JPS61265088A - Endo-beta-n-acetylglucosaminidase and production thereof - Google Patents

Abstract

NEW MATERIAL:Endo-beta-N-acetylglucosaminidase having the following characteristics. The fittest pH is 5.0-6.0; stable pH is 5.0-7.0 (under retention condition at 4 deg.C for 48hr); stable temperature range is up to 40 deg.C under retention condition at 7.0pH for 10min; molecular weight 27,000-30,000; etc. USE:An enzyme for structural analysis of sacchride chain of glycoprotein. It acts specifically on a saccharide chain of asparagine bond type and hydrolyzes specifically N,N'-diacetylchitobiose of saccharide chain. PREPARATION:A strain of Flavobacterium sp. 1022 (FERM P-8201) is cultivated preferably under an aerobic condition at 20-30 deg.C for 3-4 days.

Description

Detailed Description of the Invention [Object of the Invention] The present invention provides a novel enzyme endo-β-N-acetylglucosaminidase (hereinafter referred to as B n d O-β-GlcNAca
(sometimes abbreviated as se) and its manufacturing method. For more details, see high mannose type and mixed type N.
- An endo-β-N-acetylglucosaminidase that has broad substrate specificity for asparagine-linked sugar chains and can directly act on sugar chains of glycoproteins, and a method for producing it using microorganisms.

(Industrial Application Field) In recent years, it has become clear that the sugar chain moiety of complex carbohydrates plays an important role in the differentiation and growth of cells, intercellular recognition, and the onset of many diseases including malignant tumors in vivo. It has been. In order to elucidate these roles, it is essential to investigate their sugar chain structures.

Various glycosidases that have high substrate specificity for the sugar chain structure of complex carbohydrates are widely used for such analysis of sugar chain structures.

Endo-β-GlcNAcase acts on asparagine-linked sugar chains present in glycoproteins and hydrolyzes the NN'-diacetylchitobiose moiety of the sugar chains as described below.

--・Manβ1→4GlcNAcβl-+4GloN
Ao-+Asn1 endo-β-01cNAcase
・-Mtsnβl →4GlcNAc-4-(]IcN
Ac-+Asn like this endo-β-01cNAca
Since se can liberate the sugar chain moiety of glycoproteins from the protein moiety, it is an enzyme that provides an important means for structural analysis of glycoprotein sugar chains.

(Prior art) (Problems to be solved by the invention) Conventionally, as an e n d O-β-()lcNAcase, pneumococcus (r)iplncoccus pneum
o former ae) produced by endo f) (Koid, N
&Muramatsu%T: , J, mountain (+1.0h
eru, 2494897-4904 (1974)
), 8 trepLomycos pi 1cut
US produced J Rundo H (Tarentino,
A. L. & Maley, F. J.

Biol, Ohem, 249 811-81
7 (1974)), 0] Ostridiumper
Endo-CI, C1F produced by fringens
(8, Ito, T-Murarnatsu, A, Kob
ata, Arch, Bioclem, Biophys
.

171 78 (1975)), Flavobacter
ium meningoseptic-un production 7
．． endo F (Elder, J H, & Ale
xander.

8, PoO, Natl, Acad, 8ci-U8 794540-4544 (1982), etc. are known. These enzymes act on either or both of the high-mannose-type and mixed-type asparagine-linked sugar chains of glycopeptides, but they hardly act on natural glycoproteins, so they must be converted into glycopeptides in advance using proteases. There is. The enzyme of the present invention is a novel enzyme that differs from conventionally known enzymes in that it acts on both types of sugar chains and can also directly act on the sugar chains of natural glycoproteins including ovalbumin.

[Structure of the invention]

(Means for Solving the Problems) The present inventors specifically cleaved the sugar chain moiety bonded to the asparagine residue of glycoproteins, and endo-β- As a result of searching for bacteria that produce N-acetylglucosaminidase, we found strong enzyme activity in cultures of bacteria isolated from soil.
-β-GlcN,/% case was successfully obtained, and the present invention was completed.

That is, the present invention is a high mannose type and a mixed type.

The object of the present invention is to provide endo-β-GloNAoase that acts on any asparagine-linked sugar chain and acts on natural glycoprotein sugar chains in a 1-ntact state.

Furthermore, the present invention involves culturing a microorganism that belongs to the genus Flavobacterium and has the above-mentioned ability to produce ando-β-GlcNA, aase, and producing endo-β-01 from the culture.
ando-, which is characterized by collecting cNAcase
A method for producing β-01cNAcase is also provided.

The enzyme of the present invention has the ability to specifically cleave N-asparagine-linked sugar chains among the protein sugar chains, and is also characterized by acting on both high-mannose and mixed types of sugar chains. have. Also, conventional (7) 6n
While do-β-(-jloNAoase can hardly cleave sugar chains unless the glycoprotein is previously treated with pro6ase and converted into a glycopeptide form, the enzyme of the present invention The enzyme of the present invention has an extremely excellent property of acting directly on endo-β-GlcNAc.
This enzyme has a new type of substrate specificity different from that of Ase, and is extremely useful for analyzing the sugar chain structure of complex carbohydrates.

Furthermore, since the enzyme of the present invention is an inducible enzyme that is secreted outside the bacterial cell, multiple enzyme proteins can be easily obtained, and furthermore, it can be easily purified as a single enzyme protein preparation. It can be supplied in large quantities and at low cost as a reagent for sugar chain structure research.

Hereinafter, embodiments of the present invention will be described in more detail.

en(io-β-(i
The enzymatic chemical and physicochemical properties of laNAcasθ are as follows.

(1) Action of the enzyme The enzyme of the present invention acts on the asparaquine-linked sugar chain of a glycoprotein and hydrolyzes the N,N'-diacetylchitobiose moiety of the sugar chain as described below.

---Man 111-+401c NAcβ1-+
4(ilcNAc-+Asn...Δ(anβl-+4
GIoNAc-)-GlcNAc-+Asn (2) Substrate specificity The enzyme of the present invention can handle all different types of sugar chains contained in ovalbumin, that is, both high-mannose type and mixed type glycopeptides. Disassemble. on the other hand,
Since the enzyme of the present invention releases carbohydrates even when ovalbumin itself is used as a substrate, it also acts on natural toxin proteins. In addition to ovalbumin, this enzyme also contains RN
It was also observed that sugar chains of aseB and invertase were released.

(3) Measurement method of titer Enzyme activity was measured by dansylating the glycopeptide obtained by treating ovalbumin with pronase. ’c to carry out the reaction, 40
After stopping the reaction by adding % trichloroacetic acid, the produced 1) N8-Asparagine-N-acetylglucosamine (T)N8-Asn-GlcNAc) Total n-butanol:acetone:water (8:1:l) It was separated using paper chromatography using a developing agent, extracted with water, and then quantified using a fluorescence method. The unit of enzyme activity is l μmal DNS-Asn-G per minute.
l cNA.

The amount of enzyme liberated was defined as 1 unit.

(4) Optimum pH and stable pH range Optimum p■ is pT(+), o to 6.0 as shown in Figure 1.
The stable phi was pEI 5.0 to 7.0 under treatment conditions of 4° C. and 48 hours. The buffer used in Figure 1 was citric acid-HCl; H9 acetate →,
Potassium phosphate: H, 4 cl in Tris: k-@,
Glycine-NBOH: Shown as Q-Q.

(5) Range of suitable temperature for action The range of suitable temperature for action of the enzyme of the present invention was 25 to 60°C, and the optimum temperature for action was around 50'C. (Figure 2) (6)
Conditions for temperature-induced inactivation pff7. 1 at each temperature from 20 to 80°C in Q
After holding for 0 minutes, the remaining enzyme activity was measured. As a result, the enzyme of the present invention was stable up to 40'C, as shown in Figure 8, and showed about 80% residual activity at 80'C.

(7) Inhibition, Activation, and Stabilization Table 1 shows the results of examining the effects of various additives on the enzyme of the present invention. There were no additives that significantly activated the enzyme of the invention.

Among the metal salts, 85% inhibition was observed by kIgC12. In addition, the enzyme of the present invention can be used for other endo-β-Glo
It was not affected by mannose or mannan, which strongly inhibit NAcase.

Table 1 Additives Concentration (mIvI) Relative activity (%) None 1
000aC] 2 1
890d0121 102 0+3CI2 1 101
0r(312199 We8C)a l
89(3uSU41 121 ZnS()41 107 DT'l! 1 8
02ME 1 89
NEM 1 106N
aF' l 10
1(isHl 105 01c 1(1100(Jal
IQ 112 (41cN
Ac Ig 105 (8
) Purification method The enzyme of the present invention can be purified by an appropriate combination of salting-out methods, various chromatographic methods, and the like. Specific examples of purification are as shown in Examples.

(9) Molecular Weight The molecular weight of the enzyme of the present invention is approximately 27,000.8 as determined by gel p-filtration using Sephadex G-200, and approximately ao, oo as determined by D8-polyacrylamide gel electrophoresis.

It was calculated that

(10) The enzyme of the present invention purified by polyacrylamide gel electrophoresis shows a single band in polyacrylamide gel electrophoresis! , 7.

(11) As a result of measurement by a chromatofocusing method using isoelectric point ampholine, the pI was 4.50.

(12) Amino acid analysis The purified enzyme preparation was hydrolyzed in 6N-)104 for 24 hours, and then the amino acid composition was measured using an automatic amino acid analyzer. The results are shown in Table 2.

Table 2 Amino acids Number of residues 1 mole Asp 28'rl+r 148er
19 (1 supervision u
16r06 01 y 213Ala
26 Val 14 Met 3 He 10 Leu 15 Tyr 12 Phe 7 Lys 9 +4is 8 Arg 4 1' r P 201ialf
Cystine 2 Next, a method for producing the enzyme of the present invention by culturing microorganisms will be specifically described.

The microorganism used for producing the enzyme of the present invention may be any microorganism that belongs to the genus Flavobaoteriun and has the ability to produce the enzyme of the present invention. A specific example of such a microorganism is strain 1022, which was isolated from soil by the present inventors. The mycological properties of this strain are described below.

(1) Morphological properties ゛ Short culm size: 0.4~0.5 x 1.2~
1.5 μm Motile 2 None Flagella: None Spores: None Durham staining: Negative (2) Growth status (a) Juice agar plate culture Shape: Small circular (granular) bumps:
Inner mouth circumference Green: Full surface: Lubricated (B) Meat juice agar slant culture Growth quality: Moderate Surface: Lubricated Growth shape: Stringy color Tone: Yellow Gloss: Yes Transparency 2 Translucent (C) Meat juice liquid culture surface Growth: None Turbidity: Moderate Sedimentation: Moderate) Meat Juice Gelatin puncture culture Growth state 2 Growth only on the surface Liquefaction of gelatin: llI (ho)lidomus milk Coagulation: Negative pH/acidic (8) Physiological Properties Reduction of nitrate 2 - Denitrification reaction 2 - MR test: - VP test: + Production of indole 2 - Hydrolysis of starch 2 - (Christensen c7) medium) - Utilization of inorganic nitrogen sources (growth) Ammonium chloride - Ammonium nitrate - Potassium nitrate + W Pigment production: + (water-insoluble) Oxidase: + Catalase + Growth pH' 6.5-7.5 Optimum growth temperature
:20-80'c Attitude towards oxygen: Facultative aerobic 0-F test
: Generation of acid and gas from oxidative sugars Sugars Acid Gas D-xylose -D-glucose ± -D-mannose
-〇-Fructose -〇-Galactose - Maltose ± - Sucrose - Lactose - Glycerin - Starch Identified according to the classification of Eve Bacteriology (8th edition),
This strain belongs to the genus Flavobacterium, but since no known species that fully matches the characteristics of this strain was found, this strain was identified as a new strain, and Flavobacterium sp.
(Flavobaotarium 8P, 1022). This strain has been deposited with the Institute of Microbial Technology as Microbiological Research Institute No. 8201A.

The composition of the medium used for culturing the microorganisms used is not particularly limited as long as it is used for the cultivation of normal microorganisms.

Examples of carbon sources include glucose, sucrose,
Arabinose, fructose, mannitol, inositol, soluble starch.

Nitrogen sources such as molasses and dextrin include peptone, meat extract, yeast extract, casamino acids, cornstarch liquor, various ammonium salts, various nitrates, and urea. Other phosphoric acid, magnesium, potassium, and sodium as needed.

A medium containing an appropriate amount of inorganic salts such as calcium, manganese, zinc, copper, iron, and molybdic acid is used.

Furthermore, a sludge extract obtained by hydrolyzing activated sludge with alkali and acid can also be used as a culture medium. The enzyme of the present invention has an inducing effect when yeast extract is added to the culture medium.
An approximately 9-fold increase in activity was observed with addition of 1%. For culturing, the culture medium is sterilized by a conventional method, and the strain of the present invention is inoculated.
It is carried out aerobically by shaking or aerating at 0 to 80°C for 8 to 4 days.

The enzyme of the present invention can be collected as follows.

In other words, after the completion of culture, the culture supernatant from which bacterial cells have been removed is centrifuged, and an inorganic salt such as ammonium sulfate is added to the resulting salt precipitate. A method of concentrating the culture supernatant by ultrafiltration, etc. There is. The enzyme of the present invention is purified from these salted-out products or concentrates by conventional methods. EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the following Examples are not intended to limit the scope of the present invention in any way.

〔Example〕

Glucose 0.5%, Pefton 0.5%, ill parent material 1
Add 2 volumes of medium containing 1.0% and 0.5% sodium chloride.
Dispense 500 ml each into 19 shake flasks,
After autoclaving for 15 minutes at 20"C9, 5 ml of Flavobacterium sp. 1022 strain precultured in a medium with the same composition was inoculated and cultured with shaking at 28"C for 4 days. After completion of the culture, the bacterial cells were separated by centrifugation. This was removed to obtain a culture P solution. Ammonium sulfate was added to this F solution to reach 90% saturation, and the resulting precipitate was dissolved in 0.01 MIJ acid buffer (pH 7.0) and dialyzed against the same buffer overnight. I) EAE-cellulose column (6) equilibrated with the same buffer solution in advance.
, 0x453) and adsorption 1. 0.1M of Wli element
Elution was performed using the same buffer containing Na0J. The active fractions were collected and concentrated by ultrafiltration, and this was concentrated to 0.001M
phosphate buffer (PI-I 7.0) overnight. This dialyzed solution was passed through a hytroxylapatite column (2,6×24ffi) equilibrated with the same buffer, and the adsorbed enzyme was eluted with a 0.11 M +) acid buffer (PH7,0). The eluted active fractions were collected and concentrated by ultrafiltration, and then mixed with 0.01M phosphate buffer (PI(7,
Sephadex G-150 column (2
, OX 1183). The active fractions were collected and treated in the same manner as above, and gel filtration was performed using a Sephadex G-100 column (2, OX 118ffi). The active fractions were collected and concentrated by ultrafiltration to obtain 10.8 wg of purified endo-β-GlcNAcase (specific activity 17.2υ/η yield 31.8%).
I got it.

〔Effect of the invention〕

Since the enzyme of the present invention can release asparagine-linked sugar chains from glycoproteins, it is extremely effective for structural analysis of glycoprotein sugar chains.

[Brief explanation of drawings]

FIG. 1 shows the optimal pn range for the action of the enzyme of the present invention. In Figure 1, H is citric acid -■Cj, o-O is acetic acid,
t→ is potassium phosphate, t→ is tris-flJ. Q-→ indicates each glycine-NaOH buffer. FIG. 2 shows the optimal temperature range for the action of the enzyme of the present invention. FIG. 8 shows the stable temperature range of the enzyme of the present invention.

Claims (10)

[Claims] (1) Acting on asparagine-linked sugar chains,
An endo-β-N-acetylglucosaminidase that specifically hydrolyzes the N'-diacetylchitobiose moiety. (2) The endo-β-N- according to claim (1), wherein the asparagine-linked sugar chain is a high-mannose type or a mixed type.
Acetylglucosaminidase. (3) The endo-β-N-acetylglucosaminidase according to claim (1), wherein the asparagine-linked sugar chain is bound to a natural high-molecular protein. (4) The endo-β-N-acetylglucosaminidase according to claim (1), which has an optimum pH of 5.0 to 6.0. (5) Stable pH is 5 under the holding conditions of 4℃ and 48 hours.
．． The endo-β-N-acetylglucosaminidase according to claim (1), which has a molecular weight of 0 to 7.0. (6) The endo-β-N-acetylglucosaminidase according to claim (1), wherein the endo-β-N-acetylglucosaminidase has a stable temperature range of up to 40° C. under conditions of pH 7.0 and holding for 10 minutes. (7) The endo-β-N-acetylglucosaminidase according to claim (1), which is not inhibited by mannan and mannose. (8) Claims (1) whose molecular weight is 30,000 as measured by SDS-polyacrylamide gel electrophoresis
The endo-β-N-acetylglucosaminidase described in ). (9) Ovalbumin, ribonuclease B (bovine pancreas)
, endo-β-N-acetylglucosaminidase according to claim (1), which is capable of cleaving sugar chains of yeast invertase in an intaot state. (10) Belongs to the genus Flavobacterium and endo-β-N
- Endo- characterized by culturing a microorganism having the ability to produce acetylglucosaminidase and collecting endo-β-N-acetylglucomidase from the culture.
Method for producing β-N-acetylglucosaminidase.