JPH06189779A - Production of trehalose - Google Patents

Abstract

PURPOSE:To produce trehalose from alpha-glucose in high efficiency at a low cost. CONSTITUTION:Trehalose is produced by treating alpha-glucose-1-phosphate and glucose with a trehalose phosphorylase originated from microorganism selected from the genus Schizophyllum, Agaricus, Pleurotus, Lyophyllum and Grifola.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing trehalose.

[0002]

2. Description of the Related Art Recently, various oligosaccharides have received attention and are being developed. Trehalose is yeast, mold,
It is a disaccharide widely distributed in the microbial kingdoms such as bacteria, seaweeds, algae, and insects, plant kingdoms, and animal kingdoms. Since it is more stable than other disaccharides such as sucrose, it is a sweetener, a bulking agent, etc. 63-240758) and as a dry protector for proteins and the like (Japanese Patent Publication No. 63-500562). Conventionally, as a method for obtaining trehalose, the above natural products, yeasts, extraction methods from fungi, yeasts, Arthrobacter, fermentation methods using microorganisms using Nocardia, etc. are known, but in these methods, Purity of trehalose, problems of impurities, mass production is difficult in terms of operation and equipment. As another method, an enzymatic method is known in which maltose is used as a substrate to produce trehalose via β-glucose-1-phosphate (JP-A-63-63).
-60998). In the method for producing trehalose via β-glucose-1-phosphate, the supply of β-glucose-1-phosphate must be produced from maltose by maltose phosphorylase, the price of the substrate, the supply of the enzyme, the stability of the enzyme. There are problems such as sex. Furthermore, trehalose phosphorylase that produces trehalose from β-glucose-1-phosphate can be obtained from, for example, the green alga Euglena, but its culturing is not always easy, and this trehalose phosphorylase is not very stable. Not an enzyme. In addition, conventional α-glucose-1-
As the enzyme that produces trehalose from phosphoric acid, it is known that trehalose can be obtained by reacting an enzyme obtained from an enokitake mushroom extract with α-glucose-1-phosphate at around pH 7 (FEMS Microbiol).
ody Letters. 55.147-150 (19
88)). However, this enzyme is not suitable for mass production of trehalose because it has poor temperature stability and has problems in enzyme preparation and enzyme reactivity.

[0003]

SUMMARY OF THE INVENTION In view of the above situation, the present invention provides α-glucose-1-phosphate obtained from an inexpensive raw material and an enzyme that is easily available and can exist stably. It is an object of the present invention to provide a method for producing trehalose that is easy to use and has excellent productivity. In addition, as a method for producing α-glucose-1-phosphate, α-glucose is produced by the enzymatic action of phosphorylase.
Various methods for producing from glucan (starch, glycogen, etc.) and orthophosphate are known. For example, a method for producing α-glucose-1-phosphoric acid from dextrin and orthophosphate using potato juice has been disclosed (JP-A-63-63).
208594).

[0004]

Means for Solving the Problems The inventors of the present application have conducted earnest research on efficient and inexpensive production of trehalose, and as a result, α-glucose-1 which can be produced from inexpensive sugars such as starch and dextrin. It has been found that trehalose can be efficiently produced by reacting phosphate and glucose at a specific pH or by causing a novel trehalose phosphorylase to act.

The constitutional feature of the invention of the present application is that α-glucose-1-phosphate and glucose contain sizophiram (S
genus chizophyllum, Agaricus (Agar)
Icus), Pleurotus
A trehalose production method for producing trehalose by allowing a trehalose phosphorylase derived from a microorganism selected from the genus, the genus Lyphilum (genus Lyophllum), the genus Glyfola (genus Grifola), to be detailed.
Trehalose phosphorylase is preferably added to α-glucose-1-phosphate and glucose at a pH of 2.0 to 6.0.
It is a method for producing trehalose which is acted on by.

The invention of this application will be described in detail below. α
As a result of diligent research conducted by the present inventors on a more stable enzyme capable of producing trehalose from -glucose-1-phosphate, Schizophyllum (Schizophyllu)
m), Agaricus, Pleurotus, Lyphilum
α-in the fruiting bodies, mycelium or liquid culture mycelium of the genus hllum, the genus Grifola, etc.
It was found that there is a trehalose phosphorylase that acts on glucose-1-phosphate to produce trehalose.

To obtain the trehalose phosphorylase as described above, Schizophyllum commune (Schi
zophyllum commune), agaricus
Bisporus (Agaricus bisporu)
s), Pull Lotus Ostreatus (Pleurot)
us ostreatus), Lyophilum ulmarium, Grifola frondos
The basidiomycetes such as a) are well-known culture methods, for example, yeast extract 0.75%, malt extract 0.2%, glucose 3%, p.
Shake culture is performed at 28 ° C. for several days in a H6.0 medium, and after the culture, cells are collected by centrifugation and used as an enzyme source. Others, Schizophyllum commune
lum commune), Agaricus bisporus, Pleurotus ostreus
atus), Rifilam ulmarium (Lyophr)
It is also possible to use fruit bodies obtained by culturing basidiomycetes such as lum ulmarium) and Grifola frondosa (Grifola frondosa) in a known sawdust medium for 1 to 2 months or commercially available oyster mushrooms, beech mushrooms, mushrooms and the like as a yeast source.

The trehalose phosphorylase is prepared by crushing the basidiomycete fungus bodies or fruiting bodies in a phosphate buffer and separating and removing the crushed insoluble matter to obtain a crude enzyme. The crude enzyme can be used as it is for the production of trehalose, but can also be used after further purification. The crude enzyme can be separated and purified by the salt precipitation method using ammonium sulfate precipitation, solvent precipitation method, adsorption with ion exchange resin, separation with dialysis membrane, concentration method with ultrafiltration membrane, adsorption with hydroxyapatite, hydrophobic carrier. A general protein purification method such as separation by the method described above can be used. It is also possible to fix the enzyme in the cells by treating the cells with toluene or acetone. Further, the crude enzyme or the purified enzyme may be immobilized by a known immobilization means such as a gel encapsulation method or a microencapsulation method and used as an immobilized enzyme. In addition, it is also possible to immobilize live cells by the entrapping immobilization method using a synthetic polymer prepolymer as a raw material.

In carrying out the invention of the present application, α-glucose-1-phosphate as a substrate is added to the reaction solution in an amount of 5-500.
mM, preferably 50-200 mM and glucose 5
0-2000 mM, preferably 100-500 mM in the presence of trehalose phosphorylase, pH 2
7, preferably 2.5-6.0, reaction temperature 20-60
The reaction may be performed at a temperature of 25 ° C, preferably 25 to 50 ° C. Further, trehalose phosphorylase is used in a ratio of 0.01 unit or more, preferably 0.1 to 100 unit, per 1 gram of the substrate.

The above enzyme reaction is completed in 10 to 200 hours. After completion of the reaction, after heat treatment to deactivate the enzyme,
The precipitate is removed by centrifugation, filtration or the like. The resulting supernatant is adsorbed with trehalose produced by, for example, an activated carbon adsorption treatment, then eluted with a 20% aqueous ethanol solution, and subsequently treated with an anion-type ion exchange resin such as Dowex-1 (manufactured by Dow Chemical Co.) and CM cellulose. By doing so, it can be purified as a sugar solution. Further, trehalose can be obtained by adsorbing the sugar solution on a boric acid type ion exchange resin, eluting it with an aqueous solution of potassium borate or the like, taking out a trehalose fraction, and subjecting this fraction to a treatment such as concentration. . Further, it can be purified by recrystallization or the like, if necessary.

The enzymatic reaction of the present invention is a phosphorylase capable of producing α-glucose-1-phosphate, for example, α-glucan phosphorylase, sucrose phosphorylase, and theobiobiphosphorylase. It is also possible to simultaneously carry out an enzymatic reaction with a enzyme such as a enzyme such as a enzyme. In addition, comparing the temperature stability of various trehalose phosphorylases prepared as in Example 1 described later, the known Flammulina velupes are known.
The enzyme derived from Schizophyllum commune, Agaricus bisporus, Pultus ostretus, and Glyphora frondosa is completely inactivated by treatment at 30 ° C. for 30 minutes.
It was not deactivated after 30 minutes of treatment. Hereinafter, the present invention will be specifically described with reference to examples.

[0012]

【Example】

Example 1 (Preparation of trehalose phosphorylase) Grifola frondosa fruiting body (Grifola frondo)
sa CM-236, Mikori Kenjoyori No. 35) 200 ml of 50 mM phosphate buffer (pH 7.0, 20% glycerol, 1 μM, 1 μMEDTA, 1 μM dithiothreitol, the same applies hereinafter) is added to 100 g, and the mixture is stirred with a Waring blender. Crush the entity. The crushed insoluble matter was separated by centrifugation to obtain 175 ml of a crude enzyme solution as a supernatant. After adsorbing 165 ml of the crude enzyme solution onto a column packed with 20 ml of DEAE Toyopearl (manufactured by Tosoh), 200 ml of the same 50 mM phosphate buffer as above and 50 mM phosphate buffer containing 0.5 mol / l potassium chloride (p
H7.0) (200 ml) was used for linear concentration gradient elution. Fractionation of 3 ml resulted in fraction 10
Trehalose phosphorylase activity was observed in 20 fractions. This fraction was concentrated by ultrafiltration to obtain an enzyme solution having an enzyme activity of 4.1 units / ml and a specific activity of 0.32 units / mg protein. The activity unit of the enzyme is phosphoglucomutase, glucose-6-phosphate dehydrogenase, which is α-glucose-1-phosphate produced by the production reaction of α-glucose-1-phosphate by the phosphorolysis reaction of trehalose phosphorylase. The amount of NADPH produced from NADP by the coupling reaction of
It was measured by measuring the increase in m. Enzyme reaction solution composition is 40mM phosphate buffer (pH 7.0), 200m
M trehalose, 0.16 mM EDTA, 14 mM glutathione, 1 mM NADP, 1.3 mM magnesium chloride, 0.067 mM α-glucose-1,6-diphosphate,
2000 μl of a reaction solution containing 3.1 units / ml of phosphoglucomutase, 3.5 units / ml of glucose-6-phosphate, and an enzyme solution was reacted at 30 ° C. to give an absorbance of 340 nm.
The amount of NADPH produced was measured from the change in Under this condition, the amount of enzyme that produces 1 μmol of NADPH in 1 minute was defined as 1 unit.

(Production of trehalose) Crude enzyme solution 500 of trehalose phosphorylase prepared as described above
μl (4.1 units / ml), glucose 200 mM, α
-Glucose-1-phosphate 100 mM consisting of 100 mM
MES (2- (N-Morpholino) etha
nesulfonic acid) buffer (pH
5.25) 5 ml was used, and it was made to react at 30 degreeC for 13 hours, 22 hours, and 50 hours. After the reaction was completed, each reaction solution was transferred to a polyamine column (YMC pack Polyamii).
ne 4.6 mm I.D. D. X 250 mm), eluent acetonitrile / water (7/3), flow rate 1 ml / min,
As a result of quantifying trehalose produced by high-performance liquid chromatography with a column temperature of 35 ° C. and a differential refractometer (cell temperature of 35 ° C.) as a detection means, the produced trehalose was 54 mM in the enzyme reaction solution at a reaction time of 13 hours and a reaction time of 22 The time was 64 mM and the reaction time was 50 mM at 70 mM.

Example 2 (Preparation of Trehalose Phosphorylase) 100 g of commercially available pull lotus ostreatus fruiting body was added to 250 ml of 50 mM phosphate buffer (pH 7.0), and the fruiting body was crushed with a Waring blender. The crushed insoluble matter was separated by centrifugation to obtain 225 ml of a crude enzyme solution as a supernatant. 220 ml of this crude enzyme solution was adsorbed on a column packed with 20 ml of DEAE Toyopearl (manufactured by Tosoh), and then 200 ml of the same 50 mM phosphate buffer as above and 50 mM phosphate buffer (pH 7.0) containing 0.5 mol / liter of potassium chloride. Linear gradient elution was carried out using 200 ml. As a result of fractionation into 3 ml, trehalose phosphorylase activity was observed in fractions 10 to 20. This fraction was concentrated by ultrafiltration to obtain an enzyme solution having an enzyme activity of 11.5 units / ml and a specific activity of 0.21 units / mg protein.

(Production of trehalose) 10 μl of trehalose phosphorylase enzyme solution prepared as described above (1
1.5 unit / ml), glucose 100 mM, α-glucose-1-phosphate 100 mM, 100 mM ME
Using 500 μl of S buffer (pH 6.0),
The reaction was carried out at 0 ° C for 12 hours and 36 hours. After the reaction,
Add each reaction solution to a polyamine column (YMC pack Po
lyamine 4.6 mm I.D. D. × 250m
m), eluent acetonitrile / water (7/3), flow rate 1 m
1 / min, column temperature 35 ° C., trehalose produced by high performance liquid chromatography with a differential refractometer (cell temperature 35 ° C.) as a detection means was quantified, and as a result, the produced trehalose was 31 m in the enzyme reaction solution at a reaction time of 12 hours.
M, 50 mM at a reaction time of 36 hours.

Example 3 (Preparation of Trehalose Phosphorylase) Lyphilum ulmarium fruiting body (Lyophyllum ulma)
rumCM-506, Microtechnology Research Institute, Microbiology No. 985) 100
250 ml of 50 mM phosphate buffer (pH 7.0)
Add the mixture and crush the fruiting body with a Waring blender. The crushed insoluble matter was separated by centrifugation to obtain 235 ml of a crude enzyme solution as a supernatant. 200 ml of this crude enzyme solution was added to DE
After adsorbing to a column filled with 20 ml of AE Toyopearl (manufactured by Tosoh), 200 ml of the same 50 mM phosphate buffer as described above and 200 ml of 50 mM phosphate buffer (pH 7.0) containing 0.5 mol / liter of potassium chloride were used for linear concentration. Gradient elution was performed. As a result of fractionation into 3 ml, trehalose phosphorylase activity was observed in fractions 14 to 22. This fraction was concentrated by ultrafiltration to obtain an enzyme solution having an enzyme activity of 1.4 units / ml and a specific activity of 0.05 unit / mg protein.

(Production of trehalose) 10 μl of crude trehalose phosphorylase enzyme solution prepared as described above
(1.4 units / ml), glucose 100 mM, α-glucose-1-phosphate 100 mM 100 mM M
Using 500 μl of ES buffer (pH 6.0),
The reaction was conducted at ℃ for 111 hours. After the reaction was completed, the reaction solution was added to a polyamine column (YMC pack Polyamine).
4.6 mm I.D. D. × 250 mm), eluent acetonitrile / water (7/3), flow rate 1 ml / min, column temperature 35 ° C., trehalose produced by high performance liquid chromatography with a differential refractometer (cell temperature 35 ° C.) as a detection means. As a result, the trehalose produced was 1
It was 7.5 mM.

EXAMPLE 4 Schizophyllu commune
m commune Fr. CM-556, Micro Engineering Research Institute No. 1744), yeast extract 0.75%, malt extract 0.
Medium 100 having a composition of 2%, glucose 5%, pH 6.2
200 liters of jar fermenter,
Culture temperature 25 ° C, agitation number 290 rpm, aeration rate 0.5
It was cultured in vvm for 70 hours. After culturing, 400 ml of culture solution
The mycelium was collected by centrifugation from and the cultured mycelium was treated in the same manner as in Example 1. The enzyme solution obtained is 3.5
Unit / ml, specific activity 0.13 unit / mg protein.

(Production of trehalose) 10 μl of crude trehalose phosphorylase enzyme solution prepared as described above
(3.5 units / ml), glucose 100 mM, α-glucose-1-phosphate 100 mM 100 mM M
Using 500 μl of ES buffer (pH 5.5), 30
The reaction was carried out at ℃ for 24 hours. After the reaction was completed, the reaction solution was added to a polyamine column (YMC pack Polyamine).
4.6 mm I.D. D. × 250 mm), eluent acetonitrile / water (7/3), flow rate 1 ml / min, column temperature 35 ° C., trehalose produced by high performance liquid chromatography with a differential refractometer (cell temperature 35 ° C.) as a detection means. As a result, the trehalose produced was 11
It was mM.

Example 5 (Preparation of trehalose phosphorylase) Agaricus bisporus fruiting body (Agaricus bispo)
rus (Lange) Sing. 90 g of CM-686, Microtechnical Laboratory No. 1748) and 250 ml of 50 mM phosphate buffer (pH 7.0) are added, and the fruiting bodies are crushed with a Waring blender. The crushed insoluble matter was separated by centrifugation to obtain 200 ml of a crude enzyme solution as a supernatant. After adsorbing 200 ml of this crude enzyme solution on a column packed with 20 ml of DEAE Toyopearl (manufactured by Tosoh), 200 ml of the same 50 mM phosphate buffer as above and 50 mM phosphate buffer (pH: 0.5 mol / l potassium chloride 0.5 mol / l)
7.0) 200 ml was used for linear concentration gradient elution. As a result of fractionation into 3 ml, fractions 16-2
Trehalose phosphorylase activity was observed in the 4th fraction. This fraction was concentrated by ultrafiltration to obtain an enzyme solution having an enzyme activity of 2.5 units / ml and a specific activity of 0.14 units / mg protein.

(Production of trehalose) 10 μl of crude trehalose phosphorylase enzyme solution prepared as described above
(2.5 units / ml), glucose 100 mM, α-glucose-1-phosphate 100 mM 100 mM M
Using 500 μl of ES buffer (pH 5.5), 30
The reaction was carried out at ℃ for 12 hours. After the reaction was completed, the reaction solution was added to a polyamine column (YMC pack Polyamine).
4.6 mm I.D. D. X 250 mm), eluent acetonitrile / water (7/3), flow rate 1 ml / min, column temperature 35 ° C, trehalose generated by high performance liquid chromatography with a differential refractometer (cell temperature 35 ° C) as a detection means As a result, the produced trehalose was 1
It was 7.3 mM.

Example 6 In order to purify the trehalose produced according to Example 1, an activated carbon column (φ10 × 200 mm, about 16 ml,
Chromatograph, Wako Pure Chemical Industries, Ltd.) to adsorb trehalose, followed by washing with 20 ml of water, and then elution with 30 ml of 20% ethanol. Next, this fraction was concentrated and fractionated by high performance liquid chromatography.
Use a polyamine column (YMC pack Pol
yamine 10 mmI. D. X 250 mm), eluent acetonitrile / water (75/25), flow rate 3 ml /
min. , Column temperature 30 ° C, differential refractometer (cell temperature 3
5 ° C.). The trehalose peak at 35 minutes was collected. From this fraction, 110 mg of white powder could be obtained. 250 MHz H ¹ NMR of this white powder
The spectrum showed the same spectrum as the standard trehalose.

Example 7 Using trehalose phosphorylase obtained according to the method of Example 1, a trehalose production reaction was carried out while changing the pH as shown in Table 1. The reaction solution composition is 50 μl of 0.5 M buffer, 125 μl of 400 mM glucose, 4
62.5 μl of 00 mM α-glucose-1-phosphate and 12.5 μl of enzyme solution (18.6 units / ml) were used. As the buffer, a sodium acetate-hydrochloric acid buffer was used for pH 2 to 5.0, and a MES buffer was used for pH 5.0 to 6.5. The reaction was carried out at 30 ° C. for 4 hours, and
The same treatment was carried out and the enzyme activity was examined, and the results are shown in Table 1. In Table 1, the amount of trehalose produced at pH 4.5, which shows the best reactivity, was defined as 100, and the enzyme activity (ratio) at each pH was shown by the ratio.

[0024]

[Table 1]

[0025]

INDUSTRIAL APPLICABILITY By using the trehalose phosphorylase of the present invention, inexpensive trehalose can be produced stably from inexpensive starch, α-glucose-1-phosphate and glucose which can be produced from sugars such as sucrose, and It can be produced in high yield.

[Procedure amendment]

[Submission date] March 2, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 1

[Correction method] Change

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[Procedure 3]

[Document name to be amended] Statement

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[Correction method] Change

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[0002]

2. Description of the Related Art Recently, various oligosaccharides have received attention and are being developed. Trehalose is yeast, mold,
It is a disaccharide widely distributed in the microbial kingdoms such as bacteria, seaweeds, algae, and insects, plant kingdoms, and animal kingdoms. Since it is more stable than other disaccharides such as sucrose, it is a sweetener, a bulking agent, etc. 63-240758) and as a dry protector for proteins and the like (Japanese Patent Publication No. 63-500562). Conventionally, as a method for obtaining trehalose, the above natural products, yeasts, extraction methods from fungi, yeasts, Arthrobacter, fermentation methods using microorganisms using Nocardia, etc. are known, but in these methods, Purity of trehalose, problems of impurities, mass production is difficult in terms of operation and equipment. As another method, an enzymatic method is known in which maltose is used as a substrate to produce trehalose via β-glucose-1-phosphate (JP-A-63-63).
-60998). In the method for producing trehalose via β-glucose-1-phosphate, the supply of β-glucose-1-phosphate must be produced from maltose by maltose phosphorylase, the price of the substrate, the supply of the enzyme, the stability of the enzyme. There are problems such as sex. Furthermore, trehalose phosphorylase that produces trehalose from β-glucose-1-phosphate can be obtained from, for example, the green alga Euglena, but its culturing is not always easy, and this trehalose phosphorylase is not very stable. Not an enzyme. In addition, conventional α-glucose-1-
As the enzyme that produces trehalose from phosphoric acid, it is known that trehalose can be obtained by reacting an enzyme obtained from an enokitake mushroom extract with α-glucose-1-phosphate at around pH 7 (FEMS Microbiol).
ody Letters. 55.147-150 (19
88)). However, this enzyme is not suitable for mass production of trehalose because it has poor temperature stability and has problems in enzyme preparation and enzyme reactivity.

[Procedure amendment 4]

[Document name to be amended] Statement

[Name of item to be corrected] 0005

[Correction method] Change

[Correction content]

The constitutional feature of the invention of the present application is that α-glucose-1-phosphate and glucose contain sizophiram (S
genus chizophyllum, Agaricus (Agar)
Icus), Pleurotus
A method for producing trehalose in which trehalose is produced by causing a trehalose phosphorylase derived from a microorganism selected from the genus, Lyophilum genus, and Glyfola genus, to produce trehalose, and specifically, α-glucose-1-phosphate and For glucose, trehalose phosphorylase is preferably pH 2.0 to
This is a method for producing trehalose which is allowed to act at 6.0.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0006

[Correction method] Change

[Correction content]

The invention of this application will be described in detail below. α
As a result of diligent research conducted by the present inventors on a more stable enzyme capable of producing trehalose from -glucose-1-phosphate, Schizophyllum (Schizophyllu)
m), Agaricus, Pleurotus, Lyphilum
genus hyllum, genus Grifola,
Α in the fruiting bodies, mycelium or liquid culture mycelium
It was found that there is trehalose phosphorylase which acts on -glucose-1-phosphate to produce trehalose.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

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To obtain the trehalose phosphorylase as described above, Schizophyllum commune (Schi
zophyllum commune), agaricus
Bisporus (Agaricus bisporu)
s), Pull Lotus Ostreatus (Pleurot)
us ostreatus), Lyphyllum ulmarium, Grifola frondosa
sa) and other basidiomycetes are well-known culture methods, for example, yeast extract 0.75%, malt extract 0.2%, glucose 3%, p.
Shake culture is performed at 28 ° C. for several days in a H6.0 medium, and after the culture, cells are collected by centrifugation and used as an enzyme source. Others, Schizophyllum commune
lum commune), Agaricus bisporus, Pleurotus ostreus
atus), Rifilum ulmarium (Lyophy)
It is also possible to use fruit bodies obtained by culturing basidiomycetes such as L. lum ulmarium) and Glyfola frondosa (Grifola frondosa) in a known sawdust medium for 1 to 2 months, or commercially available oyster mushrooms, beech shimeji mushrooms, maitake mushrooms and mushrooms as the enzyme source.

[Procedure Amendment 7]

[Document name to be amended] Statement

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[Correction method] Change

[Correction content]

The trehalose phosphorylase is prepared by crushing the basidiomycete fungus bodies or fruiting bodies in a phosphate buffer and separating and removing the crushed insoluble matter to obtain a crude enzyme. The crude enzyme can be used as it is for the production of trehalose, but can also be used after further purification. The crude enzyme can be separated and purified by the salt precipitation method using ammonium sulfate precipitation, solvent precipitation method, adsorption with ion exchange resin, separation with dialysis membrane, concentration method with ultrafiltration membrane, adsorption with hydroxyapatite, hydrophobic carrier. A general protein purification method such as separation by the method described above can be used. It is also possible to fix the enzyme in the cells by treating the cells with toluene or acetone. Further, the crude enzyme or the purified enzyme may be immobilized by a known immobilization means such as a gel encapsulation method or a microencapsulation method and used as an immobilized enzyme. In addition, it is also possible to immobilize live cells by the entrapping immobilization method using a synthetic polymer prepolymer as a raw material.

[Procedure Amendment 8]

[Document name to be amended] Statement

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[Correction method] Change

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[0012]

Examples Example 1 (Preparation of Trehalose Phosphorylase) Grifola frondosa fruiting body (Grifola frondo)
sa CM-236, Mikori Kenjoyori No. 35) 200 ml of 50 mM phosphate buffer (pH 7.0, 20% glycerol, 1 mM, 1 mM EDTA, 1 mM dithiothreitol, the same applies below) was added to 100 g of the mixture, and the mixture was mixed with a Waring blender. Crush the entity. The crushed insoluble matter was separated by centrifugation to obtain 175 ml of a crude enzyme solution as a supernatant. After adsorbing 165 ml of the crude enzyme solution on a column packed with 20 ml of DEAE Toyopearl (manufactured by Tosoh), 200 ml of the same 50 mM phosphate buffer as above and 50 mM phosphate buffer containing 0.5 mol / 1 potassium chloride (p
H7.0) (200 ml) was used for linear concentration gradient elution. Fractionation of 3 ml resulted in fraction 10
Trehalose phosphorylase activity was observed in 20 fractions. This fraction was concentrated by ultrafiltration to obtain an enzyme solution having an enzyme activity of 4.1 units / ml and a specific activity of 0.32 units / mg protein. The activity unit of the enzyme is phosphoglucomutase, glucose-6-phosphate dehydrogenase, which is α-glucose-1-phosphate produced by the production reaction of α-glucose-1-phosphate by the phosphorolysis reaction of trehalose phosphorylase. The amount of NADPH produced from NADP by the coupling reaction of
It was measured by measuring the increase in m. Enzyme reaction solution composition is 40mM phosphate buffer (pH 7.0), 200m
M trehalose, 0.16 mM EDTA, 14 mM glutathione, 1 mM NADP, 1.3 mM magnesium chloride, 0.067 mM α-glucose-1,6-diphosphate,
2000 μl of a reaction solution containing 3.1 units / ml of phosphoglucomutase, 3.5 units / ml of glucose-6-phosphate, and an enzyme solution was reacted at 30 ° C. to give an absorbance of 340 nm.
The amount of NADPH produced was measured from the change in Under this condition, the amount of enzyme that produces 1 μmol of NADPH in 1 minute was defined as 1 unit.

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(Production of trehalose) Crude enzyme solution 500 of trehalose phosphorylase prepared in this manner
μl (4.1 units / ml), glucose 200 mM, α
-Glucose-1-phosphate 100 mM consisting of 100 mM
MES (2- (N-Morpholino) etha
nesulfonic acid) buffer (pH
5.25) 5 ml was used, and it was made to react at 30 degreeC for 13 hours, 22 hours, and 50 hours. After the reaction was completed, each reaction solution was transferred to a polyamine column (YMC pack Polyamii).
ne 4.6 mm I.D. D. X 250 mm), eluent acetonitrile / water (7/3), flow rate 1 ml / min,
As a result of quantifying trehalose produced by high-performance liquid chromatography with a column temperature of 35 ° C. and a differential refractometer (cell temperature of 35 ° C.) as a detection means, the produced trehalose was 54 mM in the enzyme reaction solution at a reaction time of 13 hours and a reaction time of 22 The time was 64 mM and the reaction time was 50 mM at 70 mM.

[Procedure Amendment 10]

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Example 2 (Preparation of Trehalose Phosphorylase) 100 g of commercially available pull lotus ostreatus fruiting body was added to 250 ml of 50 mM phosphate buffer (pH 7.0), and the fruiting body was crushed with a Waring blender. The crushed insoluble matter was separated by centrifugation to obtain 225 ml of a crude enzyme solution as a supernatant. 220 ml of this crude enzyme solution was adsorbed on a column packed with 20 ml of DEAE Toyopearl (manufactured by Tosoh), and then 200 ml of the same 50 mM phosphate buffer as above and 50 mM phosphate buffer (pH 7.0) containing 0.5 mol / liter of potassium chloride. Linear gradient elution was carried out using 200 ml. As a result of fractionation into 3 ml, trehalose phosphorylase activity was observed in fractions 10 to 20. This fraction was concentrated by ultrafiltration to obtain an enzyme solution having an enzyme activity of 11.5 units / ml and a specific activity of 0.21 units / mg protein.

[Procedure Amendment 11]

[Document name to be amended] Statement

[Name of item to be corrected] 0015

[Correction method] Change

[Correction content]

(Production of Trehalose) 10 μl of trehalose phosphorylase enzyme solution prepared as described above (1
1.5 unit / ml), glucose 100 mM, α-glucose-1-phosphate 100 mM, 100 mM ME
Using 500 μl of S buffer (pH 6.0),
The reaction was carried out at 0 ° C for 12 hours and 36 hours. After the reaction,
Add each reaction solution to a polyamine column (YMC pack Po
lyamine 4.6 mm I.D. D. × 250m
m), eluent acetonitrile / water (7/3), flow rate 1 m
1 / min, column temperature 35 ° C., trehalose produced by high performance liquid chromatography with a differential refractometer (cell temperature 35 ° C.) as a detection means was quantified, and as a result, the produced trehalose was 31 m in the enzyme reaction solution at a reaction time of 12 hours.
M, 50 mM at a reaction time of 36 hours.

[Procedure Amendment 12]

[Document name to be amended] Statement

[Correction target item name] 0016

[Correction method] Change

[Correction content]

Example 3 (Preparation of trehalose phosphorylase) Lyphiram ulmarium fruiting body (Lyophyllum ulm)
arium CM-506, Micro Engineering Research Institute, Microbiology No. 985) 10
0 g to 250 m of 50 mM phosphate buffer (pH 7.0)
Add 1 and crush the fruiting body with a Waring blender.
The crushed insoluble matter was separated by centrifugation to obtain 235 ml of a crude enzyme solution as a supernatant. 200 ml of this crude enzyme solution was added to D
After adsorbing to a column filled with 20 ml of EAE Toyopearl (manufactured by Tosoh), 200 ml of the same 50 mM phosphate buffer as described above and 200 ml of 50 mM phosphate buffer (pH 7.0) containing 0.5 mol / liter of potassium chloride were used for linear concentration. Gradient elution was performed. As a result of fractionation into 3 ml, trehalose phosphorylase activity was observed in fractions 14 to 22. This fraction was concentrated by ultrafiltration to obtain an enzyme solution having an enzyme activity of 1.4 units / ml and a specific activity of 0.05 unit / mg protein.

[Procedure Amendment 13]

[Document name to be amended] Statement

[Correction target item name] 0017

[Correction method] Change

[Correction content]

(Production of Trehalose) 10 μl of crude trehalose phosphorylase enzyme solution prepared above
(1.4 units / ml), glucose 100 mM, α-glucose-1-phosphate 100 mM 100 mM M
Using 500 μl of ES buffer (pH 6.0),
The reaction was conducted at ℃ for 111 hours. After the reaction was completed, the reaction solution was added to a polyamine column (YMC pack Polyamine).
4.6 mm I.D. D. × 250 mm), eluent acetonitrile / water (7/3), flow rate 1 ml / min, column temperature 35 ° C., trehalose produced by high performance liquid chromatography with a differential refractometer (cell temperature 35 ° C.) as a detection means. As a result, the trehalose produced was 1
It was 7.5 mM.

[Procedure Amendment 14]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction content]

(Production of Trehalose) 10 μl of crude trehalose phosphorylase enzyme solution prepared as described above
(3.5 units / ml), glucose 100 mM, α-glucose-1-phosphate 100 mM 100 mM M
Using 500 μl of ES buffer (pH 5.5), 30
The reaction was carried out at ℃ for 24 hours. After the reaction was completed, the reaction solution was added to a polyamine column (YMC pack Polyamine).
4.6 mm I.D. D. × 250 mm), eluent acetonitrile / water (7/3), flow rate 1 ml / min, column temperature 35 ° C., trehalose produced by high performance liquid chromatography with a differential refractometer (cell temperature 35 ° C.) as a detection means. As a result, the trehalose produced was 11
It was mM.

[Procedure Amendment 15]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction content]

Example 5 (Preparation of trehalose phosphorylase) Agaricus bisporus fruiting body (Agaricus bispo)
rus (Lange) Sing. 90 g of CM-686, Microtechnical Laboratory No. 1748) and 250 ml of 50 mM phosphate buffer (pH 7.0) are added, and the fruiting bodies are crushed with a Waring blender. The crushed insoluble matter was separated by centrifugation to obtain 200 ml of a crude enzyme solution as a supernatant. After adsorbing 200 ml of this crude enzyme solution on a column packed with 20 ml of DEAE Toyopearl (manufactured by Tosoh), 200 ml of the same 50 mM phosphate buffer as above and 50 mM phosphate buffer (pH: 0.5 mol / l potassium chloride 0.5 mol / l)
7.0) 200 ml was used for linear concentration gradient elution. As a result of fractionation into 3 ml, fractions 16-2
Trehalose phosphorylase activity was observed in the 4th fraction. This fraction was concentrated by ultrafiltration to obtain an enzyme solution having an enzyme activity of 2.5 units / ml and a specific activity of 0.14 units / mg protein.

[Procedure Amendment 16]

[Document name to be amended] Statement

[Correction target item name] 0021

[Correction method] Change

[Correction content]

(Production of trehalose) 10 μl of crude trehalose phosphorylase enzyme solution prepared as described above
(2.5 units / ml), glucose 100 mM, α-glucose-1-phosphate 100 mM 100 mM M
Using 500 μl of ES buffer (pH 5.5), 30
The reaction was carried out at ℃ for 12 hours. After the reaction was completed, the reaction solution was added to a polyamine column (YMC pack Polyamine).
4.6 mm I.D. D. X 250 mm), eluent acetonitrile / water (7/3), flow rate 1 ml / min, column temperature 35 ° C, trehalose generated by high performance liquid chromatography with a differential refractometer (cell temperature 35 ° C) as a detection means As a result, the produced trehalose was 1
It was 7.3 mM.

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Claims (3)

[Claims] 1. α-Glucose-1-phosphate and glucose are added to Schizophyllum.
Genus, genus Agaricus, genus Pleurotus, lyophilum
lum) and Glyfola genus microorganism-derived trehalose phosphorylase is allowed to act to produce trehalose. 2. A trehalose phosphorylase is a Schizophyllum commune.
commune), Agaricus bisporus (Aga)
ricus bisporus, Pleurotus ostreatus
s), Lyphilum ulmarium
ulmarium) or Grifola forasa (Grifola forasa).
The method for producing trehalose according to 1. 3. α-Glucose-1-phosphate and glucose are treated with trehalose phosphorylase at a pH of 2.0 to.
A method for producing trehalose, which is characterized in that it is allowed to act at 6.0.