JPS6244180A - Universal-type endo-beta-n-acetylglucosaminidase and production thereof - Google Patents

Abstract

NEW MATERIAL:An endo-glycosidase having the substrate specificity character ized by the activity to act to the N,N'-diacetylchitobiose structure part of an asparagine-bonded sugar chain of a glycoprotein, hydrolyze the beta-1 4 bond of N-acetylglucosamine as shown by the arrow in the figure, isolate oligosaccharide from glycoprotein, and act to a combined-type asparagine-bonded sugar chain as well as the high-mannose-type or mixed-type sugar chain. USE:A universal-type endo-beta-N-acetylglucosaminidase useful for the determination of the structure of the sugar chain of a glycoprotein. PREPARATION:The root, stalk, leaf, flower, seed, pod or juice of sword bean is extracted e.g. with an aqueous medium and the obtained extract liquid is purified by a conventional method for the purification of enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel general-purpose endo-β-N-acetylglucosaminidase (hereinafter sometimes abbreviated as the enzyme of the present invention) and a method for producing the same. More specifically, we will examine not only the high-mannose type and mixed type of asparagine-linked sugar chains of glycoproteins, but also the N, N'-
It is an endo-glycosidase that also has substrate specificity to act on the diacetylchitobiose structure and has the action of hydrolyzing the β1→4 bond within the N,N'-diacetylchitobiose structure. General-purpose endo-β-
This invention relates to N-acetylglucosaminidase and a method for collecting it from catfish.

In general, glycoproteins are widely distributed in the biological world, including enzymes, hormones, immunoglobulins, and cell surfaces, and the importance of the role of their sugar chain moieties has recently attracted particular attention, and many researchers have Although we are currently working on elucidating the functions of glycoproteins, we have not yet fully elucidated the whole picture.The reason for this is that research on the structure of sugar chains, which is the basis for elucidating their functions, is due to the fact that glycoproteins have constituent units with different properties: amino acids and sugars. It is thought that this is a hindrance. In this sense, endo-glycosidases, which cut off sugar moieties from glycoproteins as oligosaccharides, are very important enzymes when studying the structure of sugar chains or the influence of the N-chain of glycoproteins on physiological activity. It is thought that it will become.

Therefore, the present inventors investigated the end-products in various sources.
As a result of intensive research on glycosidase, a novel general-purpose endo-β-N-acetylglucosaminidase was discovered in a solution extracted from soybeans.

Conventionally, endo-β-N-acetylglucosaminidase obtained from bacteria has been known, but all of them act only 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.

Next, the formula and action site of each type are shown.

In each of the above types, conventional endo-β-N-acetylglucosaminidase acts on high-mannose type and mixed type sugar chains, but does not act on complex type with sialic acid still bound. In addition, other sources have a strong effect on high mannose type sugar chains, but on complex type sugar chains, high concentrations of 2-mercaptoethanol and nonidet-P2
It was believed that it would only work in the presence of a protein denaturing agent such as O.

However, the enzyme of the present invention obtained from Natamame strongly acts not only on high-mannose type and mixed type sugar chains, but also on complex type M chains with sialic acid still attached, and moreover, it is necessary to express activity. 2-mercaptoethanol and nonidet-P2O, which can denature proteins.
It is recognized as a novel endo-β-N-acetylglucosaminidase, which can be said to be a general-purpose type that does not require the following.

It is clear that the enzyme of the present invention will be important for research to elucidate the role that sugar chains of glycoproteins play in the physiological activity of the glycoproteins.

In other words, the enzyme of the present invention, which exhibits activity without the need for any substance that chemically denatures the protein portion of glycoproteins, is more useful than known enzymes because it has broader substrate specificity. be.

The enzyme of the present invention has 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 with sialic acid still attached. In studying the functional and physiological roles of sugar chains, it is important to examine the activity of proteins from which most asparagine-linked sugar chains, including complex types, have been removed by allowing the enzyme of the present invention to act on the glycoproteins. Therefore, the enzyme of the present invention is expected to be used in this field. Also, some.

Many reports have been published that the sugar chain structure of glycoproteins changes to a structure different from the normal sugar chain structure due to tumors. For these reasons, the structure of sugar chains has also attracted attention in medicine, biochemistry, and physiology, and the enzyme of the present invention, which has the effect of releasing sugar chains from protein moieties, is also useful in determining the structure of sugar chains. It is expected that it will be used as an enzyme.

The enzyme of the present invention is extracted and separated from the soybeans using an aqueous medium or the like. Since the extract has enzymatic activity, it can be used as is as a crudely purified enzyme.

The extract can be purified to a purification step according to the intended use according to a conventional enzyme purification method. What is the above Natamame?
Roots, stems, leaves, flowers, and seeds of the soybean.

There are no particular limitations such as pods or biological fluids, and a specific part or a mixture of several parts can be used. Moreover, the extraction method is not particularly limited and may be carried out by a conventional method. For example, extraction from soybean can usually be carried out with water or a buffer.

Generally, the seeds of Natamame are taken out, crushed with a crusher, extracted with 0.1M phosphate buffer at about 4℃ for 12 hours, and ammonium sulfate is added to the extract to give a 3o-h% ammonium sulfate,
Separate the precipitated fraction, dissolve it in water, dialyze and desalt. The dialysed fluid was adsorbed on DEAE-Toyopearl and eluted using a linear gradient method. The obtained active fraction was dialyzed against a phosphate buffer. The dialyzed fluid was adsorbed on a hydroxyapatite column and eluted using a linear gradient method. The eluate fraction obtained was dialyzed against phosphate buffer, the dialysate was concentrated, and the concentrated solution was purified with Sephacryl S-
The enzyme preparation of the present invention can be obtained by gel filtration at 200 °C, dialyzing the obtained active fraction against water, and freeze-drying the dialyzed solution.

Next, the physicochemical properties of the general-purpose endo-β-N-acetylglucosaminidase obtained in Example 5 will be shown.

1. β1 of N-acetylglucosamine, which acts on the N,N'-diacetylchitobiose structure of the asparagine-linked sugar chain of substrate-specific glycoproteins, as shown by the arrow (''>) in the figure below.
→It has the activity of hydrolyzing 4-bonds and releasing oligosaccharides from glycoproteins, and acts not only on high-mannose and mixed types of asparagine-linked sugar chains, but also on complex types.

2. Effect The method of Huang, C, -C et al. (Carbohydr,
Res, 13.127-137, 1970) when a general high-mannose sugar chain was used as a substrate,
It has the effect of hydrolyzing the part of the substrate shown by the arrow in the reaction formula below.

The activity of this enzyme is not limited to this sugar chain, but acts on all high-mannose types. Even if the substrate is a mixed or complex type with sialic acid bound to it, it is still N-acetylglucosamine bound to the riasparagine and N-acetylglucosamine bound to the 4th carbon position of the glucosamine. Hydrolyzes the bond between.

3. Molecular weight 62.000 (from the elution position of gel filtration with Sephacryl S-200) 4. Optimal pH 5.0 5. Stable pH Stable around neutrality 6. Km value Man GGlc NAc@ASN-DNS
m value is 0.18mM.

7. Method for measuring titer The titer of the enzyme of the present invention can be measured using Iwase (Iwase, I).
The method of Analytical Biochemistry (An
al, Biochem) 113.93-95.1981
).

That is, Gray's methods (Methods in Enzymology)
ynology).

0.1 M acetate buffer pH 5, 20 μQ containing 1 μg of substrate prepared according to Vol.
The titer is measured by adding 5 μQ of the enzyme solution to the solution, separating the substances contained in the solution after reaction by high performance liquid chromatography, and quantifying the hydrolyzed content. The amount of enzyme that decomposes 1 μmol of substrate per minute at 37°C is 1 unit (Un
it C (abbreviated herein as rUJ).

Examples of the present invention will be shown below. However, the present invention is not limited to this example. Example 1: 10 g of Natamama was crushed using a crusher, and the resulting crushed product was
00mQ beaker, add 0.1M phosphate buffer (pH 7,0) 40mQ, and store in a cold room (4℃).
The mixture was stirred and extracted. After 12 hours, this solution was centrifuged, and the supernatant liquid was used as a crude extract. The enzyme activity in the crude extract was 300 mU.

Example 2 20.2 ammonium sulfate was added to 36 m H of the crude extract obtained in Example 1.
g was added with stirring, and after the ammonium sulfate was dissolved, the mixture was allowed to stand at room temperature.

After 1 hour, this ammonium sulfate solution was centrifuged, and the resulting precipitate fraction was dissolved in 10 mQ water, and then dissolved in 5o+M phosphate buffer pH.
Dialyzed against 7.0. The enzyme activity in the obtained dialyzed fluid was 290 mU.

Example 3゜The dialyzed fluid obtained in Example 2 was added to 5mM phosphate buffer p
DEAE-Toyoperl (
The mixture was passed through a 15 x 200 cm column packed with Toyo Soda Co., Ltd.).

The adsorbed enzyme was eluted using a linear gradient method from 0 to 0.1M sodium chloride, and the active fraction was collected. There was 270 mU of activity in the resulting active fraction.

Example 4 The active fraction obtained in Example 3 was added to 5mM phosphate buffer (p
i(7,0), and the dialyzed solution was equilibrated with the same buffer using a hydroxyapatite column (10X 15
0 mm). The adsorbed enzyme was eluted using a linear gradient method using a pH 7.0 phosphate buffer solution with a concentration of 5 to 100 n+M, and the eluted active fraction was collected. The enzyme activity in the active fraction was 240 mU.

Example 5゜After dialysis and concentration of the active fraction obtained in Example 4, 5ephacryl S-20 was equilibrated with 5cod phosphate buffer.
0 (manufactured by Pharmacia Fine Chemicals) column (1
, 5×150 cm). The eluted active fractions were collected and concentrated, then dialyzed against water, and the dialyzed solution was freeze-dried to obtain a purified sample of endo-β-N-acetylglucosaminidase.

Claims (3)

[Claims] (1) N, N of asparagine-linked sugar chains of glycoproteins
’-Diacetylchitobiose structure, and the β of N-acetylglucosamine shown by the arrow (⇒) in the figure below
It has the activity of hydrolyzing 1→4 bonds and releasing oligosaccharides from glycoproteins, and has substrate specificity that acts not only on high-mannose and mixed types of asparagine-linked sugar chains, but also on complex types. A general-purpose endo-β-N-acetylglucosaminidase that is a glycosidase. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (2) The general-purpose endo-β-N-acetylglucosaminidase according to claim 1, wherein the glycoprotein also contains a glycopeptide or an oligosaccharide bound to an amino acid. (3) A method for producing general-purpose endo-β-N-acetylglucosaminidase, which comprises collecting general-purpose endo-β-N-acetylglucosaminidase from soy beans.