JP3026322B2 - Method for producing trehalose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing trehalose.

[0002]

2. Description of the Related Art Recently, various types of oligosaccharides have attracted attention and are being developed. Trehalose is a yeast, mold,
It is a disaccharide widely distributed in the microbial, plant, and animal kingdoms of bacteria, seaweeds, algae, insects, etc., and is more stable than other disaccharides such as sucrose. 63-240758) and as a dry protective agent such as a protein (Japanese Patent Publication No. 63-500562). Conventionally, as a method for obtaining trehalose, the above-mentioned natural product, yeast, a method of extracting from mold, yeast, Arthrobacter, a fermentation method using a microorganism using Nocardia, and the like are known.In these methods, Trehalose purity, problems of impurities, mass production is difficult in terms of operation and equipment. As another method, an enzymatic method for producing trehalose via β-glucose-1-phosphate using maltose as a substrate is known (Japanese Patent Application Laid-Open No. Sho 63).
-60998 publication). 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, and the price of the substrate, supply of the enzyme, and stability of the enzyme There are problems such as gender. Furthermore, trehalose phosphorylase that produces trehalose from β-glucose-1-phosphate can be obtained, for example, from the green alga Euglena, but its culture is not always easy, and this trehalose phosphorylase is not very stable. Not an enzyme. Conventionally, α-glucose-1-
As an enzyme that produces trehalose from phosphate, it is known that trehalose can be obtained by reacting an enzyme obtained from an enokitake extract with α-glucose-1-phosphate at around pH 7 (FEMS Microbiol).
oggy Letters. 55.147-150 (19
88)). However, this enzyme has poor temperature stability and has problems in enzyme preparation and enzyme reactivity, and is not suitable for mass production of trehalose.

[0003]

In view of the above situation, the present invention relates to α-glucose-1-phosphate obtained from inexpensive raw materials and an enzyme which is easily available and can be stably present. It is an object of the present invention to provide a method for producing trehalose easily using trehalose and having excellent productivity. As a method for producing α-glucose-1-phosphate, α-glucose-1-phosphate is produced by 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 ground juice has been disclosed (Japanese Patent Application Laid-Open No. Sho 63-163).
208594).

[0004]

The present inventors have conducted intensive studies on the efficient and inexpensive production of trehalose, and as a result, have found that α-glucose-1 which can be produced from inexpensive saccharides such as starch and dextrin. -It has been found that trehalose can be efficiently produced at a specific pH or by reacting a novel trehalose phosphorylase with phosphate and glucose.

A structural feature of the present invention is that α-glucose-1-phosphate and glucose have schizophyllam (S
chizophyllum, Agaricus (Agar)
icus), Pullotus
The present invention relates to a method for producing trehalose by generating a trehalose by reacting a trehalose phosphorylase derived from a microorganism selected from the genus, phyllum (Lyophilum), and the genus Grifola, and more specifically, α-glucose-1-phosphate and Trehalose phosphorylase is preferably added to glucose at pH 2.0 to
This is a method for producing trehalose, which is operated at 6.0.

Hereinafter, the present invention will be described in detail. α
The present inventors have conducted intensive studies on a more stable enzyme capable of producing trehalose from glucose-1-phosphate, and as a result, found that Schizophyllu.
m), genus Agaricus, genus Pleurotus, lyophilum (Lyop)
yllum) genus, Grifola genus,
In the fruiting body, mycelium, liquid culture mycelium, etc.
-Trehalose phosphorylase which acts on glucose-1-phosphate to produce trehalose was found to be present.

[0007] To obtain trehalose phosphorylase as described above, Schizophyllum commune (Schi)
zophyllum commune), Agaricus
Bisporous (Agaricus bisporu)
s), Pullotus ostreatus (Pleurot)
us ostreatus, Lyphyllum ulmarium, Grifola frontosa
basidiomycetes such as sa), for example, yeast extract 0.75%, malt extract 0.2%, glucose 3%, p
Shaking culture is performed at 28 ° C. for several days in a medium of H6.0, and after the culture, the cells are collected by centrifugation and used as an enzyme source. In addition, Schizophyllum
lum commune), Agaricus bisporus, Plurotus ostreatus
atus), Riphilum ulmarium (Lyophy)
An enzyme source may be a fruiting body obtained by cultivating basidiomycetes such as Ilum ulmarium and Grifola frontosa in a known sawdust medium for one to two months or a commercially available oyster mushroom, Bunashimeji, maitake, or mushroom.

[0008] In preparing trehalose phosphorylase, a cell obtained by crushing the basidiomycete cells or fruiting bodies in a phosphate buffer and separating and removing the crushed insolubles is used as a crude enzyme. The crude enzyme can be used as it is for the production of trehalose, but it can also be used after further purification. The separation and purification methods of the crude enzyme include salt precipitation by ammonium sulfate precipitation, solvent precipitation, adsorption by ion exchange resin, separation by dialysis membrane, etc., concentration by ultrafiltration membrane, adsorption by hydroxyapatite, etc., hydrophobic carrier General protein purification methods such as separation by HPLC can be used. Alternatively, the cells can be treated with toluene, acetone, or the like, and the enzyme can be immobilized in the cells and used. Further, the crude enzyme or the purified enzyme may be immobilized by a known immobilization means, for example, a gel entrapment method, a microencapsulation method or the like, and used as the immobilized enzyme. In addition, live cells can be immobilized and used by a comprehensive immobilization method using a synthetic polymer prepolymer as a material.

In carrying out the present invention, α-glucose-1-phosphoric acid is used as a substrate in a reaction solution in an amount of from 5 to 500 μm.
mM, preferably 50-200 mM, and glucose of 5 mM.
0-2000 mM, preferably 100-500 mM, in the presence of trehalose phosphorylase,
7, preferably 2.5-6.0, reaction temperature 20-60
C., preferably at 25 to 50.degree. Trehalose phosphorylase is used in an amount of 0.01 unit or more, preferably 0.1 to 100 units per gram of the substrate.

The above enzyme reaction is completed in 10 to 200 hours. After the reaction is complete, heat-treat 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, 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 Company) and CM cellulose. By doing so, it can be purified as a sugar solution. Further, after adsorbing this sugar solution to a boric acid type ion exchange resin, it is eluted with an aqueous solution of potassium borate or the like, a trehalose fraction is taken out, and trehalose can be obtained by subjecting this fraction to treatment such as concentration. . Further, if necessary, it can be purified by recrystallization or the like.

The enzymatic reaction of the present invention is carried out by a phosphorylase capable of producing α-glucose-1-phosphate, for example, α-glucan phosphorylase, sucrose phosphorylase, and biobiophosphate. An enzymatic reaction can also be carried out simultaneously with an enzyme such as an enzyme and a substrate of the enzyme. In addition, comparing the temperature stability of various trehalose phosphorylases prepared as described in Example 1 below, it was found that the known flamalina vertipes (Flammulina verulus) was used.
The enzymes derived from Schizophyllum commune, Agaricus bisporus, Plutus ostretas, and Grifola frondosa are completely inactivated by treatment at 30 ° C. for 30 minutes.
There was no inactivation by the treatment for 30 minutes. Hereinafter, the present invention will be specifically described with reference to examples.

[0012]

EXAMPLES Example 1 (Preparation of Trehalose Phosphorylase) The fruiting body of Grifola frondosa (Grifola f
(Rondosa CM-236, No. 35
100 g of 50 mM phosphate buffer (pH 7.0, 20
% Glycerol, 1 mM, 1 mM EDTA, 1 mM dithiothreitol; the same applies hereinafter) 200 ml, and the fruiting bodies are crushed with a Waring blender. The crushed insolubles were separated by centrifugation, and the crude enzyme solution 175 as a supernatant was separated.
ml were obtained. After adsorbing 165 ml of the crude enzyme solution to a column packed with 20 ml of DEAE Toyopearl (manufactured by Tosoh), 200 ml of the same 50 mM phosphate buffer and 200 ml of 50 mM phosphate buffer (pH 7.0) containing 0.5 mol / 1 of potassium chloride as described above. Was used to perform a linear concentration gradient elution. As a result of fractionation of 3 ml, trehalose phosphorylase activity was observed in the fractions of fractions 10 to 20. This fraction is concentrated by ultrafiltration,
Enzyme activity 4.1 units / ml, specific activity 0.32 units / ml
An enzyme solution of mg protein was obtained. The activity unit of the enzyme is α-glucose-1-phosphoric acid produced by the phosphorylation reaction of trehalose phosphorylase.
-Glucose-1-phosphate to phosphoglucomutase,
The amount of NADPH produced from NADP by the coupling reaction of glucose-6-phosphate dehydrogenase was measured by measuring the increase in absorbance at 340 nm. Enzyme reaction solution composition is 40 mM phosphate buffer (pH 7.0),
200 mM trehalose, 0.16 mM EDTA, 14
mM glutathione, 1 mM NADP, 1.3 mM magnesium chloride, 0.067 mM α-glucose-1,6-
Diphosphate, phosphoglucomutase 3.1 units / ml,
A reaction solution containing glucose-6-phosphate 3.5 units / ml and a reaction solution containing 2000 μl containing an enzyme solution was reacted at 30 ° C. to obtain an absorbance of 3
From the change of 40 nm, the amount of NADPH generated was measured. Under these conditions, the amount of the enzyme producing 1 μmol of NADPH per minute was defined as 1 unit.

(Generation of trehalose) A 100 mM MES (2-(-()-) comprising 500 μl (4.1 units / ml) of the crude enzyme solution of trehalose phosphorylase thus prepared, 200 mM glucose, and 100 mM α-glucose-1-phosphate. N-Morphor
ino) ethanesulfonic acid) buffer (pH 5.25) at 30 ° C with 1 ml
The reaction was carried out for 3, 22 and 50 hours. After completion of the reaction, each reaction solution was applied to a polyamine column (YMC pack).
Polyamine 4.6mm I.I. D. × 250
mm), eluent acetonitrile / water (7/3), flow rate 1
As a result of quantifying the amount of trehalose generated by high performance liquid chromatography using ml / min, a column temperature of 35 ° C., and a differential refractometer (cell temperature of 35 ° C.) as a detecting means, the generated trehalose was added to the enzymatic reaction solution in a reaction time of 13 hours.
mM, 64 mM at a reaction time of 22 hours, and 70 mM at a reaction time of 50 hours.

Example 2 (Preparation of Trehalose Phosphorylase) 100 g of a commercially available fruit body of Plutus ostreatus is added with 250 ml of 50 mM phosphate buffer (pH 7.0), and the fruit body is crushed with a Waring blender. The crushed insolubles were separated by centrifugation, and the crude enzyme solution 2
25 ml were obtained. 220 ml of this crude enzyme solution is added to DEAE
After adsorption to a column packed with 20 ml of Toyopearl (manufactured by Tosoh), the same 50 mM phosphate buffer 20
50 containing 0 ml and 0.5 mol / l of potassium chloride
A linear concentration gradient elution was performed using 200 ml of an mM phosphate buffer (pH 7.0). As a result of fractionation of 3 ml, trehalose phosphorylase activity was observed in the fractions of 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 (11.5 units / ml) of trehalose phosphorylase enzyme solution prepared as described above, glucose 10
500 μm of 100 mM MES buffer (pH 6.0) composed of 0 mM and α-glucose-1-phosphate 100 mM
The mixture was reacted at 30 ° C. for 12 hours and 36 hours. After completion of the reaction, each reaction solution is applied to a polyamine column (YMC
pack Polyamine 4.6 mm
D. × 250 mm), eluent acetonitrile / water (7
/ 3), a flow rate of 1 ml / min, a column temperature of 35 ° C., and trehalose produced by high performance liquid chromatography using a differential refractometer (cell temperature of 35 ° C.) as a detection means. It was 31 mM in the enzyme reaction solution, and 50 mM in a reaction time of 36 hours.

Example 3 (Preparation of trehalose phosphorylase) [0016] Lyphyllum ulmarium fruiting body (Lyophyllu)
mulmarium CM-506, 98
No. 5) 100 g of 50 mM phosphate buffer (pH 7.
0) Add 250 ml and crush the fruiting body with a Waring blender. The crushed insolubles were separated by centrifugation to obtain 235 ml of a crude enzyme solution as a supernatant. This crude enzyme solution 20
0 ml, 20 ml of DEAE Toyopearl (Tosoh)
After adsorption to a column packed with
200 ml of phosphate buffer and 0.5 mol of potassium chloride /
Liter containing 50 mM phosphate buffer (pH 7.0)
A linear concentration gradient elution was performed using 200 ml. 3m
As a result of fractionation of l, 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 units / mg protein.

(Production of trehalose) 10 μl (1.4 units / ml) of trehalose phosphorylase crude enzyme solution prepared as described above, glucose 10
500 μm of 100 mM MES buffer (pH 6.0) composed of 0 mM and α-glucose-1-phosphate 100 mM
The reaction was carried out at 25 ° C. for 111 hours. After completion of the reaction, the reaction solution was applied to a polyamine column (YMC pack P
olyamine 4.6 mm I.O. D. × 250m
m), eluent acetonitrile / water (7/3), flow rate 1m
As a result of quantifying trehalose produced by high performance liquid chromatography using 1 / min, a column temperature of 35 ° C. and a differential refractometer (cell temperature of 35 ° C.) as a detecting means, the produced trehalose was 17.5 mM.

Example 4 Schizophyllum commune
m_communeFr. CM-556, No. 1744), a yeast extract 0.75%, a malt extract 0.
Medium 100 having a composition of 2%, glucose 5%, and pH 6.2
Liters with a 200 liter jar fermenter,
Culture temperature 25 ° C, stirring number 290 rpm, aeration 0.5
The cells were cultured at vvm for 70 hours. After culturing, 400 ml of culture solution
, Mycelium was collected by centrifugation, and the cultured mycelium was treated in the same manner as in Example 1. The resulting enzyme solution was 3.5
Unit / ml, specific activity 0.13 unit / mg protein.

(Production of trehalose) 10 μl (3.5 units / ml) of trehalose phosphorylase crude enzyme solution prepared as described above, glucose 10
500 μm of 100 mM MES buffer (pH 5.5) consisting of 0 mM and α-glucose-1-phosphate 100 mM
The mixture was reacted at 30 ° C. for 24 hours. After the reaction,
The reaction solution was applied to a polyamine column (YMC pack Pol).
yamine 4.6mm I. D. × 250m
m), eluent acetonitrile / water (7/3), flow rate 1m
As a result of quantifying trehalose produced by high performance liquid chromatography using 1 / min, a column temperature of 35 ° C., and a differential refractometer (cell temperature of 35 ° C.) as a detecting means, the produced trehalose was 11 mM.

Example 5 (Preparation of Trehalose Phosphorylase) Agaricus bisporous fruiting body (Agaricus)
bisporus (Lange) Sing. CM-6
No. 86, No. 1748, manufactured by Micro Koken Co., Ltd.), 250 ml of 50 mM phosphate buffer (pH 7.0) is added, and the fruiting bodies are crushed with a Waring blender. The crushed insolubles were separated by centrifugation to obtain 200 ml of a crude enzyme solution as a supernatant. After adsorbing 200 ml of the crude enzyme solution to a column packed with 20 ml of DEAE Toyopearl (manufactured by Tosoh), the same 50 mM phosphate buffer as above and 200 mM 50 mM phosphate buffer (pH 7.0) containing 0.5 mol / l potassium chloride were used. A linear concentration gradient elution was performed using 200 ml. As a result of fractionation of 3 ml, trehalose phosphorylase activity was observed in fractions 16 to 24. 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 (2.5 units / ml) of the crude enzyme solution of trehalose phosphorylase prepared above and glucose 10
500 μm of 100 mM MES buffer (pH 5.5) consisting of 0 mM and α-glucose-1-phosphate 100 mM
The mixture was reacted at 30 ° C. for 12 hours. After the reaction,
The reaction solution was applied to a polyamine column (YMC pack Pol).
yamine 4.6mm I. D. × 250m
m), eluent acetonitrile / water (7/3), flow rate 1m
As a result of quantifying trehalose generated by high-performance liquid chromatography using 1 / min, a column temperature of 35 ° C., and a differential refractometer (cell temperature of 35 ° C.) as a detection means, the generated trehalose was 17.3 mM.

Example 6 In order to purify trehalose produced in Example 1, an activated carbon column (φ10 × 200 mm, about 16 ml,
Chromatograph, manufactured by Wako Pure Chemical Industries, Ltd.) to adsorb trehalose, then washed with 20 ml of water and then eluted with 30 ml of 20% ethanol. Next, this fraction was concentrated and fractionated by high performance liquid chromatography.
The fractionation was performed using a polyamine column (YMC pack Pol
yamine 10mmI. D. × 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 a 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, trehalose was produced by changing the pH as shown in Table 1. The composition of the reaction solution was 50 μl of 0.5 M buffer, 125 μl of 400 mM glucose,
62.5 μl of 00 mM α-glucose-1-phosphate and 12.5 μl of an enzyme solution (18.6 units / ml) were used. As a buffer, a sodium acetate-hydrochloric acid buffer was used for pH 2 to 5.0, and an MES buffer was used for pH 5.0 to 6.5. The reaction was carried out at 30 ° C. for 4 hours.
The enzyme activity was examined by treating in the same manner as described above. In Table 1, the amount of trehalose produced at pH 4.5 showing the best reactivity was set to 100, and the enzyme activity (ratio) at each pH was shown by the ratio.

[0024]

[Table 1]

[0025]

EFFECT OF THE INVENTION By using the trehalose phosphorylase of the present invention, trehalose can be stably produced from α-glucose-1-phosphate and glucose which can be produced from inexpensive saccharides such as starch and sucrose. It can be produced in high yield.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 645)

Claims (3)

(57) [Claims] 1. The method according to claim 1, wherein α-glucose-1-phosphate and glucose are added to Schizophyllum.
Genus, Agaricus, Pleurotus, Lyophilum
A method for producing trehalose, which comprises producing trehalose by reacting a trehalose phosphorylase derived from a microorganism selected from the genus Ilum and the genus Grifola. 2. The method according to claim 1, wherein the trehalose phosphorylase is Schizophyllum.
commune), Agaricus Bisporous (Aga)
ricus bisporus, Plurotus ostreatus (Pleurotus ostreatus)
s), Lyphilum ulmarium
2. The method for producing trehalose according to claim 1, wherein the trehalose is derived from M. mulmarium or Grifola frondosa. 3. A method in which trehalose phosphorylase is added to α-glucose-1-phosphate and glucose at a pH of 2.0 or more.
3. The method for producing trehalose according to claim 1, wherein the trehalose is acted on at 6.0.