JPS6158587A - Alpha-l-fucosidase and its preparation - Google Patents

Abstract

PURPOSE:To obtain alpha-L-fucosidase capable of specifically hydrolyzing the alpha- bond of fucose residue, and useful for the determination of fucose-content of a sugar-containing compound, by culturing and proliferating a specific microbial strain belonging to Fusarium genus. CONSTITUTION:A microbial strain belonging to Fusarium genus and capable of producing alpha-L-fucosidase, such as Fusarium oxysporum SA252 (FERM-P No.7762) separated from the soil under a rotten stump, is cultured in a glucose-agar medium at 4.0-9.0pH and 15-37 deg.C. The proliferated cells are transferred to a medium containing fucose, peptone, yeast extract, NaCl, etc., and cultured aerobically under aeration and agitation at about 7pH and 20-30 deg.C for 2-3 days. The supernatant liquid obtained by the filtration of the culture medium is concentrated by ultrafiltration to obtain the objective alpha-L-fucosidase having the optimum pH of 4.5-5.4, stable at 4.5-8.5pH when maintained at 4 deg.C for 48hr, losing its activity at >=60 deg.C when treated at 7pH for 10min, and having a molecular weight of 120,000+ or -5,000 (gel-filtration).

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel enzyme α-L-fucosidase (hereinafter sometimes abbreviated as the enzyme of the present invention) and a method for producing the same.

In recent years, the importance of the physiological role of complex carbohydrates present on the cell membrane surface has been recognized, and various glycosidases with different substrate specificities have been widely used for structural analysis of sugar chains of complex carbohydrates. Among these, α-L is mainly used as a fucose residue degrading enzyme due to its importance in structural research.
- Much research has been done on fucosidases.

a-L-fucosidase exists widely in f411+'14, fungi, plants, molluscs, mammals, etc. However, enzymes derived from microorganisms and plants have strict substrate specificity.
- Since it does not act on artificial substrates such as fucosides, it has the disadvantage that enzyme system activity cannot be easily measured. On the other hand, animal-derived enzymes do not have the above-mentioned drawbacks, but there are problems with obtaining materials, and large amounts of enzymes can be produced easily.

It is difficult to prepare inexpensively. The present inventors have conducted intensive research to provide α-L-fucosidase that can be easily used in research such as analysis of sugar chain structures, and as a result, we have developed α-L-fucosidase-producing bacteria newly isolated from nature. We succeeded in obtaining a-L-fucosidase as a single enzyme protein from the culture, and completed the present invention.

That is, the present invention provides human substrates such as p-nitrophenyl-α-L-7 coside, gastric mucin, Lewis blood group substances,
The present invention provides α-L-fucosidase that hydrolyzes fucose residues on natural substrates such as human red blood cells. Moreover, the present invention belongs to the genus Fusarium, and the above-mentioned a
- Cultivating a pharyngeal organism capable of producing L-fucosidase,
The present invention also provides a method for producing α-L-fucosidase, which comprises collecting α-L-fucosidase from a cultured product.

The enzyme of the present invention acts on p-nitrophenyl-a-L-fucoside, which was not a substrate for conventional enzymes derived from microorganisms, and also acts on fucose residues contained in gastric mucin, Lewis blood group substances, human red blood cells, etc. It has a wide substrate specificity, such as the ability to act as a compound, and has the advantage that it can be used to analyze the fucose content in various sugar-containing compounds, including complex carbohydrate sugar chains.

Furthermore, since the enzyme of the present invention is an inducible enzyme that is secreted outside the bacterial cells, it is possible to easily obtain a large amount of enzyme protein, and it can be extremely easily purified as a single enzyme protein preparation. Therefore, it can be supplied at low cost and in large quantities as an analytical reagent for sugar chain structure research.

The present invention will be explained in more detail below. The enzymatic and physicochemical properties of α-L-fucosidase prepared by the method of the Examples shown later are as follows.

(1) Action The enzyme of the present invention specifically hydrolyzes the α-viscosity of fucose residues that are α-viscous.

(2) Substrate specificity Table 1 shows the results of allowing the enzyme of the present invention to act on various p-nitrophenyl-glycosides.

As shown in Table 1 and above, the enzyme of the present invention is p-nitrophenyl α-
°[ shows virtually no activity against p-nitrophenyl-glycosides other than L-fucoside.

On the other hand, the enzyme of the present invention hydrolyzes fucose residues contained in gastric mucin, Lewis blood group substances, human red blood cells, etc. in addition to p-nitrophenyl-a-L-fucoside. This indicates that the enzyme of the present invention hydrolyzes the fucose residue of the αl→2 viscosity and the recombination of αl→4 bonds in the 4-fi formula. α
-A completely new type of α different from L-fucosidase
-L-fucosidase.

The Km value of the enzyme of the present invention for p-nitrophenyl-a-L-fucoside is 8.7×10M.

(8) Measurement method of titer Enzyme activity was measured using p-nitrophenyl-a-L-fucoside as a substrate in citrate buffer at pH 4.5.
The reaction was carried out at C for 20 minutes, and borate buffer (pf-I9
．． 8) to stop the reaction, the amount of free p-nitrophenol was determined by measuring the absorbance of 4QQybm. The unit of enzyme activity is 1 minute (1 μmol of p-nitrophenol). The amount of enzyme that releases phenol was defined as 1 unit.

When pig stomach munan was used as a substrate, the reaction was carried out in a citrate buffer of pH 4.5 at 37°C for 8 hours.
Reaction was carried out at 87°C for 3 hours in citrate buffer of fi,
Decreased inhibitory effect on the agglutination reaction between anti-Le saliva and Le blood cells (enzyme activity was confirmed from this).

When human erythrocytes are used as a substrate, the enzyme of the present invention is treated with type 0 erythrocytes dissolved in 2% physiological saline at 87°C for 3 hours, and the action of the enzyme is determined by reducing the agglutination ability with Urex lectin. I recognized it as a wild goose.

(4) To 1VIP H and stable pH range The optimum pH is pf″I4..5 to 5.5 as shown in FIG.
The stable pi (IJH) was 4.5 to 8.5 under the treatment conditions of 4° C. and 48 hours, as shown in FIG.

In addition, factor 1 and Figure 2 (herein, the buffer used was
Indicated by l-1-mouth.

10-(5) Conditions for inactivation due to temperature conditions After holding the enzyme at pH 7 for 10 minutes at each temperature of 20°C to 60°C, the remaining enzyme activity was measured to indicate the activity.

(6) Purification method The enzyme of the present invention can be purified by an appropriate combination of salting-out methods, various chromatographic methods, and the like.

Specific examples of purification are as shown in Examples.

(7) Molecular Weight The molecular weight of the enzyme of the present invention was determined to be 120,000±5,000 by the glue filtration method using Sephadex G-200.

(8) Polyacrylamide electrophoresis The purified enzyme of the present invention showed a phosphorus band in polyacrylamide electrophoresis.

Next, a method for producing the enzyme of the present invention by culturing microorganisms will be specifically described.

The microorganism used in the production of the enzyme of the present invention is Fusarium (F
Any substance that belongs to the genus Usarium and has the ability to produce α-L-fucosidase may be used. A specific example of such a microorganism is Fusarium oxysporum 5A252 strain, which was isolated from the soil under a decaying Basho plant by the present inventors. The mycological properties of this strain are described below.

(1) Growth status on each medium ■ Wort agar medium Growth is poor. The mycelium is thin and white. The underside of the hyphae is reddish-purple. Macroconidia are mainly type-type, but some yeast-type types are also seen. The microconidia are linked together in a pseudopygia-like structure.

■ Oatmill military medium growth is poor. Hyphae are sparse and sparse. The underside of the hyphae is white with no diffusible pigments and no lysis. The macroconidia are elongated and set;
- Many Cal conidia are yeast-like spores that are linked together like pseudohyphae.

■ Glucose agar medium glucose 1%, peptone 0.5%, yeast extract 0.5
%g! In a medium containing 0.5% sodium AN and 2% agar, the growth is very good and a dense white bacterial flora is formed. The underside of the hyphae is white and no diffusible pigments are observed.

(2) Physiological properties Growth pFT range is 4.0-9.0 and 7.0-8.
5 is optimal. The growth temperature range is 15-87℃.
The optimum temperature is 5-80°C.

Based on the following characteristics, this strain was identified as Fusarium oxysporum (Fusarium oj (ysporum)), and it was submitted to the Institute of Microbial Technology, Microbiological Research Institute No. 7762.
It has been deposited under No.

Cultivation of the microorganisms used (The medium composition used here may be any of those commonly used for the cultivation of microorganisms. Carbon sources include, for example, glucose, fucose, arabinose, sucrose, etc.) IIJ bath) Starch, molasses, carbohydrates such as dextrin, nitrogen sources include peptone and meat extract.

Examples include yeast extract, casamino acids, cornstarch liquor, various ammonium salts, various nitrates, urea, and the like.

711 (Salts such as various sodium salts, dipotassium salts, calcium salts, manganese salts, red sea bream salts, sium salts, phosphates, and nitrate r1β salts are used as machine salts, and in some cases, vitamins, etc. Please add υ11Cj) for the purpose of promotion. In addition, activated sludge can be treated with alkali and acid.
It is also possible to use the sludge extract obtained by water decomposition as a culture medium. Fucose is suitable as a carbon source in the present invention, and fucose-containing products such as alkali and acid extracts of activated sludge are also effective. For culturing, the culture medium is sterilized by a conventional method, inoculated with the strain of the present invention, and kept at 20-80°C.

Perform aerobic treatment at pH 7.0 for 2 to 811 minutes by shaking or aerating.

The enzyme of the present invention can be collected as described in F below.

That is, after the end of the culture T', inorganic salts such as ammonium sulfate are added to the culture 1-rft solution from which bacterial cells have been removed by filtration or centrifugation.
□11II There is a method of separating the salt precipitates produced in the process, and a method of concentrating the culture supernatant by ultrafiltration. #Made.

The inventive enzyme can also be used in a culture medium that does not contain fucose as a carbon source. After that, the obtained bacterial cells were collected, washed, and then transferred to a medium containing fucose, which made it more effective.
It is possible to do something. The α-L-fucosidase thus obtained was also purified in the same manner as in Section 1.

Hereinafter, the present invention will be further explained in Examples (this invention will be explained in detail).
The following Examples do not limit the scope of the present invention in any way.

Example 1 Equivalent amount of 0.6N to active 1゛L sludge (water content approximately 85%)
-After adding NaOH and heating extraction at 120°C for 30 minutes, centrifugation 1'i! The supernatant obtained by IJ was dialyzed against running water and then freeze-dried. This stuff is 0.8N-1 (12 in cl)
0℃'.

15 m of pomegranate that was heat-treated for 80 minutes and then neutralized with NaOH.
Dispense 1 into test tubes, autoclave at 120°C for 15 minutes, add Fusarium oxysporum SA 252 (
Inoculated with the microorganism strain ``Keikoken Bacteria No. 7112'' and incubated at 28°C.

Culture was carried out with shaking for 2 days. To the culture obtained by centrifugation, add ammonium sulfate to 75% saturation and remove the precipitate. Old M+)
It was dissolved in buffered acid solution (pi(7,o)) and dialyzed overnight against the same buffer.
The purified DWA was passed through a Sephadex A-500 column (1,6×5 (Ic, vl)) and the adsorbed enzyme was eluted using the same buffer containing 0.68M NaCl.

The active fractions were collected and ammonium sulfate was added to 80% saturated A11.
(pH 7.0) and dialyzed overnight against 1 dfi7 column of the same size. Add this dialysis fluid to one nail with the same edge t1 c) V-1
4j hydrodylapatite column (2,3X
1 3Cm), and the adsorbed enzyme was passed through phosphate buffer solution (3Cm).
Elution was performed using a linear gradient method of pH 7,o) t'5ool to OJ]5M. The eluted active fractions were collected and ammonium sulfate was added to reach 80% saturation, and the resulting precipitate was dissolved in 0.01 MIJ acid buffer (pH 7.0). Using a Sephadex G-150 column (1.2 x 105 cm, l) equilibrated with the same buffer solution, this solution was
[Gel sawdust was done. Add ammonium sulfate to 8°C (
) The same amount of saturated G was added, and the precipitate formed was 0.0
It was dissolved in 5M'J acid buffer (pH'7.0) and dialyzed against the same buffer overnight. This dialyzed fluid was passed through 4 concanavalin A-Sepharose 4B columns (0.5 x 10 cm) equilibrated with the same buffer solution to remove the adsorbed enzyme.
Elution was performed using a linear gradient method of methyl-mannoside θ~0.5M. The active fractions were collected and concentrated to give 800 μg of α-L-fucosidase loading sample (specific activity: 2.58
U/mg protein, yield 19.9%).

Example 2 Fucose 0.2%, peptone 0.5%, yeast extract 0.
Dispense 100ml of a medium containing 5% sodium chloride and 0.5% sodium chloride into a 1t5001L shaking flask and heat at 120°C.
.

After autoclaving for 15 minutes, 5 strains of Fusarium oxysporum SA 252 precultured in a medium with the same composition were added.
m7? It was inoculated and cultured with shaking at 28°C for 2 days.

After the culture was completed, the bacterial cells were removed by sieving to obtain a culture solution.

From this product, 1 mg (specific activity: 9.86 UAng protein, yield: 28.6%) of α-L-fucosidase was obtained in the same manner as in Example 1.

Example 3 Glucose 2%, peptone 0.5%, yeast extract 0.5
%, sodium chloride 0.5% was poured into a 500 m shake flask and heated at 120'C.
.

After autoclaving for 15 minutes, 5 strains of Fusarium oxysporum SA 252 precultured in a medium with the same composition were added.
ml incubation L was cultured at 128°C for 2 days with shaking. After culturing, collect the bacterial cells by filtration, wash well with physiological saline, and add 0.5% fucose containing the same volume as the culture solution.
lmM Tris-I4ce#Anan i('L(p i
f 8.5) The suspension of Chinese popsicles was shaken at 28°C for 24 hours. After the filtration, the lJi solution from which the bacterial cells were removed was filtered through an AE-Sephadex A-'5 (1 column (2,2 , the adsorbed enzyme was 0.
The cells were eluted with the same buffer containing 3 M NaCl. The active fraction was concentrated by Tome C ultrafiltration to obtain 1.1 mg of 4nn product of 1α-L~fucosidase (specific activity 72.51 J//ic
g protein, yield 55.8%).

[Brief explanation of drawings]

FIG. 1 shows the working pH range of the enzyme of the present invention. FIG. 2 shows the stable pH range of the enzyme of the present invention. In Figures 1 and 2, Bi is citric acid-[/- is acetic acid,
- is citric acid, L- is potassium phosphate,
Each buffer solution of I (C1 is shown. Figure 3 shows the stable temperature range of the enzyme of the present invention. Applicant: Steel Chemical Industry Co., Ltd. Representative: Hiroshi Sasaki f'HpH Saki LA'c)

Claims (11)

[Claims] (1) α-L-fucosidase that acts on artificial substrates and natural substrates having α-linked fucose residues to specifically hydrolyze α-linkages of fucose residues. (2) The α-L fucosidase according to claim (1), wherein the artificial substrate is p-nitrophenyl-α-L-fucoside. (3) Claims in which the natural substrate is porcine gastric mucin (
1) α-L-fucosidase described. (4) The α-L-fucosidase according to claim (1), wherein the natural substrate is a Lewis blood group substance. (5) Claims (1) in which the natural substrate is human red blood cells
)-L-fucosidase described in ). (6) The α-L-fucosidase according to claim (1), which has an optimum pH of 4.5 to 5.5. (7) α according to claim (1), which has a stable pH range of 4.5 to 8.5 under holding conditions of 4°C and 48 hours.
-L-fucosidase. (8) The α-L-fucosidase according to claim (1), which is inactivated at 60° C. or higher when treated at pH 7 for 10 minutes. (9) Molecular weight measured by gel filtration method is 120,00
α- according to claim (1) which is 0±5,000
L-fucosidase. (10) Cultivate a microorganism that belongs to the genus Fusarium and has the ability to produce α-L-fucosidase, and extract α-L-fucosidase from the culture.
α-L- characterized by collecting L-fucosidase
Method for producing fucosidase. (11) A microorganism that belongs to the genus Fusarium and has the ability to produce α-L-fucosidase is cultured in a nutrient medium to proliferate the bacterial cells, and then the bacterial cells are transferred to a medium containing fucose, and α-L-fucosidase is added to the medium. α-L- according to claim (10), which accumulates L-fucosidase.
Method for producing fucosidase.