JPH067161A - New endoglycoceramidase - Google Patents

Abstract

PURPOSE:To enable ready analysis of a sugar chain structure of a glycolipid by using an enzyme capable of excellently acting on a sphingogllycolipid, especially a globoside-based one and also on a cerebroside composed of a monosaccharide bonded to a ceramide. CONSTITUTION:Endoglycocermidase (an enzyme) exhibits an activity against a sphingoglycolipid and can hydrolyze the sugar-ceramide bond through the activity so as to produce an oligosaccharide or a monosaccharide and a ceramide. This enzyme exhibits an activity also on a ganglioside-, globoside- and lactoside-based sphingoglycoside and on a cerebroside but can not exhibit an activity against a sulfatide. The optimum pH is 6 to 7 and the stable pH range is 6 to 9. The optimum temperature is 40 to 44 deg.C and the thermal stability is <=40 deg.C. This enzyme is inhibited by Hg<2+>, Zn<2+> and Ag<+> and activated by Ba<2+> and Mg<2+>. The enzyme can be obtained, e.g. by culturing No. 363-2-1 strain (FERM-P-12793), i.e., a Gram-positive short bacillus separated from the sheath of a bamboo shoot produced in Shizuoka prefecture, Japan and subsequently collecting from the cultured material.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel endocrycoceramidase having a wide substrate specificity. Such an enzyme is useful for elucidating the structure and function of glycolipids, which have recently attracted attention in vivo importance.

[0002]

2. Description of the Related Art Conventionally, as an endoglycoceramidase derived from a microorganism which acts on a glycosphingolipid to produce an oligosaccharide and ceramide, an enzyme produced by a microorganism belonging to the genus Rhodococcus (see JP-A-62-122587)
And enzymes produced by microorganisms belonging to the genus Corynebacterium (Biochemistry, 63, No. 8, p. 796 (1991)
Year))) is known.

In the former, a glycosphingolipid, in which glucose in an oligosaccharide chain is directly bound to ceramide, specifically cleaves the bond between glucose and ceramide to form an oligosaccharide. An enzyme that does not decompose cerebroside, which is a combination of sugar and ceramide (molecular weight: about 230,000 (gel filtration), optimum pH 6.0, stable pH 5-9,
The optimum temperature of action 37 ° C) and the glycosphingolipid in which galactose in the oligosaccharide chain is directly bound to ceramide,
An enzyme that specifically cleaves the bond between galactose and ceramide to form an oligosaccharide, but does not decompose cerebroside in which a monosaccharide such as galactose and ceramide are combined (molecular weight: about 160,000 (gel filtration), optimum pH 5 0.0, stable pH
5-9, optimum action temperature 37 ° C).

The latter acts well on ganglio-type and lacto-type glycosphingolipids, but it hardly acts on globo-type glycosphingolipids and does not decompose cerebrosides in which monosaccharides and ceramide are bound. It includes various types of enzymes (intracellular enzyme composed of subunits having a molecular weight of about 65,000 and extracellular enzyme composed of subunits having a molecular weight of about 31,000).

Further, a microorganism belonging to the genus Rhodococcus,
Two types of endoglycoceramidase, which are enzymes produced by a mutant strain different from the above and acting on glycosphingolipids to produce oligosaccharides and ceramides, are disclosed in JP-A-1-3096.
Although it is disclosed in Japanese Patent Publication No. 77, on the other hand, the glycosphingolipid in which glucose in the sugar chain is directly bound to ceramide, specifically cleaves the bond between glucose and ceramide to produce an oligosaccharide. An enzyme that does not decompose cerebroside, which is a combination of ceramide and a monosaccharide such as glucose (molecular weight 55,900 (SDS-PAGE), optimum pH 5 to 5).
5.5, stable pH 5-9, optimum temperature of action 37 ° C), and the other specifically, the binding of galactose and ceramide of glycosphingolipid in which galactose in oligosaccharide chain is directly bound to ceramide. An enzyme (molecular weight 53,700 that cleaves to form oligosaccharides but does not degrade cerebrosides that combine monosaccharides such as galactose and ceramides
(SDS-PAGE), optimum pH 5 to 5.5, stable p
H 5-9, optimal temperature of action 37 ° C.), and both are enzymes that do not act on cerebroside in which monosaccharide and ceramide are bound.

That is, the conventionally known endoglycoceramidase is an enzyme that does not act on cerebroside in which a monosaccharide and ceramide are bound.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide an enzyme having a wider substrate specificity than a known endoglycoceramidase known in the art, particularly a globo-type glycosphingolipid and a cerebroside in which a monosaccharide and ceramide are bound. It is to provide an endoglycoceramidase that also works well.

[0008]

The present invention provides a novel endoglycoceramidase having the following properties.

Action It acts on glycosphingolipids and hydrolyzes the bond between sugar and ceramide to produce oligosaccharide or monosaccharide and ceramide. Substrate specificity It acts on ganglio-type, globo-type and lacto-type glycosphingolipids and cerebrosides in which monosaccharides and ceramide are bound and decomposes, but it does not substantially act on sulfatide. Optimum pH 6.0-7.0 Stable pH range 6.0-9.0 Optimum temperature 40-44 ° C Thermal stability Stable at 40 ° C or lower. Inhibition and activation Inhibited by Hg ²⁺ , Zn ²⁺ or Ag ⁺ , Ba ²⁺
Or activated by Mg ²⁺ .

The present invention will be described in detail below. The method for producing the endoglycoceramidase of the present invention (hereinafter sometimes referred to as “the enzyme of the present invention”) is not limited. For example, ganglioside (GM) is derived from the skin of bamboo shoots in Shizuoka prefecture.
No. 1 which is a gram-positive bacillus isolated by culturing in an accumulation medium containing 1a) as a sole carbon source and screening by determining enzyme productivity by disappearance of ganglioside. Strain 363-2-1 can be cultured and harvested from the culture. In addition, this No. 363-2-
One strain was deposited on February 25, 1992 at the Institute for Microbial Technology, National Institute of Advanced Industrial Science and Technology, with the microorganism deposit number, and the number 12
Deposited as 793 (FERM P-12793).

The enzyme of the present invention is cultivated by inoculating an appropriate medium with a microorganism producing the enzyme, and separating and removing the microbial cells from the culture (culture liquid in the case of liquid culture) after the culture (centrifugation, Aggregation separation, filtration, etc., followed by general enzyme separation and purification methods (eg, "Introduction Enzyme Chemistry", July 1, 1973, Co., Ltd.)
It is collected as an enzyme preparation with the required degree of purification by applying Nankodo.

The enzyme-producing microorganism of the present invention can be cultured by a carbon source (cattle brain acetone powder, purified sphingoglycolipid, glucose, starch, etc.) that can be assimilated by the bacterium, nitrogen source (yeast extract,
Organic nitrogen sources such as peptone, meat extract, corn steep liquor and soybean meal; inorganic nitrogen sources such as ammonium chloride, ammonium sulfate, urea and ammonium nitrate), inorganic salts (sulfates such as iron, magnesium, calcium, potassium and sodium, phosphorus) Acid salts, hydrochlorides, etc.), and optionally, a culture medium containing a surfactant by an aerobic culture method (shaking culture, aeration stirring culture, etc.) at a temperature suitable for growth for several hours to several days. Thus, the enzyme can be produced in the medium.

From the liquid culture after the liquid culture in which the bacterial cells have been separated and removed, for example, salting out with ammonium sulfate (ammonium sulfate), sodium sulfate, etc .; dialysis; precipitation with an organic solvent such as ethanol, acetone, isopropanol, tetrahydrofuran; hydroxyapatite ( Hydroxyl calcium phosphate)
Chromatographic method using anion exchangers having an exchange group such as diethylaminoethyl (DEAE) group and triethylaminoethyl group; affinity chromatography method; gel filtration chromatography method; molecular sieve membrane An enzyme preparation having a desired degree of purification can be obtained by a well-known enzyme purification method such as an ultrafiltration method by electrophoresis, etc .;

The enzyme reaction in the following measurements was basically carried out using the following reaction system.

[Basic reaction system] Reaction solution (total volume 200 μl) Substrate: 20 nmol GM1a (ganglioside; manufactured by Sigma) Enzyme solution: Purified enzyme 25 μl Buffer solution: Acetate buffer solution (in reaction solution: 50 mM, pH 6.
5) Surfactant: Triton X-100
(0.1% in reaction liquid; Wako Pure Chemical Industries, Ltd.) Reaction temperature: 37 ° C. Reaction time: 2 hours Activity measurement method: reducing group quantification method

The enzyme activity of the enzyme of the present invention can be measured by the following method. (A) Quantitative Method for Reducing Group After using the above-mentioned enzyme reaction solution containing an appropriate amount of enzyme at 37 ° C. for 2 hours, 1 ml of carbon cyanide solution was added to stop the reaction, and 2.8 ml of distilled water was added thereto. . The solution is then Park-John.
Son) method (J, Biol. Chem. 181, 149).
(1949)) to quantify free reducing groups. For the free reducing group, the absorbance at 690 nm is measured, and this is calculated as the glucose equivalent amount. One unit of enzyme activity (unit (U)) was defined as the amount of enzyme that liberates 1 μmol of reducing groups per minute.

(B) Thin Layer Chromatography (TLC) Method The enzyme reaction solution containing an appropriate amount of enzyme (however, the enzyme solution: 8
After reacting with (5 μl) at 37 ° C. for 48 hours, 20 μl was spotted on a silica gel thin layer plate, which was mixed with chloroform: methanol: water (55:45:10, V / V).
It develops in the solvent system of. The color development of glycolipids is carried out using the resozinol hydrochloric acid reagent for gangliosides and the 50% sulfuric acid reagent for globoside, lactosylceramide, cerebroside and sulfatide.
The decomposition rate of glycolipid is determined by a densitometer with reference to the spot of glycolipid in the reaction system without addition of enzyme.

[Physicochemical Properties] Next, the physicochemical properties of the enzyme of the present invention will be shown. The measurement was performed using the purified enzyme obtained in the example.

(1) It acts on a glycosphingolipid for production and hydrolyzes the bond between sugar and ceramide to produce oligosaccharide or monosaccharide and ceramide.

(2) Substrate specificity It acts on ganglio-type, globo-type, lacto-type glycosphingolipids and cerebrosides in which monosaccharides and ceramide are bound and decomposes, but it does not substantially act on sulfatide.

That is, 20 nmol of ganglio-based glycosphingolipid (GM1a, GM2, GM3, G as a substrate)
D1a, GD1b, GD3, GT1b), globo-based glycosphingolipids (Gb4Cer, Gb5Cer), lacto-sphingolipids (lactosylceramide) glycosphingolipids, and cerebrosides (galactocerebrosides, glucocerebrosides) in which monosaccharides and ceramides are linked. And a reaction solution containing sulfatide, respectively, according to the above [basic reaction system] (however, an enzyme solution, 85 μl) is reacted, and a reaction time of 2 hours is measured by a reducing group quantification method and a reaction time of 48 hours is used. Thin layer chromatography (TL
When the enzyme activity (decomposition rate and hydrolysis rate) was measured by the C) method, as shown in Table 1, ganglio-, globo-, and lacto-sphingoglycolipids and cerebrosides (galactocerebroside, monosaccharides and ceramides bound to ceramides, It was found that after 48 hours, 100% was decomposed after acting on glucocerebrosid, but it did not substantially act on sulfatide.

Further, in the 2 hour reaction, it acted on glycosphingolipids other than sulfatide as in the 48 hour reaction,
There is a difference in the hydrolysis rate depending on the type, and GM1a
Was the fastest, followed by GM2.

[0023]

[Table 1]

As described above, the enzyme of the present invention has a wider substrate specificity than the conventional endoglycoceramidase, and in particular, the conventional endoglycoceramidase is not particularly effective for cerebroside in which monosaccharides such as glucose and galactose and ceramide are bound. It does not act, but acts on cerebroside and decomposes, and in this respect, it is distinguished from conventional enzymes.

(3) Optimum pH For the enzyme of the present invention, 50 mM acetate buffer was used as a buffer solution, and the reaction was carried out at each pH of 4.5 to 7.5 according to the above [basic reaction system]. When the enzyme reaction was carried out and the enzyme activity was measured, as shown in FIG. 1, the enzyme of the present invention suitably acted at pH 6.0 to 7.0, and most suitably acted at around pH 6.5. .

(4) Stable pH range 200 mM buffer solution (acetic acid buffer solution, Tris-hydrochloric acid buffer solution, sodium carbonate-sodium hydrogen carbonate buffer solution) adjusted to each pH so that the enzyme solution of the present invention has a predetermined pH Was added for 27 hours at 4 ° C., and then 50 mM acetate buffer (pH: 0.1% Triton X-100) was added.
It was dialyzed against 6.5), and an enzyme reaction was carried out according to the above [basic reaction system] to measure the enzyme activity. As a result, as shown in FIG. 2, the enzyme of the present invention had a pH of 6.0 to 9.
It was stable at 0.

(5) Optimum temperature For the enzyme of the present invention, the enzyme reaction was carried out according to the above [basic reaction system] except that the reaction temperature was set to a predetermined temperature of 30 to 47 ° C., and the enzyme activity was measured. As shown, the enzyme of the present invention suitably acted at 40 to 44 ° C, and most suitably acted at around 42 ° C.

(6) Thermostability A 50 mM acetate buffer solution (pH 6.
5) was added, and the mixture was allowed to stand at a predetermined temperature of 0 to 60 ° C for 15 minutes, and then an enzymatic reaction was carried out according to the above [basic reaction system] to examine the residual activity. It was found that the treatment with the above treatment completely retained the activity, and the treatment at 55 ° C. had a residual activity of about 50% of the untreated treatment.

(7) Inhibition and Activation Regarding the enzyme of the present invention, an inorganic ion and an enzyme inhibitor (in the case of inorganic ion, as a salt thereof) are added to the reaction solution,
The enzyme reaction was carried out according to the above [basic reaction system] to examine the influence of the enzyme inhibitor. The results of the examination are shown in Table 2.

[0030]

[Table 2]

From Table 2, the reaction of the enzyme of the present invention is Hg ²⁺ , Zn
^Inhibited by ²⁺ or Ag ⁺ , Ba ²⁺ or Mg ²⁺
It was found that the enzyme activity was activated by addition of PCMB (P-chloromercuribenzoic acid) and EDTA (ethylenediaminetetraacetic acid).

(8) Effect of Surfactant Various surfactants shown in Table 3 below were added to the reaction solution obtained by removing Triton X-100 from the above [basic reaction system] so as to be 0.2%. The reaction was performed according to the [basic reaction system], and the enzyme activity was measured. The results are shown in Table 3.

In Table 3, Tween 2
0 is a product of Hanai Chemical Co., Ltd., and Brij is a product of Kao Atlas Co., Ltd. Also, S
DS is sodium lauryl sulfate.

[0034]

[Table 3]

From Table 3, it was found that the reaction of the enzyme of the present invention was inhibited by the addition of 0.2% of various surfactants.

Further, among the surfactants, Triton X-1
For 00, the concentration was adjusted to each concentration of 0 to 1.0% and the enzyme activity was measured in the same manner as the above-mentioned various surfactants.
As shown in FIG. 5, when 0.1% of Triton X-100 was added, the enzyme activity slightly increased, but when it was 0.2% or more, the activity did not increase, and on the contrary, it acts as an inhibitor. It has been found.

It is known that the enzyme activity of conventional endoglycoceramidase is remarkably increased by the addition of a surfactant (sodium taurodeoxycholate, etc.) (Japanese Patent Laid-Open No. 62-122587 and Japanese Patent Laid-Open No. 122587/1987). 1-
309677), the enzyme of the present invention is distinguished from conventional enzymes in that the activity is inhibited by the addition of a relatively high concentration of a surfactant.

(9) Molecular weight Regarding the enzyme of the present invention, the gel filtration method (superrose (S
upperose) 12 column (Pharmacia); high performance liquid chromatography; flow rate 0.4 ml / min)
When the molecular weight was examined by, it was about 95,000,
It was about 90,000 when examined by polyacrylamide gel electrophoresis (Ready gel (manufactured by Bio-Rad); 200 V constant current).

(10) Isoelectric point With respect to the enzyme of the present invention, the isoelectric point (pI) is determined by isoelectric focusing (Ampholitic isoelectric focusing column (LKB); 400 V constant current, 72 hours). The pI was 4.05 as determined by.

[0040]

EXAMPLES Next, the present invention will be described in more detail by way of examples, which are examples of the present invention and are not limited thereto.

Beef brain acetone powder (manufactured by Sigma) 1.0
%, Peptone 0.5%, yeast extract 0.1%, sodium chloride 0.2%, pH 7.0 5 liters of production medium
1000 ml was dispensed into the Erlenmeyer flask and autoclaved. No. After inoculating the 363-2-1 strain, the cells were shake-cultured at 30 ° C. for 4 days using a rotary shaker.

The supernatant (culture supernatant; 890 ml) of the culture solution centrifuged (9000 rpm, 10 minutes) was cooled to 8
Ammonium sulfate was added to 0% saturation, and the mixture was allowed to stand at 4 ° C. overnight. The precipitate was filtered using a paper filter, and the precipitate on the paper was buffered with an appropriate amount of buffer A (0.1% Triton X. 5 including -100
Dissolve in 0 mM acetate buffer, pH 6.5) to obtain buffer A
Against it, it was dialyzed at 4 ° C. for two nights.

Next, the dialysate (45 ml) was concentrated to 10 ml with an ultrafiltration device (UHP-25, manufactured by Toyo Roshi Co., Ltd.), and the whole amount was separated by Sephadex G.
-100 (Pharmacia) column (1.8 x 60)
cm) and the enzyme was eluted at a flow rate of 10 ml / hour.

The eluted active fraction (84 ml) was added to DE
AE-Sepharose CL-6B
It was adsorbed on a column (manufactured by Pharmacia) (1.8 × 10 cm), and the enzyme was eluted with a linear concentration gradient of 0 to 0.5 M sodium chloride at a flow rate of 10 ml / hour.

Further, the eluted active fraction (41 ml) was concentrated to 12 ml with an ultrafilter, and then the total amount was 11
It was packed in 0 ml of an ampholite isoelectric focusing column (manufactured by LKB) and electrophoresed at 2 ° C. and constant current (400 V) for 72 hours. After the electrophoresis, 2 ml of each fraction was fractionated, the pH was measured, and each fraction was dialyzed against the buffer solution A for 48 hours, and then the enzyme activity was measured.

Then, the active fraction (8 ml) was concentrated to 2 ml with an ultrafilter to obtain a purified enzyme solution.

Table 4 shows the enzyme activity, the amount of protein, the yield, etc. for each purification step of the obtained purified enzyme.

[0048]

[Table 4]

[0049]

EFFECTS OF THE INVENTION According to the present invention, the substrate specificity is wider than that of the conventionally known publicly known endoglycoceramidase, and in particular, the conventional endoglycoceramidase binds monosaccharides such as glucose and galactose with ceramide. It does not act on the cerebrosides, but it acts on the cerebrosides to decompose it, and in this respect it is distinguished from conventional enzymes and is a reagent useful for research such as analysis of the structure and function of sugar chains of glycolipids. It is expected to be utilized for such purposes.

Further, unlike the conventional endoglycoceramidase, it has a novel property that is distinguished from the conventional enzyme such that the activity is inhibited by the addition of a relatively high concentration of surfactant.

[Brief description of drawings]

FIG. 1 is a pH-enzyme activity curve showing the optimum pH of the enzyme of the present invention.

FIG. 2 is a pH-enzyme activity curve showing the stable pH of the enzyme of the present invention.

FIG. 3 is a temperature-enzyme activity curve showing the optimum temperature of the enzyme of the present invention.

FIG. 4 is a temperature-residual activity curve showing a stable temperature range of the enzyme of the present invention.

FIG. 5 is an addition concentration-enzyme activity curve showing the influence of Triton X-100 on the enzyme of the present invention.

Claims (1)

[Claims] 1. A novel endoglycoceramidase having the following physicochemical properties: Action It acts on glycosphingolipids and hydrolyzes the bond between sugar and ceramide to produce oligosaccharide or monosaccharide and ceramide. Substrate specificity It acts on ganglio-type, globo-type and lacto-type glycosphingolipids and cerebrosides in which monosaccharides and ceramide are bound and decomposes, but it does not substantially act on sulfatide. Optimum pH 6.0-7.0 Stable pH range 6.0-9.0 Optimum temperature 40-44 ° C Thermal stability Stable at 40 ° C or lower. Inhibition and activation Inhibited by Hg ²⁺ , Zn ²⁺ or Ag ⁺ , Ba ²⁺
Or activated by Mg ²⁺ .