JPH0616705B2 - General-purpose endo-β-N-acetylglucosaminidase and method for producing the same - Google Patents

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, in addition to the high mannose type and mixed type of asparagine-linked sugar chains of glycoprotein, N, N ′ of complex type sugar chains
An endo-glycosidase which also has a substrate specificity to act on the diacetylchitobiose structure and which has the effect of hydrolyzing the β1 → 4 bond part in the N, N′-diacetylchitobiose structure. A general-purpose end-β-
The present invention relates to N-acetylglucosaminidase and a method for collecting the same from lentils.

In general, glycoproteins are widely distributed in the living world such as enzymes, hormones, immunoglobulins and cell surfaces, and the importance of the role of the sugar chain portion has recently been particularly noted. I have been engaged in the elucidation of the function of, but the whole picture has not yet been elucidated. The reason for this is considered to be an obstacle to research regarding the structure of sugar chains, which is the basis for elucidation of functions, because glycoproteins have structural units of different properties, amino acids and sugars. In this sense, when studying the sugar chain structure and when studying the effect of sugar chains of glycoproteins on physiological activity, the endo-glycosidase that cuts the sugar moiety from the glycoprotein as an oligosaccharide is a very important enzyme. It is supposed to become.

Therefore, the present inventors have found that in various sources,
As a result of diligent research on glycosidases, a novel general-purpose endo-β-N-acetylglucosaminidase has been found in a solution extracted from lentils.

Conventionally, endo-β-N-acetylglucosaminidase is known to be obtained from bacteria and the like, but all of them act only on a specific sugar chain.

That is, the constitution of the asparagine-linked sugar chain is classified into high mannose type, mixed type and complex type. Next, the formula of each type and the site of action are shown.

1. High mannose type 2. Mixed type 3. Composite type In each of the above types, the conventional endo-β-N-acetylglucosaminidase acts on the high-mannose type and mixed type sugar chains, but does not act on the complex type in which sialic acid remains bound. Also, another source strongly acts on high-mannose type sugar chains, but on complex type sugar chains in the presence of high concentrations of 2-mercaptoethanol and protein denaturants such as nonidet-P40. If it wasn't, it wouldn't work.

However, the enzyme of the present invention obtained from Nameame strongly acts not only on high-mannose type or mixed type sugar chains, but also on complex type sugar chains in which sialic acid remains bound, and for the purpose of activity expression. A new end that can be said to be a general-purpose type that does not require 2-mercaptoethanol or nonidet-P40 that denatures proteins.
It is recognized as β-N-acetylglucosaminidase.

It is clear that the enzyme of the present invention is important for the research to elucidate the role of the sugar chain of glycoprotein in the physiological activity of the glycoprotein.

That is, the enzyme of the present invention that is active without any substance that chemically denatures the protein portion of glycoprotein is more useful because it has a wider substrate specificity than known enzymes. is there.

The enzyme of the present invention has a wide substrate specificity that it acts not only on the high-mannose type or mixed type of the asparagine-linked sugar chain of glycoprotein, but also on the complex type sugar chain of sialic acid as it is bound. In order to study the functional and physiological role of sugar chains of Escherichia coli, the activity of the protein from which most of the asparagine-linked sugar chains including the complex type is removed by acting the enzyme of the present invention on the glycoprotein Therefore, the enzyme of the present invention is expected to be used in this direction. In addition, many reports have been published that the sugar chain structure of some glycoproteins changes to a structure different from the normal sugar chain structure depending on the tumor. From these facts, the structure of the sugar chain has been attracting attention medically, biochemically, and physiologically, and the enzyme of the present invention having the action of releasing the sugar chain from the protein part is also useful in determining the structure of the sugar chain. Expected to be used as an enzyme.

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

The extract can be purified by a conventional enzyme purification method at a purification step according to the purpose of use. What is the above name beans?
The roots, stems, leaves, flowers, seeds, pods, biological fluids, etc. of the namelets are not particularly limited, and specific parts or a mixture of several parts can be used. Moreover, the extraction method is not particularly limited, and an ordinary method is used. For example, extraction from lentils can usually be performed with water or buffer.

Generally, the seeds of lentil are taken out, crushed with a crusher, extracted with 0.1 M phosphate buffer at 4 ° C for 12 hours, and ammonium sulphate is added to the extract to give 30-40% ammonium sulphate. The precipitate fraction is separated, dissolved in water, dialyzed and desalted. The dialysis inner solution was adsorbed on DEAE-Toyopearl and eluted by the linear gradient method, the obtained active fraction was dialyzed against phosphate buffer solution, the dialysis inner solution was adsorbed on the hydroxyapatite column, and the linear gradient method was used. Elute, dialyzing the obtained elution section against phosphate buffer,
It is possible to obtain the enzyme preparation of the present invention by concentrating the dialysis inner solution, subjecting the concentrate to gel filtration with Cefacrylic S-200, dialysis of the obtained active fraction against water, and lyophilizing the dialysis inner solution. it can.

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

1. Substrate specificity It acts on the N, N'-diacetylchitobiose structure part of the asparagine-linked sugar chain of glycoprotein, and the arrow in the figure below Hydrolyze the β1 → 4 bond of N-acetylglucosamine, which has the activity of releasing oligosaccharides from glycoproteins, and acts not only on the high-mannose type or mixed type of asparagine-linked sugar chains, but also on the complex type. To do.

2. Work Huang.C.-C et al. Method (Carbohydr.Res. 13 , 127-137,
When a general high-mannose type sugar chain prepared according to 1970) is used as the substrate, the arrow of the substrate is as shown in the reaction formula below. It has the effect of hydrolyzing the portion indicated by.

The activity of this enzyme is not limited to this sugar chain but acts on any high-mannose type. Even if the substrate is a mixed type or a complex type with sialic acid bound, N-acetylglucosamine still bound to asparagine and N-acetylglucosamine bound to the 4-position carbon of the glucosamine Hydrolyze the bonds between.

3. Molecular weight 62,000 (from elution position of gel filtration with Cefacrylic S-200) 4. Optimum pH 5.0 5. Stable pH Stable near neutral 6. Km value The Km value for Man ₆ Glc NAc ₂ ASN-DNS is 0.18 mM.

7. Method for Measuring Titer The titer of the enzyme of the present invention can be measured by the method of Iwase.I et al. (Analytical Biochemistry (Anal.Bioch).
em) 113 , 93-95, 1981). That is, Gray.WR (Methods in Enzymology), Vol. 11 , pp. 139-15
0.1M acetate buffer pH containing 1 μg of the substrate prepared in 1)
The titer is measured by adding 5 μl of the enzyme solution to 5 and 20 μl, separating the substances contained in the solution after the reaction by high performance liquid chromatography, and quantifying the hydrolyzed content. 37
One unit (Unit: abbreviated as “U” in the present specification) is the amount of enzyme that decomposes 1 μmol of substrate at 1 ° C. for 1 minute.

Hereinafter, examples of the present invention will be described. However, the present invention is not limited to this embodiment.

Example 1. 10 g of ground bean is crushed with a crusher, and the obtained crushed product is 100
Add it to a beaker of ml and add 0.1M phosphate buffer (pH
7.0) 40 ml was added, and the mixture was stirred and extracted in a cold room (4 ° C.). 1
After 2 hours, this solution was centrifuged, and the supernatant was used as a crude extract. The enzyme activity in the crude extract was 300 mU.

Example 2. Ammonium sulfate (20.2 g) was added to 36 ml of the crude extract obtained in Example 1 with stirring, and the mixture was allowed to stand at room temperature after the ammonium sulfate was dissolved. After 1 hour, the ammonium sulfate solution was centrifuged, and the resulting precipitate was collected for 10 m.
After dissolving in 1 l of water, it was dialyzed against 5 mM phosphate buffer pH 7.0. The enzyme activity in the obtained dialysate was 290 mU.

Example 3. The dialysis inner solution obtained in Example 2 was treated with 5 mM phosphate buffer pH 7.
The mixture was passed through a 15 × 200 cm column filled with DEAE-Toyoperl (manufactured by Toyo Soda Co., Ltd.) equilibrated with 0. The adsorbed enzyme was eluted by a linear gradient method with 0 to 0.1 M salt and the active fractions were collected. There was 270 mU of activity in the resulting activity compartment.

Example 4. The active fraction obtained in Example 3 was treated with 5 mM phosphate buffer (pH 7.
The solution was dialyzed with (0) and the dialyzed solution was passed through a hydroxyapatite column (10 × 150 mm) equilibrated with the same buffer solution. Adsorbed enzyme pH 7.0 phosphate buffer solution concentration 5-100 mM
The enzyme activity was 240 mU in the active fractions obtained by collecting the eluted active fractions by elution by the linear gradient method.

Example 5. The active fraction obtained in Example 4 was dialyzed and concentrated, and then gel filtration was performed using a Sephacryl S-200 (Pharmacia Fine Chemicals) column (1.5 × 150 cm) equilibrated with 5 mM phosphate buffer. The eluted active fractions were collected, concentrated, dialyzed with water, and the dialyzed solution was freeze-dried to obtain a purified preparation of endo-β-N-acetylglucosaminidase.

Claims (3)

[Claims] 1. A general-purpose endo-β-N-acetylglucosaminidase which is an endo-glycosidase having the following properties. A: Substrate specificity It acts on the N, N'-diacetylchitobiose structure part of the asparagine-linked sugar chain of glycoprotein, and the arrow in the figure below Hydrolyze the β1 → 4 bond of N-acetylglucosamine, which has the activity of liberating oligosaccharides from glycoproteins, and acts not only on the high-mannose type or mixed type of asparagine-linked sugar chains, but also on the complex type. To do. B: General high-mannose type sugar chain (according to Huang.C.-C et al.)
When using as a substrate, the arrow in the figure below It has the effect of hydrolyzing the portion indicated by. C: Optimal pH 5.0 D: Stable pH Stable near neutral 2. The general-purpose endo-β-N-acetylglucosaminidase according to claim 1, wherein the glycoprotein also contains an oligosaccharide to which a glycopeptide or an amino acid is bound. 3. An end having the following properties based on a lentil:
A method for producing a general-purpose endo-β-N-acetylglucosaminidase, which comprises collecting a general-purpose endo-β-N-acetylglucosaminidase that is a glycosidase. A: Substrate specificity It acts on the N, N'-diacetylchitobiose structure part of the asparagine-linked sugar chain of glycoprotein, and the arrow in the figure below Hydrolyze the β1 → 4 bond of N-acetylglucosamine, which has the activity of releasing oligosaccharides from glycoproteins, and acts not only on the high-mannose type or mixed type of asparagine-linked sugar chains, but also on the complex type. To do. B: General high-mannose type sugar chain (according to Huang.C.-C et al.)
When using as a substrate, the arrow in the figure below It has the effect of hydrolyzing the portion indicated by. C: Optimal pH 5.0 D: Stable pH Stable near neutral