JP3003009B2 - Mannose isomerase and method for producing mannose using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel mannose isomerase for interconverting D-mannose (hereinafter abbreviated as mannose) and D-fructose (hereinafter abbreviated as fructose), a process for producing the same, and a process for preparing fructose using the same. It relates to a method for producing mannose.

[0002]

BACKGROUND ART It has recently been revealed that mannose has a function of inhibiting the growth of intestinal harmful Salmonella [R. H. Brown, Foodstuff, June
12, 10 (1989)], and its use as a feed additive for poultry such as chickens and as a functional food material has been considered. However, mannose is usually very expensive because it is usually produced by decomposing mannan contained in plants such as wood and konjac.

[0003] The inventors of the present invention used mannose isomerase for interconverting mannose and fructose to produce mannose from abundant and inexpensively produced fructose, and a method for producing mannose isomerase and glucose isomerase (enzyme for interconverting glucose and fructose). The purpose of this study was to search for microorganisms that produce mannose isomerase, which can be used industrially, as a result of isolating from the soil and producing Pseudomonas sp. It was confirmed that the bacterium identified as a strain produced mannose isomerase with significantly higher thermostability than conventionally known mannose isomerase.

[0004] Mannose isomerase was introduced in 1956.
According to Palleroni and Doudroff et al., Pseudomonas saccharophila (Pseudomonas saccharophila) was used as the first enzyme of free hexose isomerase.
charophia] [J. Biol.
Chem. 218, 535 (1956)]. Then, the present inventors separated from the soil, and Xanthomonas rubrillinance (Xanthomonas rub).
urineeans) produces mannose isomerase [Japanese Journal of Agricultural Chemistry, 37
Vol. 524-528 (1963), Agric. Bi
ol. Chem. 28, pp. 601-604 (196
4)]. Also, Streptomyces aerocolorines
Genes) also confirmed the presence of the enzyme [Report from the Institute of Fermentation Research, Industrial Technology Institute, Vol. 28, pp. 89-94 (1966)].
However, each of these enzymes has an optimum temperature of 35 to
Since it is at 40 ° C. and has poor thermal stability, it cannot be used industrially.

[0005]

DISCLOSURE OF THE INVENTION The present inventors have conducted a search for microorganisms for the purpose of establishing a technique for industrially producing mannose from fructose-containing substances, and as a result, have been isolated from soil. Mannose isomerase produced by a bacterium identified as Pseudomonas has an optimum temperature of about 55 ° C., which is 15 to 20 ° C. higher than conventionally known mannose isomerase. In addition, it was found that the present enzyme has advantageous properties for industrial use, such that the reaction proceeds efficiently without inhibition even under a high concentration of the substrate of 20 to 30%. Such a mannose isomerase has not been known so far.
The present invention has been made based on such findings, and the present inventors have produced and purified mannose isomerase, and further established the use thereof to complete the present invention.

Hereinafter, the physicochemical properties of the mannose isomerase of the present invention will be described. (A) Action Isomerizes mannose to fructose and fructose to mannose. It also acts on D-lyxose,
-Isomerizes to xylulose.

(B) Substrate specificity It acts on D-mannose and D-lyxose, and acts on D-rhamnose, D-fucose, D-glucose, D-ribose, D-xylose, D-arabinose, L-xylose, L-xylose. It does not substantially act on arabinose, L-rhamnose or L-fucose.

(C) Working pH and Optimum pH The pH is in the range of about 6 to 11, and the optimum pH was found to be around 8 (3% at 50 ° C. under 0.1 M phosphate buffer).
0 minute reaction). The result is shown in FIG.

(D) Working temperature and optimum temperature The compound works up to about 70 ° C., but the optimum temperature was observed at about 55 ° C. [under 0.1 M mannose and 0.05 M Tris buffer (pH 7.0). For 30 minutes. Figure 2 shows the result.
Shown in

(E) Thermal stability 60 ° C. under 0.05M Tris buffer (pH 7.0)
The remaining activities when heated for 5, 10, 15, 20, and 30 minutes are about 90%, about 68%, about 52%, respectively.
About 42% and about 30%. The result is shown in FIG.

(F) pH stability After standing for 3 hours at room temperature (25 ° C.) in a 0.1 M buffer (acetic acid or phosphate buffer), the residual activity was measured. As a result, it was stable in the pH range of 6 to 9. The result is shown in FIG.

(G) Inhibitor 5 × 10 ⁻³ M HgCl ₂ , FeSO ₄ , AgNO ₃ , Al
Cl ₃ , p-chloromercurybenzoate (pCM
B).

Among the above physicochemical properties, the main properties are enzymes derived from Pseudomonas saccharophila (hereinafter referred to as enzyme A), enzymes derived from Xanthomonas rubrilianeans (hereinafter referred to as enzyme B) and strept. The properties of enzymes derived from Myces aerocolorigenes (hereinafter referred to as enzyme C) were compared, and the results were shown in Table 1.
Shown in

The activity was measured by the following method. 0.
2M mannose dissolved in 0.1M phosphate buffer (pH
7.0) An appropriate amount of enzyme is added to 0.5 ml, the total amount is adjusted to 1.0 ml with water, and the mixture is reacted at 50 ° C. Then, the generated fructose is quantified by the cysteine / carbazole method. Under these conditions, the amount of enzyme that produces 1 micromol of fructose per minute is defined as one unit.

[0015]

[Table 1]

The enzyme of the present invention has an optimum temperature as high as about 15 to 20 ° C. as compared to enzymes B and C, and a stable temperature of 10 ° C. or more. Thus, the thermal stability of the enzyme is clearly different from the conventionally known enzymes B and C. Further, when the enzyme of the present invention and the enzyme A of the same genus are compared, they differ in substrate specificity. That is, the enzyme of the present invention acts on D-mannose and D-lyxose, and the enzyme A acts on D-mannose, D-lyxose and D-rhamnose. In addition, the enzyme of the present invention is superior in the degree of inhibition by glucose as compared with the inhibition in the case of enzyme A, and therefore is practical in the production of mannose using mannose isomerase from isomerized saccharide, and It has excellent properties.

Further, in terms of the optimum pH and the inhibitor, there is a difference between the mannose isomerase of the present invention and the known enzymes A, B and C.

As described above, the enzyme of the present invention is a novel mannose isomerase completely different from conventionally known enzymes.

Examples of the microorganism that produces the mannose isomerase of the present invention include Pseudomonas sp. AM-9582 (Pseudomonas sp.)
p. AM-9582) or a mutant thereof can be advantageously used in the present invention.

Pseudomonas sp. AM-958
2 (Pseudomonus sp. AM-958)
2) is newly discovered and isolated from soil, and its bacteriological properties are as follows. This bacterium has been deposited with the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology as No. 3207 of Microbial Research Laboratories.

(1) Morphology Shape and size of cells: 0.6-0.8 × 1.0-2.
0μc bacillus Polyplasia of cells: not observed Motility: motility with two or more polar flagella Spores: no formation Gram stain: negative Acid-fast: negative

(2) Growth state in each medium Broth agar plate culture: Colonies are cultivated at 37 ° C. for 48 hours and have a circular shape with a diameter of 0.5 to 0.8 mm, a smooth surface, semi-lens-like bulges, and a full edge shape. Yellow-yellowish brown, translucent, resinous glossy broth agar slant culture: good growth, straight swelling, growth, yellowish-brown, metallic-like gloss, green-brown water-soluble pigment diffuses into agar under slope Broth liquid culture: litmus milk culture that does not form a pellicle, is moderately turbid and yellowish:
Liquefies but does not solidify

(3) Physiological properties Reduction of nitrate: negative Gas from nitrate: negative MR test: negative VP test: negative Production of indole: negative Production of hydrogen sulfide: negative Hydrolysis of starch: negative Use of citric acid: Used both in Simmons and Christen's medium Pigment formation: No formation of pigment in King A, King B medium Urease: negative Oxidase: negative (if any) Catalase: positive OF test: oxidized arginine hydrolase: positive acyl Amidase: Positive amino acid decarboxylase (lysine, ornithine): Negative DNase: Negative Growth temperature range: Does not grow above 41 ° C Attitude to oxygen: Aerobic Growth to salt: 0-4 (%) Acid production from saccharides Presence / absence: galactose + glucose + saccharose + Trehalose + L-arabinose + cellobiose + xylose + lactose + maltose + mannose + mannitol + fructose + salicin + starch-Degradation of gelatin (Fraizer method): positive TSI agar medium (slope acid):-(high acid): -SS agar: does not grow NAC agar: does not grow MacConky agar: grows Triphenyl tetrozolium (0.1%) agar:
Growing 10% lactose produces acid: positive Esculin degradation: positive (weak) phenylalanine deaminase: negative Tween 80 degradation: positive 0.1% methylene blue milk reaction: reduced liquefaction ONPG reaction: positive Levan production: negative mucoid Outbreak of settlement: Negative

Regarding the above mycological properties, Berge
y's Mannualof Systematic
Bacteriology, Vol. 2, (1986), the properties of which are compared, and the bacterium is a straight rod bacterium that produces yellow pigment and has polar flagella, and is negative for reduction of nitrate, negative for oxidase, positive for degradation of Tween 80, and resistant to salt. Low,
Since the produced pigment was water-soluble, this bacterium was recognized as a bacterium belonging to the genus Pseudomonas.
This bacterium also has physiological characteristics such as negative for levan production ability, positive for arginine hydrolase, negative for phenylalanine deaminase, negative for starch degradation, and positive for acylamidase.
Further, the bacterial width is 0.3 to 0.6 μm. Based on the above, the strain was determined to be a new strain, and Pseudomonas sp. AM-
9582.

To produce mannose isomerase by culturing this bacterium, meat extract, peptone,
Organic nitrogen sources usually used for culturing microorganisms, such as corn steep liquor and casein, and inorganic nitrogen sources such as ammonium chloride and ammonium sulfate are used.
Also, as carbon sources, the substrates mannose and fructose are good carbon sources, but they do not necessarily need to be these sugars and are inexpensive, sucrose, isomerized sugar, glucose, maltose, lactose, galactose, sorbitol, In addition to various sugars and sugar alcohols such as mannitol and glycerin, starch, dexothrin and the like can also be used. In addition to the nitrogen source and the carbon source, various metal salts such as phosphates, magnesium salts, and copper salts are added as supplementing medium components.

The cultivation is carried out at pH 5 to 9, preferably at pH 6 to
8. The reaction is performed at a temperature of 25 to 50 ° C, preferably 30 ° C, for about 2 to 4 days.

Since mannose isomerase is an enzyme produced in the cells, after culturing, the cells are collected by filtration or centrifugation and used as they are or after being subjected to an appropriate immobilization treatment. The enzyme is extracted and concentrated by digestion. If necessary, precipitate with ammonium sulfate, acetone, methanol, ethanol, etc., and store by drying.

The present enzyme can be purified electrophoretically to a single enzyme by a conventional enzyme purification method. For example, the cells are extracted with cetyltrimethylammonium bromide, etc.
It can be purified by centrifugation, salting out, DEAE-Sepharose column chromatography, Sephadex G-150 column chromatography, or the like.

The reaction for producing mannose from fructose using the present enzyme is carried out under the conditions of 10 to 50% fructose or a fructose-containing solution, at pH 6 to 8, at a temperature of 40 to 60 ° C.
Done in Analysis of the sugar composition after the reaction was performed by high performance liquid chromatography. The ratio of mannose to fructose at equilibrium was about 25:75.

Hereinafter, the present invention will be described in detail with reference to examples.

EXAMPLES Example 1 1% fish meat extract, 1% carbon source listed in Table ₂ , K ₂ HPO ₄
0.2%, MgSO ₄ · 7H ₂ put O consisting of 0.05% medium (pH 7.0) 4 ml test tube diameter 18 mm, sterilized by a conventional method, Pseudomonas sp A
Inoculated with M-9582 (Microtechnical Laboratories No. 3207)
Shaking culture was performed at 30 ° C. for 2 days. After the cultivation, pH 8.0, 3,
After treatment at 0 ° C. for 6 hours, the enzyme was extracted by centrifugation. Mannose isomerase produced from the enzyme solution thus obtained was measured. Table 2 shows the obtained results.

[0031]

[Table 2]

As is evident from Table 2, mannose or fructose, which is a substrate of mannose isomerase, is a good carbon source, but mannose isomerase can be produced using various carbon sources such as inexpensive sucrose and glucose. all right.

Further, the obtained supernatant is salted out with 60% ammonium sulfate to obtain a precipitate. Further dialysis was performed, and gel filtration using DEAE Sepharose column chromatography and Sephadex G150 was performed three times. The resulting mannose isomerase is electrophoretically single and has a specific activity of about 290.
Purified 2-fold.

Example 2 In Example 1, Pseudomonas sp. AM-9582 was added to a medium containing 1.5% of sucrose, fructose, glucose or a mixture thereof as a medium carbon source.
(Microtechnical Laboratory No. 3207) and cultured with shaking at 30 ° C. for 2 days. After the culture, the produced mannose isomerase was measured according to Example 1. Table 3 shows the results.

[0035]

[Table 3]

As is clear from Table 3, the best results were obtained when a mixture of fructose and glucose, particularly a carbon source having a composition of about 80% fructose and about 20% glucose, was used as the culture carbon source. .

Example 3 Fish Extract 2.5%, Glucose 2.5%, Fructose 1.25%, K ₂ HPO ₄ 0.15%, MgSO ₄ .7
A medium consisting of 0.05% H ₂ O and CuSO ₄
Of Pseudomonas sp. AM-9 in the same manner as in Example 1 for a medium containing 2 × 10 ⁻⁴ to 8 × 10 ^−4.
582 (Microtechnical Laboratories No. 3207) at 30 ° C
For 24 hours, and the produced mannose isomerase was measured. Table 4 shows the obtained results.

[0038]

[Table 4]

As is clear from Table 4, the addition of copper ions significantly increased the production of mannose isomerase.

Example 4 Cells obtained by culturing in accordance with Example 3 were treated with 20KC ultrasonic waves for 1 minute to use an enzyme solution (0.42 units / ml) from which mannose isomerase was extracted. Production test.

Fructose was added to the mannose isomerase (0.30 units) in each of 50, 100, 200, 3
00 mg, and the total amount was adjusted to 1.0 ml with water.
The reaction was performed at 0.45 ° C. A certain amount was taken over time, and the amount of produced mannose and residual fructose was quantified by high performance liquid chromatography. The results obtained are shown in Table 5 and FIG.

[0042]

[Table 5]

As is clear from Table 5, the present enzyme efficiently produced mannose even under a high concentration of substrate, and finally obtained mannose in a yield of 24 to 26%.

Example 5 Fish meat extract 2.5%, glucose 2.5%, fructose 1.25%, K ₂ HPO ₄ 0.15%, MgSO ₄
・ 7H ₂ O 0.05%, CuSO ₄ 6 × 10 ^-4 M
Pseudomonas SP AM
-9582 (Microbial Lab. No. 3207)
The cells were cultured at 0 ° C. for 24 hours to obtain 250 g of wet cells. Next, 100 g of the obtained wet cells was added to 30% fructose solution 1
L, and reacted at pH 7.0 and 45 ° C. for 16 hours. The activity of the wet cells was 7 units per gram of wet matter.

When the produced mannose was measured, 5
6.3 g. Conversion rate from fructose is 18.
8%.

Example 6 100 g of the wet cells obtained in Example 5 was added to 25 mM, pH
Suspended in 5 L of 7.0 phosphate buffer, and used as a carrier for immobilizing bacterial cells, "Celite Catalyst Carrie"
rs "No.R-620 (manufactured by U.S.A.)
S. A. ) Packed column (diameter 5cm, height 30c)
Under m), the solution was circulated from below at a flow rate of 2.5 L / hour for 7 hours. Thereafter, 25 mM, pH 7.0 phosphate buffer was added to 2.
The solution was passed for 10 hours at a flow rate of 5 L / hour, and the cells not adsorbed on the immobilization carrier were washed out to prepare an immobilized cell column having mannose isomerase activity.

When a 30% fructose solution (pH 7.0, 45 ° C.) was continuously passed through the prepared column from below at a flow rate of 60 ml / hour (SV = 0.1), 68 mg of mannose per ml was continuously passed. Could be produced.

After reacting continuously for 7 days, 65 mg of mannose could be produced per ml.

[0049]

Industrial Applicability The mannose isomerase of the present invention is thermostable and has an excellent property that the reaction proceeds efficiently without being inhibited even in the presence of a high concentration of substrate of 20 to 30%. For the first time, mannose can be produced abundantly and industrially at low cost.

[Brief description of the drawings]

FIG. 1 shows the optimum pH of the mannose isomerase of the present invention.

FIG. 2 shows the optimum temperature of the mannose isomerase of the present invention.

FIG. 3 shows the thermostability of the mannose isomerase of the present invention.

FIG. 4 shows the pH stability of the mannose isomerase of the present invention.

FIG. 5 shows the amount of mannose produced when the present enzyme was allowed to act on fructose. In the figure,-●-represents 5% fructose,-○-represents 10%,-■-
Indicates the result at 20% and-□-indicates the result at 30%.

Claims (5)

(57) [Claims] 1. A mannose isomerase having the following physicochemical properties: (A) Action Isomerizes mannose to fructose and fructose to mannose. (B) Substrate specificity It acts on D-mannose and D-lyxose, and acts on D-rhamnose, D-fucose, D-glucose, D-ribose, D-xylose, D-arabinose, L-xylose, L-arabinose, L -Has substantially no effect on rhamnose or L-fucose. (C) Optimum pH Near pH 8 at 50 ° C reaction. (D) Optimum temperature: pH 7.0, around 55 ° C for 30 minutes reaction. (E) Thermal stability pH 7.0, around 55 ° C. in 10 minutes. (F) pH stability A pH of about 6 to 9 at 25 ° C. for 3 hours. (G) Inhibition Inhibited by mercury ions, iron ions, silver ions, aluminum ions and p-chloromercurybenzoate. 2. A microorganism belonging to the genus Pseudomonas, which produces the mannose isomerase according to claim 1, which is cultured.
A method for producing mannose isomerase, comprising producing the mannose isomerase and collecting the mannose isomerase. 3. A method for producing mannose, wherein a mannose isomerase having the following physicochemical properties is allowed to act on a fructose-containing solution, and the resulting mannose-containing solution is collected. (A) Action Isomerizes mannose to fructose and fructose to mannose. (B) Substrate specificity: acting on D-mannose and D-lyxose; D-rhamnose, D-fucose, D-glucose, D-ribose, D-xylose, D-arabinose, L-xylose, L-arabinose, L -Has substantially no effect on rhamnose or L-fucose. (C) Optimum pH Near pH 8 at 50 ° C reaction. (D) Optimum temperature: pH 7.0, around 55 ° C for 30 minutes reaction. (E) Thermal stability pH 7.0, around 55 ° C. in 10 minutes. (F) pH stability A pH of about 6 to 9 at 25 ° C. for 3 hours. (G) Inhibition Inhibited by mercury ions, iron ions, silver ions, aluminum ions and p-chloromercurybenzoate. 4. A mannose obtained by culturing, collecting and immobilizing a microorganism belonging to the genus Pseudomonas and having the ability to produce mannose isomerase according to claim 1, and then allowing the immobilized cells to act on a fructose-containing solution. A method for producing mannose, comprising collecting a contained solution. 5. Pseudomonas having the physiological properties of arginine hydrolase positive, phenylalanine deaminase negative, starch degradation negative, acyl amidase positive and levan production negative, and having the ability to selectively produce heat-resistant mannose isomerase at 55 ° C. for 10 minutes. SP AM95
82.