JPH0349682A - Novel alpha-l-fucosidase and its preparation - Google Patents

Abstract

PURPOSE:To advantageously prepare alpha-L-fucosidase useful for the structural analysis of saccharide chains and the research for the functions of saccharides by culturing a specific microorganism belonging to the genus Bacillus. CONSTITUTION:A microorganism belonging to the genus Bacillus having an activity to produce alpha-L-fucosidase outside the cell is cultured and the alpha-L- fucosidase is collected from the cultured product. The microorganism is separated from a soil in Tatsuno city, Hyogo prefecture as the result of an extensive research of the alpha-L-fucosidase-producing activity-having microorganism in the natural field, has a wide aglicone specificity and produces the alpha-L-focosidase capable of acting on both glycoproteins and glycolipids. The alpha-L-fucosidase never acts on synthetic substrates such as p-nitrophenyl alpha-L-fucoside and methyl alpha-L-fucoside. The alpha-L-fucosidase decomposes both of alpha-1 2, alpha-1 3 and alpha-1 4 fucosyl bonds in natural substrates to liberate L-fucose.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention provides α
-L-fucosidase and its production method.

(Prior art) α-L-fucosyl groups are frequently found at the non-reducing ends or branched portions of sugar chains of complex carbohydrates such as glycoproteins and glycolipids, and in recent years, studies have been made on the metabolism and cellular functions of this group. It has been suggested that there is a deep relationship with cancer. For example, CA19-9
Most ganglioside tumor markers, such as L-fucosyl group, contain an L-fucosyl group. Furthermore, it has been observed in various cancer patients that the amount of L-fucose in the blood increases as cells become cancerous. α-L-fucosidase is such a α-L
-It is an enzyme that decomposes fucosyl bonds, and has important significance as a modification reagent that specifically removes fucose in the structural analysis of sugar chains of complex carbohydrates and in the study of the relationship between structure and function. α-L-fucosidase is widely distributed from bacteria to fungi, plants, molluscs, and mammals. Turban shells and mullets (J, Biochem., 75
, 509. (1974)) have broad aglycone specificity, and have a broad aglycone specificity for synthetic substrates such as p-nitrophenyl α-L-fucoside and for porcine submandibular gland mucin and 2'-fucosyllactose. α− that exists
1→2 fucosyl bond, α-1→3 fucosyl bond present in orosomucoid (α1 monoacid glycoprotein), α-1→4 fucosyl bond present in lacto-N-fucopentaose derived from human breast milk, or bovine immunofluorescence. Decomposes the α-1→6 fucosyl bond present in glopurin G, etc. However, these are disadvantageous for industrial production in terms of stable supply of materials.

On the other hand, the genus Bacillus (J, Biochem., 74,
fl41. (1973)), Aspergillus (J
, Biol, chem, , 245, 299.
(1970), Biochem, Biopb7t
Res. Con+mon. , 136, 563.
(1986)) and enzymes derived from microorganisms such as Almond (J. Bin1.chew., 257.
8205. The enzyme (19g2)) has strict aglycone specificity and does not act on synthetic substrates such as p-nitrophenyl α-L-fucoside, but only breaks down one type of bond. Therefore, it is very disadvantageous when it is desired to remove fucose regardless of the binding mode. Furthermore, it has hardly been investigated whether enzymes of either origin act on glycolipids. Therefore, it is desired to develop a microbial-derived α-L-fucosidase that has broad aglycone specificity and can act on both glycoproteins and glycolipids.

(Problems to be Solved by the Invention) In recent years, microorganisms belonging to the genus Corynebacterium or Flavobacterium (Japanese Unexamined Patent Publication No. 62-155086) and the genus Fusarium (Japanese Unexamined Patent Publication No. 61-58587) have been It has been found to produce α-L-fucosidase with broad substrate specificity, acting not only on synthetic substrates such as -nitrophenyl αL-fucoside, but also on natural substrates. However, the enzyme produced by the former is an intracellular enzyme, which is disadvantageous for industrial production, and the latter enzyme has a weaker effect on large molecular substrates such as porcine gastric mucin than p~nitrophenyl α-L-fucoside. being weak,
Furthermore, it has not been investigated whether both enzymes act on glycolipids, and neither is satisfactory.

(Means for solving the problem) In order to solve the problem, the present inventors extensively searched the natural world for microorganisms capable of producing α-L-fucosidase. As a result, bacteria isolated from soil in Tatsuno City, Hyogo Prefecture were found to produce α-L-fucosidase in the culture medium, which has broad aglycone specificity and can act on both glycoproteins and glycolipids. The present invention has been completed.

The bacterial strain used in the present invention has the following mycological properties.

(Morphological findings) 1. Cell morphology and size: Bacillus 0.5X4-6μm2.
Polymorphism: None3. Motility: Yes4. Spores: Yes l. OX2.57zm5. Spore part: quasi-end 6. Gram staining: Positive (growth status) 1. The shape of the colony cultured on the broth agar plate is circular, the periphery is gold-rimmed, and the surface ridges are flat. The color tone of the village is milky white to white yellow.

2. Meat juice liquid culture It grows and becomes cloudy.

3. Gelatin puncture culture Does not liquefy.

(Physiological properties) 1. Reduction of octopus salt: negative 2, production of hydrogen sulfide: negative 3. Presence of indole: Negative 4. Decomposition of starch: Positive 5. Casein degradation: Negative6. Use of citric acid: Negative7. Urease: Negative 8. Catalysis: Positive9. Oxidase: Negative 10, MR test: Negative 11. V P
Test: Negative12. Growth temperature: 10-44°C Optimal temperature 30-35°C 13. Attitude towards oxygen; aerobic14. Production of acids and gases from sugars (+: produced, -: not produced) Acid Gas D-glucose + D-arabinose + D-xylose + D-mannitol + inositol + sorbitol + sugar Decamelibiose + trehalose + 15 ．． VP medium - pH in (pH 6, O) pH 5
．． 48 (6th) 16. Due to the above characteristics, 5% growth in salt-containing medium, 7% growth, and no growth, Bergey's Manual o
froeter-minative Bacleriol
og7, 8th edition, this strain was identified as belonging to Bacillus, and Bacillus spicii M2 8 (
Bacillus sp. M2g).

This strain was submitted to the Institute of Microbial Technology, No. 107.
It has been deposited as No. 88.

Next, the enzymatic and physicochemical properties of α-L-fucosidase produced by this strain will be described.

! ) Action of the enzyme The enzyme of the present invention specifically hydrolyzes α-L-fucoside to liberate L-fucose.

2) Substrate specificity The action of this enzyme on various α-L-fucoside-containing substrates is shown in Table 1. This enzyme is p-nitrophenyl α-L
-It has no effect on synthetic substrates such as fucoside and methyl α-L-fucoside. On the other hand, α-1→2 in the natural substrate,
Both α-1→3 and α-1-4 fucosyl bonds are broken down, liberating L-fucose. Furthermore, the synthetic glycolipid lacto-N-fucopentaose I-PA. (Lact-N
A phenylalkyl group is bonded to fucopentaose I) or O
It also decomposes type O active glycolipids on type red blood cell membranes. Therefore,
The enzyme of the present invention is a new type of α-I7-fucosidase that is completely different from α-L-fucosidase of any origin, not just conventional enzymes of microbial origin.

The Km values of the enzyme of the present invention for pig stomach mucin, 2'-fucosyllactose, and 3-fucosyllactose are 8. OXIO-'M, 6.3X10-4M
and 1.2×10 −3 M.

3》Measurement method for titer Enzyme activity was measured using porcine gastric mucin as a substrate, pH 6.0.
The reaction was carried out for 10 minutes at 37°C in a potassium phosphate buffer of This was done by measuring using fucose dehydrogenase. The enzyme unit was defined as the amount of enzyme that releases 1 μmol of L-fucose per minute.

The effect on O-type active glycolipids present on the membrane of human O-type red blood cells was determined by treating O-type red blood cells with the enzyme of the present invention at 37°C for 1 hour, extracting the glycolipids from the red blood cells, and expressing the anti-I-1 activity. This was confirmed by the TLC-immunostaining method using a monoclonal antibody and +2'' l-Protein A.p
- When nitrophenyl glycoside is used as a substrate, p
The reaction was carried out at 37°C in I-T6.0 potassium phosphate buffer, and the reaction was stopped by adding borate buffer (pH 9.8), and the amount of free p-nitrophenol was reduced to 400°C.
This was done by measuring the absorbance at nm. In addition, the effects on various natural substrates and synthetic glycolipids were performed in the same manner as for pig stomach mucin.

4) Optimal pH and stable pH range The optimal pH is pI 15.5-6 as shown in Figure 1.
5, and the stable pH range is pH 4.5 as shown in Figure 2 under treatment conditions of 4°C and 63 hours. It was 5-9.5. The buffers used in Figures ↑ and 2 were citric acid-tlcl: ○○, acetic acid: ▲-▲, citric acid 2010, potassium phosphate ●-●, tris-HCI:
Indicated by △-△, glycine-NaOH: ■-■.

5) Optimal reaction temperature and stable temperature range The optimal temperature for the reaction was 50°C. Stable temperature range is pH
7. The remaining activity was measured by incubating in 0.10 mM phosphate buffer at each temperature for 10 minutes. As a result, as shown in FIG. 3, the enzyme of the present invention was stable up to 45°C and showed about 50% residual activity at 50°C.

6) Purification method The enzyme of the present invention can be purified by appropriately combining 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 approximately 285,000 by gel filtration using Toyopearl IW65F.

8) Polyacrylamide electrophoresis The purified enzyme showed a single band in polyacrylamide electrophoresis.

9) Effects of inhibitors, etc. When we investigated the effects of various additives on this enzyme, it was found that it was significantly inhibited by mercury (1mM), but other metal ions (1mM), S1I reagent (1mM), and sugar (10mM) was hardly affected.

Next, the present invention will be explained in more detail.

The microorganism used in the present invention may be any microorganism as long as it belongs to the genus Bacillus and has the ability to produce α-L-fucosidase having the above properties.

Among them, a preferred strain is M28 strain, which was isolated from soil by the present inventors.

In order to produce α-L-fucosidase using this bacterium, there is no particular limitation as long as it is used for the cultivation of ordinary microorganisms.

Examples of carbon sources include carbohydrates such as glucose, fucose, arabinose, sucrose, soluble starch, dextrin, molasses, human breast milk, and puta gastric mucin. Examples of nitrogen sources include peptone, yeast extract, meat extract, casamino acids, and corn. Steeply force, various ammonium salts, various salt salts, urea, etc. are used. Furthermore, a medium containing only porcine gastric mucin can also be used as a medium. α of the present invention
Since L-fucosidase is an inducible enzyme, if carbohydrates containing α-L-fucoside, such as oligosaccharides prepared from pig stomach mucin or human breast milk, are added to the medium, the amount of enzyme produced can be significantly increased. For example, by adding 1.0% pig stomach mucin, compared to adding glucose, the amount of
0 times more αL-fucosidase of the present invention is produced. For culturing, the medium is sterilized by a conventional method, and the strain of the present invention is inoculated.
25-35°C, pI{6.0-7. It is carried out aerobically by shaking or aerating at 0 for 2-3 days. This enzyme can also be obtained by induced production using cultured bacterial cells.

After completion of the culture, α-L-fucosidase can be collected from the culture solution and purified by a combination of known methods. Since this enzyme is produced in the culture solution, it can be purified by removing the bacterial cells by centrifugation, etc., and fractionating the culture supernatant with ammonium sulfate, followed by chromatography such as ion exchange, gel filtration, or adsorption.

Examples will be described in detail below, but the present invention is not limited thereto.

(Example) Example 1 Glucose 1%, peptone 0.5%, yeast extract 0.5
%, sodium chloride 0. 100 ml of a medium containing 5% (pl 16.5) was dispensed into a 500 ml shaking flask, and after sterilization, Bacillus subicii M28 was inoculated and cultured with shaking at 30°C to prepare a seed culture. Next, pig stomach mucin 2.0%, peptone 0.5%, yeast extract 0
．． 5%, 0 sodium chloride.

8 L of a medium consisting of 5% (pII 6.5) was placed in an IOL jar fermenter and sterilized at 120° C. for 30 minutes. After cooling, inoculate the above seed culture solution and incubate at 30°C for 3 days.
Culture was carried out under conditions of an aeration rate of 3.5 L/min and a stirring speed of 350 revolutions/min. After completion of the culture, bacterial cells and insoluble matter were removed by centrifugation to obtain a culture filtrate. Ammonium sulfate was added to the filtrate while keeping it at O-4°C, and a precipitate fraction between 33 and 80% saturation was collected. The obtained precipitate was dissolved in 10 mM phosphate buffer (pH 7.0) and dialyzed against the same buffer overnight.

This dialysate solution was passed through a DEAE-cell fine AM force ram (5.6 x 45 cm) that had been equilibrated with the same buffer solution.
The active fraction eluted from the non-adsorbed fraction with the same buffer was collected, and ammonium sulfate was added to achieve 35% saturation. The generated precipitate was removed by centrifugation, and Toyopearl I was equilibrated with 10 mM phosphate buffer (p}17.0) containing 35% saturated ammonium sulfate.
It was passed through a W65C column (6.OX26 cm), and the adsorbed enzyme was eluted with the same buffer containing 10% (w/v) ammonium sulfate. The active fractions were collected and ammonium sulfate was added to achieve 80% saturation. The generated precipitate was diluted with 1mM phosphate buffer (pI-1
7.0) and dialyzed against the same buffer overnight. This dialyzed solution was passed through a hydroxylapatite force column (2.6 x 16 cm) equilibrated in advance with the same buffer, and the adsorbed enzyme was eluted using a linear gradient method from 1 to 200 mM phosphate buffer (p117.O). . The eluted active fractions were collected and concentrated, and prepared in advance with 10 mM phosphate buffer (pH?, O
) equilibrated with Toyopearl HW65F column (1,5X
Gel filtration was performed using a filter (120 cm). After collecting and concentrating the active fractions, 10 mM containing 20% (w/v) ammonium sulfate
Dialyzed overnight against phosphate buffer (pl{7.0). The dialyzed fluid was passed through a Butil Toyopearl 650 column (1.2 x 16 cm) that had been equilibrated with the same buffer solution, and 20-
Elution was performed using a linear gradient method using O% (W/V) ammonium sulfate. After collecting and concentrating the active fractions, they were dialyzed overnight against 10 mM phosphate buffer (pH 7.0) to obtain 700 μg of purified α-L-fucosidase (specific activity: 20 units).
/mg, yield 0.9%).

Example 2 Glucose 0.5%, peptone 0.5%, yeast extract 0
．． Dispense 500 ml of a medium containing 5% sodium chloride and 0.5% (pH 6.5) into a 2 L shake flask,
After sterilizing at 120°C for 20 minutes, add 5 ml of Bacillus spicii inoculum precultured in the same medium.
It was inoculated and cultured overnight at 30°C with shaking. After culturing, collect the bacterial cells by centrifugation, wash thoroughly with physiological saline,
The enzyme was suspended in 500 ml of 20 mM phosphate buffer containing 2% pig stomach mucin and shaken at 30°C for 24 hours to induce enzyme production. After the induction, the bacterial cells were removed by centrifugation, and the
An induced enzyme solution with an activity of 0.9 units/ml was obtained.

(Effects of the Invention) As explained above, the α-L-fucosidase of the present invention acts not only on glycoproteins but also on glycolipids and has broad aglycone specificity, so it can be used for structural analysis and functional analysis of sugar chains. Extremely useful for research.

Furthermore, since the enzyme of the present invention is secreted outside the bacterial cells, it can be supplied in large quantities and at low cost as a modification reagent that specifically removes fucose.

[Brief explanation of drawings]

FIG. 1 is a graph showing the optimum p I-1 of this enzyme, FIG. 2 is a graph showing the stable pH of this enzyme, and FIG. 3 is a graph showing the stable temperature of this enzyme.

Claims (2)

[Claims] (1) α-L-fucosidase [1] action having the following enzymatic properties It acts well on the α-L-fucoside bonds of complex carbohydrates such as glycoproteins and glycolipids, and liberates L-fucose. [2] Substrate specificity This enzyme does not act at all on synthetic substrates such as p-nitrophenyl α-L-fucoside and methyl α-L-fucoside. On the other hand, α-1→2, α-1→3 and α in natural substrates
- Breaks any of the 1→4 fucosyl bonds, liberating L-fucose. Furthermore, it also decomposes the synthetic glycolipid lacto-N-fucopentaose I-PA_3 (a phenylalkyl group is bonded to lacto-N-fucopentaose I) and the O-type active glycolipid on the O-type red blood cell membrane. [3] Optimal pH and stable pH range The optimal pH is pH 5.5-6.5, and the stable pH range is pH 4.5-9.5 under the holding conditions of 4°C and 63 hours.
It is. [4] Optimal reaction temperature and stable temperature range The optimal temperature for the reaction is 50°C, and when treated at pH 7.0 for 10 minutes, it is stable up to 45°C and is inactivated at 60°C or higher. [5] Molecular weight The molecular weight measured by gel filtration is approximately 285,000. [6] Influence of inhibitors, etc. Although it is significantly inhibited by mercury, it is hardly affected by other metal ions, SH reagents, and sugars. (2) α-L-fucosidase is characterized by culturing a microorganism belonging to the genus Bacillus and having the ability to produce α-L-fucosidase extracellularly, and collecting α-L-fucosidase from the culture.
Method for producing L-fucosidase.