We investigated the storage stability of As shown in Figure 5, the results were 96% after 3 weeks and 9% after 6 weeks.
It maintained a high activity of 1%.

(12) Fructokinase activity measurement method 1. Reaction solution composition 100mM Tris-HCl buffer (pH 8.0) 5
mM MgCl2 30mM D-fructose (manufactured by Wako Pure Chemical Industries) 1
0mM ATP (manufactured by Oriental Yeast Co., Ltd.) 0.2
% Bovine serum albumin (manufactured by Sigma) 1mM
NADP+ (manufactured by Oriental Yeast Co., Ltd.) 5U/ml
Diaphorase (manufactured by Toyo Jozo Co., Ltd.) 5 U/ml Phosphoglucose isomerase (manufactured by Boehringer Mannheim Yamanouchi Co., Ltd.) 20 U/ml Glucose 6-phosphate dehydrogenase (manufactured by Toyo Jozo Co., Ltd.) 0.025% Nitrotetrazolium Blue (manufactured by Wako Pure Chemical Industries, Ltd.) 2 ．． Activity measurement method: Put 1 ml of the above reaction solution into a small test tube, incubate at 37°C for 2 minutes, and then add 0.0 mL of the appropriately diluted enzyme solution.
Add 02 ml to start the reaction. After incubating for exactly 10 minutes, the reaction is stopped by adding 2.0 ml of 0.1N hydrochloric acid, and the absorbance A1 is determined by measuring A550nm. At the same time, the absorbance A0 is determined by using the sample containing no enzyme as a blank. 3. Calculation formula U/ml = (A1-A0)/19.3 ×
1/10 × 3.02 /0.02 × Z
In the formula, 19.3; molecular extinction coefficient cm2/μmol,
Z; dilution ratio

[0023]

[Examples] Next, the present invention will be specifically explained with reference to Examples, but the present invention is not limited thereto. Example 1 [Pseudomonas sp. no. 921 culture]
Polypeptone 1%, yeast extract 0.5%, casamino acid 1
%, malt extract 1%, mannose 1%, KH2PO4
100 ml of liquid medium (pH 7.0) containing 0.3% and 0.05% MgSO4 7H2O was added to 500 ml
Dispense into 20 Erlenmeyer flasks, heat sterilize at 120°C for 20 minutes, and add Pseudomonas sp.
．． Inoculate 1 platinum loop of 921 bacteria and incubate for 120r at 28°C.
．． p. m. The cells were cultured in a shaking incubator for 40 hours to obtain 1.9 L of culture with an enzyme activity of 0.1 U/ml.

Example 2 [Separation and purification of enzyme] 1.9 L of the culture solution obtained in Example 1
The cells were collected by centrifugation, and the obtained cells were washed once with 1 L of 20 mM Tris-HCl buffer (pH 8.0). The washed bacterial cells were suspended in 20mM Tris-HCl buffer (pH 8.0) to make a total volume of 50ml, and then washed using a Kubota ultrasonic disruptor (
INSONATOR 201M) 180W,
The mixture was crushed for 10 minutes to obtain a crushed liquid. 15,000r. p. m. 42 ml (enzyme activity 3.5 U/ml) of supernatant was obtained by centrifugation for 20 minutes. 11 g of ammonium sulfate was dissolved in this supernatant, the resulting precipitate was removed by centrifugation, 8 g of ammonium sulfate was again dissolved in the resulting supernatant, the resulting precipitate was centrifuged, and the resulting precipitate was removed. Dissolved in 20 ml of 20 mM Tris-HCl buffer (pH 8.0), dialyzed overnight against 2 L of 20 mM Tris-HCl buffer (pH 8.0) using a dialysis tube, and dissolved in 25 m
1 of enzyme solution (enzyme activity 4.7 U/ml) was obtained. The obtained enzyme solution was passed through a 50 ml column of DEAE-Sepharose CL-6B (manufactured by Pharmacia) buffered with 20 mM Tris-HCl buffer (pH 8.0), and 20 mM Tris-HCl buffer (pH 8.0) containing 0.2 M potassium chloride was passed through the column. pH8.0) 1L
Then, elute with 20mM Tris-HCl buffer (pH 8.0) containing 0.3M KCl, and add 100ml of enzyme solution.
(enzyme activity 1.2 U/ml) was obtained. The obtained enzyme solution was dialyzed overnight against 5 L of 20 mM Tris-HCl buffer (pH 6.5). The enzyme solution obtained by dialysis was passed through a 5 ml column of Blue Sepharose CL-6B (manufactured by Pharmacia) buffered with 20 mM Tris-HCl buffer (pH 6.5), and 110 ml of the passed solution (enzyme activity 1.0 U/ml) was collected. I got it. There were no contaminating enzymes such as hexokinase, NADH oxidase, or lactate dehydrogenase in this pass-through. This passed liquid was concentrated using Amicon Reflow CF25 (manufactured by Amicon) to 2 ml.
(enzyme activity 54 U/ml) enzyme solution was obtained.

Example 3 Quantification of D-fructose using the present enzyme 100mM
Tris-HCl buffer (pH 8.0) 2U/ml Fructokinase (this enzyme obtained in Example 2) 5U/ml Phosphoglucose isomerase 20U/ml
ml glucose 6-phosphate dehydrogenase 5mM
ATP 1mM NADP + 5mM MgCl2 Prepare the above reaction solution, dispense 1ml each into small test tubes, incubate at 37°C for 2 minutes, then add 1, 2
A340nm of NADPH generated after adding 10 μl of each D-fructose solution prepared to , 3, 4, and 5 mM and incubating at 37°C for 10 minutes.
The absorbance was measured. At this time, a sample without D-fructose was measured as a blank, and the result of ΔA340nm after subtracting this blank is as shown in Figure 6, which shows a straight line passing through the origin, which is almost the theoretical amount of D- Fructose 6-phosphate was produced.

[0026]

[Effects of the Invention] The present invention facilitates the production of fructokinase, which has excellent properties such as high specificity for fructose, no substantial action on other sugars, high phosphorylation activity, and good stability. can be obtained efficiently. Also,
By using the fructokinase obtained in this way, it becomes possible to accurately and easily quantify fructose even in the presence of large amounts of other sugars, and the industrial value of the present invention is extremely high. .

[0027]

[Brief explanation of drawings]

FIG. 1 is a graph showing the thermostability of the present enzyme.

FIG. 2 is a graph showing the optimum temperature of this enzyme.

FIG. 3 is a graph showing the pH stability of this enzyme.

FIG. 4 is a graph showing the optimum pH of this enzyme.

FIG. 5 is a graph showing the storage stability of this enzyme in an aqueous solution.

FIG. 6 is a graph showing a calibration curve when quantifying D-fructose using this enzyme.

Claims (2)

[Claims] 1. A method for producing fructokinase, which comprises culturing a fructokinase-producing bacterium belonging to the genus Pseudomonas and collecting fructokinase from the resulting culture. 2. A fructokinase-producing bacterium belonging to the genus Pseudomonas is Pseudomonas sp.
Claim 1: 921 (FERM BP-3310)
Manufacturing method described.