JP3014950B2 - Method for producing trehalose - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to trehalose (O-α-D-glucopyranosyl- (1 → 1) -D-glucopyra) obtained by an enzymatic method.
Noside). More specifically, the present invention relates to a novel maltose phosphorylase derived from a novel microorganism belonging to the genus Plesiomonas and a method for producing trehalose using trehalose phosphorylase.

[0002]

2. Description of the Related Art Trehalose has the effect of protecting cells and intracellular substances during freezing or drying, and serves as a preservative and stabilizer for medicines, cosmetics, foods and the like. Is a substance that is expected. Thus, many attempts have been made to industrially produce trehalose. These techniques can be broadly classified into three categories. One of them is a method of extracting and purifying trehalose from a microorganism having a property of accumulating trehalose in the cells [Journal of American Chemical.
J. Am. Chem. Soc. 72, 2059, 1950, German Patent No. 266584, JP-A-3-1300084
JP-A-5-91890, JP-A-5-184353
No. JP-A-5-292986]. This method comprises a microorganism culturing step, a microorganism separating step, a trehalose extraction step from the microorganism, and a purification and crystallization step of the extracted trehalose, and the production step is very complicated.
In addition, the trehalose productivity is not only low compared to other methods, but also a large amount of microorganism extraction residue is generated as waste, so that it cannot be said to be an economically efficient method.

As another method, microorganisms that produce trehalose extracellularly (in a culture medium) have been searched, and Brevibacterium and Corynebacterium (C) have been identified.
A fermentation method has been developed in which a microorganism of the genus Orynebacterium) or the like is cultured to produce trehalose outside the cells (in the medium) (Japanese Patent Application Laid-Open No. 5-218882). However, also in this method, the accumulation rate of trehalose in the medium is about 3%.
Since the yield is as low as about (w / v), mass production of trehalose on an industrial scale requires a large-capacity fermenter and a corresponding purification equipment, which is economically problematic.
In addition, in this method, not only the eradication operation is required to obtain purified trehalose, but also complicated steps are required for removing contaminants other than trehalose or medium components produced by the strain used during culture. And

On the other hand, an enzymatic method has already been developed as a method for solving all of the various problems of the fermentation method at once. This includes maltose phosphorylase from microorganisms (Malto
se: orthophosphate β-D-glucosyltransferase) and algal trehalose phosphorylase (α, α-Trehalos
e: orthophosphate β-D-glucosyltransferase) on maltose in the presence of phosphoric acid to produce trehalose (Patent No. 1513517, Agri. Biol. Chem., 49).
Volume, 2113, 1985), and bacterial sucrose phosphorylase (Sucrose: orthophosphate α-D-glucose).
syltransferase) and basidiomycete-derived trehalose phosphorylase (α, α-Trehalosep: orthophosphate α-D-glucose
syltransferase) on sucrose in the presence of phosphoric acid to obtain trehalose (Abstracts of the 2006 Annual Meeting of the Japanese Society of Agricultural Chemistry, 3Ra14).

According to these methods, it has been reported that trehalose is produced from maltose or sucrose in a high yield of 60 to 70%. In addition, since the method used is a purified high-purity saccharide, the trehalose obtained by the enzymatic reaction is easily purified, and is considered to be an industrially advantageous method as compared with other methods. Have been. However, the enzymes used in these methods,
In particular, sources of trehalose phosphorylase are algae and basidiomycetes, such as Euglena and Maitake, and producing enzymes from these has not only been an economic problem but also technically difficult. In addition, the obtained trehalose phosphorylase and the sucrose phosphorylase and maltose phosphorylase used in combination with the trehalose phosphorylase are extremely difficult to control pH when used in combination because the optimal pH range of each enzyme is greatly different. , Very low temperature stability,
The trehalose formation reaction could only be performed at a low temperature of about 25 to 37 ° C. This suggests that microbial contamination may occur during the enzymatic reaction performed using the open-type reaction tank, which requires strict hygiene control to prevent secondary reactions. have. Furthermore, when these known enzymes are used in combination, a high-concentration raw material cannot be used because of the substrate concentration dependence of these enzymes. For this reason, this method was not economically efficient.

[0006] From the above, if a new maltose phosphorylase and trehalose phosphorylase which are easy to produce and purify, have high thermostability and do not depend on the substrate concentration can be found, maltose which can be easily obtained in large quantities can be obtained. Trehalose can be easily and efficiently produced in a high yield from the starting material.

Accordingly, an object of the present invention is to provide an enzyme reaction at a relatively high temperature and a relatively high substrate concentration using a novel maltose phosphorylase and trehalose phosphorylase satisfying the above various conditions and using maltose as a substrate. It is an object of the present invention to provide a method for producing trehalose, which is capable of adjusting pH and easily adjusting pH.

[0008]

Means for Solving the Problems The present inventors have attempted to obtain a microorganism capable of producing an enzyme having these properties required for maltose phosphorylase and trehalose phosphorylase to be used industrially. I searched the natural world widely. As a result, novel maltose phosphorylase and trehalose phosphorylase were obtained from a novel microorganism belonging to the genus Plesiomonas, and by using these maltose phosphorylase and trehalose phosphorylase, it was found that the above object was achieved, and the present invention was completed. .

That is, the present invention relates to a method for producing trehalose, which comprises reacting maltose and trehalose phosphorylase having the following physicochemical properties with maltose in the presence of phosphoric acid. Maltose phosphorylase (a) action: reversibly phosphorolytically decomposes α-1,4-glucopyranoside bond in maltose in the presence of phosphoric acid to produce glucose and β-D-glucose 1-phosphate. (B) Substrate specificity (decomposition reaction): acts on maltose,
Does not act on other disaccharides. (C) Optimum pH and stable pH range: optimal p for decomposition reaction
H is from 7.0 to 7.5, and the optimum pH for the synthesis reaction is 6.
0. Under heating conditions of 50 ° C. for 10 minutes, pH5.
It is stable within the range of 5-7.0. (D) Stability to temperature: Stable up to 45 ° C. under heating conditions of pH 6.0 and 15 minutes. (E) Range of suitable temperature for operation: The optimum temperature for the decomposition reaction is around 50 ° C, and the optimum temperature for the synthesis reaction is 50 to 55 ° C. (F) Deactivation condition: p under heating conditions of 50 ° C. for 10 minutes
Completely inactivated at H5.0 and 8.0. Also, pH
It is completely inactivated at 55 ° C in the treatment for 6.0 and 15 minutes. (G) Inhibition: copper, mercury, cadmium, zinc, N-bromosuccinimide, p-chloromercurybenzoate,
Inhibited by sodium dodecylbenzenesulfonate. (H) Isoelectric focusing by isoelectrofocusing method: 3.8 (ii) Molecular weight by SDS polyacrylamide electrophoresis: about 92,000 (molecular weight by gel filtration is about 20
0000, and is composed of two subunits).

Trehalose phosphorylase (a) action: Reversibly phosphorolytically decomposes the α-1,1-glucopyranoside bond in trehalose to produce glucose and β-D-glucose 1-phosphate. (B) Substrate specificity (decomposition reaction): It acts on trehalose and does not act on other disaccharides. (C) Optimum pH and stable pH range: The optimum pH for the decomposition and synthesis reactions is 7.0. Under heating conditions of 50 ° C. for 10 minutes, the pH is in the range of 6.0 to 9.0. (D) Temperature stability: stable up to 50 ° C. under heating conditions of pH 7.0 and 15 minutes. (E) Range of suitable temperature for operation: The suitable temperature for the decomposition reaction and the synthesis reaction is 50 to 55 ° C. (F) Deactivation condition: p under heating conditions of 50 ° C. for 10 minutes
Completely inactivated at H5.0 and 9.5. Also, pH
It is completely inactivated at 55 ° C. in the treatment for 7.0 and 15 minutes. (G) Inhibition: Inhibited by copper, mercury, cadmium, zinc, N-bromosuccinimide, p-chloromercurybenzoate. (H) Isoelectric focusing by isoelectrofocusing method: 4.5 (i) Molecular weight by SDS polyacrylamide electrophoresis: about 88,000 (molecular weight by gel filtration is about 20
0000, and is composed of two subunits). Hereinafter, the present invention will be described.

The two enzymes used in the present invention are derived from a strain newly discovered by the present inventors. This new strain was newly isolated by the present inventors from sludge on the Tago-no-Ura coast in Fuji City, Shizuoka Prefecture. This strain is known as Bergey's Mannual of Determinative Bac
teriolgy), identified in accordance with the 8th edition, volume 1, the mycological properties of the isolated strain belonged to the genus Pleseiomonas, as shown in Table 1 below.
loides were identified as related bacteria. However, the VP test and urease test are positive, and
D-mannose, D-galactose, L-arabinose, D-fructose can be assimilated, and pH
9 differs from the description in that it can grow even under alkaline conditions, and is different from the known strains in that both trehalose phosphorylase and maltose phosphorylase are produced and accumulated in the cells and in the medium (extracellular). Significantly different. In addition, the above strain Plesiomonas SH-35
Is with the National Institute of Advanced Industrial Science and Technology.
No. (FERM BP-5144).

[0012]

[Table 1]

The above-mentioned new strain was screened as follows. First, suspending the collected coastal sludge in the saline solution,
One drop of the suspension was spread on an agar medium having the following composition. The agar plate medium used was agar 2% (w / v), trehalose or maltose 1%, polypeptone 0.5%, yeast extract 0.5%, dipotassium phosphate 0.1%, magnesium sulfate heptahydrate 0. Contains 02%. Thus, the agar plate was cultured aerobically at 37 ° C. to obtain each colony that appeared on the plate, and each colony was cultured at 37 ° C. for 24 to 72 hours in a liquid medium having the above composition without agar. 180r. p.
m. With shaking. Then, each culture was added to 12,000
The cells were centrifuged at 4 ° C for 10 minutes at × g to separate the cells from the supernatant. The cells thus obtained were suspended in a small amount of 0.1 M phosphate buffer (7.0), and the activity was measured by the method described below. As a result, strains having the above-mentioned mycological characteristics could be isolated.

The above-mentioned novel microorganism is a bacterium which produces novel maltose phosphorylase and trehalose phosphorylase. Next, a method for producing these enzymes will be described. The above microorganism (FERM P-14465) is inoculated into an appropriate medium and cultured. From the viewpoint of the growth temperature of the cells, the cultivation is suitably performed at a temperature in the range of 25 to 42 ° C. and aerobically cultured for 15 to 70 hours. By culturing the above microorganism, trehalose phosphorylase and / or maltose phosphorylase used in the present invention are produced. Most of the produced enzyme is accumulated in the cells, and a part is accumulated outside the cells (in the medium). Then, maltose phosphorylase and / or trehalose phosphorylase produced and accumulated in the cells or outside the cells (in the medium) are collected. The culturing can be carried out by either a batch system or a continuous system.

The medium used for the above culture will be described.
As the carbon source, trehalose, maltose, and saccharides containing these can be used. Various organic and inorganic nitrogen compounds are used as the nitrogen source, and the medium can further contain various inorganic salts. When trehalose or a saccharide containing the substance is used as a carbon source,
The microorganism of the present invention produces trehalose phosphorylase preferentially. When maltose or a saccharide containing the substance is used as a carbon source, the microorganism of the present invention simultaneously produces maltose phosphorylase and trehalose phosphorylase. However, in this case, the production amount of trehalose phosphorylase tends to be smaller than when trehalose is used as a carbon source. Furthermore, when both trehalose and maltose or a saccharide containing these substances is used as a carbon source, trehalose phosphorylase and maltose phosphorylase can be produced simultaneously, and by controlling the amounts of trehalose and maltose, trehalose phosphorylase can be obtained. And maltose phosphorylase production ratio can also be controlled.

Also, corn steep liquor is used as a nitrogen source,
Inexpensive compounds generally used for culturing microorganisms such as soybean meal, an organic nitrogen source such as various peptones, and an inorganic nitrogen source such as ammonium sulfate, ammonium nitrate, phosphorus phosphorus, and urea can be used. It goes without saying that urea and organic nitrogen sources can also be carbon sources.

As a medium suitable for use in culturing the microorganism, for example, when preferentially producing trehalose phosphorylase, trehalose 1-2
% (W / v), yeast extract 2%, ammonium phosphate 0.1%.
Liquid medium of pH 7.0-7.5 containing 15%, urea 0.15%, salt 1%, dipotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.02% and calcium carbonate 0.2% It is appropriate to use When it is desired to simultaneously produce maltose phosphorylase and trehalose phosphorylase, maltose 1-2% (w / v), polypeptone S (manufactured by Nippon Pharmaceutical) 2-3%, ammonium phosphate 0.15%, urea 0.15% It is appropriate to use a liquid medium containing sodium chloride, 1%, dipotassium phosphate 0.1%, magnesium sulfate heptahydrate 0.02% and calcium carbonate 0.2%. As described above, when maltose is used as a carbon source, not only maltose phosphorylase but also trehalose phosphorylase is produced in a certain amount. Therefore, in order to obtain a crude enzyme (a mixture of maltose phosphorylase and trehalose phosphorylase) for trehalose production used in the present invention, it is convenient and economical to use maltose as a carbon source.

In the above culture, each enzyme is accumulated in the culture cells or in the culture supernatant. In the production method of the present invention,
Enzymes in the cells can be used as crude enzymes together with the cells. Further, a crude enzyme obtained by crushing the cells from the cells and extracting the enzyme can also be used. Further, the enzyme is also contained in the culture supernatant outside the cells, and the remaining culture solution obtained by separating the cells can be used as a crude enzyme-containing solution. Furthermore, these crude enzymes are subjected to a solvent precipitation method using ethanol, acetone, isopropanol, or the like,
It can also be used in the production method of the present invention after purification using ordinary methods such as ammonium sulfate fractionation, ion exchange chromatography, gel filtration chromatography and the like. Since maltose phosphorylase and trehalose phosphorylase have different isoelectric points, they can be separated by anion exchange chromatography. Maltose phosphorylase and trehalose phosphorylase can be used as crude enzymes or purified enzymes, respectively. Further, it is also possible to use cells having the activity of both enzymes and immobilized cells obtained by entrapping, adsorbing or chemically binding the cells in a suitable carrier. Moreover,
Each enzyme can be used as an immobilized enzyme immobilized by a known method. For example, cells can be transformed into alginic acid or κ-
It can be immobilized with carrageenan, or a product obtained by adsorbing an extraction enzyme to an anion exchange resin.

The enzymes thus obtained are maltose phosphorylase and trehalose phosphorylase having the above-mentioned physicochemical properties. The activity of these enzymes is determined by the above-mentioned Plesiomonas strain SH-35, which does not produce α-glucosidase (maltase), glucoamylase, trehalase, or the like that hydrolyzes maltose or trehalose. A simple method in which maltose or trehalose is used as a substrate and glucose produced by an enzymatic reaction in the presence of phosphate is measured by a glucose oxidase method is applicable. The synthesis reaction can be performed by using a mixed solution of β-D-glucose monophosphate and glucose as a substrate and measuring the inorganic phosphoric acid generated by the enzymatic reaction by a conventional method.

Enzyme activity measurement method (1) Decomposition reaction: 0.01 ml of the enzyme solution was added to 0.5 ml of a 20 mM maltose or trehalose solution dissolved in a 50 mM phosphate buffer (pH 7) and reacted at 50 ° C. for 15 minutes. After that, the enzyme reaction is stopped by heating for 3 minutes in a boiling water bath. Next, after cooling in running water, the produced glucose is measured by a glucose oxidase method (manufactured by Wako Pure Chemical Industries, Ltd., glucose C-II test Wako). Here, one unit of enzyme activity is 1 minute per minute under the same conditions.
It is defined as the amount of enzyme that produces μmole (micromoles) of glucose. (2) Synthesis reaction: 0.05 ml in 0.15 ml of a mixed solution of 27 mM β-D-glucose monophosphate Na salt dissolved in 70 mM HEPES buffer (pH 7.0) and glucose of the same concentration Add enzyme solution at 50 ℃
, And then heated in a boiling water bath for 2 minutes to stop the enzyme activity. Next, the produced inorganic phosphoric acid is measured using P-test Wako manufactured by Wako Pure Chemical Industries, Ltd. Here, one unit of enzyme activity is defined as the amount of enzyme that produces 1 micromol (μmole) of inorganic phosphoric acid per minute under the same conditions.

In the production method of the present invention, the two enzymes are allowed to act on maltose in the presence of phosphoric acid. Phosphoric acid is necessary for maltose to undergo phosphorolysis,
It is appropriate that the concentration is in the range of 0.1 to 500 mmol / L, preferably 5 to 10 mmol / L in the reaction solution. As the phosphoric acid, for example, inorganic phosphoric acid such as orthophosphoric acid, sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, and salts thereof can be used. Maltose is used as a substrate. However, maltose may be a purified product or a sugar containing maltose. The maltose concentration is suitably in the range of 10 to 600 g / l, preferably 200 to 400 g / l, from the viewpoints of a solution having a viscosity that is easy to operate and the yield per operation, that is, economy.

The concentration of the enzyme can be appropriately determined in consideration of the trehalose production rate, reaction time, and the like. However, it is usually appropriate to use 0.1 to 50 units / g-substrate. The reaction temperature is appropriately determined in consideration of the optimum temperature of the enzyme, the reaction time, etc., and is preferably in the range of 30 to 65 ° C., preferably 45 to 55 ° C. in consideration of prevention of contamination by various bacteria. . The reaction pH is 5.0 to 9.0, preferably 6.0 to 7.0, in consideration of the optimum pH of the enzyme.
Suitably, it is in the range of 0. The reaction time is appropriately determined depending on the production rate of trehalose, the amount of the enzyme added, the capacity of the reaction vessel, and the like. However, in general, it is appropriate that the range is about 50 to 80 hours on an industrial scale.

The trehalose produced by the above reaction is
In the same manner as in the method for purifying starch sugar, it can be separated and purified by diatomaceous earth filtration, desalting with an ion exchange resin, fractionation by ion exchange chromatography, concentration and crystallization.

[0024]

EXAMPLES The present invention will be further described below with reference to Examples and Reference Examples. Reference Example 1 Production of intracellular and extracellular trehalose phosphorylase Trehalose 1% (w / v), yeast extract (manufactured by Difco)
2%, ammonium phosphate 0.15%, urea 0.15%,
It was obtained by previously culturing overnight in 20 liters of a pH 7.5 liquid medium containing 1% of sodium chloride, 0.1% of dipotassium phosphate, 0.02% of magnesium sulfate / heptahydrate and 0.2% of calcium carbonate in the same medium as above. Plesiomonas SH-35 strain (FE
RM BP-5144) Aseptically add 1 liter, 37 ℃, 3
00 rpm, ventilation rate 1v. v. Under aeration conditions, aeration and agitation culture was performed for 24 hours. The trehalose phosphorylase activity of the main culture was measured and found to be 1.5 units per ml of the culture. Maltose phosphorylase activity was also measured, but the activity was trace. Then, 12,0
After centrifugation at 00 × g and 4 ° C. for 10 minutes, about 160 g of cells (when wet) and 19.5 liters of supernatant were obtained, and concentrated with a Romicon UF membrane (YM-30). , About 1
One liter (about 6,400 units) of extracellular concentrated crude enzyme was obtained. As a result of measuring the enzyme activity in the supernatant, the activity was about 25% (7.5 × 10 ³ units) of the total activity (about 30 × 10 ³ units). In addition, about 10m
M was thoroughly washed with a phosphate buffer (pH 7), suspended in 500 ml of the same buffer, and disrupted with an ultrasonic cell disrupter. The trehalose phosphorylase activity was measured by a conventional method. About 75% (22.5 × 10 ³ units) of the activity was contained in the cells.

Reference Example 2 (Production of Intracellular and Extracellular Enzymes Containing Maltose Phosphorylase and Trehalose Phosphorylase) Reference Example 1
5% of trehalose and maltose in the medium components used in the above and yeast extract in polypeptone FC (manufactured by Nippon Pharmaceutical Co., Ltd.)
(W / v), and SH-3 in a similar manner.
Five strains (FERM BP-5144) were cultured. As a result of measuring the maltose phosphorylase activity and the trehalose phosphorylase activity of the main culture solution, 0.5 unit of maltose phosphorylase activity and 0.45 unit of trehalose phosphorylase were contained per 1 ml of the culture solution. Then, the cells were centrifuged similarly to about 150 g of cells (when wet) and 1
9.6 liters of supernatant was obtained. Then, as a result of measuring maltose phosphorylase activity in the cells and supernatant,
About 78% (about 1%) of the total activity of maltose phosphorylase
(000 units) was contained in the cells, and about 22% was contained extracellularly (culture supernatant). About 82% (about 9,000 units) of the total activity of trehalose phosphorylase was contained in the cells, and about 18% was contained extracellularly (culture supernatant). The culture supernatant was concentrated in the same manner as in Reference Example 1,
About 1 liter of concentrated enzyme is obtained, in which about 1,700 units of maltose phosphorylase and 1,43
0 units of trehalose phosphorylase were included.

Reference Example 3 Purification of Trehalose Phosphorylase 500 ml of the disrupted cell suspension obtained in Reference Example 1
The mixture was adjusted to 30% saturation with ammonium sulfate and left at 4 ° C. overnight.
Next, the supernatant was centrifuged to remove the precipitate, and ammonium sulfate was further added to the supernatant to obtain 70% saturation. The precipitate formed by standing at 4 ° C. overnight was collected by centrifugation, and dissolved in 20 mM phosphate buffer (pH 7) containing 0.4 M sodium chloride.
Dialyzed thoroughly against the same buffer.

Next, the solution was passed through a DEAE-Fractogel (manufactured by Merck) column equilibrated with the same buffer solution to adsorb the enzyme. The adsorbed enzyme was eluted by a concentration gradient method of 0 M to 0.6 M salt contained in the same buffer solution, and then concentrated by a UF membrane (YM-30, manufactured by Amicon). The concentrated enzyme was purified by gel filtration chromatography using a Sephacryl S-300 (Pharmacia) column equilibrated with the same buffer containing 0.2 M sodium chloride. The obtained active fractions were collected, sufficiently dialyzed against the same buffer containing 1.5 M ammonium sulfate, and passed through a phenyl toyopearl (Tosoh Corporation) column equilibrated with the same buffer containing 1.5 M ammonium sulfate. Then, the enzyme was adsorbed. The adsorbed enzyme is contained in the buffer as described above.
After elution by a concentration gradient method of 5M to 0M ammonium sulfate, the obtained active fractions were collected and sufficiently dialyzed against the same buffer containing 0.2M sodium chloride. After concentrating using the UF membrane described above, 0.
The gel filtration chromatography was performed again with Superdex 200 (manufactured by Pharmacia) equilibrated with the same buffer solution containing 2M salt, and the active fraction obtained was concentrated by the method described above, and the polyacrylamide gel disk was used. In the electrophoresis method (PAGE) and the SDS-PAGE method, a homogeneous intracellular trehalose phosphorylase purified enzyme (5
ml, about 7,880 units, activity yield about 35%). The extracellular enzyme is also purified and concentrated in the same manner as described above, and the trehalose phosphorylase purified enzyme (5
ml, about 2,400 units, activity yield about 32%).

Reference Example 4 Purification of Maltose Phosphorylase The maltose phosphorylase-containing bacterial cells and the culture supernatant obtained in Reference Example 2 were purified in the same manner as in Reference Example 3 to obtain PAG.
Uniform intracellular maltose phosphorylase (5 ml, about 2,800 units, activity yield about 28%) and extracellular trehalose phosphorylase (5 ml, about 425 units, activity yield about 25%) in the method E and SDS-PAGE method Was obtained respectively.

Reference Example 5 Maltose phosphorylase and trehalose phosphoryl
Enzyme-chemical properties of the enzyme Plesiomonas strain SH-35 obtained in Reference Examples 1 and 2
The general enzymatic properties of the novel maltose phosphorylase and trehalose phosphorylase produced by (FERM P-14465) are shown below. As a result of the preliminary experiment, both the intracellular and extracellular enzymes showed almost the same physicochemical properties. Therefore, here, the properties of the intracellular enzymes are shown.

(A) Action 1% dissolved in 10 mM phosphate buffer (pH 7.0)
To the (w / v) maltose and trehalose solution, maltose phosphorylase and trehalose phosphorylase were added in an amount of 5 units (decomposition reaction) per 1 g of the substrate, and reacted at 50 ° C. for 5 hours, followed by heating in a boiling water bath for 3 minutes. As a result of measuring the sugar in the saccharified solution obtained by inactivating the enzyme by high performance liquid chromatography, glucose and glucose monophosphate were respectively detected. Also, 10m
Mixed solution of 1% (w / v) glucose and β-D-glucose monophosphate sodium salt or α-D-glucose monophosphate sodium salt dissolved in M Tris (HCl) buffer (pH 7.0) Was used as a substrate, maltose phosphorylase and trehalose phosphorylase were added in 5 units each to 1 g of the substrate, reacted at 50 ° C. for 5 hours, treated as described above, and the sugar composition was measured. As a result, glucose and β-D- Maltose and trehalose were detected from glucose monophosphate, respectively, but no synthesis reaction of disaccharide from glucose and α-D-glucose monophosphate was detected.

The analysis of the produced sugar was performed by the following method. That is, the insoluble matter in the saccharified solution obtained by heat inactivation is reduced by 0.4%.
A filtrate obtained by filtration with a 5 μm membrane filter was used as a test sugar solution, and was subjected to YMC-Pack, ODS-AQ (A
(Q-304, manufactured by YMC) column. In addition, water was used for the mobile phase, the column temperature was 30 ° C., and a differential refractometer was used for detection. (B) Substrate specificity (decomposition reaction) The activity when various saccharides were used in place of the substrate (maltose or trehalose) shown in the activity measurement method (decomposition reaction) described above was expressed as a relative activity. Table 2 shows the results
Shown in

[0032]

[Table 2]

(C) Optimum pH and stable pH range The optimum pH for the decomposition and synthesis reactions was measured using the purified enzyme. As a result, as shown in FIG. 1A, the optimum pH for the maltose phosphorylase decomposition reaction (indicated by a white circle) is 7.0 to 7.5, and the synthesis reaction (indicated by a black circle) is 6.
0 was optimal. As shown in FIG. 1B, trehalose phosphorylase was optimally 7.0 for both the decomposition reaction (open circles) and the synthesis reaction (black circles). In the case of a pH decomposition reaction, a 20 mM phosphate buffer was used. In the case of a synthesis reaction, MES (pH 5.5 to 6.5) and MOPS (pH
Values 6.5-7.0), HEPES (pH 7.0-8.
0) and Tris-HCl (pH 7.5 to 9.0). In addition, both purified enzymes were treated in each buffer for 10 minutes at 50 ° C., and their residual enzyme activities were measured by a decomposition reaction. As shown in FIG. 2, maltose phosphorylase (open circles) had a pH of 5.5. In the range of 5-6.5, trehalose phosphorylase (solid circle) indicates p
H was stable up to 6.0 to 9.0. The pH was acetic acid (pH 5.0 to 5.5) and phosphoric acid (pH 6.0 to 8.0).
0) and carbonic acid (pH 8.9).

(D) Appropriate temperature of action As a result of measuring the appropriate temperature of the decomposition reaction and the synthesis reaction of both enzymes, as shown in FIG. 3, maltose phosphorylase (A, open circle: decomposition reaction, closed circle: synthesis reaction), trehalose phosphorylase (B, open circles: decomposition reaction, black circles: synthesis reaction) In both cases, the decomposition reaction was at 50 ° C, and the synthesis reaction was at 50 to 55 ° C. (E) Conditions for inactivation by temperature: Treat with maltose phosphorylase and trehalose phosphorylase at a stable pH of pH 6.0 and 7.0, respectively, for 15 minutes, and compare the remaining inactivation with untreated one by a conventional method. Was measured by As a result, as shown in FIG. 4, maltose phosphorylase (open circle) was 55%.
° C, trehalose phosphorylase (solid circle)
It was completely inactivated at 0 ° C. (F) Inhibitors The activity in the synthesis reaction of both enzymes was measured in the presence of various inhibitors, and the results are shown in Table 3 as relative activities with the activity without addition being 100%.

[0035]

[Table 3]

(G) Isoelectric focusing method using isoelectric point isogel (manufactured by FMC BioProducto). The isoelectric points of both enzymes were measured. As shown in FIG. 5, maltose phosphorylase was 3.8, and trehalose was trehalose. The phosphorylase was 4.5. (H) Molecular weight The molecular weight of both purified enzymes was measured by SDS-PAGE and gel filtration using Sephacryl S-200.
As a result, as shown in FIG. 6, in the gel filtration method, the molecular weights of both enzymes were about 200,000, but SDS-PA
In the GE method, maltose phosphorylase is about 92,000.
Since the amount of trehalose phosphorylase was about 88,000, it was expected that each of these enzymes was composed of two subunits. Tables 4 and 5 compare the above physicochemical properties with known maltose phosphorylase and trehalose phosphorylase.
Summarized in

[0037]

[Table 4]

[0038]

[Table 5]

Example 1 In the same manner as in Reference Examples 3 and 4, a 10, 20, 30, and 40% (w / v) maltose solution dissolved in 10 ml of 10 mM phosphate buffer (pH 6) was added. The prepared intracellular purified trehalose phosphorylase and intracellular purified maltose phosphorylase were each added at 5 units (degrading activity) per 1 g of substrate weight, and reacted at 55 ° C. for 70 hours. After the completion of the reaction, the reaction solution was heated at 100 ° C. for 5 minutes to inactivate the enzyme, and the trehalose content in the saccharified solution obtained by measurement was measured. As a result, 58.2, 58.1, and 58.6.57.9% of trehalose were formed with respect to the substrate weight, respectively. The quantification of trehalose was performed by the following method. That is, water was added to the heat-inactivated saccharified solution to make it about 1% (W / V), and 0.5 ml of the saccharified solution was added to glucoamylase (Pure Grade 30 manufactured by Seikagaku Corporation).
U / mg) at 50 ° C., pH 5.0.
For 1 hour to completely decompose unreacted maltose into glucose. Then, the glucoamylase was inactivated by heating in a boiling water bath at 100 ° C. for 5 minutes, and the resulting insoluble protein was removed with a 0.45 μm membrane filter.
Liquid chromatography using a Pack ODS-AQ (AQ-304, manufactured by YMC) column (HPLC
Method). The measurement was performed using water as the mobile phase, the column temperature was set to 30 ° C., and the differential refractometer was used for detection.

Example 2 2 dissolved in 10 ml of 5 mM phosphate buffer (pH 6)
0% (W / V) high maltose syrup (manufactured by Nippon Shokuhin Kako Co., Ltd., trade name MC-95, sugar composition: glucose 2.5
%, Maltose 95.2%, maltotriose 0.8
%, Maltotetraose 1.5%), 5 units of trehalose phosphorylase extracellularly purified and 4 units of extracellularly purified maltol phosphorylase prepared in the same manner as in Reference Examples 3 and 4 were added per 1 g of substrate weight. 1
The reaction was carried out in the same manner as described above. HPLC generated trehalose
As a result of measurement by the method, 54.3 with respect to the weight of the substrate used.
%Met.

Example 3 Approximately 1 kg of cells containing trehalose phosphorylase and maltose phosphorylase (when wet) were prepared in the same manner as in Example 2. As a result of measuring the enzymatic activity (decomposing activity) by a conventional method, the cells showed 55 units / g (when wet) of trehalose phosphorylase and 68 units / g.
(When wet) with maltose phosphorylase activity. 1 kg of the prepared cells is added to a 10 mM phosphate buffer (pH
The mixture was added to 250 liters of a 30% (W / V) maltose solution contained in 6.5), reacted at 50 ° C. for 80 hours, and centrifuged to remove the cells. A part of the obtained supernatant was treated with glucoamylase, and the sugar composition was measured by HPLC. As a result, trehalose was 58.1%, glucose was 39.6%, and glucose monophosphate was 2.3%. Then, about 240 liters (solids 72 kg) of the obtained saccharification supernatant liquid, 0.1% of a solid crude glucoamylase (manufactured by Shin Nippon Chemical Co., Ltd., trade name Sumiteam # 3,000) was added to the solids weight. PH 5.5, 20 at 55 ° C.
The maltose remaining after the time saccharification was almost completely hydrolyzed to glucose. Then, the purified saccharified solution (about 65% solids) having a concentration of about 75% (W / V) is purified by decolorization of activated carbon, desalting with an ion exchange resin, or the like, and concentrated under reduced pressure.
kg).

[0042]

According to the present invention, trehalose can be produced enzymatically using novel maltose phosphorylase and trehalose phosphorylase. In the method for producing trehalose of the present invention, pH can be easily adjusted, and trehalose can be obtained in high yield even at a high substrate (maltose) concentration. Furthermore, the reaction temperature can be increased, and trehalose can be obtained in high yield by selecting a higher reaction temperature. Further, since the enzyme reaction at a high temperature is possible, it is possible to avoid contamination by various bacteria during the reaction.

[Brief description of the drawings]

FIG. 1 shows a reaction optimal curve of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas strain SH-35 [symbols: decomposition reaction (○), synthesis reaction (●)].

FIG. 2 shows the pH stability of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas strain SH-35 [symbols: maltose phosphorylase (）), trehalose phosphorylase (●)].

FIG. 3 shows the optimal temperature for maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas strain SH-35 [symbols: decomposition reaction (○), synthesis reaction (●)].

FIG. 4 shows the temperature stability of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas strain SH-35 [symbols: maltose phosphorylase (○), trehalose phosphorylase (●)].

FIG. 5 shows isoelectric points of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas strain SH-35.

FIG. 6 shows the molecular weights of maltose phosphorylase and trehalose phosphorylase produced by Plesiomonas strain SH-35.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ identification code FI (C12N 9/10 C12R 1:01) (58) Investigated field (Int.Cl. ⁷ , DB name) C12P 19/00-19 / 64 C12N 9/00-9/99 C12N 1/00-1/38 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A process for producing trehalose, which comprises reacting maltose and trehalose phosphorylase having the following physicochemical properties with maltose in the presence of phosphoric acid. Maltose phosphorylase (a) action: reversibly phosphorolytically decomposes α-1,4-glucopyranoside bond in maltose in the presence of phosphoric acid to produce glucose and β-D-glucose 1-phosphate. (B) Substrate specificity (decomposition reaction): acts on maltose,
Does not act on other disaccharides. (C) Optimum pH and stable pH range: optimal p for decomposition reaction
H is from 7.0 to 7.5, and the optimum pH for the synthesis reaction is 6.
0. Under heating conditions of 50 ° C. for 10 minutes, pH5.
It is stable within the range of 5-7.0. (D) Stability to temperature: Stable up to 45 ° C. under heating conditions of pH 6.0 and 15 minutes. (E) Range of suitable temperature for operation: The optimum temperature for the decomposition reaction is around 50 ° C, and the optimum temperature for the synthesis reaction is 50 to 55 ° C. (F) Deactivation condition: p under heating conditions of 50 ° C. for 10 minutes
Completely inactivated at H5.0 and 8.0. Also, pH
It is completely inactivated at 55 ° C in the treatment for 6.0 and 15 minutes. (G) Inhibition: copper, mercury, cadmium, zinc, N-bromosuccinimide, p-chloromercurybenzoate,
Inhibited by sodium dodecylbenzenesulfonate. (H) Isoelectric focusing by isoelectrofocusing method: 3.8 (ii) Molecular weight by SDS polyacrylamide electrophoresis: about 92,000 (molecular weight by gel filtration is about 20
0000, and is composed of two subunits). Trehalose phosphorylase (a) action: reversibly phosphorolytically decomposes the α-1,1-glucopyranoside bond in trehalose to produce glucose and β-D-glucose 1-phosphate. (B) Substrate specificity (decomposition reaction): It acts on trehalose and does not act on other disaccharides. (C) Optimum pH and stable pH range: The optimum pH for the decomposition and synthesis reactions is 7.0. Under heating conditions of 50 ° C. for 10 minutes, the pH is in the range of 6.0 to 9.0. (D) Temperature stability: stable up to 50 ° C. under heating conditions of pH 7.0 and 15 minutes. (E) Range of suitable temperature for operation: The suitable temperature for the decomposition reaction and the synthesis reaction is 50 to 55 ° C. (F) Deactivation condition: p under heating conditions of 50 ° C. for 10 minutes
Completely inactivated at H5.0 and 9.5. Also, pH
It is completely inactivated at 55 ° C. in the treatment for 7.0 and 15 minutes. (G) Inhibition: Inhibited by copper, mercury, cadmium, zinc, N-bromosuccinimide, p-chloromercurybenzoate. (H) Isoelectric focusing by isoelectrofocusing method: 4.5 (i) Molecular weight by SDS polyacrylamide electrophoresis: about 88,000 (molecular weight by gel filtration is about 20
0000, and is composed of two subunits). 2. The method according to claim 1, wherein the maltose phosphorylase and the trehalose phosphorylase are intracellular enzymes or extracellular enzymes obtained by culturing a microorganism described in No. 5144 of Life Science Research Institute. 3. The method according to claim 1, wherein the enzymatic reaction is performed in a temperature range of 30 to 65 ° C.