JPH08280396A - Production of trehalose - Google Patents

Abstract

PURPOSE: To readily and efficiently produce trehalose useful for medicines, food preservatives, low-caloric sweeteners, etc., by reacting a microorganism, etc., belonging to the genus Pseudomonas and having the ability to convert maltose into trehalose. CONSTITUTION: A microbial cell or its treated substance of a microorganism, belonging to the genus Pseudomonas and having the ability to convert maltose into trehalose [e.g. Pseudomonas sp. F-1 (FERM P-14747)] is added to a 0.1M phosphoric acid buffer solution (pH 7.0) containing maltose in the presence of a suppressant suppressing interfering enzym contaminant (e.g. ethylenediaminetetraacetic acid), reacted at 37 deg.C for 24hr and then centrifuged. The resultant supernatant liquid is treated by preparative high-performance liquid chromatography to separate and purify the product. Thereby, the objective trehalose useful as a medicine, a diagnostic agent, an enzyme or a preservative for foods, a water holding agent for cosmetics, a low-calorie sweetener, etc., is readily and efficiently be obtained.

Description

Detailed Description of the Invention

[0001]

The present invention relates to trehalose (O-α
-D-glucopyranosyl- (1 → 1) -α-D-glucopyr
anoside), and more particularly to a method for producing trehalose from maltose using a microbial cell or a treated product of the microbial cell.

[0002]

2. Description of the Related Art Trehalose has two glucose molecules α
(1,1) Non-reducing disaccharide linked together in nature, widely used in insects, yeasts, molds, bacteria, basidiomycetes, red algae, lichens, plant mannan, rotifers, invertebrates, flowering plants, etc. It is distributed. Since this trehalose has a structure similar to that of overlapping water molecules, it acts as a substitute for water, which is indispensable for life in living organisms, and protects cells, proteins, nucleic acids, etc. from drying and freezing as a bioprotective function and natural preservative. It has a function. Therefore, trehalose can be used as a drug, a diagnostic agent, a hormone, a fertilized egg, a sperm, a physiologically active substance, an enzyme, a useful substance such as a microorganism or a food preservative, and can also be used as a water retention agent by being added to cosmetics. it can.

Further, trehalose does not produce insoluble glucan or acid due to dental caries bacteria such as Streptococcus mutans, and has antitussive properties such as suppressing production of insoluble glucan due to sucrose. Moreover, trehalose can be effectively used as a low-calorie sweetener because it has a lower sweetness than sugar and is hardly absorbed by the human body. Furthermore, trehalose has an action of suppressing the accumulation of cholesterol in serum and liver, and also has a physiologically active function as a growth factor of bifidobacteria in the intestine. Healthy food is expected.

Conventionally, as a method of producing trehalose, a method of extracting from natural products of baker's yeast, Nocadia (Noca)
rdia), a fermentation method using a microorganism belonging to the genus, a culturing method using a microorganism belonging to the genus Brevibacterium (JP-A-5-211882), the genus Micrococcus or the dynococcus (Deinococc).
us) Cultivation method using microorganisms belonging to the genus (Japanese Patent Laid-Open No. 6-319578), Lactobacillus brevis
evis) -derived maltose phosphorylase and Euglena gracilis-derived trehalose phosphorylase-derived method from maltose (JP-A-63-60998), α-glucose-1-phosphate and glucose Enzymatic method for producing trehalose by reacting trehalose phosphorylase
No. 189779) is known.

However, in the above-mentioned culture method, the amount of trehalose accumulated in the culture solution is as small as 2% or less, and in the above-mentioned enzyme method, it is difficult to supply a large amount of enzyme, and the enzyme activity lacks long-term stability. However, it was not suitable for continuous mass production of trehalose using the enzyme as a bioreactor.
In the invention described in JP-A-63-60998 using maltose phosphorylase and trehalose phosphorylase, trehalose is produced from maltose by using them as a bioreactor, but different microorganisms are used for each enzyme. However, there is a problem that it is very complicated.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for easily and efficiently producing trehalose, and further a method for inexpensively mass-producing trehalose by utilizing a bioreactor. .

[0007]

As a result of earnest research in view of the above problems, the present inventors have found that a bacterial cell or a treated product of a microorganism belonging to the genus Pseudomonas having an enzyme capable of converting maltose to trehalose in the bacterial cell. By using, it was found that trehalose can be easily and efficiently produced, and that by using the cells of the microorganism or a treated product of the microorganism as a bioreactor, trehalose can be mass-produced at low cost, and the present invention has been completed. .

[0008] That is, the present invention is a method for producing trehalose, which comprises reacting maltose with a microbial cell or a treated product of a microorganism belonging to the genus Pseudomonas and having the ability to convert maltose to trehalose. . Further, in the present invention, the microorganism is Pseudomonas sp. F-1.
(Pseudomonas sp. F-1), which is a method for producing trehalose. Furthermore, the present invention is a method for producing trehalose, characterized in that the microbial cells or the treated product of the microbial cells are allowed to act on maltose in the presence of a contaminant-interfering enzyme inhibitor.

The present invention will be described in detail below. The microorganism used in the present invention may be any strain as long as it belongs to the genus Pseudomonas and has the ability to convert maltose to trehalose. Such strains may be known strains or may be strains newly isolated from soil, seawater, etc., and further improved trehalose synthesis ability by subjecting these strains to mutation treatment or the like. It may be a mutant strain. Specific examples of preferable strains include Pseudomonas sp. F-
1) is mentioned.

Pseudomonas sp. F-1
sp. F-1) is a strain having the ability to synthesize trehalose, which was newly isolated from the soil by the present inventors. Pseudomonas
An example of the screening method for sp. F-1 (Pseudomonas sp. F-1) is shown below. A soil sample containing the strain is suspended in sterilized water, appropriately diluted, and then smeared on a glucose agar plate medium (glucose 0.5%, peptone 0.5%, yeast extract 0.3% and agar 1.5%). This medium is kept at about 30 ° C. for about 24 hours, and the colonies that grow well are picked up on an agar plate medium and isolated. Then, the grown bacteria are shake-cultured for about 20 hours in a liquid medium (glucose 1%, Nutrient Broth (manufactured by DIFCO) 4%), and the culture solution is centrifuged to collect the bacterial cells. About 1/20 volume of toluene is added to the cells and shaken vigorously for about 15 seconds.

Maltose is added to the treated cells, reacted at about 37 ° C. for about 20 hours, and then centrifuged. Then, a strain can be selected by high performance liquid chromatography in which a peak whose retention time coincides with that of standard trehalose can be confirmed. The mycological properties of Pseudomonas sp. F-1 thus obtained are shown in Tables 1 to 3.

[0012]

[Table 1]

[0013]

[Table 2]

[0014]

[Table 3]

The strain F-1 having the above-mentioned mycological properties is
Bergey's Manual of Systematic Bacter
As a result of a search according to Volume 2, 1986, this strain was identified as a strain belonging to the genus Pseudomonas. This Pseudomonas sp. F-1 (Pseu
domonas sp. F-1) was established on January 30, 1995 by FERM P-147 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry.
Deposited as 47.

The medium used for culturing the above microorganisms is
Any carbon source can be used, and as the carbon source, glucose, fructose, maltose, starch, crude sugar, molasses, etc. can be used alone or in combination. As the nitrogen source, in addition to organic nitrogen sources such as peptone, meat extract, yeast extract, corn steep liquor and urea, sulfuric acid,
Inorganic nitrogen sources such as nitric acid and ammonium salts such as phosphoric acid can be used alone or in combination. As the inorganic salts, sulfates such as potassium, sodium, calcium, magnesium, manganese, and iron, hydrochlorides, carbonates, nitrates, phosphates and the like can be used alone or in combination.

The culture can be carried out under aeration conditions using a shaking culture or a jar fermenter. Medium pH
Is preferably in the range of 5 to 8, the culturing temperature is preferably in the range of 20 to 35 ° C., and the culturing time is preferably 1 to 3 days. The bacterial cells may be recovered from the culture medium after completion of the culture by filtration separation such as centrifugation.

The above-mentioned microbial cells obtained by culturing are used as they are as live cells, lyophilized or treated with toluene, acetone or the like, and further known immobilization methods such as inclusion method and carrier. It may be immobilized by a binding method, a cross-linking method or the like and used as an immobilized cell. Examples of the encapsulation method include a method using a natural polymer such as carrageenan and alginic acid, or a synthetic polymer including a monomer and a prepolymer. As a carrier binding method, there is a method of adsorbing it on chitosan or the like. Glutaraldehyde or the like can be used for the crosslinking method.

The cells are crushed by a known cell crushing method, for example, ultrasonic crushing method, French press crushing method, glass bead crushing method, dynomill crushing method, etc. to extract the enzyme,
The extract can be used as a crude enzyme solution, immobilized as in the case of bacterial cells, as an immobilized enzyme. The crude enzyme solution may be purified by a combination of salting-out by ammonium sulfate precipitation, concentration by ultrafiltration membrane, ion exchange chromatography, hydrophobic interaction chromatography or gel filtration chromatography to be used as a purified enzyme. it can. In the present invention, the treated microbial cell refers to a crushed product of the microbial cell as described above, a ground product, an immobilized microbial cell, or a crude or purified enzyme isolated from them, an immobilized enzyme having those enzymes immobilized, and the like. Shall be said.

In the present invention, trehalose is produced by allowing the above-mentioned bacterial cells or a treated product of bacterial cells to act on maltose. What is important in the present invention is that, unlike the method of extracting and purifying trehalose from the culture solution or from the inside of the microbial cells, maltose as a substrate is directly reacted with the microbial cells or a treated product of the microbial cells to convert to trehalose. .
Since most of the components in the reaction solution are sugars and do not contain other organic substances, etc., separation and purification of trehalose from the reaction solution is very easy as compared with the prior art.

Further, since its enzymatic activity is high, trehalose can be efficiently produced, and one kind of microorganisms for maltose phosphorylase and another kind of microorganisms for trehalose phosphorylase as in the conventional enzymatic method. Since trehalose can be easily produced because there is no need to use the above method.

The concentration of maltose as a substrate in this reaction is preferably 10 mM to 2M. The reaction temperature is 30
The temperature is preferably -60 ° C and the pH is preferably 5-9, and the reaction is preferably carried out for 1-200 hours. To keep the pH constant, it is preferable to use a phosphate buffer. When it is used as immobilized cells or immobilized enzyme, it can be carried out by a continuous method. For example, they are packed in a column and used as a bioreactor under continuous reaction conditions of 1 month to 1 year under the reaction conditions similar to those of the batch system. By doing so, trehalose can be continuously mass-produced at low cost.

In the present invention, the enzyme activity for converting maltose to trehalose can be improved by adding a contaminant-interfering enzyme inhibitor to the reaction solution. As a contaminant inhibitor, EDTA (Ethylenediaminet)
chelating agents such as etraacetic acid), especially E
It is preferable to use DTA. The amount added is 0.
1-10 mM is preferable.

After completion of the reaction, the bacterial cells or the treated product of bacterial cells are removed from the reaction solution by centrifugation or filtration separation such as ultrafiltration. A trehalose fraction is collected from the obtained supernatant or filtrate using activated carbon or ion exchange resin chromatography. Ion exchange resin chromatography is based on Na type or Ca
It is preferably carried out by the simulated moving bed method using a cation exchange resin of the type. Next, the obtained fraction is concentrated and trehalose is crystallized by a crystallization method with ethanol or the like. If necessary, recrystallization can be performed for purification. In the present invention, by immobilizing bacterial cells or crude or purified enzyme on a carrier, the immobilized bacterial cells or immobilized enzyme can be repeatedly used, and as a result, trehalose can be continuously produced in large amounts from maltose. it can.

[0025]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

(Example 1) Isolation of strains 1 g of a soil sample collected from soil in Shizuoka prefecture was suspended in 10 ml of sterilized water and diluted appropriately, and 0.1 ml of the suspension was diluted with glucose agar plate medium (glucose 0.5%, peptone 0.5%). , Yeast extract 0.3% and agar 1.5%). This plate medium was kept warm in an incubator at 30 ° C. for 24 hours, and the colonies that grew well in the colony were picked up on an agar plate medium and isolated. Next, 1 platinum loop of the grown bacteria was added to 10 ml of liquid medium (glucose 1%, nutrient broth (manufactured by DIFCO) 4
%) Was inoculated into a 50 ml large-sized test tube, shake culture was carried out at 30 ° C. for 20 hours, and the culture solution was centrifuged to recover bacterial cells. 1/20 volume of toluene was added to the cells and shaken vigorously for 15 seconds.

To the treated cells, 1 ml of 50 mM maltose was added, reacted at 37 ° C. for 20 hours and then centrifuged, and the supernatant was subjected to high performance liquid chromatography (column; YMC-03 (Yamamura Chemical Co., Ltd.) for elution. Liquid; acetonitrile: water = 77: 23,
Flow rate: 1 ml / min, temperature: 30 ° C, detector: differential refractometer)
Was analyzed. As a result of the analysis, a strain in which a peak whose retention time coincided with that of the standard (trehalose manufactured by Wako Pure Chemical Industries, Ltd.) was confirmed was selected. The present inventors have designated this strain as Pseudomonas sp. F-1.

Production of trehalose by freeze-dried cells Pseudomonas sp. F-1
Was cultured in a 5 liter Jafamentor containing 3 liters of liquid medium (1% glucose, 4% Nutrient Broth (DIFCO)) at 30 ° C. for 20 hours. After culturing,
The operation of collecting the bacterial cells by centrifugation, suspending the bacterial cells in distilled water, and collecting the bacterial cells by centrifugation was repeated twice to wash the bacterial cells. The collected bacterial cells were freeze-dried to obtain powder bacterial cells.

As a reaction liquid serving as a substrate, 100 mM maltose (0.1 M KH ₂ PO ₄ -K ₂ HPO ₄ pH 7.0) was used. Powdered bacterial cells were added to this reaction solution, and after reacting at 37 ° C for 24 hours,
High-performance liquid chromatography (preparative column; Asahi Pack NH2 (manufactured by Asahi Kasei Corp.), eluent; acetonitrile: water: phosphoric acid = 80: 17: from the supernatant obtained by centrifugation.
3. The trehalose fraction was collected using a column temperature of 40 ° C. and a flow rate of 1 ml / min.

As a result of analyzing this trehalose fraction with a nuclear magnetic resonance apparatus and a mass spectrometer, it was in agreement with the spectrum of trehalose (manufactured by Wako Pure Chemical Industries, Ltd.). In addition, microorganisms (extracellular trehalase-producing bacteria Chaetomium aureu
When reacted with trehalase separated and purified from m), production of glucose could be confirmed. From this result, the substance produced from maltose by this microorganism was trehalose (O-α-D-glucopyranosyl- (1 → 1) -α-
D-glucopyranoside). Also,
When the amount of trehalose in the reaction solution in Example 1 was measured, the reaction proceeded to 60 mM trehalose. From this, the conversion rate from maltose to trehalose was calculated to be 60%.

(Example 2) Production of trehalose using immobilized cells Pseudomonas sp. F-1 (Pseudo prepared in Example 1)
2 g of frozen cells of monas sp. F-1) was dissolved in a 0.9% NaCl solution to 8 ml and heated at 45 ° C. To this, 45 ℃ in advance
Carrageenan solution warmed to (2 g of carrageenan 45 g
Dissolved in 0.3 M KCl) and mixed well, then cooled in a water bath. After cooling, 500 ml of 0.3M KCl was added and left overnight at 4 ° C. 3 mm of immobilized gel cells
It was cut into horns and washed with 500 ml of 0.3 M KCl and 1000 ml of 0.1 M phosphate buffer pH 7.0 to obtain 39.6 g of immobilized microbial cells.

To 8 g of the immobilized cells thus obtained, 6.25 ml of 1M
A maltose solution (containing 0.1 M phosphate buffer pH 7.0) was added and a shaking reaction was performed at 45 ° C. After the reaction was completed, the supernatant was quantified by high performance liquid chromatography. As a result, it was found that 200 mM trehalose with a conversion of about 50% was produced in 7 days.

(Example 3) Production of trehalose by trehalose synthesis crude enzyme Pseudomonas sp. F-1 (Pseudomonas sp.
After thoroughly washing the cells of (F-1) with distilled water, add 5 ml of cells to 30 ml.
0.05 M phosphate buffer (pH 7.0) was added to the cells, and the cells were disrupted with an ultrasonic disrupter in ice. The precipitate was removed from the crushed solution by centrifugation to obtain 28 ml of a crude enzyme as a supernatant. 13.5 g of ammonium sulfate was gradually added to this crude enzyme solution,
It was left overnight at 4 ° C. The crude enzyme solution precipitated with ammonium sulfate was recovered by centrifugation, then redissolved in 0.1 M phosphate buffer pH 7.0 and dialyzed against the same buffer to obtain 12.5 ml (2.5 units) of crude enzyme.

After washing 50 ml of Chitopearl BCW2603 (manufactured by Fuji Spinning Co., Ltd.) with 4 liters of distilled water, 135 ml of 0.1 M phosphate buffer pH 7.0, 2.25 ml of distilled water and 20 ml of 14% glutaraldehyde solution are added and mixed. Then, a shaking reaction was performed for 2 hours. After completion of the reaction, the product was washed with 6 liters of distilled water to obtain activated chitopearl. 10 ml of the crude enzyme solution was added to 10 ml of activated chitopearl, and shaken overnight at 4 ° C. to immobilize it. After completion of the immobilization, the enzyme was thoroughly washed with 0.1 M phosphate buffer pH 7.0 to obtain an immobilized enzyme. 10 ml of the obtained immobilized enzyme
7.5g maltose and 0.5M phosphate buffer pH 7.0
Was added to make 20 ml with distilled water, and a shaking reaction was carried out at 37 ° C. After the reaction was completed, the supernatant was quantified by high performance liquid chromatography. As a result, it was found that 412 mM trehalose with a conversion of about 40% was produced in 4 days.

(Example 4) Effect of EDTA, a contaminant-interfering enzyme inhibitor , Pseudomonas sp. F-1 (Pseudo) prepared in Example 1
Monas sp. F-1) 0.02 g of frozen cells, 0.75 ml of 0.1 M phosphate buffer pH 7.0 and 0.25 ml of 0.4 M maltose,
A reaction solution for trehalose synthesis was prepared by adding 0.02 ml of 0.5 M EDTA. A reaction solution having the same composition was prepared except that distilled water was used instead of 0.5M EDTA. Both reaction solutions were reacted at 37 ° C. for 20 hours and then centrifuged, and the amount of trehalose produced was quantified from the resulting supernatant using high performance liquid chromatography.

After the completion of the reaction, the cells were washed with 0.1 M phosphate buffer pH 7.0 and reacted again under the same conditions. The reaction was repeated three times in this way, and the amounts of trehalose produced by the respective reaction solutions were compared. The results are shown in Table 4.

[0037]

[Table 4]

As is clear from Table 4, in the reaction solution containing EDTA, the amount of trehalose produced did not decrease even after the cells were used three times.

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, trehalose can be easily and efficiently produced, and trehalose can be mass-produced at low cost by utilizing a bioreactor.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location (C12N 1/20 C12R 1:38) (72) Inventor Takafumi Sano 1759 Minamimatsuno, Fujikawa-cho, Anbara-gun, Shizuoka -4 (72) Inventor Kazuo Oishi 7-1 Sanen-cho, Numazu-shi, Shizuoka 307 Work-occupation 303 (72) Masaaki Yamagishi 256-2 Ogawa, Yaizu-shi, Shizuoka (72) Inventor Toshiya Ota Shimizu-cho, Sunto-gun, Shizuoka Kakita 178-3 Employment housing 301

Claims (5)

[Claims] 1. A method for producing trehalose, which comprises reacting maltose with a microbial cell or a treated product of a microorganism belonging to the genus Pseudomonas and having the ability to convert maltose to trehalose. 2. The microorganism is Pseudomonas sp. F-
1 (Pseudomonas sp. F-1),
The method for producing trehalose according to claim 1. 3. The method for producing trehalose according to claim 1, wherein the microbial cell or the treated product of the microbial cell is allowed to act on maltose in the presence of a contaminant-interfering enzyme inhibitor. 4. The method for producing trehalose according to claim 3, wherein the contaminant-interfering enzyme inhibitor is a chelating agent. 5. The method for producing trehalose according to claim 4, wherein the chelating agent is EDTA.