JPS6360998B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for producing trehalose. Trehalose, also called mycose, is a disaccharide widely distributed in natural products such as yeast, mold, and seaweed. Since it is extremely stable compared to other disaccharides such as sucrose, it is widely used as a sweetener, bulking agent, etc., and as an energy source. Conventionally, methods for obtaining this substance include extraction from the above-mentioned natural products or microorganisms belonging to the genus Arthrobacter [Agric.Biol.
Chem., 33 , No. 2, 190, 1969, Suzuki T.
Tanaka K & Kinoshita S] and microorganisms belonging to the genus Nocardia [Unexamined Japanese Patent Application Publication No. 1973-
154485], but these methods are difficult to mass produce or require operational, equipment, and energy costs to refine and obtain until they can be safely used as food. A large amount of investment is required, and currently no technology has been established for supplying trehalose at low cost and in large quantities. The present inventors have conducted extensive research with the aim of solving all the drawbacks of the conventional methods described above and providing a new method that can obtain trehalose in large quantities at low cost and with high yield. As a result, it has been found that trehalose can be easily produced in high yield when maltose, which is available at low cost and in large quantities, is used as a raw material and maltose phosphorylase and trehalose phosphorylase are combined to act on it. The present invention was completed based on this new knowledge. That is, the present invention relates to a method for producing trehalose, which is characterized in that trehalose is produced by treating maltose with maltose phosphorylase and trehalose phosphorylase. According to the method of the present invention, trehalose can be easily and efficiently obtained from maltose as a raw material in high yield by using the above two different enzymes in combination. The conversion rate (ie, yield) of maltose to trehalose by the method of the present invention is actually around 60%, which is completely unexpected from the known properties of each of the enzymes mentioned above. In the present invention, the enzyme treatment of raw maltose with a combination of maltose phosphorylase and trehalose phosphorylase is usually carried out in the presence of phosphoric acid in a suitable solvent. Each enzyme used here may be a known commercial product or one obtained by culturing microorganisms that produce the enzyme. In particular, examples of maltose phosphorylase include Neisseria meningitidis, (J.Biol.Chem.
199, 153-163 (1952) (and Lactobacillus
Enzymes produced by Lactobacillus brevis and the like are preferred. In addition, Euglena glatilis (Trehalose phosphorylase)
gracilis) (J.Biol.Chem., 247 , 3223-3228
(1972)) is preferred. The amounts of these enzymes used in combination are not particularly limited and are determined as appropriate. Maltose phosphorylase per mole of maltose, which is usually used as a raw material, is 0.1 unit or more,
Preferably about 350 units or more, and trehalose phosphorylase about 0.05 units or more, preferably about 200 units
It is better to consider it as more than a unit. The units expressing the amount of each enzyme are defined by the method described below. In addition, the phosphoric acid present in the enzyme treatment system is
In addition to orthophosphoric acid, various ordinary inorganic phosphoric acids and their salts such as sodium phosphate, potassium phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, etc. can be used. Among these, potassium dihydrogen phosphate is preferred. The phosphoric acid is also usually preferably used in the form of a phosphate buffer.
Therefore, the solvent constituting the enzyme treatment system is usually the water constituting the slow solution. Furthermore, various solvents that do not adversely affect the enzyme reaction, such as preferably imidazole-hydrochloric acid solution, can be added to the enzyme treatment system. The usage ratio (concentration) of the above phosphoric acid is not particularly limited, but there is usually about 0.1 mol or more, preferably about 0.5 to 1.5 mol of phosphoric acid (or its salt) per 1 mol of maltose as a raw material. It is best to define the amount (concentration) as The enzymatic reaction is usually completed in about 24 hours at a temperature of about 20 to 50°C, preferably about 37°C. The pH of the enzyme treatment reaction system is within a range in which none of the enzymes used is inactivated, usually preferably about 5 to 8, more preferably about 6 to 7. After the enzyme treatment reaction of the present invention is completed, the reaction solution is heated to inactivate the enzyme, and then centrifuged.
The precipitate is removed by any conventional means, and the resulting supernatant is purified by e.g.
1), manufactured by The Dow Chemical Company), CM-cellulose, etc., can be purified as a sugar solution by treatment with an anion type ion exchange resin such as CM-cellulose. type anion exchange resin, then 10
By elution with an aqueous solution of about mM potassium borate, trehalose is eluted first, and after the elution of trehalose is completed, unreacted maltose and by-product glucose are sequentially eluted. The trehalose fraction eluted in the above manner is passed through a cation-type ion exchange resin, the effluent is returned to near neutrality with ammonia, and after concentration, lower alcohols such as methanol and ethanol are added and distilled to remove boric acid. By removing , columnar trehalose crystals can be obtained. The method of the present invention also includes the immobilization of the above-mentioned two enzymes separately or in advance by mixing them in the above-mentioned appropriate proportions, and then immobilizing them according to known immobilization methods such as gel entrapment method, microcapsule method, etc. If each immobilized enzyme is immobilized separately, the resulting immobilized enzymes may be mixed in an appropriate ratio). According to this continuous method using an immobilized enzyme, there is very little contamination of impurities in the reaction system, and not only can even higher purity trehalose be easily obtained, but also the immobilized enzyme used can be used repeatedly, and the above-mentioned There is an advantage that trehalose can be produced more efficiently. The method using the above-mentioned immobilized enzyme is carried out by packing it into a suitable column and passing the raw material maltose dissolved in a phosphate buffer solution through this, and the passing liquid (enzyme-treated liquid) is It is purified using an ion exchange resin in the same way. Thus, according to the present invention, trehalose can be obtained at low cost, in large quantities, and in high yield. Examples will be given below to explain the present invention in more detail. Each enzyme used in each example was prepared by the following method. Preparation of maltose phosphorylase Lactobacillus brevis
brevis) IFO3345 cultured cells were crushed in a cell mill using glass beads (0.1 to 0.2 mm in diameter) and then centrifuged to collect 535 ml of supernatant (enzyme activity 2600 units). Next, add 2% to 515 ml of this supernatant.
Add 80ml of protamine sulfuric acid, stir at 12℃ for 30 minutes, centrifuge, and 580ml of supernatant (enzyme activity
2395 units) were recovered. Add 140 g of ammonium sulfate (40% saturation), stir at 2°C for 30 minutes, centrifuge to remove the precipitate, add 170 g of ammonium sulfate (80% saturation),
The mixture was allowed to stand at ℃ for 12 hours, and the resulting precipitate was obtained by centrifugation. Dissolve the precipitate in 100ml of 5mM citrate buffer (PH6.6) and incubate at 2°C with a large amount of the same buffer.
Dialysis was performed for 20 hours under cooling. DEAE was equilibrated with 5mM citrate buffer (PH6.6).
- passed through a cellulose column (4 x 27 cm in diameter);
It was adsorbed. 5mM citrate buffer (PH6.6)
After washing with 0-1M NaCl (citrate buffer,
Protein was eluted using a linear gradient of pH 6.6). Perform fractionation in 20 ml portions, collect active fractions, and add again to 5 mM citrate buffer (PH6.6).
20 hours of dialysis was performed on DEAE-
Column chromatography using cellulose was performed. Ammonium sulfate was added to the enzyme solution obtained in this operation to make it 80% saturated, and the mixture was left at 4°C overnight. The precipitate was collected by centrifugation and
It was dissolved in mM citrate buffer (PH6.6) to obtain 20 ml of crude enzyme preparation. Enzyme activity was 1638 units. The units in the above maltose phosphorylase were determined as follows. That is,
0.2 molar _K2HPO4 -citrate buffer ( _PH
Add 0.6ml of enzyme solution to 0.ml of 5.2) and incubate at 37°C.
Let stand for a minute to preheat. At this time, a control using water instead of the enzyme solution was prepared at the same time, and the same procedure was repeated. Next, add 0.2 ml of 0.2M maltose and react at 37°C for 10 minutes. The reaction is then stopped by immersing it in a boiling water bath. Next, the amount of glucose produced in this reaction solution is determined by the glucose oxidase method. The amount of enzyme that produced 1 μmol (180 μg) of glucose per minute under these conditions was defined as 1 unit. Preparation of trehalose phosphorylase Euglena glatilis
gracilis var.
Suspended in 600ml. 200ml of this was cooled in ice water and treated with ultrasound to crush the bacterial cells.
A supernatant was obtained by centrifugation at 8,000 rpm, and the precipitate was further washed, and the supernatant and washing solution were combined to obtain 1,610 ml of a crude enzyme extract. This solution was treated with protamine, and the precipitated enzyme was collected and resuspended in the same buffer as above to obtain 300 ml of a crude enzyme preparation. Its enzyme activity was 86 units. The units in the above trehalose phosphorylase were determined as follows. That is,
20ml of substrate solution (100mM imidazole-HCl buffer (PH7.0), 100mlM phosphate buffer (PH7.0),
Add 20μ of enzyme solution to 100mM trehalose and react at 37°C for 10 minutes, then add 0.5ml of Somogi reagent and incubate in a boiling water bath.
Heat for 15 minutes. Next, after cooling, add 0.5 ml of Nelson's reagent and leave at room temperature for 20 minutes. Next, add 4ml of water and compare the colors at 500nm.
Determine the amount of glucose produced in the reaction system. The amount of enzyme that produced 1 μmol of glucose per hour under these conditions was defined as 1 unit. Example 1 A liquid mixture having the following composition was prepared. Maltose phosphorylase 0.2 units/ml Trehalose phosphorylase 0.158 units/ml Potassium dihydrogen phosphate/citrate buffer 40mM
(PH7.0) Imidazole/hydrochloric acid buffer 40mM (PH7.0) Add 5g of maltose to 50ml of this mixture and heat at 37°C.
Stirred for 24 hours. As a result, 2.95 g of trehalose was produced, and 1.0 g of unreacted maltose. Next, this was heated at 100° C. for 10 minutes to stop the reaction, and the precipitate was removed by centrifugation to obtain a supernatant. The trehalose concentration of this supernatant was 59 mg/ml. Next, water was added to the supernatant liquid to make 100 ml, and the mixture was applied to a column of Dowex 1 (OH ^- type, φ2×23, approximately 72 ml, manufactured by Dow Chemical Company), which had been thoroughly washed with water. Next, approximately 140 ml of water was poured into the column to separate the passing liquid and washing liquid. combined. Potassium tetraborate was added to this solution and the concentration was adjusted to 1 mM. Add this solution to Dowex1 (boric acid type, φ
x33, approximately 103 ml, Dow Chemical Company) column to adsorb trehalose, and then eluted with 10 mM potassium tetraborate. The eluate was fractionated into 18 ml portions. Trehalose was obtained in elution fractions No. 3 to 72. Collect this No. 3 to 72 fractions and use Dowex50W.
(H ⁺ type, φ2×33, approx. 103ml, manufactured by Dow Chemical Company)
column, washed with about 200 ml of water, and combined with the previous passed-through solution. The pH was returned to neutral with ammonia, and the mixture was concentrated using a rotary evaporator. After adding methanol and repeating distillation to remove boric acid, the mixture was concentrated under reduced pressure until it became syrupy, and 48 ml of ethanol was added to obtain a precipitate. The weld was heated to 50°C and a small amount of water was added to dissolve the precipitate. then 4
After 2 days, the resulting crystals were collected, washed with a small amount of cold ethanol, and dried under reduced pressure with a drying agent (silica gel + phosphorus pentoxide) overnight to obtain about 2.4 g of columnar trehalose crystals. The thus obtained crystals (hereinafter referred to as below) were subjected to various confirmation tests (1
When steps 5) to 5) were carried out, it was confirmed that the crystals obtained in this example were highly pure trehalose as shown below. Confirmation Test 1 Melting point: mp = 133-134°C The same value was also shown in the mixed melting test. Confirmation test 2 Optical rotation: [α] ²¹ _D = +176.4° (Cl water): [α] ²¹ _D = +176.1° Confirmation test 3 Comparison of IR with IR shows 2400 to 2100 cm ^-1
There were slight differences in the results, but otherwise they were completely consistent.
The IR diagram is shown in Figure 1 and the IR diagram is shown in Figure 2. Confirmation Test 4 Gas Chromatography A comparison of the two gas chromatography results revealed complete agreement. The purity of is 118 compared to
%. A gas cochromatography analysis diagram is shown in Figure 3. In the figure, 1 indicates a 50:50 mixture of , 2 indicates a , and 3 indicates a 50:50 mixture of and . The measurement conditions in FIG. 3 are as follows. Column: Metal column (1m) Stationary phase: 5% SE-30/Chrom sorb Column temperature: 160→250℃, heating at 5℃/min Inlet temperature and detection temperature: 285℃ Flow rate: 30ml/min Internal standard: 1 = Sucrose, 2 = Trehalose Confirmation Test 5 The reducing power of the crystals of purity was measured by the Somgyi-Nelson method, and it was found that the crystals had no reducing power. Add 4 ml of an aqueous solution to 0.5 ml of 0.1M acetate buffer (PH5.6), and
Add 0.1 ml of 100 μg/ml trehalase aqueous solution 37
Glucose produced by reacting at ℃ for 30 minutes was mixed with glucose oxidase solution (glucose oxidase
102 % purity. Example 2 Add 800 g of maltose to 50 ml of a mixture consisting of the following composition:
When 475 mg of trehalose and 17.5 mg of maltose were obtained, 475 mg of trehalose and 17.5 mg of maltose were obtained.
Crystals of trehalose were prepared in the same manner as in Example 1.
Got 400mg. Maltose phosphorylase 0.2 units/ml Trehalose phosphorylase 0.1148 units/ml Phosphate-citrate buffer 40mM (PH6.3) Imidazole-hydrochloric acid solution 40mM (PH6.3)

[Brief explanation of drawings]

FIG. 1 is an infrared absorption spectrum analysis diagram of trehalose obtained by the present invention, FIG. 2 is the same analysis diagram of commercially available trehalose, and FIG. 3 is a gas chromatography analysis diagram of each of the above-mentioned trehaloses.

Claims (1)

[Claims] 1. A method for producing trehalose, which comprises producing trehalose by treating maltose with maltose phosphorylase and trehalose phosphorylase.