JP2001299338A - Laminmaribiose phosphorylase, method for producing the same and method for producing laminarioligosaccharide using the same enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for safely producing an inexpensive laminarioligosaccharide. SOLUTION: A laminarioligosaccharide is obtained by reacting a thermostable laminaribiose phosphorylase prepared by culturing a microorganism belonging to the genus Bacillus with α-glucose-1-phosphate and glucose. The laminarioligosacharide is obtained even by adding a starch and a glucan phosphorylase in place of the α-glucose-1-phosphate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel laminaribiose phosphorylase. More specifically, the present invention relates to a laminaribiose phosphorylase, a method for producing the same, and a method for producing a laminarioligosaccharide.

[0002]

2. Description of the Related Art Laminarioligosaccharide is a general term for oligosaccharides in which glucose is bound by β1,3 bonds from several molecules to several tenths of molecules, and includes, for example, laminaribiose, laminaritriose, laminaritetraose and the like. . β1,3
As the polysaccharide having a bond, curdlan, pakiman, and the like are known, and among them, substances having an anticancer activity are known. Therefore, various physiological activities are also expected for laminari oligosaccharides, which are oligosaccharides having a β1,3 bond.

[0003] As a method for producing laminari-oligosaccharides, β
Methods in Carbohydrate Decomposition of 1,3 Glucan (Methods in Carbo
hydrate Chemistry, 1, 330-333, 1962), a method by enzymatic decomposition (JP-A-59-162897), a method of performing a condensation reaction using β-glucosidase using a high-concentration glucose solution (Biotechnology Letter 9, 4, 243-248 (1987)). ) Are known.

[0004] However, the method of acid-decomposing β1,3 glucan has the disadvantages that the raw material β1,3 glucan is expensive, has poor handling due to its viscosity, and has a low reaction yield, making it difficult to perform specific synthesis. Therefore, it cannot be a means for producing laminari-oligosaccharides at low cost. In addition, in the condensation reaction using β-glucosidase, specific laminari-oligosaccharides cannot be produced, and genthio-oligosaccharides having β1,6-bonds are usually produced as a main component. Therefore, there is a problem that a high-level separation technique is required.

Further, as a method for producing a laminari-oligosaccharide which can be synthesized specifically with high reaction yield without using expensive β-glucan, laminaribiose phosphorylase acts on glucose and α-glucose-1-phosphate. A method for causing this is known (JP-A-4-66092).
This laminaribiose is derived from Euglena,
The only known one. However, this laminaribiose phosphorylase has low thermostability (temperature stability of 4
At 5 ° C., about 30% or more is inactivated), and is not suitable for industrial production (Archives of Biochemistry and Biophysics, vol.3)
04 (2), 508-514, 1993).

[0006]

Therefore, there is a demand for an inexpensive method for producing laminari-oligosaccharides at low cost.

[0007]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems. INDUSTRIAL APPLICABILITY According to the present invention, laminaribiose phosphorylase having heat resistance is provided, and laminarioligosaccharide, which is a very expensive saccharide at present, can be produced at low cost, and its use in foods and drinks is intended.

The present invention has the following properties: (1) substrate specificity: produces laminarioligosaccharide from α-glucose-1-phosphate and glucose; (2) optimal pH: pH 6.5 to 7.0; 3) optimum temperature: 55 ° C .; (4) pH stability: pH 5.5 to 7.0; and (5) temperature stability: 57.5 ° C. or less.

In a preferred embodiment, the laminaribiose phosphorylase further has the following properties: (6) molecular weight: about 170 KDa (gel filtration HPLC); and (7) isoelectric point: pH about 4.7 (isoelectric Point electrophoresis).

[0010] The present invention further relates to a method for producing laminarioligosaccharide, which comprises a step of reacting a fungus having the ability to produce laminaribiose or a treated product thereof with α-glucose-1-phosphate and glucose.

In a preferred embodiment, the treated product has the following characteristics: (1) substrate specificity: forms laminari-oligosaccharide from α-glucose-1-phosphate and glucose; (2) optimal pH: pH6. (3) optimum temperature: 55 ° C .; (4) pH stability: pH 5.5 to 7.0; and (5) temperature stability: 57.5 ° C. or less. It is a phosphorylase.

In a more preferred embodiment, the laminaribiose phosphorylase further has the following characteristics: (6) molecular weight: about 170 KDa (gel filtration HPLC); and (7) isoelectric point: pH about 4.7 (e.g., Electrofocusing).

[0013] In another preferred embodiment, the fungus is a microorganism belonging to the genus Bacillus.

Further, in a preferred embodiment, the microorganism belonging to the genus Bacillus is Bacillus subtilis K2145, Bacillus circulans IAM12462 (Bacillus circulans IAM1246).
2) or Bacillus coagulans IAM1194 (Bacil
lus coagulans IAM1194).

[0015] The present invention further relates to a method for producing laminaribiose phosphorylase, which comprises a step of culturing a fungus having the ability to produce laminaribiose and a step of collecting laminaribiose phosphorylase from the culture.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The laminaribiose phosphorylase of the present invention has been obtained from fungi for the first time. Heretofore, it has been discovered for the first time that laminaribiose phosphorylase, which was known only in Euglena, is also produced in microorganisms.

Here, in the present specification, a microorganism is
A taxonomically refers to a microorganism belonging to a bacterium, a transformant, a trichome, or a fungus.

We have found that glucose and α-glucose-1
-The microorganism which produces laminarioligosaccharide from phosphoric acid was searched from soil, and the new microorganism which produces laminaribiose phosphorylase was obtained for the first time. This microorganism was identified as a microorganism belonging to the genus Bacillus on the basis of its mycological properties, and the present inventors have identified the microorganism as Bacillus subtilis K2145 (Bacillussu).
btilis K2145) and has been deposited with the National Institute of Advanced Industrial Science and Technology as a FERMP-17710.

The bacteriological properties of Bacillus subtilis strain K2145 are as follows. (1) Shape Cell shape and size: 0.8 × 2 to 3 μm bacilli Motility: + Sporulation: + Colony shape: irregular, curly margin, convex, matte,
Cream (2) Physiological properties β-galactosidase: + arginine dihydrolase:-lysine decarboxylase:-ornithine decarboxylase:-availability of citric acid: + hydrogen sulfide production:-urease:-tryptophan deaminase:-indole production: + acetoin Production: + gelatinase: + nitrate reduction: + catalase: + oxidase: + O / F test:-

Bacillus subtilis has long been eaten as fermented soybeans such as natto, and there is no problem in safety when used in the food industry.

The laminaribiose phosphorylase of the present invention has an optimum temperature around 55 ° C.
It has the characteristic of producing laminari-oligosaccharides from phosphoric acid and glucose. It is characterized by higher heat resistance than conventional laminaribiose phosphorylase from Euglena.

The laminaribiose phosphorylase of the present invention has the following characteristics: (1) The following reaction: α-glucose-1-phosphate + glucose ← → catalyzes laminarioligosaccharide + phosphate. (2) Optimum pH: pH 6.5 to 7.0; (3) Optimum temperature: 55 ° C; (4) pH stability: pH 5.5 to 7.0; (5) Temperature stability: 57.5 (6) Molecular weight: about 170 kDa (gel filtration HPLC); and (7) Isoelectric point: pH about 4.7 (isoelectric focusing).

The laminaribiose phosphorylase of the present invention is, for example, Bacillus subtilis K2145 (Bacillus sub
Tilis K2145), for example, can be obtained by the following method. First, Bacillus subtilis K2145
(Bacillus subtilis K2145) with fructose, glucose, laminaribiose, galactose, isomerized sugar,
Various sugars, such as sucrose, maltose, xylose, sorbitol, mannitol, and starch, sugar alcohols, starch and dextrin, etc., are used as carbon sources, and organic substances such as yeast extract, fish meat extract, peptone, and amino acids necessary for the growth of microorganisms are used. A nitrogen source, or ammonium sulfate,
In a medium supplemented with an inorganic nitrogen source such as ammonium chloride, urea or ammonium nitrate, and minerals if necessary,
The cells are cultured under aerobic conditions at 0 to 40 ° C, preferably around 30 ° C, for 1 to 5 days, preferably 1 to 3 days.

The cells obtained by culturing are used as they are or after washing to produce laminari-oligosaccharides.

The cells obtained by culturing are further processed,
The resulting processed product can also be used for producing laminari-oligosaccharides. Here, the processed product is 1) a microorganism cell immobilized by a method usually used by those skilled in the art; 2) mechanically or enzymatically treating the microorganism cell, or a surfactant or an organic compound. Those treated with solvents, etc.
Or those obtained by immobilizing them; 3) those obtained by crushing microbial cells to remove crushed residues; and those obtained by immobilizing them; 4) enzymes obtained by further purifying from 2) or 3), Or an immobilized version thereof. For enzyme purification, means commonly used by those skilled in the art are used alone or in an appropriate combination.

The immobilization of the treated material is performed by using polyacrylamide,
Calcium alginate, carrageenan, ion exchange carriers, chitosan beads and the like are used.

The obtained microbial cells and processed products thereof (including purified enzymes and partially purified enzymes) are mixed with a reaction solution containing α-glucose-1-phosphate and glucose, and used for the production of laminari-oligosaccharides. Can be

It is preferable that α-glucose-1-phosphate and glucose are contained in a ratio of about 100: 1 to 1: 100. It is preferable that glucose and α-glucose-1-phosphate are contained in a total amount of 1 to 75% by weight in the reaction solution. Preferably, it is 5 to 50% by weight, more preferably 20 to 40% by weight.

The pH of the reaction solution is not particularly limited, but may be about 5.
It is preferably from 0 to 8.0, and more preferably a pH of about 6.
0 to 7.0. The reaction temperature ranges from about 40 to 65 ° C, preferably about 50 to 60 ° C, more preferably
About 50-55 ° C. Considering industrial practicality, a range of about 50 to 55 ° C and a pH of about 6.0 to 7.0 is preferable.

The substrate used in the present invention, α-glucose-
1-phosphate can also be obtained from oligosaccharides or polysaccharides having α-1,4 bonds. Examples of the oligosaccharide or polysaccharide having an α-1,4 bond include, but are not limited to, amylose and starch. Α-Glucose-1-phosphate is produced by reacting amylose, starch or the like with glucan phosphorylase or the like. In the case of using starch, α-glucose-1-phosphate is produced by previously treating with a branching enzyme or amylase, or by simultaneously reacting these enzymes with glucan phosphorylase.

Also, sucrose phosphorylase can act on sucrose to obtain α-glucose-1-phosphate, and α-glucose-1-phosphate derived from root products such as potatoes and natural products such as milk. Can also be used.

The α-glucose-1-phosphate and glucose may be added at the start of the laminari-oligosaccharide production reaction or may be added sequentially. By changing the concentration of both substrates or the timing of addition, a product containing a relatively large amount of a specific polymerization degree component can be obtained.

Further, laminarioligosaccharides can be produced using oligosaccharides or polysaccharides having α-1,4 bonds and glucose as substrates. In this case, laminarioligosaccharide is produced by the presence of glucan phosphorylase together with laminaribiose phosphorylase. The reaction conditions at this time are not particularly limited, and the above reaction conditions are applied.

Furthermore, when laminaribiose phosphorylase is allowed to act using sucrose phosphorylase and glucose isomerase in combination with sucrose as a substrate to produce laminarioligosaccharides, the action conditions of glucose isomerase (pH 6.5-7.0, temperature 60 ° C.).

By the above reaction, laminari-oligosaccharide is obtained, and the obtained laminari-oligosaccharide is separated into components having a specific polymerization degree by combining a stationary phase type chromatographic separator, a pseudo mobile phase type chromatographic separator, and the like. You can also.

[0036]

The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples. Unless otherwise indicated, percentages are W / V%.

Example 1 Preparation of Laminaribiose Phosphorylase and Synthesis of Laminarioligosaccharide 500 ml of a medium (pH 7.0) based on 2% polypeptone, 1% yeast extract and 2% NaCl
The mixture was placed in an Erlenmeyer flask equipped with a baffle, sterilized by a conventional method, inoculated with Bacillus subtilis K2145 (FERM P-17710), and cultured with shaking at 37 ° C. for 24 hours. 6 cultures
After centrifugation at 000 rpm, the cells were collected and washed twice with 25 ml of 20 mM sodium acetate buffer (pH 7.0). 20 ml of sodium acetate buffer (pH 7.
After suspending the cells in 0), the cells were physically crushed with glass beads and a crude enzyme solution was obtained by centrifugation.

The obtained crude enzyme was appropriately diluted to give 20
0 μl was added to 200 μl of a 2% laminaribiose solution dissolved in a 20 mM phosphate buffer (pH 7.0).
The enzyme reaction was performed at 5 ° C. Next, the reaction solution was boiled for 10 minutes to stop the enzymatic reaction, and the sugar composition was analyzed by HPLC. Under these conditions, the amount of enzyme that decomposes 1 μmol of laminaribiose per minute was defined as 1 unit. Laminaribiose phosphorylase activity of the crude enzyme solution was 0.17 units / ml.

To 500 μl of the crude enzyme solution, 250 μl of a 100 mM α-glucose-1-phosphate solution and 250 μl of a 100 mM glucose solution were added and reacted at 55 ° C. for 24 hours. The reaction was analyzed by HPLC (FIG. 1).
The HPLC conditions are shown below. When the laminarioligosaccharide in the reaction solution was quantified, the conversion rate from glucose to laminaribiose was 50%, and the conversion rate from laminarioligosaccharide of three or more saccharides was 10%. HPLC conditions Pump: JASCO Corporation PU-980 Column: Showa Denko Corporation NH ₂ P50 column Detector: Showa Denko Corporation SE-61 type differential refractometer Eluent: acetonitrile: water = 75: 25 Flow rate: 0.8 ml / min

Example 2 Properties of Laminaribiose Phosphorylase The crude enzyme solution derived from Bacillus subtilis K2145 prepared in Example 1 was subjected to ammonium sulfate fractionation (40-60% ammonium sulfate precipitation fraction).
Hydrophobic chromatography (butyl toyopearl: Tosoh), anion exchange chromatography (DEAE-Toyopearl: Tosoh), gel filtration chromatography (Supe)
A single protein was purified by various types of chromatography such as rdex200: manufactured by Pharmacia and anion exchange chromatography (MonoQ: manufactured by Pharmacia). Using this, various properties of the following laminaribiose phosphorylase were confirmed.

(Optimal temperature) 200 μm of a 100 mM phosphate buffer (pH 7.0) containing 2% laminaribiose
Add 200 μl of appropriately diluted enzyme solution to
The reaction was performed at 5, 50, 55, 60, 62.5, and 65 ° C. for 30 minutes. Laminaribiose phosphorylase activity is 1
After inactivating the enzyme by boiling for 0 minutes, glucose was quantitatively determined. The results are shown in FIG.

As apparent from FIG. 2, the optimum temperature of the present enzyme was 55 ° C. In addition, there is activity even at 62.5 ° C, and when the activity at the optimal temperature is 100%,
The relative activity was about 85%.

(Optimal pH) 200 μl of an appropriately diluted enzyme solution was added to 200 μl of 100 mM various buffer solutions containing 2% laminaribiose, and reacted at 55 ° C. for 30 minutes. Laminaribiose phosphorylase activity was determined by quantifying glucose after inactivating the enzyme by boiling for 10 minutes. The results are shown in FIG. As is clear from FIG. 3, the optimum pH of the present enzyme was 6.5 to 7.0,
Substantial use is also possible at 6.0 to 7.5.

(Temperature stability) Appropriately diluted enzyme solution 20
0 μl of 40, 45, 50, 55, 57.5, 60, 6
After incubation at 5 ° C. for 10 minutes, 100 mM phosphate buffer containing 2% laminaribiose (pH 7.0).
0) 200 μl was added and reacted. Laminaribiose phosphorylase activity was determined by quantifying glucose after inactivating the enzyme by boiling for 10 minutes. Fig. 4 shows the results.
Shown in As is clear from FIG. 4, this enzyme was substantially stable up to 57.5 ° C.

(Stable pH) After incubating 200 μl of an enzyme solution appropriately diluted with each buffer of pH 3 to 9 at 25 ° C. for 3 hours, 100% containing 2% laminaribiose was added.
200 μl of an mM phosphate buffer (pH 7.0) was added and reacted. Laminaribiose phosphorylase activity is 1
After inactivating the enzyme by boiling for 0 minutes, glucose was quantitatively determined. FIG. 5 shows the results. As is clear from FIG. 5, the present enzyme was stable up to pH 5.5 to 7.0.

(Molecular Weight and Isoelectric Point) The molecular weight of the enzyme purified by gel filtration chromatography was about 170 kDa, and the isoelectric point by isoelectric focusing was about pH 4.7.

Example 3 According to Example 1, B. circulan
A crude enzyme solution of s (IAM12462) and B. coagulans (IAM1194) was prepared. 20 mM phosphate buffer (pH 6.0) containing 2% laminaribiose per 100 μl of crude enzyme solution
100 µl were mixed and reacted at 55 ° C for 24 hours. The reaction solution was analyzed by HPLC, and α-glucose-1-phosphate was measured. As a result, B.circulans (IAM12462), B.coa
gulans (IAM1194) contains α-glucose-
1-phosphate was detected, and laminaribiose phosphorylase activity was observed.

[0048]

Industrial Applicability The laminaribiose phosphorylase of the present invention has higher heat resistance and a wider pH range of action than those known hitherto, and efficiently converts laminarioligosaccharide from glucose and α-glucose-1-phosphate. To produce. In addition, the enzyme laminaribiose phosphorylase of the present invention can be produced from a microorganism derived from Bacillus genus which has high food safety. By using this enzyme, laminari-oligosaccharides can be produced inexpensively and in large quantities from starchy materials under industrial production conditions.

[Brief description of the drawings]

FIG. 1 is a diagram showing the synthesis of laminari-oligosaccharide.

FIG. 2 is a graph showing the optimum temperature of laminaribiose phosphorylase of the present invention.

FIG. 3 is a graph showing the optimum pH of laminaribiose phosphorylase of the present invention.

FIG. 4 is a diagram showing the temperature stability of laminaribiose phosphorylase of the present invention.

FIG. 5 is a diagram showing the pH stability of laminaribiose phosphorylase of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1: 125) C12R 1: 125) (C12N 9/10 (C12N 9/10 C12R 1:07) C12R 1 : 07) (C12P 19/18 (C12P 19/18 C12R 1:09) C12R 1:09) (C12P 19/18 (C12P 19/18 C12R 1: 125) C12R 1: 125) (C12P 19/18 (C12P 19/18 C12R 1:07) C12R 1:07) (72) Inventor Shinsuke Miyoshi 1-16-1 Sakura, Tsukuba-shi, Ibaraki Showa Sangyo Co., Ltd. F-term in Bio-Research Center (Reference) 4B050 CC01 DD02 FF11E FF12E LL05 4B064 AF04 CA21 CB30 CC03 CD09 CD15 DA05

Claims (8)

[Claims] 1. The following properties: (1) Substrate specificity: produces laminarioligosaccharide from α-glucose-1-phosphate and glucose; (2) optimal pH: pH 6.5 to 7.0; (3) (4) pH stability: pH 5.5 to 7.0; and (5) Temperature stability: 57.5 ° C. or less; laminaribiose phosphorylase. 2. The composition of claim 1, further comprising: (6) a molecular weight of about 170 KDa (gel filtration HPLC); and (7) an isoelectric point of about pH 4.7 (isoelectric focusing). 2. The laminaribiose phosphorylase according to 1. 3. A method for producing a laminarioligosaccharide, comprising a step of reacting a fungus having the ability to produce laminaribiose or a treated product thereof with α-glucose-1-phosphate and glucose. 4. The treated product has the following properties: (1) substrate specificity: produces laminari-oligosaccharide from α-glucose-1-phosphate and glucose; (2) optimal pH: pH 6.5 to pH 7. 0; (3) optimal temperature: 55 ° C .; (4) pH stability: pH 5.5 to 7.0; and (5) temperature stability: 57.5 ° C. or less. A method for producing a laminari-oligosaccharide according to claim 3. 5. The laminaribiose phosphorylase further has the following properties: (6) molecular weight: about 170 KDa (gel filtration HPLC); and (7) isoelectric point: pH about 4.7 (isoelectric focusing). The method for producing a laminari-oligosaccharide according to claim 4, comprising: 6. The method for producing a laminari-oligosaccharide according to claim 3, wherein the fungus is a microorganism belonging to the genus Bacillus. 7. The microorganism belonging to the genus Bacillus is Bacillus subtilis K214.
5), Bacillus circulans IAM12462 (Bacillus ci
rculans IAM12462), or Bacillus coagulans
The method for producing a laminari-oligosaccharide according to claim 6, which is IAM1194 (Bacillus coagulans IAM1194). 8. A method for producing laminaribiose phosphorylase, comprising a step of culturing a fungus having the ability to produce laminaribiose, and a step of collecting laminaribiose phosphorylase from the culture.