JPH04126077A - Endo-beta-n-acetylglucosaminidase and production thereof - Google Patents

Abstract

PURPOSE:To obtain an endo-beta-N-acetylglucosaminidase acting to sugars bonded to asparagine residue of a glycoprotein and specifically hydrolyzing the diacetylchitobiose segment of a sugar chain to isolate an oligosaccharide by culturing a microbial strain belonging to genus Acinetobacter. CONSTITUTION:A microbial strain belonging to genus Acinetobacter and capable of producing an endo-beta-N-acetylglucosaminidase (e.g. FERM P-11699) is cultured and the subject enzyme is separated from the cultured product. The enzyme acts on a sugar bonded to an asparagine residue of a glycoprotein (a composite type including high-mannose type, a mixed type, sialic acid, etc., and a sugar bonded to a peptide) to specifically hydrolyze the N,N'-diacetylchitobiose segment of the sugar and isolate an oligosaccharide. The enzyme has a stable pH range of 6.0-8.0 (4 deg.C, 5 days), a stable temperature range of <=45 deg.C (pH 7.0, 10 min) and a molecular weight of about 35,000.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a novel endo-β-N-acetylglucosaminidase (hereinafter abbreviated as endo-β-GlcNAcase) and a method for producing the same using a microorganism. . More specifically, e
This invention relates to ndo-β-GlcNAcase and its production method using microorganisms.

Glycoproteins are widely distributed in animal organs, plant tissues, and cell membranes and walls of microorganisms.

In recent years, it has become clear that sugar chains of glycoproteins play an important role in molecular identification phenomena in living organisms, such as cell differentiation and intercellular recognition. In order to elucidate these roles, it is important to investigate the structure and function of the sugar chains. Various highly specific glycosidases produced by microorganisms have attracted attention as a means of elucidating the structure and function of such sugar chains, and research using this enzymatic method has been widely conducted. . However, since the sugar chains of glycoproteins usually have a complex structure, analysis using exo-type glycosidase alone is extremely difficult. Therefore, endo-type glycosidases, which separate sugar moieties from glycoproteins as oligosaccharides, have been considered to be extremely important enzymes.

Endo-β-GlcNAcase acts on asparagine-linked sugar chains present in glycoproteins and cleaves the N,N1-diacetylchitobiose moiety present at the bond between the sugar chain and protein as shown below. Release sugar chains.

(Oligosaccharide...Manβ1→4G1cNAcβ
1→4G 1cNAc-Asn-Protein) 8endo
- Effect of β-G1cNAcase (oligosaccharide-Manβ
I-4GlcNAc)+(GlcNAc-As
n-protein) This enzyme is a very important enzyme for analyzing the structure and function of glycoprotein sugar chains because it can release the sugar chain moiety of glycoproteins from the protein moiety.

[Problems to be solved by conventional technology/invention] Conventionally, e
ndo-β-G1cNAcase is a diplococcus pneumoniae (Di
Endo-D produced by Plococcus pneumoniae.

Cl0StridiUIIl perfringens
Endo-CI and Cl produced by strept
Endo produced by Omyces plicatus
Although enzymes such as H are known and used for research on sugar chains of glycoproteins, these enzymes only act on specific sugar chains.

That is, the structure of asparagine-linked sugar chains can be divided into high-mannose type, mixed type, and complex type as shown below.

(High mannose type) KA Manal-2 Manal” (Mixed type) ±Fucαl → 4GICNACβl-4GlcNAc-Asn The recently discovered Flavo
bacterium meningosepticum
endo-β-G1cNAcase (Endo-F
) acts on high-mannose type and complex type sugar chains, or on complex type sugar chains in the presence of high concentrations of 2-mercaptoethanol and protein denaturants such as nonidet-P2O. Without it, it won't work.

On the other hand, the present inventors previously filed an application (Japanese Unexamined Patent Publication No. 1-3096
No. 85) en produced in the culture solution by a strain of Mucor genus
do-β-GlcNAcase acts not only on high-mannose-type and mixed-type sugar chains, but also on complex-type sugar chains bound to sialic acid. This is a novel endo-β-GlcNAcase that does not require a protein denaturant. However, the enzyme derived from the genus Mucor is not easy to purify, and the proteolytic enzymes present therein have strong activity, so they have drawbacks from the viewpoint of enzyme utilization.

[Means to solve the problem]

The present inventors have discovered that they have broad substrate specificity, acting not only on high-mannose and mixed-type asparagine-linked sugar chains of glycoproteins, but also on complex-type sugar chains that bind sialic acid. endo-β- which can directly act on sugar chains without denaturing existing glycoproteins.
As a result of searching for bacteria that produce GlcNAcase, specific enzyme activity was found in the culture solution of bacteria isolated from soil in Sakyo Ward, Kyoto City. Endo-β-GlcNAcas can be obtained as a single protein from the culture solution of such microorganisms.
We succeeded in obtaining e and completed the present invention.

That is, the present invention provides a novel endo-linker that acts on high-mannose type, mixed type, and complex type asparagine-linked sugar chains, and acts on natural glycoprotein sugar chains in a 1-ntact state. β-GlcNAcase
It provides: Furthermore, the present invention relates to the endo-β-GlcNAcas belonging to the genus Acinetobacter.
By culturing microorganisms capable of producing e, en is extracted from the culture solution.
The present invention also provides a method for producing endo-β-GlcNAcase, which comprises collecting dq-β-GlcNAcase.

The enzyme of the present invention can release most asparagine-linked sugar chains including complex types present in glycoproteins, and there is no need to denature proteins by adding protein denaturants. It has an extremely excellent property of being able to release sugar chains from proteins. Therefore, in studying the functional and physiological roles of sugar chains and proteins of glycoproteins, it is possible to clarify them by allowing the enzyme of the present invention to act on the glycoproteins. That is, by allowing the enzyme of the present invention to act on a glycoprotein, it is possible to obtain a protein in which almost all asparagine-linked sugar chains, including complex types, have been removed, and its physiological activity can be investigated. In addition, many reports have recently been published that the structure of asparagine-linked sugar chains in cancer cells changes to a structure different from that in normal cells, and the structure of sugar chains in glycoproteins is attracting attention.

The enzyme of the present invention, which can release sugar chains from protein moieties under such circumstances, is expected to be used as an enzyme useful for structural analysis of sugar chains. Furthermore, since the enzyme of the present invention is secreted outside the bacterial cells by culturing the bacterial cells in an ordinary inexpensive medium, it is possible to easily obtain a large amount of enzyme tannokuri, and it is relatively simple. Since purified enzymes can be obtained through manipulation, they can be supplied in large quantities and at low cost as research reagents.

The present invention will be explained in more detail below. The enzymatic chemical and physicochemical properties of endo-β-GlcNAcase obtained by the method of the Examples shown later are as follows.

(1) Action The enzyme of the present invention has the action of hydrolyzing the N,N1-diacetylchitobiose moiety in the vicinity of the protein bond of the asparagine-linked sugar chain of glycoprotein.

(Oligosaccharide...Manβl→4G1cNAcβ
l-+ 4G 1cNAc-Asn-Protein) 8 e
Action of ndo-β-GlcNAcase (oligosaccharide-・
-・Manβ1 →4G1cNAc )+ (Glc
(NAc-Asn-Protein) (2) Substrate Specificity The enzyme of the present invention is capable of absorbing all different types of sugar chains contained in the glycopeptide prepared from ovalbumin, i.e., both high-mannose type and mixed type. It also breaks down types of sugar chains. It also degrades complex sugar chains contained in glycopeptides prepared from transferrin in human plasma and fetuin in calf plasma. Complex sugar chains can be hydrolyzed even if they have sialic acid bonded to them. Furthermore, since the enzyme of the present invention has been shown to release sugar chains even when transferrin itself is used as a substrate, it can also act on natural glycoproteins and can act regardless of the presence or absence of sialic acid. be able to.

(3) Method for measuring titer The activity of this enzyme was measured using a dansyl compound of a glycopeptide (asialoglycopeptide) obtained by thoroughly treating human plasma transferrin with pronase and then removing sialic acid with sialidase. Ta. The reaction was carried out at 37°C in a potassium phosphate buffer with a pH of 6.0, the reaction was stopped by adding 40% trichloroacetic acid (final concentration 5%), and the decomposition products were dissolved in n-butanol-acetone-water (n-butanol-acetone-water). 3:
1:1) as a developing agent, extracted with water, and then quantified using a fluorescence method. 1μ per minute for reaction at 37℃
The amount of enzyme that decomposes mol of substrate is defined as 1 unit (Unit: herein abbreviated as "U").

(4) Optimal pH and stable pH The optimal pH is pH 4.0 to 5.0 (Figure 1), and the stable pH is pH 6.0 to 8.0 when treated at 4°C for 5 days.
It was 0 (Figure 2).

(5) Optimal temperature and stable temperature The optimum temperature was 50-55°C (Figure 3).

In addition, the remaining enzyme activity was measured after holding at each temperature for 10 minutes at pH 7.0. As a result, it was stable up to 45°C, and about 50% was inactivated at 55°C (Figure 4).

(6) Inhibition, Activation, and Stabilization As a result of examining the effects of various additive substances on the enzyme of the present invention, there was no additive substance that significantly activated the enzyme. Among metal salts, HgcI! , but no significant effects were observed on other substances.

A stabilizer for the enzyme of the present invention has not yet been found.

(7) Km value The Km value when using dansyl asialotransferrin glycopeptide as a substrate is 6.8X10-'M, and the Km value when using dansyl ovalbumin glycopeptide as a substrate is 3.8X10-'M. be.

(8) Purification method For the purification of the enzyme of the present invention, known purification methods may be used alone or in combination. For example, the culture solution can be filtered or centrifuged to remove bacterial cells, and then salting out, various chromatographic methods, etc. can be carried out in an appropriate combination.

An example of a purification method is shown below.

(Step C) After removing the bacterial cells from the culture solution by centrifugation, the culture solution is salted out with ammonium sulfate (50% saturation).

After removing the generated precipitate by centrifugation, the supernatant is further subjected to salting out with ammonium sulfate (80% saturation).

(Step b) The generated precipitate was collected by centrifugation, dissolved in 10 mM potassium phosphate buffer (pH 7,0), dialyzed overnight against the same buffer, and the dialyzed fluid was pre-equilibrated with the same buffer using DEAE.
- Pass through the cellulose column and elute using the same buffer.

(Step C) Collect the enzyme active fractions contained in the non-adsorbed fractions and add ammonium sulfate (80%
% saturation). The resulting precipitate was left at 4°C overnight, and the resulting precipitate was added to 20mM potassium phosphate buffer (pH 7.0.
) and dialyzed overnight with the same buffer, the dialyzed solution is passed through a hydroxylapatite force ram equilibrated with the same buffer and eluted with the same buffer.

(Step d) The non-adsorbed enzyme active fractions are collected and salted out with ammonium sulfate (80% saturation). The generated precipitate is dissolved in 10 mM potassium phosphate buffer (pH 7.0), and gel filtration is performed using a Sephadex G-150 column equilibrated with the same buffer in advance.

(e process) The active fractions are collected and salted out with ammonium sulfate (80% saturation).

The generated precipitate is collected and subjected to gel filtration again using the same Sephadex G-150 column equilibrated with 10 mM potassium phosphate buffer (pH 7.0). The active fractions are collected and concentrated by salting out with ammonium sulfate (80% saturation) to obtain a purified enzyme preparation.

(9) Molecular weight The molecular weight of the enzyme of the present invention was determined to be approximately 35 by gel filtration using Sephadex G-150 and 5DS electrophoresis.
,000.

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

The microorganism used for producing the enzyme of the present invention may be any microorganism as long as it belongs to the genus Acinetobacter and has the ability to produce the enzyme of the present invention. Specific examples of such microorganisms include:
Acinetobacter isolated from soil by the present inventors
・Subii Seeds B-43 stock may be mentioned. The mycological properties of this bacterium are described below.

(1) Morphological properties - Short culm size 20.8~1. OX1.2-1.5μm Motility: None Flagella: None Spores: None Durham staining: Negative (2) Growth status (a) Juice agar plate culture Shape: Perfect round (granular) Ridge peripheral surface: Juice agar Quality of growth on slant culture Surface growth Shape Color Gloss Transparency (c) Growth on the surface of juice liquid culture: None Turbidity: Moderate Sedimentation: Moderate Growth status of meat juice gelatin puncture culture 2 Growth only on the surface Liquefaction of gelatin: Positive (3) Physiology Reduction of nitrate: Denitrification reaction: Convex circular metal edge lubrication, moderate viscosity lubrication, regular circle, orange-yellow, translucent (2) (3) MR test: VP test: Production of indole Production of two pigments: + Oxidase : Catalase 20 Growth pE (・ 5.0-7.5 Optimum growth temperature = 15-25℃ Attitude towards oxygen 2 Facultative aerobic 0-F test: Oxidative Production of acid and gas from saccharides Sugar Acid D-glucose + D-mannose + D-fructose D-galactose + maltose + sucrose lactose + arabinose + gas (water-insoluble) Regarding bacteria with the above mycological properties, Virginie's Manual Systematic System When identified according to the classification of Acinetobacter Bacteriology Vol. 1 (1984), this strain was identified as a strain of the genus Acinetobacter, and Acinetobacter Acinetobacter B-43 (Ac
inetobacter 5pecies B-43). This strain has been deposited with the Institute of Microbial Technology as Microtechnology Research Institute No. 11699.

The medium composition used for culturing the microorganism may be of any composition as long as it is commonly used for culturing microorganisms. Carbon sources include carbohydrates such as glucose, galactose, lactose, sucrose, soluble starch, molasses, and dextrin; nitrogen sources include peptone, meat extract, yeast extract, casamino acids, cornstarch liquor, and various ammonium salts. , various nitrates, urea, etc. As inorganic salts, various salts such as sodium salts, potassium salts, calcium salts, manganese salts, magnesium salts, phosphates, and nitrates are used, and in some cases, vitamins are added for the purpose of promoting the growth of bacteria. May be added.

The culture is carried out aerobically by sterilizing the medium by a conventional method, inoculating it with the strain of the present invention, and shaking or aerating it at 20 to 30°C and pH 6 and 5 to 70 for 3 days.

〔Example〕

The enzyme of the present invention can be collected according to the following examples. The following examples are not intended to limit the scope of the invention in any way.

Example I Glucose 0.5%, Peptone 0.5%, Yeast Extract 0
．． 500 ml of a medium containing 5% was dispensed into a 21-volume shake flask, autoclaved at 120°C for 115 minutes, and then inoculated with 5 strains of Acinetobacter sp. B-43 that had been precultured in a medium with the same composition. The cells were cultured with shaking at 28°C for 13 days. After culturing, remove the bacterial cells by centrifugation.
Ammonium sulfate was added to the obtained culture supernatant to 50% saturation, and the mixture was left at 4°C overnight. After removing the generated precipitate by centrifugation, ammonium sulfate was further added to the supernatant to achieve 80% saturation (Step C).

The generated precipitate was collected by centrifugation, dissolved in 10 mM potassium phosphate buffer (pH 7.0), and dialyzed against the same buffer overnight. This dialyzed solution was passed through a DEAE-cellulose column (2,6×41an) equilibrated in advance with the same buffer and eluted using the same buffer (step b).

The enzyme active fraction contained in the non-adsorbed fraction was collected and ammonium sulfate was added to the
The mixture was added to % saturation and left overnight at 4°C. The generated precipitate was added to 20mM potassium phosphate buffer (pH 7,
0) and dialyzed overnight with the same buffer, the dialyzed solution was passed through a hytroxylapatite column (2.3 x 10ao) equilibrated with the same buffer and eluted with the same buffer (Step C). .

The non-adsorbed enzymatically active fractions were collected and ammonium sulfate was added to achieve 80% saturation, and the resulting precipitate was dissolved in 10 mM potassium phosphate buffer (pH 7.0).

This solution was subjected to gel filtration using a Sephadex G-150 column (1.2 x 120 cm) equilibrated in advance with the same buffer (step C).

The active fractions were collected and ammonium sulfate was added to 80% saturation, and the resulting precipitate was collected and applied to the same Sephadex G-150 column (pre-equilibrated with 10mM potassium phosphate buffer (pH 7,0)). 1.2 x 120 cm). The active fractions were collected and concentrated by adding ammonium sulfate to 80% saturation to obtain a purified enzyme preparation (Step C).

Table 1 shows the degree of enzyme purification in each step when purified through these steps.

Table 1 Example 2 The present invention was applied to dansylated glycopeptides obtained from opalbumin having high-mannose type and mixed type sugar chains, transferrin having double-chain complex type sugar chains, and asialotransferrin excluding sialic acid. The reaction solution was subjected to thin layer chromatography after the enzyme was applied, and the specificity of the enzyme of the present invention to glycoprotein sugar chains was investigated. The results are shown in FIG.

In the figure, lane I shows the results when the enzyme of the present invention was applied to each substrate, and lane 2 shows the results when the endo-β-GLcNAcase derived from Mucor sp. 3 indicates untreated substrate.

As is clear from the figure, the enzyme of the present invention can act not only on high-mannose type and mixed type sugar chains, but also on complex type sugar chains, and can also decompose complex type sugar chains bound to sialic acid. I can do it.

Example 3 Regarding the effects of the enzyme of the present invention on natural glycoproteins, the effects on asialotransferrin were investigated.

1 Omg of asialotransferrin was treated with 3 mU of the enzyme of the present invention at 37° C. for 46 hours, and the reaction solution was subjected to gel filtration chromatography using Sephadex G-100. The results are shown in FIG. As can be seen from the figure, in the reaction solution treated with the enzyme for 46 hours, a sugar peak was found on the lower molecular side than the elution position of asialotransferrin, indicating that sugar chains were liberated by the enzyme reaction. Therefore, it is clear that the enzyme of the present invention can also act on natural glycoproteins.

〔Effect of the invention〕

Conventionally known endo-β-GlcNAcases act on high-mannose and mixed asparagine-linked sugar chains, but do not act on complex-type sugar chains (e.g.
JP-A No. 61-265088), it acts on high mannose type and complex type sugar chains, but does not act on complex type sugar chains unless in the presence of a denaturing agent.

The endo-β-GlcNAcase of the present invention acts not only on high-mannose and mixed-type sugar chains, but also on complex-type sugar chains. No denaturing agents required. In this respect, it has properties in common with the above-mentioned enzyme derived from the genus Mucor, which was discovered by the present inventors.

However, the endo-β-GlcNAca of the present invention
Since se can be purified more simply and easily than enzymes derived from the genus Mucor, it has the feature that it is possible to obtain purified enzymes that do not contain proteolytic enzymes.

It is highly expected that the enzyme of the present invention obtained by such a method will be used as a powerful means for elucidating in-vivo molecular identification phenomena, including structural analysis of sugar chains on the surface of cancer cells.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the optimum pH of the enzyme of the present invention. FIG. 2 is a diagram showing the stable pH of the enzyme of the present invention. FIG. 3 is a diagram showing the optimum temperature of the enzyme of the present invention. FIG. 4 is a diagram showing the stable temperature of the enzyme of the present invention. FIG. 5 is a diagram showing the results of examining the specificity of the enzyme of the present invention for glycoprotein sugar chains by thin layer chromatography. FIG. 6 is a diagram showing gel filtration chromatography showing the release of sugar chains when an enzymatic reaction is carried out using asialotransferrin as a substrate, regarding the action of the enzyme of the present invention on natural glycoproteins.

Claims (11)

[Claims] (1) An endo-glycoprotein that has the activity of acting on sugar chains bound to asparagine residues of glycoproteins, specifically hydrolyzing the N,N'-diacetylchitobiose moiety of the sugar chains, and releasing oligosaccharides. β-N-acetylglucosaminidase. (2) Claim (1) in which the sugar chain bonded to the asparagine residue acts not only on high mannose type and mixed type but also on complex type.
The endo-β-N-acetylglucosaminidase described in ). (3) The endo-β-N-acetylglucosaminidase according to claim 2, wherein the sugar chain bonded to the asparagine residue is a complex type containing sialic acid. (4) The sugar chain bonded to the asparagine residue is a peptide,
The endo-β-N-acetylglucosaminidase according to claim 1, which is in a form bound to an amino acid or a natural high-molecular protein. (5) The endo-β-N-acetylglucosaminidase according to claims (1) to (4), wherein the optimum pH at which the enzyme acts is pH 4.0 to 5.0. (6) Stable p of the enzyme under the holding conditions of 4℃ and 5 days.
Claims (1) to (4) wherein the H range is pH 6.0 to 8.0.
The endo-β-N-acetylglucosaminidase described in ). (7) Claims (1) to (4) wherein the stable temperature range of the enzyme is up to 45°C under the conditions of pH 7.0 and 10 minutes.
The endo-β-N-acetylglucosaminidase described in ). (8) The molecular weight of the enzyme measured by gel filtration method is approximately 35.0
The endo-β-N according to claims (1) to (4), which is 00
-Acetylglucosaminidase. (9) A bacterial strain No. 11699 belonging to the genus Acinetobacter having the ability to produce endo-β-N-acetylglucosaminidase according to claims (1) to (4). (10) Acinetobacter
r) Endo-β-, which is characterized by culturing a microorganism belonging to the genus endo-β-N-acetylglucosaminidase and having the ability to produce endo-β-N-acetylglucosaminidase in a medium in which the microorganism can grow, and collecting the target product from the culture. Method for producing N-acetylglucosaminidase. (11) The method for producing endo-β-N-acetylglucosaminidase according to claim (10), wherein the microorganism is the strain according to claim (9).