WO2001079475A1

WO2001079475A1 - Laminaribiose phosphorylase, process for producing the same and process for producing laminarioligosaccharide by using the enzyme

Info

Publication number: WO2001079475A1
Application number: PCT/JP2001/003253
Authority: WO
Inventors: Kouhei Yamada; Katsuyuki Okamoto; Shinsuke Miyoshi
Original assignee: Showa Sangyo Co., Ltd.
Priority date: 2000-04-19
Filing date: 2001-04-16
Publication date: 2001-10-25
Also published as: AU4692501A; JP2001299338A

Abstract

A laminarioligosaccharide is obtained by reacting thermotolerant laminaribiose phosphorylase obtained by culturing a microorganism belonging to the genus Bacillus with α-glucose-1-phosphate and glucose. A laminarioligosaccharide can be obtained too by adding starch and glucan phosphorylase as a substitute for α-glucose-1-phosphate. Thus, an inexpensive and safe process for producing a laminarioligosaccharide is provided.

Description

Specification

Laminariviose phosphorylase, method for producing the same, and method for producing laminarioligosaccharide using the enzyme

Technical field

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

Background art

Laminari-oligosaccharide is a generic name of oligosaccharides in which glucose is bound by several molecules to several tens of tens of 子₃ bonds, and includes, for example, laminaribiose, laminaritriose, laminaritetraose and the like. Curdlan, pakiman, and the like are known as polysaccharides having 31 and 3 bonds, and among them, substances having anticancer activity are known. Therefore, various biological activities are also expected for laminari oligosaccharides, which are oligosaccharides having 31 and 3 bonds.

Laminari-oligosaccharides can be produced by methods such as acid decomposition of 1,3 glucan (Methods in Carbohydrate Chemistry, 1, 330-333, 1962), enzymatic decomposition (JP-A-59-162897), and high-concentration glucose. A method of performing a condensation reaction using a solution and using / 3 dal cosidase (Biotechnology Letter 9, 4, 243-248 (1987)) is known.

However, the method based on the acid decomposition of 131,3 glucan has the disadvantages that the raw material / 31,3 glucan is expensive, has poor handling due to its viscosity, and has a low reaction yield, making it difficult to make a special synthesis. Therefore, it cannot be used as a means for inexpensively producing laminari-oligosaccharides. In addition, in the condensation reaction with j3 dalcosidase, Laminari-oligosaccharides cannot be produced efficiently, and genyo-oligosaccharides with i3 1,6-linkage are usually produced as the main component, which requires a high level of separation technology.

Furthermore, as a method for producing laminarioligosaccharides that can be synthesized specifically with high reaction yields without using expensive glucans, there is known a method in which laminaribiose phosphorylase acts on glucose and _α- glucose-1-phosphate. (JP-A-4-66092). This laminaribiose is derived from Medilimushi and is currently the only known one. However, this Raminaribio over scan phosphorylase low heat resistance (temperature stability 4 5 ° C, to force ^s inactivate at least about 30%), industrial production [This suitable such Rere (Archives of Biochemistry and Bioph ysics, vol .304 (2), 508-514, 1993).

Therefore, an inexpensive method for producing laminari-oligosaccharide is desired. Disclosure of the invention

The present invention has been made to solve the above problems. INDUSTRIAL APPLICABILITY The present invention provides a heat-resistant laminaribio-phosphorylase, which can produce laminari-oligosaccharide, which is a very expensive saccharide, at a low cost, and is used for foods and drinks.

The invention has the following characteristics:

(1) substrate specificity: producing laminari-oligosaccharides from α-glucose-1-phosphate and glucose;

(2) Optimum ρΗ: ρΗ6.5 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;

Laminaribiose phosphorylase having the formula: In a preferred embodiment, laminaribiose phosphorylase has the following additional properties:

(6) Molecular weight: about 17 OKDa (gel filtration HPLC); and

(7) Isoelectric point: about 4.7 (isoelectric focusing);

Having.

The present invention further relates to a method for producing laminarioligosaccharide, comprising a step of reacting a fungus having the ability to produce laminaribiose or a treated product thereof with ct-glucose-monophosphate and glucose.

In a preferred embodiment, the processed material has the following characteristics:

(1) Substrate specificity: produces laminari-oligosaccharides from α-glucose-1-phosphate and glucose;

(2) Optimum ρΗ: ρΗ6.5 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;

Is a laminaribiose phosphorylase having the formula:

In a more preferred embodiment, the laminaribiose phosphorylase further has the following characteristics:

(6) Molecular weight: about 17 OKDa (gel filtration HPLC); and

(7) Isoelectric point: about 4.7 (isoelectric focusing);

Having.

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 IA 12462), or Bacillus circulans IA12462. This is Bacillus coagulans I AMI 194. 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. BRIEF DESCRIPTION OF THE FIGURES

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

FIG. 2 is a diagram 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. BEST MODE FOR CARRYING OUT THE INVENTION

The laminaribiose phosphorylase of the present invention has been collected for the first time from a fungus. It has been discovered for the first time that laminaribiose phosphorylase, which was previously known only in Euglena, is also produced in microorganisms.

Here, in the present specification, a microorganism refers to a microorganism belonging to a bacterium, a deformed bacterium, a trichome, or a fungus in taxonomic terms.

The present inventors searched the soil for microorganisms that produce laminarioligosaccharides from glucose and _α- glucose-1-phosphate, and obtained for the first time a novel microorganism that produces laminaribiose phosphorylase. This microorganism was identified as a microorganism belonging to the genus Bacillus on the basis of its bacteriological properties. Named K2145 (Bacillus subtilis K2145), deposited at National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, 1-1-1, Tsukuba, Higashi, Ibaraki, Japan. Date: February 2, 2000).

Bacteriological properties of Bacillus subtilis strain K2145 are as follows.

(1) Form

Cell shape and size: 0.8 x 2-3 μm bacilli

Mobility: +

Sporulation: +

Colony shape: irregular, curly hair, convex, matte, cream

(2) Physiological properties

i3 galactosidase: +

Arginine hydrolase: one

Lysine decarboxylase: one

Ordinine decarboxylase: I

Availability of citrate: +

Hydrogen sulfide production: I

ゥ Lease: One

Tryptophan deaminase: I

Indole production: +

Acetin production: +

Gelatinase: +

Nitrate reduction: +

Power Tarase: +

Oxidase: +

O / F test: I Bacillus and Sacillus have long been eaten as fermented soybeans such as natto, and there is no safety problem when used in the food industry.

The laminaribiose phosphorylase of the present invention has an optimum temperature around 55 ° C., and is characterized in that laminarioligosaccharide is produced from _α- glucose monoi-phosphate and glucose. It is characterized by higher heat resistance than conventional laminaribiose phosphorylase from Euglena.

The laminaribiotis phosphorylase of the present invention also has the following characteristics:

(1) The following reaction:

It catalyzes α-glucose-1-monophosphate + glucose * ~~ laminarioligosaccharide + phosphate.

(2) Optimum ρΗ: ρΗ6.5 to 7.0;

(3) Optimal temperature: 55 ° C;

(4) pH stability: pH 5.5 to 7.0;

(5) Temperature stability: 57.5 ° C or less;

(6) molecular weight: about 170 kDa (gel filtration HPLC); and

(7) Isoelectric point: about 4.7 (isoelectric focusing).

The laminaribiose phosphorylase of the present invention can be obtained by the following method, for example, using Bacillus subtilis K2145 as an example. First, Bacillus subtilis K2145 was replaced with fructose, glucose, laminaribiose, galactose, isomerized sugar, sucrose, maltose, xylose, sorbitol, mannitol, Various sugars such as starch, sugar alcohols, starch or dextrin, etc. as a carbon source, and organic nitrogen sources such as yeast extract, fish meat extract, peptone, amino acid, etc. necessary for the growth of microorganisms, or ammonium sulfate, ammonium chloride Aerobic conditions at 20-40 ° C, preferably around 30 ° C, in a medium supplemented with inorganic nitrogen such as ammonium, urea, ammonium nitrate, etc., and minerals as required The culture is performed for 1 to 5 days, preferably 1 to 3 days.

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

The cells obtained by culturing can be further processed, and the resulting processed product can also be used for the production of lamina oligosaccharides. Here, the processed material is: 1) microbial cells immobilized by a method commonly used by those skilled in the art; 2) mechanical or enzymatic treatment of the microbial cells, or a surfactant Alternatively, those treated with an organic solvent or the like, or those immobilized on them; 3) Crushed microbial cells to remove crushed residues, or those immobilized on them; 4) 2) or 3 ), Or an enzyme obtained by further purifying the enzyme. For purification of the enzyme, means commonly used by those skilled in the art are used alone or in an appropriate combination.

For immobilization of the treated material, polyacrylamide, calcium alginate, carrageenan, an ion exchange carrier, chitosan beads, etc. are used.

The obtained microbial cells and processed products thereof (including purified enzymes and crudely purified enzymes) are mixed with a reaction solution containing ct gnorecose-i-phosphate and glucose, and used for the production of laminarioligosaccharide.

a Glucose-1-phosphate and glucose are about 100 ::! ~ 1: It is preferable that they are contained at a ratio of 100. The total amount of glucose and _α- glucose-monophosphate is 1 to 75% by weight in the reaction solution. /. Preferably, it is included. Preferably, it is 5 to 50% by weight, more preferably 20 to 40% by weight. Although ρΗ of the reaction solution is not particularly limited, it is preferably about 5.0 to 8.0, and more preferably ρΗ about 6.0 to 7.0. Reaction temperatures range from about 40-65 ° C, preferably about 50-60 ° C, more preferably about 50-55 ° C. Considering industrial practicality, about 50-55 ° C, pH about 6.0-7.

A range of 0 is preferred. The α-glucose-1-phosphate as a substrate used in the present invention can also be obtained from an oligosaccharide or polysaccharide having a ct — 1,4 bond. Oligosaccharides or polysaccharides having a 1,4-linkage include, but are not limited to, amylose and starch. By reacting amylose, starch and the like with glucan phosphorylase and the like, glucose-1-monophosphate is produced. When starch is used, a glucose-11-phosphate can be produced by simultaneously treating a branching enzyme or an amylase with these enzymes and simultaneously reacting these enzymes with glucan phosphorylase.

Further, even when allowed to act sucrose phosphorylase sucrose, _a glucose _ 1 to be able to obtain a monophosphate, _a glucose one 1 monophosphate derived from such a top Shikamono root vegetables and milk, such as Jiyagaimo Acids can also be used.

aGlucose-11-monophosphate 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.

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

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

By the above reaction, laminarioligosaccharide is obtained. By combining them, it is possible to separate components having a specific polymerization degree. Example

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. Unless otherwise specified, percentages are WZV%. Example 1

Preparation of laminaribiose phosphorylase and synthesis of laminarioligosaccharide Medium based on polypeptone 2%, yeast extract 1%, NaC 1 2% (pH 7.0) 10 Om 1 50 Om 1 volume Erlenmeyer flask with baffle After sterilization by a conventional method, Bacillus' Sacillus K2145 (FERM P-17710) was inoculated and cultured with shaking at 37 ° C for 24 hours. The culture was centrifuged at 6000 rpm, and the cells were collected and washed twice with 25 ml of 2 OmM sodium acetate buffer (pH 7.0). After suspending the cells in a 2 Om 1 sodium acetate buffer (pH 7.0), the cells were physically disrupted with glass beads, and a crude enzyme solution was obtained by centrifugation.

The obtained crude enzyme was appropriately diluted, and 200 μl thereof was added to 200% 2% laminaribiose solution 2001 dissolved in 20 mM phosphate buffer (pH 7.0), and the enzyme reaction was carried out at 55 ° C. Was. Next, the reaction solution was boiled for 10 minutes to stop the enzyme reaction, and the sugar composition was analyzed by HP LC. Under these conditions, the amount of the enzyme that degrades 1 // mol 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 100 mM α-glucose-1-phosphoric acid solution and 250 μl of a 10 OmM glucose solution were added and reacted at 55 ° C. for 24 hours. The reaction was analyzed by HP LC (Figure 1). HPLC conditions Is shown below. When the amount of laminari-oligosaccharide in the reaction solution was quantified, the conversion rate from Darcos to laminari-biose was 50%, and the conversion rate to laminari-oligosaccharides of three or more sugars was 10%.

HP LC conditions

Pump: JASCO Corporation PU-980

Column: NH ₂ P50 column manufactured by Showa Denko KK

Detector: Showa Denko KK SE-61 type 1 differential refractometer

Eluent: acetonitrile: water = 75: 25

Flow velocity: 0.8 m 1 Z m in Example 2

Properties of Laminari Biose Phosphorylase

After 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 mouth chromatography (Butyl Topearl: Tosoh), anion exchange chromatography ( A single protein was purified by DEAE-Toyopar (manufactured by Tosoh Corporation), gel filtration chromatography (Superdex200: manufactured by Pharmacia), and anion exchange chromatography (MonoQ: manufactured by Pharmacia). Using this, the following properties of laminaripiose phosphorylase were confirmed.

2.1 Optimum temperature

Add 200 μl of appropriately diluted enzyme solution to 200 μl of 10 OmM phosphate buffer (pH 7.0) containing 2% laminaribiose, and add 40, 45, 50, 55, 60, 62.5, The reaction was performed at 65 ° C for 30 minutes. The laminaribiophosphorylase activity was determined by quantifying the dulose after inactivating the enzyme by boiling for 10 minutes. The result is shown in figure 2. As is clear from FIG. 2, the optimum temperature of the enzyme was 55 ° C. The activity was also observed at 62.5 ° C. When the activity at the optimum temperature was 100%, the relative activity was about 85%. 2.2 Optimal pH

An enzyme solution (200 μl) appropriately diluted in 100 mM of various buffer solutions (200/1) containing 2% laminaribiose was added, 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 Figure 3. As is evident from FIG. 3, the optimum pH of this enzyme was 6.5 to 7.0, but it can be practically used at 6.0 to 7.5.

2.3 Temperature stability

Incubate 200 μl of the appropriately diluted enzyme solution at 40, 45, 50, 55, 57.5, 60, and 65 ° C for 10 minutes, and then add 2% laminar bios. 10 OmM phosphate buffer (pH 7.0) 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. As is clear from FIG. 4, the enzyme was substantially stable up to 57.5 ° C.

2.4 Stable pH

After incubating 200 μl of the enzyme solution appropriately diluted with each buffer at pH 3 to 9 at 25 ° C. for 3 hours, 10 mM OmM phosphate buffer containing 2% laminaribiose (pH 7.0) 200 μl was added and reacted. Laminaribiose phosphorylase activity was determined by quantifying darcose after inactivating the enzyme by boiling for 10 minutes. Fig. 5 shows the results. As is evident from Fig. 5, the yeast The element was stable up to pH 5.5-7.0.

2.5 Molecular weight and isoelectric point

The molecular weight of the purified enzyme by gel filtration chromatography was about 170 kDa, and the isoelectric point by isoelectric focusing was about 4.7. Example 3

According to Example 1, crude enzyme solutions of B. circulans (IAM12462) and B. coagulans (IAM1194) were prepared. The crude enzyme solution (10 μl) was mixed with 2 OmM phosphate buffer (ρΗ6.0) 100 // 1 containing 2% laminaribiose and reacted at 55 ° C for 24 hours. The reaction solution was analyzed by HP LC, and glucose-1 monophosphate was measured. As a result, α-glucose-1-phosphate was detected in both the reaction solutions of B. circulans (IAM12462) and B. coagulans (IAM1194), and laminaribiose phosphorylase activity was observed. Industrial applicability

The laminaribiose phosphorylase of the present invention has higher thermostability and wider action ρ Η region than those known so far, and efficiently produces laminarioligosaccharides from glucose and hydarco-su-1-phosphate. . Further, the enzyme laminaribiose phosphorylase of the present invention can be produced from microorganisms derived from the genus Bacillus, which has high food safety. By using this enzyme, laminari-oligosaccharides can be produced from starchy material at low cost and in large quantities under industrial production conditions.

Claims

The scope of the claims

1. The following characteristics:

(1) Substrate specificity: producing laminari-oligosaccharides from glucose-1-monophosphate and glucose;

(2) Optimum 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;

Laminaribiose phosphorylase having the formula:

2. Further characteristics:

(6) molecular weight: about 17 OKDa (gel permeation HPLC); and

(7) Isoelectric point: about 4.7 (isoelectric focusing);

The laminaribiose phosphorylase according to claim 1, comprising:

3. A method for producing laminario-oligosaccharide, comprising a step of reacting a fungus having the ability to produce laminaribiose or a processed product thereof with α-glucose-1-phosphate and glucose.

4. The processed material has the following characteristics:

(1) Substrate specificity: produces laminari-oligosaccharide from α-glucose-1-phosphate and glucose;

(2) Optimum ρΗ: ρΗ6.5 to 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;

4. The method for producing a laminarioligosaccharide according to claim 3, which is a laminaribiose phosphorylase having the following formula:

5. The laminaribiose phosphorylase further has the following characteristics:

(6) molecular weight: about 170 KDa (gel filtration HPLC); and

(7) Isoelectric point: 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 'Bacillus subtilis K2145, Bacillus' circulans IAM12462 (Bacillus circulans IAM12462), or Bacillus coagulans IAM1194 (Bacil lus coagulans IAM1194, claim 6). 2. The method for producing a laminari-oligosaccharide according to item 1.

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.