JPH0787783B2 - Novel endo-β-galactosidase and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel endo-β galactosidase and a method for producing the same, and more specifically, endo-β-derived from an endo-β-galactosidase-producing bacterium belonging to the genus Clostridium.
It relates to galactosidase and a method for producing the same.

[Prior art and its problems] With the recent progress of researches on the functions of various living cells, the relationship between glycoproteins and glycolipids present on the cell surface and cell functions is important medically and pharmacologically. Sex is increasing. Endoglycosidase, which is a means for elucidating the sugar chain structure constituting these glycoconjugates, has been attracting attention as an inexpensive and simple method. That is, it is also known that these glycoconjugates include functions related to promotion and suppression of abnormal growth of cancer cells and growth of normal cells. One of the factors that play an important role in these functions is the sugar composition of sugar chains and their sequences. As a means of studying the relationship between these structures and functions, a large number (multiplicity) of glycosidases with extremely high specificity are required.

Regarding endo-β-galactosidase, an enzyme having the following types of substrate specificity is conventionally known.

Escherichia freundii, Flavobacterium keratolyticus
And Bacteriodes fragilis-derived endo-β-galactosidase acts on a wide range including polylactosaminoglycan and keratan sulfate, and the enzyme from Diplococcus pneumoniae (endo-β-galactosidase DII) does not cleave keratan sulfate and is a narrow substrate. Shows specificity. Pseudomona
Enzymes from s-genus cells are highly specific for keratan sulfate.

Compared with these enzymes, blood group endo-β-galactosidase derived from Diplococcus pneumoniae has Fuc α1 → 2 and GalNAc (Gal) α1 → 3 as galactosyl residues to be recognized.
It is known to have substrate specificity such that the substitution of both residues is essential.

On the other hand, it is known that a natural substrate having the Gal α1 → 3Gal β1 → 4GlcNAc sequence exists in many glycoproteins and glycolipids, and blood group substances of these human erythrocytes, antigen determining substances of the lectin binding site, Mouse lymphoma cells, Frie
It constitutes the glycoconjugate of the nd leukemia virus. If the sugar sequences of these natural substrates can be modified using a specific enzyme,
It is thought that it will be possible to enhance and control the functions of cells, such as division, proliferation, differentiation, etc., but it will act on the Gal α1 → 3Gal β1 → 4GlcNAc sequence present at the terminal and bind Galβ1 → 4GlcNAc. An enzyme that selectively cleaves is not yet found.

Therefore, the inventors of the present invention have conducted extensive studies to find a novel endo-β-galactosidase, and as a result, Clostrid
We succeeded in collecting a novel endo-β-galactosidase from the culture of the genus bacterium, and completed the present invention.

[Structure of the Invention] The endo-β-galactosidase of the present invention has the following physicochemical properties: Action: endo-β-galactosidase Substrate specificity: at least Gal α1 → 3 Gal β1 → 4 at the end
Acting on the part with GlcNAc sequence, Gal β1 → 4GlcNAc
The bond is cleaved, but at least Gal α1 → 3 (Fu
c α1 → 2) Gal β1 → 4 Does not act on the portion having the GlcNAc sequence.

Optimum pH and stable pH range: Optimum pH: about 7.5 At pH 5.0 and pH 8.5, enzyme activity decreases by about 50%.

It is characterized by having.

Hereinafter, the present invention will be described in more detail.

The microorganism used in the present invention may be any microorganism as long as it belongs to the genus Clostridium and has the ability to produce endo-β-galactosidase. Such strains include Clostridium perfringens (ATCC 10
873 has been deposited with the Micro Type Research Institute as the American Type Culture Collection; ) And the like.

In addition, as the microorganism, for example, X-ray, γ
Radiation, ultraviolet rays, or treatment with ethyl methanesulfonate, nitrosoguanidine, etc., transformation,
You may use what improved the productivity of endo- (beta) -galactosidase by the commonly used strain mutation treatment methods, such as transduction, conjugation, and gene manipulation.

The endo-β-galactosidase of the present invention is, for example, Cl
An endo-β-galactosidase-producing bacterium belonging to the genus ostridium or a mutant strain thereof is cultured, and endo-β-
It can be produced by collecting galactosidase.

The culturing method may be any method as long as it is in accordance with a general anaerobic bacterium culturing method, but the static culturing method using a liquid medium is usually advantageous.

The medium used for the culture may be any medium component that allows the endo-β-galactosidase-producing bacterium to be used as a nutrient source.

For example, as the carbon source, glucose, mannose, starch, molasses, liquefied starch, glycerin and the like can be used.

Further, as the nitrogen source, for example, heart extract, meat extract,
Yeast extract, peptone, casein hydrolyzate, gelatin, corn meal, soybean powder, ammonium phosphate, ammonium sulfate, urea and the like can be used.

In addition, various inorganic salts such as disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, calcium carbonate, manganese chloride, magnesium chloride, sodium sulfate, sodium sulfite, etc. Can be added.

The above-mentioned medium components may be appropriately combined or added during the culture.

A suitable culture temperature is around 37 ° C and a culture time of 10-100
Time is appropriate.

Good results can be obtained by appropriately performing seed culture as the culture volume increases.

The culture conditions described above are applied by selecting the optimum conditions according to the characteristics of the strain used.

The cells were cultured in this manner, and endo-β was obtained from the obtained culture.
-Galactosidase is separated and purified.

Since the enzyme is mainly contained outside the cells in the culture,
The culture may be separated into bacterial cells and a culture solution by filtration, centrifugation or the like, and the enzyme may be purified by a general enzyme purification method.

That is, vacuum concentration, freeze-drying, dialysis, ammonium sulfate fractionation, ultrafiltration, centrifugation, chromatography using an ion exchanger, gel filtration, separation of enzymes such as fractional precipitation using an organic solvent, a purification means alone or Any combination,
In addition, endo-β-galactosidase is separated, purified and collected from the culture solution by repeatedly using it as necessary.

[Examples of the Invention] Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the scope of the present invention.

Example 1 (1) Culturing of Clostridium perfringens ATCC 10873 strain A stock culture of Clostridium perfringens ATCC 10873 strain was grown at 37 ° C. in a Cooked meat medium manufactured by Difco and used as a seed medium.

Proliferation culture for enzyme preparation is Difco Todd-Hewitt medium 35.6g Yeast extract 1.0g NaCl 2.5g K ₂ HPO ₄ 1.8g Phenylmethylsulfonyl fluoride 0.2g Cysteine hydrochloride 0.05g Glucose 1.5g Was sterilized in an autoclave using a medium containing bacterium and cultured in a 2 l Erlenmeyer flask at 37 ° C. for 72 hours.

Bacterial growth took 18-24 hours to reach the stationary phase, but it took 48 hours for enzyme production. The culture medium at this stage was cooled and centrifuged at 10,000 rpm at 60 ° C. for 60 minutes to obtain a culture supernatant. The following operations were performed at 4 ° C.

(2) Purification of endo-β-galactosidase (i) Salting out with ammonium sulfate The centrifugal supernatant was salted out with 80% saturation with ammonium sulfate, and the obtained precipitate was dissolved in a small amount of water and dialyzed against cold water to prepare an enzyme solution. And

(Ii) Apply the gel enzyme solution to a Sephadex G-200 column (2.7 x 100 cm) equilibrated with Dulbecco's phosphate buffered saline and collect 3 ml aliquots to collect fractions 26-32. -Ten
It was concentrated to 3 ml by ultrafiltration through a membrane.

The chromatogram of gel filtration is shown in FIG. In FIG. 1, the solid line represents the decay rate of the radioactive disaccharide released by the action of the enzyme on the labeled glycan, and the broken line represents the absorbance (protein absorption) at 280 nm.

(Iii) Gel ion exchanger chromatography The concentrated solution obtained in (ii) was mixed with 0.01 M Tris-HCl buffer (pH 8.
0; containing 0.1 mM dithiothreitol), then adsorbed on DEAE-Sephadex A-25 column (1.0 x 10 cm), and 0-0.4 MN with Tris-HCl buffer.
A linear gradient elution of aCl was performed. Fractions 9 to 14 (22.2 ml) were collected and concentrated to 3.8 ml by ultrafiltration using an Amicon PM-10 membrane to obtain a purified endo-β-galactosidase solution.

The gel ion exchanger chromatogram is shown in FIG. Second
In the figure, (▲) mark represents the activity of endo-β-galactosidase, (◯) mark represents the activity of β-galactosidase, and (●) mark represents the activity of β-N-acetylglucosaminidase.

Table 1 shows the results of the purification in the above (i) to (iii).

(3) Preparation of Substrate (i) High molecular weight glycoprotein is used for teratocarcinom
a) Glycoprotein preparation method from F9 strain stem cells [J. Biochem., 9
4 , 799 (1983)], and T from the membrane of the teratocarcinoma OTT 6050 strain.
It was prepared by solubilization with riton X-100, digestion with papain, digestion with pronase and gel filtration with Sephadex G-50.

Sephadex G-50 Elution fraction close to the flow-through fraction is 1M NaB
Treat with H ₄ in 0.05N NaOH solution at 50 ℃ for 15 hours and re-chromatography with Sephadex G-50 (0.5M CH ₃ COON
Fractions in the high molecular weight region with a molecular weight of 5,000 or more were collected with H ₄ buffer (pH 6.0).

OTT 6050 strain 200 g → glycan 12.3mg sugar composition (molar ratio) galactose (1.0) mannose (0.16) fucose (0.08) glucosamine (0.81) galactosamine (0.16) sialic acid (0.21) The method of this glycan Fukuda [BBA, 780, 119 (198
5)], it was found to have a poly-N-acetyllactosamine type sugar sequence.

(Ii) Galactose oxidase NaB ³ H ₄ labeling method According to the method of Fukuda et al. [J. Biol. Chem., 254 , 5458 (1979)], the non-reducing end galactosyl group and N-acetylgalactosaminyl group were labeled with tritium. .

(4) Analytical method (i) Sugar composition After each saccharide was subjected to methanolysis, N-acetylneuraminic acid was used as a standard for sialic acid, and the method of Bhatti, T. et al. [Biocim.Biophys.Acta., 222 , 339 (1970) )].

(Ii) Amino acid analysis After the sample was hydrolyzed with 6N-hydrochloric acid at 105 ° C for 24 hours,
It was performed using Hitachi's amino acid analyzer model 835.

(Iii) Methylation analysis The Hakomori method [J. Biocem., 55 , 205 (1964)] was used. The partially methylated sugar was Hitachi Gas-Mass Spectrometer 80B.
It was detected as alditol acetate by using a mold.

(Iv) Paper chromatography Whatman No. 1 paper was used and developed with solvent system I (ethyl acetate: pyridine: water = 12: 5: 4).

(V) Thin layer chromatography Solvent system II (n-propanol: ethyl acetate: water = 7: 2: 1) or solvent system III using a Merck silica gel plate.
It was developed with (chloroform: methanol: water = 65: 25: 4).

(Vi) Exoglycosidase activity measurement method 0.2 ml of the reaction mixture was mixed with 0.1 M citrate-phosphate buffer (pH
6.0) contains 0.2 mg of various p-nitrophenyl glycosides. The reaction was carried out at 37 ° C. for 15 minutes and stopped with 2 ml of 0.1 M Na ₂ CO ₃ aqueous solution.

(5) Endo-β-galactosidase activity measurement method (3) [ ³ H] -labeled glycan (25,000) prepared in (ii)
dpm, 20 μg) and purified enzyme solution and 0.1 M Tris-HCl buffer (pH 7.5; containing 50 μg of galactono-1-5-lactone) 25
Incubated for 30 minutes at 37 ° C. together with μg. The reaction was stopped by adding 50 μl of ethanol, and the reaction mixture was analyzed by paper chromatography. The radioactivity in the disaccharide region is shown as the radioactive material released by the enzyme (see Figure 3). In FIG. 3, the mark (●) represents the change in radioactivity of the product of endo-β-galactosidase digestion, and Lac and Gal represent the positions to which lactose and galactose, which were used as standard substances, migrate.

Enzyme activity is 30 d
Within minutes, the amount of product is proportional to the amount of enzyme and the reaction time.
The amount of enzyme required to obtain 2,500 dpm / min was 0.1 unit.

(6) Action and substrate specificity (i) The [ ³ H] -labeled glycan prepared in (3) (ii) was used.
When hydrolyzed with 1N-hydrochloric acid at 100 ° C for 4 hours and then analyzed by paper chromatography, 90
% Were galactose sites and the rest were N-acetylgalactosamine sites.

The endo-β-galactosidase of the present invention was allowed to act on the labeled glycan according to the activity measurement method of (5), and the radioactivity mobility of the disaccharide region was confirmed. P-Nitrophenyl glycoside was used as a substrate. In the purified endo-β-galactosidase, the presence of β-galactosidase, β-N-acetylglucosaminidase, α-N-acetylgalactosaminidase, α-mannosidase, α-galactosidase and α-L-fucosidase was not observed. .
In addition, α-L- acting on Fuc α1 → 2Gal β1 → 4Glc
The presence of fucosidase was also not recognized.

From the above, it was found that the purified endo-β-galactosidase was sufficiently pure in terms of substrate specificity.

(Ii) When the purified endo-β-galactosidase was allowed to act on the high molecular weight glycoprotein, the following results were obtained.

(A) Paper chromatography released product sugars having the same mobility as lactose.

(B) A disaccharide-like substance having a hexosyl unit was confirmed by gel filtration using a Biogel P-4 column.

(C) The produced disaccharide-like substance was completely hydrolyzed by α-galactosidase (derived from coffee beans).

(D) The produced disaccharide-like substance was not affected by β-galactosidase (derived from jack bean).

From the above, it was revealed that the non-reducing end of the produced disaccharide-like substance had an α-galactosyl residue.

(Iii) Purified endo-β on high molecular weight glycoprotein (unlabeled)
-Galactosidase was allowed to act to obtain unlabeled disaccharide. It was confirmed by methanolysis and gas chromatography that galactose was the only sugar component. By methylation analysis
2,3,4,6-Tetra-O-methylgalactose and 2,4,6-tri-O-methylgalactose were detected in a ratio of 1: 1.

From the above, it was revealed that the disaccharide has a structure of Gal α1 → 3Gal.

(Iv) Complex glycopeptide derived from bovine thyroglobulin (Gal α1 → 3Gal was added to the trimannosulcore in this substrate)
There is a β1 → 4GlcNAc sequence. ) To purified endo-β-
When galactosidase was allowed to act, 2.24 μg of the enzyme
90% of the radioactivity was found in the product. This product showed the same behavior as Gal α1 → 3Gal by chromatography on Biogel P-4 column and paper chromatography, and was confirmed to be Gal α1 → 3Gal by being completely hydrolyzed by α-galactosidase. .

From the above, the endo-β-galactosidase of the present invention acts on the Gal α1 → 3Gal β1 → 4GlcNAc sequence to produce Ga
It was found to cleave the lβ1 → 4GlcNAc bond.

(V) The purified endo-β-galactosidase was converted to pentaglycosylceramide (Gal α1 → 3Gal β1 → 4GlcNAcβ1 →
When treated with 3Gal β1 → 4Glc-Cer, it was completely triglycosylceramide (GlcNAcβ1 → 3Gal β1 → 4Glc-
Cer) was converted to.

However, the purified endo-β-galactosidase is poly-N-acetyllactosamine-type glycan derived from human type B erythrocytes and Gal α1 → 3 (Fucα1 → 2) Gal β1 → 4Gl.
It did not act on cNAcβ1 → 3 hexene-1,2,5,6 tetrol.

From the above, the endo-β-galactosidase of the present invention is different from the endo-β-galactosidase acting on blood group antigens A and B, and globotriose (Gal α1 → 4Gal β1 → 4Glc) is an enzyme of the present invention. It was found that it could not be a substrate of.

EFFECTS OF THE INVENTION According to the present invention, a novel endo-β-galactosidase can be provided.

[Brief description of drawings]

FIG. 1 shows a gel permeation chromatogram, FIG. 2 shows a gel ion exchanger chromatogram, and FIG. 3 shows an analysis result by paper chromatography.

Claims (2)

[Claims] 1. The following physicochemical properties: Substrate specificity: At least Gal α1 at the terminal of glycoconjugate →
3Gal β1 → Gal β acting on 4GlcNAc sequence
1 → 4 GlcNAc bond is cleaved, but at least Gal at the end
α1 → 3 (Fuc α1 → 2) Gal β1 → 4 Does not act on the part with GlcNAc sequence. Optimal pH and stable pH range: Optimal pH: about 7.5 Enzyme activity is about 50% at pH 5.0 and pH 8.5. An endo-β-galactosidase characterized by having a decrease. 2. An endo-β characterized in that an endo-β-galactosidase-producing bacterium belonging to the genus Clostridium or a mutant strain thereof is cultured, and endo-β-galactosidase having the following physicochemical properties is collected from the culture. -A method for producing galactosidase. Substrate specificity: at least Gel α1 at the end of glycoconjugate →
3Gal β1 → Gal β acting on 4GlcNAc sequence
1 → 4 GlcNAc bond is cleaved, but at least Gal at the end
It does not act on the portion having α1 → 3 (Fuc α1 → 2) Gal β1 → 4GlcNAc sequence. Optimum pH and stable pH range: Optimum pH: Approximately 7.5 At pH 5.0 and pH 8.5, enzyme activity decreases by approximately 50%.