JPH08131166A - Heat-resistant trehalose phosphorylase, its production, bacterum to be used for its production, and production of trehalose using the enzyme - Google Patents

Abstract

PURPOSE: To obtain the subject new enzyme active at elevated temperatures, thus capable of giving trehalose in an industrially advantageous way in a short reaction time with reduced sundry germ contamination, by culturing trehalose phosphorylase-productive microbes belonging to the genus Bacillus. CONSTITUTION: Trehalose phosphorylase-productive microbes belonging to the genus Bacillus [e.g. Bacillus stearothermophilus SK-1 (FERM P-14567)] is inoculated into a medium and subjected to aerating agitation culture at 55 deg.C for 72hr followed by centrifugal separation to take out the supernatant; ammonium sulfate is then dissolved in the supernatant so as to come to 40-60% in the degree of saturation, and the resultant protein precipitate is recovered by centrifugal separation and then dissolved in a 10mM potassium phosphate-citric acid buffer solution (pH6.0) followed by purification through e.g. ion exchange chromatography, hydrophobic chromatography, etc., thus obtaining the objective new heat-resistant trehalose phosphorylase retaining >=95% activity after treated in a pH6.0 buffer solution at 50-65 deg.C for 15min and capable of producing trehalose in an industrially advantageous way.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel trehalose phosphorylase, a method for producing the same, a bacterium used for the production,
And a use thereof, and more specifically, a novel trehalose phosphorylase having high thermostability produced by a thermophilic bacterium belonging to the genus Bacillus, a method for producing the same, the thermophilic bacterium, and production of trehalose using the enzyme. Regarding the method.

[0002]

BACKGROUND OF THE INVENTION Trehalose is a disaccharide widely distributed in yeast, fungi, bacteria, insects and the like, and is more stable than other disaccharides, so that it is a dry protectant for proteins and the like (Japanese Patent Publication No. 63-500).
It is a useful carbohydrate which is considered to be used as 562). Conventionally, as a method for preparing trehalose, an extraction method from yeast (JP-A-5-292986), a fermentation method using bacteria (JP-A-5-21882), and the like are known. However, trehalose prepared by these methods
The manufacturing cost is high and the cost is very high because the mass production is difficult in terms of operation and facilities, and the impurity removal process is complicated.

On the other hand, an enzymatic method can be mentioned as an effective method for preparing trehalose at low cost. One of them is the mushroom, Grifola f.
Rondosa (Journal of the Japanese Society of Agricultural Chemistry, 68, 580,
1994) and Schizophyllum Commune (Schizo)
phyllum commune, Agaricus bisporus, Pleurotus o Pleurotus o
streatus), refillum ulmarium (Ly
ophyllum ulmarium) (JP-A-6-1)
89779), there is a method for producing trehalose from α-glucose-1-phosphate and glucose using trehalose phosphorylase. However, the enzyme used in this production method has low thermostability and the reaction temperature is 25 to 50.
Since it is ℃, there is a problem that there is a high possibility that contamination of various bacteria will occur in the manufacturing process.

As another enzymatic method, there is a method of producing trehalose using maltose phosphorylase and trehalose phosphorylase and using maltose as a substrate and β-glucose-1-phosphate (Japanese Patent Publication No. 63-60).
998). However, a known trehalose phosphorylase that can be used in the present production method is Euglena gracilis (J.
Biol. Chem. , 274, 3223 to 3228,
1972), the optimum temperature for this enzyme is 4
There is a problem that the temperature stability at 0 ° C. is poor and the preparation is very difficult, so that it is difficult to industrially utilize.

[0005]

The enzymatic method using trehalose phosphorylase is considered to be the most effective method for producing trehalose at a low cost on an industrial scale. On the other hand, when industrially producing by an enzyme reaction, a high reaction temperature is generally adopted for the purpose of reducing contamination of various bacteria. Further, increasing the reaction temperature has the advantages that the solubility of the substrate and the product can be increased to increase the amount charged per unit volume, the enzyme reaction rate can be increased, and the reaction time can be shortened. It is also cost effective. Thus, in order to produce trehalose by an enzymatic reaction at a high temperature, a thermostable trehalose phosphorylase having high thermostability is required. However, the above-mentioned trehalose phosphorylase derived from mushrooms and green algae does not necessarily have high thermostability. Therefore, it has been demanded to provide an enzyme suitable for an actual high temperature enzyme reaction, specifically, an enzyme stable at 55 ° C or higher.

The present inventors have searched for a thermostable trehalose phosphorylase having a high thermostability and producing trehalose from glucose and β-glucose-1-phosphate from the natural world. As a result, a thermophilic bacterium belonging to the genus Bacillus was found. The present inventors have completed the present invention by finding that they often produce an enzyme that meets the above-mentioned purpose.

[0007]

The properties of the thermostable trehalose phosphorylase preparation according to the present invention are as follows. The thermostable trehalose phosphorylase preparation used was that obtained in Example 1. The trehalose phosphorylase activity was measured as follows. 0.4 ml of enzyme solution and 0.06 ml of 0.5 M potassium phosphate / citrate buffer (pH 6.0), 2 g / dl trehalose.
6 ml and 0.14 ml of distilled water were mixed and reacted at 60 ° C. for 20 minutes and then boiled for 10 minutes to stop the reaction. Next, 0.02 ml was taken from this reaction stop solution, 3 ml of a glucose test reagent (glucose CII-Test Wako; Wako Pure Chemical Industries, Ltd.) was added, and after reacting at room temperature for 20 minutes, the absorbance at 505 nm Was measured using a spectrophotometer, and the amount of glucose produced was determined from the measured value. Regarding the definition of trehalose phosphorylase activity, the amount of the enzyme that phosphorolytically decomposes 1 μmol of trehalose per minute under the above measurement conditions was defined as 1 unit.

(1) Action As shown by the formula (1), trehalose is reversibly phosphorolytically decomposed. That is, when acting on trehalose in the presence of phosphoric acid, equimolar glucose and β-glucose-
Produces 1-phosphate, glucose and β-glucose-1
-When it acts on phosphoric acid, it produces equimolar trehalose and phosphoric acid.

[0009]

Embedded image

(2) Substrate specificity Trehalose, maltose, isomaltose, neotrehalose, cellobiose, sucrose, p-nitrophenyl-α-D-glucoside, p-nitrophenyl-
When a phosphorolysis reaction was carried out using β-D-glucoside as a substrate, almost no production of glucose was observed except for trehalose (Table 1). (3) Optimum temperature 40 mM potassium phosphate / citrate buffer (pH 6.
When the reaction was carried out at various temperatures (40 to 90 ° C) in 0), the optimum temperature for the trehalose-phosphorylation reaction was 70 ° C.
At about 75 ° C, about 50% or more of the maximum activity was shown in the range of 60 ° C to 75 ° C (Fig. 1). (4) Thermal stability 10 mM potassium phosphate / citrate buffer (pH 6.
When it was incubated in 0) and the residual activity was measured, it showed 95% or more of the activity of the untreated sample at 65 ° C. for 15 minutes (FIG. 2).

(5) Optimum pH 25 mM potassium phosphate / citrate buffer (pH 4.0)
~ 7.7) and 25 mM Tris / HCl buffer (pH 7.
When the reaction was carried out at 60 ° C. using 7 to 9.0), the optimum pH was 6.5 to 7.5 (FIG. 3). (6) pH stability 100 mM potassium phosphate / citrate buffer (pH 4.
0-8.0) and 100 mM Tris / HCl buffer (pH
The enzyme was stable at pH 6.0 to 8.0 when the residual activity at each pH was measured by incubating the enzyme with 7.5 to 9.0) at 60 ° C. for 24 hours (FIG. 4). (7) Deactivation 100% deactivation by heating at 100 ° C for 10 minutes. (8) Molecular weight As a result of gel filtration chromatography using Superdex 200 pg (Pharmacia Biotech Co., Ltd.), the molecular weight was calculated from the relative elution retention time with various standard proteins. As a result, the molecular weight of this enzyme was 110,000 to 150,000. It was (9) Isoelectric point By isoelectric focusing, the isoelectric point was 4.6 to 5.2 from the relative mobility with various standard proteins. (10) Inhibitor 99% inhibition of activity was observed with 1 mM HgCl ₂ and 80% inhibition with ZnSO ₄ (Table 2).

Table 3 shows a comparison of the enzymatic properties of the thermostable trehalose phosphorylase of the present invention and the conventionally known microorganism-derived trehalose phosphorylase. As is clear from Table 3, the thermostable trehalose phosphorylase of the present invention was judged to be novel, because it differs from the known trehalose phosphorylase in at least the source bacterial species, optimum temperature and thermostability.

The thermostable trehalose phosphorylase of the present invention belongs to the genus Bacillus and is produced by culturing a microorganism having a thermostable trehalose phosphorylase-producing ability in a nutrient medium and collecting the thermostable trehalose phosphorylase produced from the culture.

The microorganism used in the present invention may be any microorganism as long as it belongs to the genus Bacillus and has the ability to produce thermostable trehalose phosphorylase. Suitable microorganisms include Bacillus stearothermophili.
and a microorganism having a thermostable trehalose phosphorylase-producing ability. Specifically, the present inventors isolated Bacillus stearothermophilus SK-1 (FERM P-1456) isolated from soil in the mountains of Kanagawa prefecture.
7).

The mycological properties of this bacterium are shown in Table 4. Due to these characteristics, "Burger's Manual of Determinating Bacteriology", No. 8
Based on the version, this bacterium was identified as Bacillus stearothermophilus. However, among the microorganisms identified as Bacillus stearothermophilus, those producing trehalose phosphorylase have not been known so far. Therefore, the thermostable trehalose phosphorylase-producing bacterium used in the present invention is considered to be novel. To confirm this, trehalose phosphorylase-producing ability of a strain of the same species held by a public strain organization was examined, but none of them produced trehalose phosphorylase (Comparative Example 1).

The microorganisms used in the present invention are not limited to wild strains, but wild strains such as the above-mentioned SK-1 strain can be treated with ultraviolet rays, X-rays, radiation, chemicals [NTG (N-methyl-N'-nitro-N-nitrosoguanidine), A mutant strain mutated by a known artificial mutagenesis method such as EMS (ethyl methanesulfonate)] can also be used as long as it has a thermostable trehalose phosphorylase-producing ability.

The nutrient medium used in the present invention may be either a natural medium or a synthetic medium as long as it appropriately contains a carbon source, a nitrogen source, an inorganic substance and, if necessary, a trace nutrient required by the strain used. As the carbon source, maltose, trehalose, glucose, fructose, sugar, dextrin, starch, carbohydrates such as glycerin and the like are used. As the nitrogen source, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, amino acids such as glutamic acid, and inorganic organic nitrogen compounds such as uric acid are used.

As the nitrogen source, nitrogen-containing natural products such as peptone, polypeptone, meat extract, yeast extract, corn steep liquor, soybean flour, soybean meal, dried yeast, casamino acid, soluble vegetable protein and the like can also be used.
As inorganic substances, monopotassium phosphate, dipotassium phosphate,
Magnesium sulfate, ferrous sulfate, manganese sulfate, zinc sulfate, sodium chloride, potassium chloride, calcium chloride and the like are used. In addition, micronutrients such as biotin and thiamine are used as needed.

Next, in the present invention, it is possible to produce thermostable trehalose phosphorylase without allowing trehalose or maltose to exist in the medium as an inducer of trehalose phosphorylase, but the presence of trehalose or maltose makes it possible to produce thermostable trehalose. In some cases, the amount of phosphorylase produced can be increased.

As the culture method, a liquid culture method (shaking culture method or aeration stirring culture method) is preferable, and the aeration stirring culture method is most suitable industrially. The culture temperature can be 40 to 65 ° C, but 45 to 60 ° C is preferable. The pH is preferably 6.5 to 7.5. Although the culturing period varies depending on the culturing conditions, it is usually about 24 to 84 hours, and the culturing is stopped when the production of thermostable trehalose phosphorylase is confirmed, preferably when the production reaches the maximum.

To collect the thermostable trehalose phosphorylase of the present invention from the thus obtained culture,
First, the culture is fractionated into a culture solution fraction and a bacterial cell fraction by centrifugation or filtration. Although thermostable trehalose phosphorylase is detected in both of the above-mentioned fractions, since it is mainly obtained from the culture solution fraction, this fraction is further subjected to ultrafiltration, salting out,
By subjecting well-known isolation / purification methods such as dialysis, solvent precipitation, ion exchange chromatography, hydrophobic chromatography, gel chromatography, adsorption chromatography, and isoelectric point precipitation, either alone or in combination, heat-resistant trehalose phosphorylase A concentrated or purified standard can be obtained. A specific example of isolation and purification of the thermostable trehalose phosphorylase of the present invention is shown in Example 1.

The present invention also provides glucose and β-glucose-1 in the presence of the above thermostable trehalose phosphorylase.
-A method for producing trehalose, which comprises reacting with phosphoric acid. This reaction is carried out in an aqueous medium.
The thermostable trehalose phosphorylase used in this case has a pH of 50 in a buffer solution such as 10 mM potassium phosphate / citrate buffer solution (pH 6.0).
~ 65 ° C, preferably 55-65 ° C
Any of the above, more preferably any temperature of 60 to 65 ° C., particularly 65 ° C., and those having an activity of 95% or more of non-treatment after 15 minutes treatment are suitably used. Specifically, among the enzyme chemical properties (1) to (10) above, an enzyme having at least the properties (1) to (6) and (8) can be mentioned. These enzymes may be purified enzymes or crude enzymes containing other enzymes that do not adversely affect the above-mentioned trehalose production method. As the crude enzyme, a crude enzyme precipitated by salting out or solvent precipitation from a heat-resistant trehalose phosphorylase-containing fraction such as the above culture solution fraction, or a crude enzyme in the middle of purification, which is further purified by the above-mentioned purification means, is used. Can be mentioned. Further, it is also possible to use an immobilized enzyme in which these enzymes are immobilized on a carrier by a conventional method.

Glucose and β-glucose-1-phosphate used in this reaction may be reagents or the like, or known maltose phosphorylase produced by bacteria of the genus Lactobacillus or Streptococcus (Japanese Patent Laid-Open No. 9778/1989). 60-54036) and glucose and β- produced by reacting maltose phosphorylase on the market with maltose.
It may be glucose-1-phosphate. Examples of the aqueous medium include water and buffer solutions. As the buffer solution, acetate buffer solution, potassium phosphate / citrate buffer solution, citrate buffer solution, succinic acid / sodium hydroxide buffer solution, Tris / hydrochloric acid buffer solution and the like can be used.

The molar ratio of glucose to β-glucose-1-phosphate used is 3: 1 to 1: 3, preferably 1 :.
1 is suitable, and 0.05 to 10 units, preferably 0.1 to 1 unit of the enzyme is suitable for 0.1 mol of the minor component of both. The above reaction is carried out at a temperature
In order to further avoid contamination by various bacteria and to increase the yield, it is suitable to carry out at 60 to 70 ° C., pH generally 4.5 to 9.0, preferably 5.5 to 8.5. The reaction is terminated when sufficient trehalose production is observed under the above conditions, but the reaction is usually terminated in 30 minutes to 72 hours.

After completion of the reaction, the reaction is stopped by an appropriate means such as deactivating the enzyme by heating the reaction solution and deactivating the enzyme by lowering the pH (adding an acid such as hydrochloric acid), and treating with activated carbon or an ion exchange resin. Trehalose can be obtained by appropriately combining isolation / purification means such as treatment and ethanol crystallization treatment.

[0026]

Next, the present invention will be described in more detail with reference to examples. Example 1 Bacillus stearothermophilus SK-1 (FERM
The thermostable trehalose phosphorylase according to P-14567) was produced and purified as follows. (Culture) SK-1 strain was autoclaved at 121 ° C. for 20 minutes in a liquid medium consisting of 0.5 g / dl of yeast extract, 1 g / dl of polypeptone, and 1 g / dl of maltose (pH 7.0). After culturing with aeration and stirring at 55 ° C. for 72 hours, the cells were separated from the culture solution by centrifugation.

(Preparation of Crude Enzyme) Ammonium sulfate was dissolved in the separated culture solution so as to be 40 to 60% saturated, and the resulting protein precipitate was recovered by centrifugation to obtain 10 mM potassium phosphate / citrate buffer solution ( After being dissolved in pH 6.0), it was dialyzed against the same buffer solution to obtain a crude enzyme solution. (Ion exchange chromatography) The crude enzyme solution was added to a column packed with TSKgel DEAE Toyopearl 650M (Tosoh Corporation) equilibrated with 10 mM potassium phosphate / citrate buffer (pH 6.0),
Elution was performed with an increasing concentration gradient of 0 to 0.4 mM NaCl in the column volume, and the eluate was collected. The active fractions were combined, concentrated, desalted, and then subjected to the same series of chromatographic operations to raise the degree of purification.

(Hydrophobic chromatography) TSKgelPhenyl Toyopearl 65 equilibrated with 10 mM potassium phosphate / citrate buffer (pH 6.0) in which ammonium sulfate was dissolved to 40% saturation.
The above partially purified enzyme solution was added to a column packed with 0M (Tosoh Corp.), and eluted with a descending concentration gradient of 40 to 0% saturated ammonium sulfate solution with 8 column volumes to separate the eluate. The active fractions were combined and concentrated and desalted. (Adsorption chromatography) C at 0.3 mM
PENTA equilibrated with 10 mM potassium phosphate-citrate buffer (pH 6.0) containing dissolved aCl _2.
The above partially purified enzyme solution was added to an XGH-0810M column, and the column was eluted with an increasing concentration gradient of 10 column volumes of 10 to 300 mM potassium phosphate / citrate buffer (pH 6.0), and the eluate was fractionated. . The active fractions were combined and concentrated and desalted.

(Gel filtration chromatography) 0.2M
The partially purified enzyme solution was added to a column packed with Superdex 200 pg (Pharmacia Biotech Co., Ltd.) equilibrated with 10 mM potassium phosphate / citrate buffer solution (pH 6.0) in which NaCl was Elution was performed with the same buffer, and the eluate was collected. The active fractions were combined and concentrated and desalted. (Native acrylamide gel electrophoresis) When the purified enzyme solution was subjected to native acrylamide gel electrophoresis and the gel was stained with CBB to examine the protein bands, only one band was detected, indicating that it was a single protein. It could be confirmed.

Example 2 (Preparation of thermostable trehalose phosphorylase) Yeast extract 2 g / dl, polypeptone 1 g / dl, maltose 1 g /
500 ml of 100 ml of dl-containing medium (pH 6.5)
One platinum loop of SK-1 strain was inoculated into a 1-Meyer flask and sterilized by autoclaving at 121 ° C. for 20 minutes, and cultured with aeration and stirring at 55 ° C. for 72 hours. After culturing,
The culture was subjected to centrifugation to separate the cells, ammonium sulfate was dissolved in the supernatant so as to be 40 to 80% saturated, and the precipitated protein was collected by centrifugation. After dissolving this in 10 mM potassium phosphate / citrate buffer (pH 6.0),
Dialysis was performed against the same buffer solution to obtain 8 units of 12 units / ml crude enzyme solution.

(Production of trehalose) 0.3 heat-resistant trehalose phosphorylase crude enzyme solution thus prepared
ml (1.5 units / ml), 6 g / dl glucose 0.12
55 ml of a reaction liquid consisting of ml (40 mM), 6 g / dl β-glucose-1-phosphate sodium salt 0.2 ml (40 mM), 0.5 M acetate buffer (pH 6.0) 0.1 ml, and distilled water 0.28 ml. Incubated at 1 ° C, 1 hour, 2 hours, 16 hours, and 70 ° C, 1 hour. To stop the reaction,
It was performed by boiling for 10 minutes. After the reaction was completed, each reaction solution was added to a TSKgel Amido 80 column (250 × 4.
6 mmφ) (Tosoh Corporation), eluent acetonitrile /
By subjecting to water (76/24 (v / v)), flow rate 0.8 ml / min, column temperature 80 ° C., high-performance liquid chromatography using a differential refractometer Shodex (Showa Denko KK) (detection means), The trehalose produced was quantified. The results are shown in Table 5. The trehalose produced by the reaction at 55 ° C. for 16 hours was 24.3 mM.

Example 3 (Preparation of thermostable trehalose phosphorylase) Example 1
100 ml of the crude thermostable trehalose phosphorylase enzyme solution prepared in 1. was added to TSKgel DEAE TO-YOPEARL 65
After adsorbing to a column filled with 160 ml of 0M (Tosoh Corp.), 10m containing 10mM potassium phosphate / citrate buffer (pH 6.0) and 0.4M sodium chloride
800 ml with M citrate buffer (pH 6.0)
The linear concentration gradient elution was carried out. When 8 ml of the eluate was collected, the desired thermostable trehalose phosphorylase activity was observed in the fractions 55 to 75.
This fraction was concentrated and desalted by ultrafiltration to obtain enzyme unit 1
20 ml of partially purified enzyme solution with 5.5 units / ml was obtained.

(Production of Trehalose) About 1 part of the crude thermostable trehalose phosphorylase enzyme solution thus prepared was used.
0.3 ml diluted 0.4 times (0.4 units / ml),
6g / dl glucose 0.12ml (40mM), 6g / dlβ
-Glucose-1-phosphate sodium salt 0.2 ml (4
0 mM), 0.5 M acetate buffer (pH 6.0) 0.1
reaction solution consisting of 0.28 ml of distilled water at 65 ° C.
Incubated for hours. The reaction was stopped by boiling for 10 minutes. After completion of the reaction, the reaction solution was subjected to high performance liquid chromatography under the same conditions as in Example 1 to quantify the produced trehalose. The trehalose produced was 10.9 mM.

Example 4 (Preparation of thermostable trehalose phosphorylase and maltose phosphorylase) The thermostable trehalose phosphorylase was obtained from the culture solution of the SK-1 strain by ammonium sulfate precipitation in the same manner as in Example 1. The activity was 1.5 units / ml. On the other hand, known maltose phosphorylase (Agric. Bi
ol. Chem. , 37, (12), 2813 to 281
9, 1973) was prepared as follows. Yeast extract 0.2 g / dl, peptone 1 g / dl, maltose 1 g / dl, sodium acetate 1 g / dl, magnesium sulfate heptahydrate 0.0
100 ml of a medium (pH 7.0) containing 2 g / dl and manganese sulfate tetrahydrate (0.0002 g / dl) was placed in a 500 ml Meyer flask and sterilized by autoclaving at 121 ° C. for 20 minutes, and Lactobacillus brevis IFO33.
Forty-five platinum loops were inoculated with 45 and cultured at 30 ° C. for 72 hours with aeration and stirring. After culturing, the culture is subjected to centrifugation to collect the bacterial cells, and 10 mM potassium phosphate / citrate buffer solution (pH
After suspending in 6.0), the cells were disrupted by an ultrasonic cell disruptor, the disrupted cells were subjected to centrifugation, and the supernatant was used as a crude enzyme solution. The activity of the obtained crude enzyme solution was 0.8 unit / ml.

(Production of trehalose) First, 0.5 parts of the maltose phosphorylase crude enzyme solution thus prepared was used.
ml (0.4 units / ml), 7.2 g / dl maltose 0.
2 ml (40 mM), 0.5 M potassium phosphate / citrate buffer (pH 6.0) 0.08 ml, distilled water 0.22
The reaction solution consisting of ml was incubated at 37 ° C. for 1 hour. The reaction solution contained 7.0 mM β-glucose-1.
-Phosphoric acid was produced. Further, 0.1 ml of the crude heat-resistant trehalose phosphorylase enzyme solution was added to 1 ml of this reaction solution.
1 was added and incubated at 70 ° C. for 1 hour, then 10
The reaction was stopped by boiling for 1 minute. After the reaction was completed, the reaction solution was subjected to high performance liquid chromatography under the same conditions as in Example 1 to quantify the produced trehalose. As a result, 4.6 mM trehalose was produced in the reaction solution.

Comparative Example 1 There has been no report of Bacillus stearothermophilus producing trehalose phosphorylase, and the presence or absence of activity was examined for the homologous strains preserved by Fermentation Research Institute, but trehalose phosphorylase activity was detected. There wasn't.
That is, Bacillus stearothermophilus IFO1
2550, IFO12983, IFO13737 and S
K-1 in liquid medium (yeast extract 1 g / dl, polypeptone 2
g / dl, trehalose or maltose 2g / d as C source
The cells were inoculated into a cell culture medium and a culture solution at 55 ° C. for 72 hours with aeration and stirring, and separated into cells and a culture solution by centrifugation. The bacterial cells were washed and then ultrasonically disrupted to prepare a crude enzyme solution in the bacterial cells. Ammonium sulfate was dissolved in the culture solution to 80% saturation, and the resulting protein precipitate was collected by centrifugation. This was dissolved in a phosphate buffer solution and dialyzed to obtain a crude extracellular enzyme solution. The reaction of each crude enzyme solution was performed under the following conditions, and the presence or absence of trehalose phosphorylase activity was determined by examining the synthesis of trehalose. The results are shown in Table 6.

Reaction solution: 0.3 ml of crude enzyme solution, 0.12 ml (40 mM) of 6 g / dl glucose, 0.2 ml (40 mM) of 6 g / dl β-glucose-1-phosphate sodium salt,
0.5 M acetate buffer (pH 6.0) 0.1 ml, distilled water 0.28 ml Reaction: 55 ° C., 16 hours Detection: Trehalose peak by high performance liquid chromatography using TSKgel Amido 80 column (Tosoh Corp.) Was detected.

[0038]

The trehalose phosphorylase provided by the present invention has thermostability and produces trehalose from glucose and glucose-1-phosphate. By carrying out an enzymatic reaction using this enzyme at a high reaction temperature, trehalose can be industrially advantageously produced while reducing contamination of various bacteria and shortening the reaction time.

[0039]

[Table 1]

[0040]

[Table 2]

[0041]

[Table 3]

[0042]

[Table 4]

[0043]

[Table 5]

[0044]

[Table 6]

[Brief description of drawings]

1 shows the optimum temperature of the thermostable trehalose phosphorylase obtained in Example 1.

FIG. 2 shows thermostability of thermostable trehalose phosphorylase obtained in Example 1.

FIG. 3 shows the optimum pH of the thermostable trehalose phosphorylase obtained in Example 1.

FIG. 4 shows the pH stability of the thermostable trehalose phosphorylase obtained in Example 1.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area // (C12N 9/12 C12R 1:07) (C12N 1/20 C12R 1:07)

Claims (7)

[Claims] 1. 50 to 65 ° C. in a buffer having a pH of 6.0
A thermostable trehalose phosphorylase having an activity of 95% or more after being treated for 15 minutes at any temperature. 2. A thermostable trehalose phosphorylase having the following enzymatic chemistry: (1) Action Reversibly phosphorolytically decomposes trehalose. That is,
When it acts on trehalose in the presence of phosphoric acid, it produces equimolar glucose and β-glucose-1-phosphate, and when it acts on glucose and β-glucose-1-phosphate, it produces equimolar trehalose and phosphate. To generate. (2) Substrate specificity It acts specifically on trehalose. (3) Optimum temperature The optimum temperature for the phosphorolysis reaction of trehalose is 70 ° C to 7 ° C.
Around 50 ℃, maximum activity of about 50 ℃ in the range of 60 ℃ to 75 ℃
% Or more. (4) Thermal stability 10 mM potassium phosphate / citrate buffer (pH 6.
In 0), after treatment at 65 ° C. for 15 minutes, it has an activity of 95% or more of that of untreated. (5) Optimum pH 6.5-7.5. (6) pH stability Stable at pH 6.0 to 8.0. (7) Molecular weight 110,000-150,000 (by gel filtration chromatography using Superdex 200 pg). 3. A thermostable trehalose phosphorylase having the following enzymatic chemical properties: (1) Action Reversibly phosphorolytically decomposes trehalose. That is,
When it acts on trehalose in the presence of phosphoric acid, it produces equimolar glucose and β-glucose-1-phosphate, and when it acts on glucose and β-glucose-1-phosphate, it produces equimolar trehalose and phosphate. To generate. (2) Substrate specificity Acts on trehalose, but does not act on maltose, isomaltose, neotrehalose, cellobiose, sucrose, p-nitrophenyl-α-D-glucoside, p-nitrophenyl-β-D-glucoside. . (3) Optimum temperature The optimum temperature for the phosphorolysis reaction of trehalose is 70 ° C to 7 ° C.
Around 50 ℃, maximum activity of about 50 ℃ in the range of 60 ℃ to 75 ℃
% Or more. (4) Thermal stability 10 mM potassium phosphate / citrate buffer (pH 6.
In 0), after treatment at 65 ° C. for 15 minutes, it has an activity of 95% or more of that of the untreated. (5) Optimum pH 6.5-7.5. (6) pH stability Stable at pH 6.0 to 8.0. (7) Deactivation 100% deactivation by heating at 100 ° C for 10 minutes. (8) Molecular weight 110,000-150,000 (by gel filtration chromatography using Superdex 200 pg). (9) Isoelectric point 4.6-5.2. (10) Inhibitors Hg ²⁺ and Zn ²⁺ remarkably inhibit the activity. 4. A method for producing trehalose phosphorylase, which comprises culturing in a nutrient medium a microorganism belonging to the genus Bacillus and capable of producing trehalose phosphorylase, and collecting trehalose phosphorylase produced from the culture. 5. A Bacillus stearothermophilus capable of producing trehalose phosphorylase. 6. Bacillus stearosa-Mophilus SK
-1 (FERM P-14567). 7. A buffer solution having a pH of 6.0 and a temperature of 50 to 65 ° C.
Of trehalose characterized by reacting glucose with β-glucose-1-phosphate in the presence of untreated thermostable trehalose phosphorylase having 95% or more activity after treatment for 15 minutes at any temperature Method.