JPH0789914B2 - Endo type glycolipid degrading enzyme - Google Patents

Description

TECHNICAL FIELD The present invention relates to a glycolipid degrading enzyme.

(Prior Art) Glycosphingolipid, which is known to exist widely in the animal kingdom, is a glycosidic bond between a primary alcoholic hydroxyl group of a sphingo base and a monosaccharide or an oligosaccharide, and a fatty acid is a sphingoglycone. The base is an acid amide bond, and the part consisting of this sphingo base and fatty acid is called ceramide. (Lipid Chemistry, p. 365, 1974
Year, issued by Tokyo Kagaku Doujinsha).

Glycosphingolipids are major constituents of the cell surface layer, and attention has been focused on their relationship with important membrane functions such as cell membrane antigens, blood group substances, mutual discrimination, and differentiation, and the structure and function of sugar chains have not been elucidated. Although being investigated, highly specific enzymes play an important role for this purpose.

(Problems to be Solved by the Invention) Conventionally, as enzymes for cleaving sugar chains of glycosphingolipids,
Only exo type glycolipid degrading enzyme is known, and endo type glycolipid degrading enzyme is not known.

The present inventors, while searching for a glycolipid degrading enzyme that cleaves the sugar chain of glycosphingolipid, found that a certain actinomycete produces highly specific endo-type glycolipid degrading enzyme. Has been achieved.

(Means for Solving Problems) In order to explain the present invention in detail, the endoglycolipid-degrading enzyme of the present invention can be used, for example, in Rhodococcus sp. G isolated from soil collected in Machida City, Tokyo. −74 (Rhodoc
occus sp. G-74). This strain has been deposited as Microorganism Research Institute No. 8424 (FERMP-8424).

The morphological characteristics, properties on various media and physiological and biochemical properties of Rhodococcus sp. G-74 are as shown below.

(1) Morphological characteristics (cultivated on Heart Infusion agar medium at 30 ° C. for 6 to 48 hours) (a) Cell morphology: After culturing, the cells grow nonuniformly up to about 7 hours and grow into mycelium However, bifurcation is rarely observed. After that, the mycelial cell bends in a zigzag pattern and repeats gradual division. In older cultures after 24 hours, hyphae segment (fragme
ntation) and rod-shaped (1.6-6.6 × 1.0-1.2 μm)
To spherical (1.0 to 1.2 μm).

(B) Division mode: snapping type (c) Motility: None (d) Spore formation: None (e) Gram stainability: Positive (f) Anti-acidity: Weak anti-acidity (2) Properties on various media (A) Sucrose-nitrate agar medium at 30 ° C: weak growth; colony color is colorless; no aerial hyphae; no diffusible pigment (b) glucose-asparagine agar medium at 30 ° C: weak growth; colony color is colorless; No aerial hyphae; no diffusible pigment (c) 30 ° C glycerin-asparagine agar medium: weak growth; colony color is colorless; no aerial hyphae; no diffusible pigment (d) 30 ° C starch agar medium: weak growth Colony color is colorless; no aerial hyphae; no diffusible pigment (e) Tyrosine agar at 30 ° C: weak growth; colony color is colorless; aerial hyphae (f) normal agar at 30 ° C: good growth; Colony color is yellowish white Near pale orange; no aerial hyphae; no diffusible pigment (g) 30 ° C yeast-malt agar medium: good growth; colony color is nib orange; no aerial hyphae; no diffusible pigment (H) 30 ° C Oatmeal agar medium: weak growth; colony color is colorless; no aerial hyphae; no diffusible pigment (3) Physiological properties (a) Growth temperature range (heart infusion agar medium): 10 to 37 ° C (b) in air Growth: Good (c) Growth under anaerobic conditions: No growth (d) Catalase: Positive (e) Oxidase: Positive (f) OF test: Negative (to) Acid production (glucose): Negative (H) Liquefaction of gelatin: Negative (R) Hydrolysis of starch: Negative (N) Coagulation of skim milk, peptone formation: Negative (L) Litmus milk: No change (E) Formation of melanin-like pigment (tyrosine agar medium and Peptone-E Iron iron agar): Negative (wa) Assimilation of sugars (cultivated on Pridham-Gotlieve agar for 30 days at 30 ° C): L-arabinose-D-xylose-D-glycose + D-fructose ± sucrose- Inositol-L-rhamnose-raffinose-D-mannite- (4) Biochemical properties (a) Base composition of DNA GC content: 69.5% (b) Major amino acid composition of cell wall: DL-diaminopimelic acid (c) Glyco Rate test: Total carbon number of glycolyl type (d) mycolic acid: C _{40 to} C ₄₆ (5) Taxonomical consideration [Higher taxonomic position] This strain has many cell cycles. It exhibits morphology, grows in a hyphal shape at the initial stage of culture, and in old cultures, hyphal breakage occurs to form rod-shaped to spherical cells, and the cell division mode shows a snapping type. The Gram stain shows a strong positive, and the acid-fast stain shows a weak positive. Furthermore, the GC content of the DNA of this bacterium is 69.5%, which is a high GC content, the major amino acid composition of the cell wall has DL-diaminopimeridic acid, and the sugar chain of the peptidoglycan exhibits a glycolyl type.

Based on the morphological, physiological and biochemical characteristics shown above, this strain (G-74) is a mycobacterial family (Mycobacteria).
ceae) or a member of the Nocardiceae family.

Meanwhile, Journal of General Microbiology, 71, 77 (1
972) and Journal of General Microbiology, 88.
Vol. 200, p. 1975 (1975) mentions the presence or structure of mycolic acid in the classification of actinomycetes related groups, which is widely accepted today. According to this classification system,
Containing mycolic acid carbon number of bacteria C ₆₀ -C ₉₀ mycobacterial family (Mycobacteriaceae), Nocardia family (NOCARD
iaceae) contains mycolic acid having a carbon number of C _{40 to} C ₆₀ .

As a result of analysis of mycolic acid for this strain, it was found that this strain contains mycolic acid having a carbon number of C _{40 to} C ₆₀ . Therefore, it is clear that this bacterium belongs to the family Nocardia.

[Identification of genus level] Berjay's Manual [Bergey's Manual 8th Edition (197
According to 4), the genera belonging to the family Nocardia include two genera of the genus Nocardia and the genus Pseudonocardia.

In particular, the genus Nocardia was roughly classified into three types according to the morphology in the early stage of culture and the presence or absence of aerial hyphae, but today, most of these strains belonging to type I and type II have morphological characteristics. , Cell wall composition, phospholipids, menaquinone type and structures of mycols
cus) genus. [International Journal of
Systematic Bacteriology, 24, pp. 278 (1970), and Journal of General Microbiology, 100, 99
See page (1977)].

This strain (G-74) is easily distinguished from the genus Pseudonocardia based on the cell division mode and contains mycolic acid having a carbon number of C ₄₀ -C _{46. 46-} C ₅₂ ).

As described above, this strain is 1) aerobic Gram bacteria, 2) polymorphic, and 3) primitive (prim) in the early stage of culture.
Positive hyphae are formed, and they are immediately segmented into rod-like to spherical shapes. 4) containing mycolic acid _{(C 40 -C 46), 5} ) and because such that with GC content 69.5% of the features, this strain was well consistent with the Rhodococcus (Rhodococcus) genus of.

Therefore, this strain (G-74) was identified as Rhodococcus sp.

The endoglycolipid-degrading enzyme of the present invention has been found by the present inventors to be produced from the above-mentioned bacterial cells.

This enzyme cleaves the bond between the sugar chain and ceramide using glycosphingolipid as a substrate to produce an oligosaccharide, but not a monosaccharide. Further, cerebroside in which monosaccharide and ceramide are bound is not decomposed.

More specifically, (1) endo-type glycolipid degradation in which glucose in an oligosaccharide chain is directly bonded to ceramide to form a glycosphingolipid as a substrate, and the bond between the glucose and ceramide is cleaved to produce an oligosaccharide. Enzyme (hereinafter referred to as Enzyme 1), and (2) Endo type that uses oligosaccharidin in which galactose in oligosaccharide chain is directly bound to ceramide as a substrate to cleave the bond between the galactose and ceramide to produce oligosaccharide. It is composed of a glycolipid degrading enzyme (hereinafter referred to as enzyme 2).

Since the enzyme 1 and the enzyme 2 of the present invention are secreted into the medium by culturing the above-mentioned strains, they can be easily purified and separated from the medium.

That is, as specifically shown in the Examples below, first, the culture solution of the above strain is centrifuged to collect the supernatant, and for example, the precipitate formed by adding isopropanol is collected and extracted with a suitable solvent. Then, the purification treatment by column chromatography is repeated, and the fractions containing the respective enzymes are collected.

Next, the physicochemical properties of the enzyme 1 and the enzyme 2 will be described in detail. The enzyme activity was measured by the following method.

[Measurement of enzyme activity] Enzyme solution 100 μl Substrate 100 μl [5 mg / ml substrate ¹⁾ and 1 mg in 0.1 M acetate buffer (pH 6.0)
/ ml TDC (sodium taurodeoxycholate, surfactant) added] After mixing the above enzyme solution and substrate and reacting at 37 ° C for 1 hour to 1 night depending on the strength of the enzyme activity, 100 μl of the reaction solution is sampled and the reducing power is measured by the Park-Johnson method, and the amount of the enzyme that produces the reducing power corresponding to 1 μmol of glucose or galactose per minute is defined as 1 unit. The reaction solution is analyzed by thin layer chromatography to confirm whether oligosaccharides and ceramide are produced.

Substrate ¹⁾ : In the case of enzyme 1, G _M1 [Gal isolated from bovine brain
β1 → 3GalNacβ1 → 4 (NeuAcα2 → 3) Galβ1 → 4Gl
cβ1 → 1Cer], and in the case of enzyme 2, neogalalatriaosylceramide, G
alβ1 → 6Galβ1 → 6Galβ1 → 1Cer) was used.

[Physical and Chemical Properties of Enzyme 1] (a) Acts on the glycosphingolipid for production, cleaving the bond between the sugar chain and ceramide to produce oligosaccharide and ceramide.

At this time, the substrate is an acidic sphingoglycolipid of the ganglioside series in which sialic acid is bound to the oligosaccharide chain, in which glucose in the oligosaccharide chain is directly bound to ceramide, such as G _T1a , G _D1a , G _D1b , G _M1 , G _M2 , G
_M3, etc. (Lipid Chemistry, pages 389-391, published in 1974 by Tokyo Kagaku Dojinsha). Further, a neutral glycosphingolipid in which sialic acid is not bound to an oligosaccharide chain, in which glucose in the oligosaccharide chain is directly bound to ceramide, for example, lactosylceramide (Galβ1 → 4Glcβ1 → 1Cer),
Neolactraosylceramide (Galβ1 → 4GlcNACβ1 →
3Galβ1 → 4Glcβ1 → 1Cer), fucosylpentaglycosylceramide [Fucα1 → 3GalNAcα1 → 3 (Fucα1 →
2), Galβ1 → 4Glcβ1 → 1Cer] and the like.

(B) Substrate specificity As described above, oxygen 1 specifically cleaves the bond between glucose and ceramide in the glycosphingolipid in which glucose in the oligosaccharide chain is directly bonded to ceramide to form an oligosaccharide. To generate. That is, it has a so-called endoglucosylceramidase activity, but does not produce a monosaccharide. In other words, it has no exoglycosidase activity.

Also, cerebroside, which is a combination of ceramide and a monosaccharide such as glucose, is not decomposed.

(C) Optimum pH 6.0 (c) Stable pH 5-9 (e) The optimum action temperature is 37 ° C, and it is applicable in the range of 30-40 ° C.

(F) Stable growth Stable at 37 ° C overnight, and freeze-dried products are stable at -25 ° C for at least several months.

(G) Inhibition The enzyme activity is inhibited by copper ion and mercury ion, but is not affected by calcium ion, magnesium ion, barium ion, zinc ion and ethylenediaminetetraacetic acid (EDTA).

(H) Activation The activity of the enzyme on the substrate is remarkably increased by the addition of the surfactant. That is, during the reaction,
When TDC is added at 0.5 mg / ml, the reaction is increased to 2.5 times as compared to the case where TDC is not added.

(I) Column of molecular weight "Sefadex G-200" (1.
5 × 61 cm) with 0.2 M NaCl and 0.1% “Triton X-10
Flow rate of a buffer (pH 6.0) consisting of 20 mM acetic acid containing 0 ", 5
It had a molecular weight of about 230,000 as measured by gel filtration with circulation at ml / hr.

[Physical and Chemical Properties of Enzyme 2] (a) Acts on the glycosphingolipid used for production, cleaves the bond between the sugar chain and ceramide to produce oligosaccharide and ceramide.

At this time, as a substrate, galactose in oligosaccharide chains such as neogalatriaosylceramide (Galβ1 → 6Galβ1 → 6Galβ1 → 1Cer) and galaviosylceramide (Galα1 → 4Galβ1 → 1Cer) directly binds to ceramide. Glycosphingolipids are known.

(B) Substrate specificity As described above, oxygen 2 specifically cleaves the bond between galactose and ceramide in the glycosphingolipid in which the galactose in the oligosaccharide chain is directly bonded to ceramide to form an oligosaccharide. To generate. That is, it has a so-called endogalactosylceramidase activity,
It does not produce monosaccharides. In other words, it has no exogalactosidase activity.

In addition, cerebroside, which is a combination of ceramide and a monosaccharide such as galactose, is not decomposed.

(C) Optimum pH 5.0 (c) Stable pH 5-9 (e) Optimal action temperature is 37 ° C, and it is applicable in the range of 30-40 ° C.

(F) Stability It is stable at 37 ° C overnight, and the freeze-dried product is stable at -25 ° C for at least several months.

(G) Inhibition The enzyme activity is inhibited by copper ion and mercury ion, but is not affected by calcium ion, magnesium ion, barium ion, zinc ion and ethylenediaminetetraacetic acid (EDTA).

(H) Molecular weight As in the case of the enzyme 1, the molecular weight measured by gel filtration using "Sefadex G-200" was about 160,000.

(Operation) The following items can be clarified by using the enzyme 1 or the enzyme 2 of the present invention.

(1) To understand the role of sugar chains in glycosphingolipids.

(2) The oligosaccharide cut out with this enzyme can be labeled with [ ³ H] -label or a fluorescent substance such as 2-aminopyridine by a simple method. The structure of the sugar chain can be determined.

(3) Since this enzyme specifically acts on glycosphingolipids as described above and does not act on glycoproteins, whether a trace amount of sample is a glycoprotein or a glycolipid. Can be easily identified.

(Example) Below, the preparation method of this enzyme is demonstrated more concretely about an Example.

Example (1) Culture of bacteria and preparation of culture supernatant Rhodococcus sp. G-7 [Rhodococcus sp. G-7
4, Micro-Technology Research Institute No. 8424 (FERM P-8424)], polypeptone 0.5%, yeast extract 0.1%, sodium chloride 0.
2000 ml liquid medium containing 2% and attractant (ganglioside 0.1% or bovine brain acetone powder 1%) (pH 7.0-7.2)
After culturing at 28 to 30 ° C. for 4 days, the culture solution was centrifuged (6000 rpm for 30 minutes) and the supernatant was collected.

(2) Preparation of enzyme Isopropanol was added to 1800 ml of the supernatant obtained above so as to have a concentration of 60%, and then the mixture was centrifuged (8500 rpm for 15 minutes) to collect a precipitate, which was collected in 0.5% Triton X
Extraction treatment was performed with -100 "(nonionic surfactant).

The extract was applied to a column of "Sephadex G-100" (5 x 85c
Gel filtration was performed using m) and the eluate was collected and concentrated by ultrafiltration (using "Amicon XM 50" membrane).

The concentrate was added to 10 volumes of isopropanol at -20 ° C and then centrifuged as above to obtain 0.5% precipitate.
A column of DEAE Sepharose FF (1.7 ×, which had been subjected to extraction treatment with “Triton X-100” and equilibrated with 50 mM acetate buffer (pH 6.0, containing 0.1% “Triton X-100”), was used. 2
0 cm). The column was washed with 3 times its volume of the same buffer, and then eluted with a 0-0.3M linear gradient of sodium chloride, and the eluate was collected sequentially to 3.4 ml, 110-127.
The enzyme 1 of the present invention was obtained from this fraction. In addition, the enzyme 2 of the present invention was obtained from the 128 to 142 fractions.

The specific activities of enzyme 1 and enzyme 2 thus obtained are 9.
It was 0 m unit / mg and 15.8 m unit / mg, and could be sufficiently used as a structural analysis reagent.

The specific activity of the above enzyme was measured by the above measuring method.

(Effects of the Invention) By using the present enzyme, for example, structural determination of viral receptors on the cell surface, receptors for cytotoxins (eg cholera toxin), specific cancer antigens, specific antigens associated with development / differentiation, and sugar chain Since you can check the role etc.,
It is useful as a reagent, and development of diagnostic agents, therapeutic agents, etc. for them is expected.

Claims (3)

[Claims] 1. An endo-type glycolipid-degrading enzyme having the following physicochemical properties. (B) Work It acts on glycolipids and cleaves the bond between sugar chains and ceramide to produce oligosaccharides and ceramides. (B) Substrate specificity The bond between the sugar chain of glycolipid and ceramide is cleaved to form an oligosaccharide, but a monosaccharide is not formed. In addition, cerebroside in which monosaccharide and ceramide are bound is not decomposed. (C) Optimum pH 5-6 (d) Stable pH 5-9 (e) Optimum temperature 30-40 ° C (f) Stability It is stable overnight at 37 ° C, and the freeze-dried product is at -25 ° C. It is stable for at least several months. (G) Inhibition The enzyme activity is inhibited by copper ion and mercury ion, but is not affected by calcium ion, magnesium ion, barium ion, zinc ion and ethylenediaminetetraacetic acid. (H) Molecular weight "Sephadex G-200 (trademark)"
The column (1.5x61 cm) was run with a buffer (pH 6.0) consisting of 20 mM acetic acid containing 0.2 M NaCL and 0.1% "Triton X-100 (trademark)" at a flow rate of 5 ml / hr. The molecular weight measured by filtration is about 230,000 or about 160,000. 2. The endoglycolipid-degrading enzyme according to claim 1, wherein the glycolipid is a glycosphingolipid in which glucose and ceramide in the sugar chain are directly bound. 3. The endoglycolipid-degrading enzyme according to claim 1, wherein the glycolipid is a glycosphingolipid in which galactose and ceramide in the sugar chain are directly bound.