JPH10201472A - Method for producing α-galactosidase and galactooligosaccharide - Google Patents

Abstract

(57) [Summary] The present invention relates to an aqueous solution containing a microorganism-derived α-galactosidase having an action of selectively synthesizing Galα1-3Gal, and an aqueous solution containing a galactooligosaccharide or a galactose derivative and a sugar or alcohol. The enzymatic reaction with α-galactosidase of the present invention
This is a method for producing a saccharide having a galactoside bond. The compound obtained by the production method of the present invention is a carbohydrate linked to α1-3 galactoside such as Galα1-3Galα-OpNP, and is useful as a drug such as a rejection inhibitor for xenografts and a raw material for synthesizing the same. .

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism-derived α-galactosidase and a method for producing a saccharide having an α1-3 galactoside bond.

[0002]

2. Description of the Related Art In recent years, the results of organ transplantation have been dramatically improved due to advances in immunosuppressants and the like. Along with this, the scope of application for organ transplantation has expanded tremendously, and a sudden shortage of donors has emerged, mainly in Europe and the United States. As a countermeasure, so-called "discordant" xenotransplantation using pigs, which are immunologically and physiologically similar to humans, is being studied.

[0003] By the way, as means for suppressing hyperacute rejection which occurs in pigs as donors, 1) introduction of human complement inhibitory factors (DAF, HRF-20, MCP-1), 2) human Inactivation of the natural humoral antibody (mostly against the galactose sugar chain antigen of pig: Galα1-3Gal) is considered. As an example of the inactivation of a humoral antibody against the carbohydrate antigen Galα1-3Gal (human anti-Galα1-3Gal antibody), administration of a carbohydrate having a Galα1-3Gal structure neutralizes the antibody and causes hyperacute rejection. (DKC Cooper, E. Koren, and R.O.
riol, Xeno, vol. 2, 22-25 (1994). That is, Galα1-3
Administration of an effective amount of a saccharide having a Gal structure has an effect of suppressing rejection in a xenograft patient.

As for the method for synthesizing a saccharide having an α1-3 galactoside bond, there is a report on a method for synthesizing by a transfer reaction using α-galactosidase (PM Dey, Phy
tochemistry, 1979, vol.18, 35-38 and Tokuhei 8-2459
2) However, in the transfer reaction using α-galactosidase in this report, α1-6-linked galactooligosaccharides are obtained as the main product, and it is not possible to synthesize α1-3-linked galacto-oligosaccharides inexpensively and stably. It is possible.

[0005]

Accordingly, an object of the present invention is to provide a novel α-galactosidase derived from a microorganism for inexpensively and stably synthesizing a saccharide having an α1-3 galactoside bond, and to use the enzyme. The present invention provides a method for synthesizing a saccharide.

[0006]

Means for Solving the Problems As a result of various studies to solve the above problems, the present inventors have found that a novel microorganism-derived α-galactosidase which can selectively synthesize the saccharide having the α1-3 galactoside bond [ EC 3.2.1.22], and completed the present invention. That is, the present invention includes the claims described in the claims. Hereinafter, the present invention will be described in detail.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION

[0008] As microorganisms producing the α- galactosidase present invention, for example, Penicillium (Penicillium)
Genus Aspergillus (Aspergillus) genus, or a Streptomyces (Streptomyces) bacteria belonging to the genus, and the like. Of these, penicillium funiculosum or Aspergillus oryzae is preferred. In order to obtain α-galactosidase from these microorganisms, a known method, that is, a method of culturing the microorganism and obtaining it from a culture supernatant or microbial cells can be applied. Alternatively, commercially available enzymes, for example, α mixed in cellulase preparations and the like
-Galactosidase can be purified and used.

Next, a method for producing a saccharide having an α1-3 galactoside bond will be described.

Although α-galactosidase is a hydrolase in nature, increasing the substrate concentration catalyzes the transglycosylation. As the sugar donor, natural saccharides such as raffinose and melibiose, and synthetic substrates such as paranitrophenyl-α-galactopyranoside (pNP-α-Gal) can be used. Any carbohydrate can be used as long as it has a reducing terminal. As the sugar acceptor, compounds having a hydroxyl group such as galactose, galactooligosaccharide, various sugars and various alcohols can be used. When synthesizing a saccharide having an α1-3 galactoside bond, the ratio between the galactose donor and the galactose acceptor is 1: 0.1 to 1:10, preferably 1: 0.2 to 1: 5. Note that the sugar donor and the sugar acceptor may be the same compound without any problem. For the production of α1-3 galactoside-linked saccharide, it is preferable to use a purified enzyme,
The partially purified enzyme or the culture solution may be used as it is, or a crude enzyme obtained by pulverizing the culture solution may be used. Further, an immobilized enzyme may be used.

Hereinafter, embodiments of the present invention will be shown and described in more detail, but the present invention is not limited to these embodiments.

[0012]

【Example】

Example 1 Purification of Enzyme Penicillium funiculos
um ) 8.37 g of cellulase (Sigma) from the original enzyme
It was suspended in 5.1 ml and stirred at 4 ° C. After about 12 hours, centrifugation (10,000 × g, 10 minutes) was performed, and the supernatant was added to a Q-Sepharose FF column (2.5 cm × 25 cm) equilibrated with 10 mM sodium phosphate buffer (pH 7.4). Then, α-galactosidase was eluted with a concentration gradient of 10 mM sodium phosphate buffer (pH 7.4) containing 1 M sodium chloride. Flow rate is 1ml /
min.

Action:

[0014]

Embedded image

R-OH is a compound having a hydroxyl group such as galactose, galactooligosaccharide, various sugars, various alcohols and various phenols.

Optimum pH and stable pH Buffer containing 1 μl of the α-galactosidase of the present invention (pH 1.8
1-11.1 is Briton-Robinson buffer, pH 3.34-6.22
Is acetate buffer, pH 1.17 to 3.72 is glycine buffer) 2 ml and 5 m
500 μl of M pNP-α-Gal was mixed and reacted at 37 ° C. Ten
After a minute, 2 ml of a 0.2 M sodium carbonate solution was added to stop the reaction. FIG. 1 shows the specific activity using the amount of paranitrophenol produced by hydrolysis as an index. High enzyme activity was observed between pH 3.0 and 3.5.

To determine the stable pH, 100 μl of Briton-Robinson buffer at each pH containing 1 μl of the α-galactosidase of the present invention was kept at 37 ° C. Twenty hours later, the amount of paranitrophenol produced by hydrolysis was measured in the same manner as in the measurement of the optimum pH. The results are shown in FIG.

The enzyme activity was not inactivated even after 10 minutes of treatment at 37 ° C. at pH 3.5 to 6.5.

Optimum temperature range and optimum temperature range for action 100 μl of Briton-Robinson buffer (pH 2.49) containing 1 μl of α-galactosidase of the present invention and 500 μm of 5 mM pNP-α-Gal
were mixed and reacted at 20-70 ° C. After 10 minutes, 2 ml of a 0.2 M sodium carbonate solution was added to stop the reaction. FIG. 3 shows the specific activity using the amount of paranitrophenol produced by hydrolysis as an index.

FIG. 3 shows that the optimum temperature of the α-galactosidase of the present invention was 50 ° C., and that it was completely inactivated at 70 ° C.

Further, 1 μl of the α-galactosidase of the present invention
100 μl of Briton-Robinson buffer (pH 3.48) containing 25
Hold at 6060 ° C. for 30 minutes. Thereafter, the amount of paranitrophenol produced by hydrolysis was measured in the same manner as in the measurement of the optimum pH. FIG. 4 shows the results.

At pH 3.48, the treatment was stable at 25 to 40 ° C. for 30 minutes.

Molecular weight TSK gel G300SW (manufactured by Tosoh Corporation) has a gel filtration method of 44.
000.

Example 2 Method for Producing Oligosaccharide 100 mg of pNp-α-Gal was dissolved in a solution consisting of 300 μl of dimethylformamide and 700 μl of 0.1 M potassium phosphate buffer (pH 6.0), and 13.3 units were added thereto. Α-galactosidase (of Penicillium funiculosum origin) and 37
Shake at ℃. Six days later, the reaction solution was heated at 100 ° C. for 5 minutes to inactivate the enzyme. Sephadex G
The product was purified on a -10 column (5 cm × 100 cm) to obtain 6.1 mg of Galα1-3Galα1-OpNP. The paranitrophenyl group of the disaccharide derivative can be obtained by a known method (Japanese Patent Application No. 7-3190).
22) and free disaccharide Galα1-3Ga
l.

[0025] The elution pattern of the sugar in Figure 5, the product ¹³ C
FIG. 6 shows the NMR (D ₂ O, 125 MHz).

In addition, the enzyme preparation of Aspergillus oryzae (trade name "Orientase ONS (manufactured by Hankyu Bioindustry)") or Streptomyces genus (trade name "glucose isomerase (produced by Nagase Seikagaku Corporation)") is used. FIG. 7 shows HPLC of the reaction solution when the transfer reaction was performed using the included α-galactosidase. further,

Industrial Applicability The novel α-galactosidase derived from the microorganism of the present invention can efficiently synthesize a disaccharide Galα1-3Gal derivative which can effectively suppress hyperacute rejection in xenograft transplantation.

[0027]

[Brief description of the drawings]

FIG. 1: Penicillium funiculosum origin α of the present invention
-Shows the pH dependence of galactosidase. ○, □ and △
Indicates the specific activity as measured using a Briton-Robinson buffer, an acetate buffer, and a glycine buffer, respectively.

FIG. 2: Penicillium funiculosum origin α of the present invention
-Shows the pH stability of galactosidase.

FIG. 3. Penicillium funiculosum origin α of the present invention
-Shows the temperature dependence of galactosidase.

FIG. 4: Penicillium funiculosum origin α of the present invention
-Shows the temperature stability of galactosidase.

FIG. 5 shows an elution pattern of galactooligosaccharides in Example 2.

FIG. 6 shows ¹³ C-NMR of Galα1-3Galα1-OpNP.

FIG. 7 shows HPLC of a reaction solution obtained by performing a transfer reaction using α-galactosidase contained in (a) Aspergillus oryzae or (b) an enzyme preparation derived from Streptomyces in Example 2.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:69) (C12N 9/40 C12R 1: 465)

Claims (8)

[Claims] 1. A microorganism-derived α-galactosidase having an action of selectively forming an α1-3 galactoside bond from a galactoside compound having α-linked galactose and a sugar receptor. 2. The α-galactosidase according to claim 1, which is derived from a microorganism belonging to the genus Penicillium . 3. The α-galactosidase according to claim 1, which is derived from a microorganism belonging to the genus Aspergillus . 4. The α-galactosidase according to claim 1, which is derived from a microorganism belonging to the genus Streptomyces . 5. The α-galactosidase according to claim 2, wherein the microorganism belonging to the genus Penicillium is a Penicillium funiculosum species. 6. A microorganism belonging to the genus Aspergillus is Aspergillus oryzae (oryzae) are species according to claim 3, wherein the α- galactosidase. 7. The α-galactosidase action according to claim 5, which has the following physicochemical properties: R-OH is galactose, galactooligosaccharide, various sugars,
3 shows compounds having hydroxyl groups such as various alcohols and various phenols. Substrate specificity: Generates Galα1-3Gal-OR in the transfer reaction. Optimal pH and stable pH: The optimal pH is pH 3.0 to 3.5. pH3.5 ~
Inactivation at 37 ° C for 10 minutes at 6.5 does not deactivate. Optimum temperature and optimal temperature range: optimal temperature is 50 ℃, pH
Stable at 3.48 at 25-40 ° C for 30 minutes. Molecular weight: 44000 (gel filtration method using TSK gel G300SW). 8. A method for producing a saccharide having an α1-3 galactoside bond, comprising using the α-galactosidase according to any one of claims 1 to 7.