Vol. 55, No. 12

Induction and Purification of Endo-3-N-Acetylglucosaminidase fromArthrobacter protophormiae Grown in Ovalbumin

KAORU TAKEGAWA,1* MASANAO NAKOSHI,1 SHOJIRO IWAHARA,l KENJI YAMAMOTO,2AND TATSUROKURO TOCHIKURA2

Department of Bioresource Science, Faculty ofAgriculture, Kagawa University, Miki-cho, Kagawa 761-07,1 andDepartment ofFood Science and Technology, Faculty ofAgriculture, Kyoto University, Kyoto 606,2 Japan

Received 8 June 1989/Accepted 25 September 1989

Arthrobacter protophormiae produced a high level of extraceliular endo-o-N-acetylglucosaminidase whenceUls were grown in a medium containing ovalbumin. The enzyme was induced by the glycopeptide fraction ofovalbumin prepared by pronase digestion. Production of the enzyme was also induced by glycoproteins such as

yeast invertase and bovine ribonuclease B but not by monosaccharides such as mannose, N-acetylglucosamine,and galactose. The enzyme was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresisand has an apparent molecular weight of about 80,000. The enzyme showed a broad optimum pH in the rangeof pH 5.0 to 11.0. The enzyme hydrolyzed all heterogeneous ovalbumin glycopeptides, although the hydrolysisrates for hybrid type glycopeptides were very low. The substrate specificity of A. protophormwae endo-P-N-acetylglucosaminidase was very similar to that of Endo-C. from Clostridium perfringens. Therefore, theenzyme induction by A. protophormiae seems to have a close relation to the substrate specificity of the enzyme.

Endo-,-N-acetylglucosaminidase (endo-p-GlcNAc-ase,EC 3.2.1.96) hydrolytically cleaves the N,N'-diacetylchito-biose moiety of asparagine-linked sugar chains of variousglycoproteins. The purification of this enzyme has beenreported from some microorganisms, and the substrate spec-ificities of the enzyme have been elucidated (5, 9, 13, 34).Because of its characteristics, endo-p-GlcNAc-ase has beenused as an indispensable tool for structural studies onglycoproteins.

Endo-,B-GlcNAc-ase is also found in animal tissues, andthe enzyme in human tissues plays an important role in thecatabolism of asparagine-linked sugar chains of glycopro-teins (28). Although many microorganisms produce endo-f3-GlcNAc-ase, no studies on the physiological roles of theenzyme in microorganisms have been described to date.Recently, we reported that a component of yeast cellsremarkably induced the production of endo-i-GlcNAc-asefrom a Flavobacterium sp. isolated from soil (12). Therefore,we investigated culture conditions favoring the production ofmicrobial endo-,-GlcNAc-ase. Then we found that Arthro-bacter protophormiae highly induced endo-,-GlcNAc-ase inculture fluid when grown in medium containing ovalbumin.

In this report, we describe the induction of an extracellularendo-,-GlcNAc-ase from A. protophormiae grown on vari-ous glycoproteins. We also report the purification of theenzyme and its substrate specificities toward various aspar-agine-linked oligosaccharides.

MATERIALS AND METHODSChemicals. DEAE-Toyopearl 650M was purchased from

Tosoh Co., Tokyo, Japan. Molecular weight markers were

obtained from Pharmacia Fine Chemical Co. (Piscataway,N.J.). Pronase and dansyl (dns) chloride were purchasedfrom Kakenkagaku Co. and Tokyo Kasei Co., respectively.Bovine serum albumin, bovine RNase B, yeast invertase,human transferrin, yeast mannan, and azocasein were pur-chased from Sigma Chemical Co. (St. Louis, Mo.). Pressedbaker's yeast was obtained from the Oriental Yeast Co.

* Corresponding author.

Di-N-acetylchitobiose and tri-N-acetylchitotriose were

kindly donated by Katokichi Co. Ltd., Japan. Colloidalchitin was prepared from commercial chitin (Wako PureChemical Co.) by the method of Berger and Reynolds (2).p-Nitrophenyl glycosides were purchased from SeikagakuKogyo Co. All other chemicals were of analytical reagentgrade.

Microorganisms and cultivation. A. protophormiae (AKU0647) was used throughout this investigation (Brevibacte-rium protophormiae was recently reclassified as A. proto-phormiae) (27). The bacterium was cultured in a liquidmedium composed of 0.5% peptone, 0.5% yeast extract,0.5% NaCl, and 1.0% lyophilized ovalbumin powder, pH7.5. A loopful of cells was inoculated into 5 ml of the mediumin a test tube, and the seed culture was grown at 28°C for18 h with shaking. The culture was then transferred to a

500-ml Erlenmeyer flask containing 150 ml of the medium,and cultivation was continued at 28°C on a reciprocal shaker.

Preparation of substrates. Ovalbumin was prepared fromegg whites by the method of Rhodes et al. (24). Glycopep-tides (GP) from both ovalbumin and transferrin were ob-tained by exhaustive pronase digestions of glycoproteins,followed by separation on Sephadex G-25 gel by the proce-dure of Huang et al. (8). Asparaginyl oligosaccharides ofovalbumin (GP-I to GP-V) were prepared from ovalbuminglycopeptides as described by Huang et al. (8) by usingDowex 50W-X2 column chromatography. Dns derivatives ofasparaginyl glycopeptides were prepared by the method ofGray (6), followed by paper chromatography to remove dnssulfonic acid. Dns-GP-IIA, -GP-IIB, -GP-IIIA, -GP-IIIB,and -GP-IIIC were separated by high-performance liquidchromatography by using a reversed-phase column (InertsilODS, 4.6 by 250 mm; Gasukuro Kogyo, Japan) by themethod of Iwase et al. (10). The structures of the heteroge-neous ovalbumin glycopeptides obtained were reported byKobata and co-workers (29, 30, 37) to be as follows: GP-V,(Man)5(GlcNAc)2-Asn; GP-IV, (Man)6(GlcNAc)2-Asn; GP-IIIA, (GlcNAc)2(Man)5(GlcNAc)2-Asn; GP-IIIB, (Man)7(GlcNAc)2-Asn; GP-IIIC, (GlcNAc)3(Man)4(GlcNAc)2-Asn;GP-IIA, (Gal)(GlcNAc)3-(Man)4(GlcNAc)2-Asn; GP-IIB,

3107

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1989, p. 3107-31120099-2240/89/123107-06$02.00/0Copyright © 1989, American Society for Microbiology

3108 TAKEGAWA ET AL.

(GlcNAc)3(Man)5(GlcNAc)2-Asn; and GP-I, (Gal)(GlcNAc)3(Man)5(GlcNAc)2-Asn; Man represents mannose, and Galrepresents galactose.Enzyme assays. Endo-p-GlcNAc-ase activity was assayed

with (Man)6(GlcNAc)2 Asn-Dns (Dns-GP-IV) as the sub-strate. The reaction mixture was composed of 50 nmol of(Man)6(GlcNAc)2-Asn-Dns, 1 FLmol of sodium acetate buffer(pH 6.0), and the enzyme solution in a total volume of 35 RI.After incubation for an appropriate time at 37°C, the reactionwas terminated by the addition of 5 1±l of 10% trichloroaceticacid. A portion of the reaction mixture was directly analyzedby high-performance liquid chromatography (Hitachi modelL6200 chromatograph equipped with an F-1000 fluorescencespectrophotometer) by using a reversed-phase column (11)(Merck RiChrospher 100 RP-18, 4.6 by 250 mm). The columnwas eluted with 9% acetonitrile in 25 mM sodium boratebuffer, pH 7.5, at 1 ml/min. The product, GlcNAc-Asn-Dns,which was separated from (Man)6(GlcNAc)2-Asn-Dns, wasdetermined by fluorescence (excitation, 313 nm; emission,540 nm). One unit was defined as the amount of enzymeyielding 1 ,umol of GlcNAc-Asn-Dns per min at 37°C underthe assay conditions.

Exoglycosidases were assayed with the appropriate p-nitrophenyl glycosides. One unit was defined as the amountyielding 1 ,umol of p-nitrophenol per min at 37°C. Proteaseactivity was assayed by the method of Krauspe and Scheer(14), with azocasein as the substrate. a-Mannanase activitywas determined by using yeast mannan as the substrate, andliberated reducing sugar was measured by the Nelson-Somogyi procedure (23, 25).

Analytical methods. Protein was determined by the methodof Lowry et al. (18), with crystalline ovalbumin as thestandard. The protein content via column chromatographywas determined by measuring absorbance at 280 nm. Neutralsugars were assayed by means of the phenol-H2SO4 reaction(4).

Gel electrophoresis. Polyacrylamide slab gel electrophore-sis was carried out by the method of Davis (3) by using a7.5% polyacrylamide gel with Tris-glycine buffer, pH 8.3.Sodium dodecyl sulfate-polyacrylamide gel electrophoresiswas performed in 10% acrylamide and 0.1% sodium dodecylsulfate, with a discontinuous Tris-glycine buffer system, bythe method of Laemmli (15). Protein was stained withCoomassie Brilliant Blue R-250.

Molecular weight determination. The molecular weight ofthe enzyme was determined by gel filtration on SephadexG-100 (1.8 by 113 cm) by the method of Andrews (1). Elutionwas carried out with 10 mM potassium phosphate buffer (pH7.0). The following proteins were used as molecular weightmarkers: chymotrypsinogen (molecular weight, 25,000),ovalbumin (43,000), bovine serum albumin (67,000), andaldolase (158,000).

Purification of endo-13-GlcNAc-ase. The purification of theendo-p-GlcNAc-ase from the culture fluid of A. proto-phormiae is described below. All procedures in the enzymepurification were carried out at 5°C unless otherwise noted.

(i) Ammonium sulfate precipitation. Solid ammonium sul-fate was added to 90% saturation to the clarified culture fluid(1,600 ml) obtained after 72 h of cultivation. After standingovernight, the precipitate was collected by centrifugation(10,000 x g for 30 min at 4°C) and dissolved in 10 mMpotassium phosphate buffer (pH 7.0). This solution wasdialyzed against the same buffer overnight.

(ii) First DEAE-Toyopearl 650M column chromatography.The dialyzed solution was applied to a column (3.5 by 20 cm)of DEAE-Toyopearl 650M equilibrated with 10 mM phos-

phate buffer (pH 7.0). The column was washed with the samebuffer (500 ml) and then with buffer containing 0.2 M NaCl(300 ml), and the enzyme was eluted with buffer supple-mented with 0.3 M NaCl. The active fractions (100 ml) werecombined and dialyzed against 10 mM phosphate buffer (pH7.0) containing 0.1 M NaCl.

(iii) Second DEAE-Toyopearl 650M column chromatogra-phy. The dialyzed solution was applied to a column (1.0 by5.0 cm) of DEAE-Toyopearl equilibrated with 10 mM phos-phate buffer (pH 7.0) containing 0.1 M NaCl. The columnwas washed with the same buffer (50 ml) and then elutedwith a linear gradient of 0.1 to 0.3 M NaCl in the same buffer(300 ml). The active fractions (120 ml) were combined anddialyzed against 10 mM phosphate buffer (pH 7.0) including1.0 M ammonium sulfate overnight with two changes of thebuffer.

(iv) Phenyl-Sepharose CL-4B column chromatography. Theenzyme solution was applied to a Phenyl-Sepharose CL-4Bcolumn (1.0 by 30 cm) equilibrated with 1.0 M ammoniumsulfate in 10 mM phosphate buffer (pH 7.0). The enzyme waseluted with a linear gradient formed between 100 ml of 1.0 Mammonium sulfate in 10 mM phosphate buffer (pH 7.0) and100 ml of 40% ethylene glycol in 10 mM phosphate buffer(pH 7.0). The active fractions (40 ml) were combined andconcentrated by pressure dialysis by using a collodion bag(Sartorius).

(v) Sephadex G-100 column chromatography. The concen-trated enzyme solution was applied to a Sephadex G-100column (1.6 by 104 cm) previously equilibrated with 10 mMphosphate buffer (pH 7.0). The enzyme was eluted with thesame buffer in 3-ml fractions. The active fractions (33 ml)were combined and concentrated by using a collodion bag.The resulting enzyme preparation was stored at -20°C.

RESULTS

Effect of ovalbumin on endo-I-GlcNAc-ase production.Enzyme production in the supernatant of A. protophormiaeculture broth at different stages of growth was investigatedwhen the bacterium was grown in medium with or without0.3% ovalbumin. As shown in Fig. 1, both culture fluidsshowed similar profiles for pH and cell growth. However,endo-p-GlcNAc-ase activity in the culture fluid with 0.3%ovalbumin was more than 10 times higher than that withoutovalbumin. Maximum enzyme activity was observed after 2to 4 days. On the other hand, as shown in Table 1, all otherglycosidase activities were weak, except for ,B-galactosidase,after 3 days of cultivation and did not markedly increaseafter the addition of ovalbumin to the medium.For further examination of endo-p-GlcNAc-ase produc-

tion, the effect of the medium ovalbumin concentration onenzyme production was investigated after 3 days of cultiva-tion. As shown in Fig. 2, the enzyme activity increasedlinearly with increasing ovalbumin added to the medium.However, additions greater than 2% ovalbumin to the me-dium were not so effective.

Effect of various compounds on enzyme production. Thebacterium was cultivated in media containing various com-pounds, and the enzyme activity in each culture fluid wasinvestigated after 72 h of cultivation. As shown in Table 2,besides the addition of ovalbumin, the enzyme was inducedwith the addition of glycoproteins, such as ribonuclease Band yeast invertase, to the medium. These glycoproteinshave high-mannose type oligosaccharides (16, 17). We foundthat GP-III, -IV, and -V derived from ovalbumin remarkablyinduced enzyme production. These glycopeptides also have

APPL. ENVIRON. MICROBIOL.

ENDO-0-N-ACETYLGLUCOSAMINIDASE FROM A. PROTOPHORMIAE 3109

(A)

73(B)

cL

1-1

ElScn

cElO-re)

0 5

> L

i_:

1 2 3 4 5

CuLtivation time (day)

E

C0

(0

(L)C)

FIG. 1. Growth and enzyme production in cultures of A. protophormiae. The cultures were carried out in 500-ml Erlenmeyer flaskswithout (A) or with (B) 0.3% ovalbumin at 28°C. Symbols: 0, growth; 0, endo-p-GlcNAc-ase; and U, pH.

high-mannose type oligosaccharides. On the other hand,human transferrin, which has complex type oligosaccharides(26), and GP-I and -II, which have hybrid type oligosaccha-rides, hardly induced enzyme production. Moreover,monosaccharides such as mannose, galactose, and N-acetyl-glucosamine did not induce the enzyme either.Enzyme purification. The purification of endo-,B-GlcNAc-

ase from A. protophormiae grown in 1% ovalbumin mediumis summarized in Table 3. The enzyme was purified with anoverall recovery of about 66%.Enzyme purity and molecular weight. The purified endo-

P-GlcNAc-ase preparation showed a single protein band onpolyacrylamide gel and sodium dodecyl sulfate-polyacryl-amide gel electrophoreses. The molecular weight of theenzyme was estimated to be 81,000 + 2,000 by gel filtration.Upon sodium dodecyl sulfate gel electrophoresis, the en-zyme exhibited a single band corresponding to a molecularweight of 80,000 + 1,600. These results indicate that theenzyme has a monomeric structure. The final enzyme prep-aration was free of other exoglycosidase, chitinase, andprotease activities tested.

Effects of pH and temperature. The activity of the purifiedenzyme at different pHs was examined under the standardassay conditions. All assays were carried out with 100 mMbuffers. The enzyme exhibited a very broad optimum pH in

TABLE 1. Various glycosidase activities in two culturesof A. protophormiaea

Enzyme activity (10- U/ml)Substrates

-Ovalbumin +Ovalbumin

p-Nitrophenyl:a-Glucose 1.35 1.10p-Glucose 1.56 0.88a-Gal 0.97 0.50p-Gal 4.80 7.10a-Man 0.49 0.39p-Man 0.09 0.10a-Fucose 0.15 0.10p-Fucose 0.12 0.11P-Xylose 0.87 0.34P-GlcNAc 0.17 0.10

Yeast mannan 0 0

a A. protophormiae was grown in media containing 0.5% peptone, 0.5%yeast extract, and 0.5% NaCI, with or without L.0o ovalbumin, for 72 h.

the range of pH 5.0 to 11.0 (Fig. 3A). Even at pH 12.0, theenzyme showed about 90% of the maximum activity. Theeffect of pH on the stability of the enzyme was investigatedby storing the enzyme in 50 mM buffers of various pHs at5°C for 48 h. The stable pH range of the enzyme was from 5.0to 7.0, and it was also fairly stable at alkaline pHs (Fig. 3B).The purified enzyme was stable up to 60°C after incubation

for 10 min in 100 mM acetate buffer (pH 6.0). The optimumtemperature for the enzyme reaction was about 60°C underthe assay conditions.

Effects of various compounds. The effects of various metalions and sulfhydryl reagents on the enzyme activity wereexamined by preincubating the enzyme with the compoundat 37°C for 10 min and assaying the residual activity. Theactivity of the enzyme was not significantly affected by thefollowing compounds at 1 mM: MgSO4, MnSO4, ZnSO4,CaCl2, CoC12, CuS04, EDTA, glutathione, L-cysteine, 2-mercaptoethanol, p-chloromercuribenzoic acid, N-ethyl-maleimide, iodoacetic acid, and o-phenanthroline. However,HgCl2 (24% of the original activity), FeSO4 (44%), andCd(NO3)2 (54%) inactivated the enzyme.

Substrate specificity. The susceptibility of each asparaginyl

C-.E

1-1

0

c0

60

40

20

0.5 1.0 1.5 2.0

OvaLbumin conc. (%)FIG. 2. Effects of ovalbumin concentration on enzyme produc-

tion in a culture of A. protophormiae. Each amount of ovalbuminwas added to medium containing 0.5% yeast extract, 0.5% peptone,and 0.5% NaCl. The culture was carried out for 72 h.

VOL. 55, 1989

3110 TAKEGAWA ET AL.

TABLE 2. Effects of various compounds on enzyme productionby A. protophormiaea

Addition to medium Concn (%) Enzyme activity (%)

Ovalbumin 1.0 100Ovalbumin-GP-:

I 0.2 11II 0.2 12III 0.2 156IV 0.2 210V 0.2 210

Bovine serum albumin 1.0 4Yeast invertase 1.0 39Bovine ribonuclease B 1.0 25Yeast mannan 1.0 9Human transferrin 1.0 6Chitotriose 0.2 6None 4

a A. protophormiae was grown in media containing 0.5% peptone, 0.5%yeast extact, 0.5% NaCl, and the added compound at the concentrationindicated. After 72 h of cultivation, endo-p-GlcNAc-ase activity in the culturefluid was determined relative to that in 1.0% ovalbumin, which was setat 100%. Other sugars such as glucose, galactose, mannose, N-acetylglu-cosamine, glucosamine, and chitobiose had little or no effect on enzymeproduction.

oligosaccharide of ovalbumin to purified endo-p-GIcNAc-ase was examined by using dansylated derivatives as sub-strates. About 7 nmol of each of Dns-GP-I to -V wasincubated at 37°C with 0.64 mU of the enzyme in 50 mMacetate buffer, pH 6.0 (total volume, 35 [ul). The time coursesfor hydrolysis are shown in Fig. 4A. Under these conditions,Dns-GP-IV and -V were completely hydrolyzed into Dns-Asn-GlcNAc and oligosaccharides, and about 70% of theDns-GP-III mixture was also hydrolyzed after 6 h of incu-bation. On the other hand, the enzyme did not hydrolyzeDns-GP-I or -II. Then, for further examination of the hydrol-ysis of the Dns-GP-III mixture, about 7 nmol of each ofDns-GP-IIIA, -IIIB, and -IIIC, which were separated byhigh-performance liquid chromatography, was incubatedwith the enzyme at 37°C. As shown in Fig. 4B, the enzymecompletely hydrolyzed Dns-GP-IIIB and -IIIA after 6 h ofincubation. However, the enzyme did not hydrolyze Dns-GP-IIIC. At a high concentration of the enzyme (64 mU),96% of Dns-GP-III and 48% of Dns-GP-I and -II werehydrolyzed after 6 h of incubation. This enzyme did not acton (Gal)2(GlcNAc)2(Man)3(GlcNAc)2-Asn-Dns complextype oligosaccharide derived from human transferrin.

DISCUSSION

Glycoproteins having asparagine-linked sugar chains areubiquitous in nature and are found in living organisms, with

TABLE 3. Purification of endo-p-N-acetylglucosaminidasefrom A. protophormiae

Source of enzyme or Total Total Sp act Yieldprocedure for purification (mg) (U) (U/mg) (

Culture fluid 3,498a 91.6 (0.026) 100Ammonium sulfate 176 89.3 0.51 971st DEAE-Toyopearl 14.5 78.0 5.4 852nd DEAE-Toyopearl 11.0 67.0 6.1 73Phenyl-Sepharose CL-4B 6.1 63.5 10.4 69Sephadex G-100 1.8 60.0 33.3 66

a Undigested ovalbumin was contained in the culture fluid.

100

Q)

0

50

100

5C

3 5 7 9 11

pHFIG. 3. Effects of pH on the activity and stability of the enzyme.

(A) Enzyme assayed in 50 mM buffers of various pHs under standardconditions. (B) Enzyme stored at 5°C for 48 h in various 50 mMbuffers, followed by assay of remaining activity. Buffers: 0, sodiumacetate-acetic acid; 0, potassium phosphate; O, glycine-NaOH.

the exception of bacteria. Although most bacteria do notproduce glycoproteins (20), some of them are good sourcesof endo-,-GlcNAc-ase. Therefore, we examined for thesubstrates of this bacterial enzyme. There are many reports

100 (A)

50-

C,)

100 (B)0

I

50-

15 30 45 60

Incubation time (min)FIG. 4. Hydrolysis of dansylated ovalbumin glycopeptides by

the enzyme. The rates of hydrolysis by the enzyme were analyzed asdescribed in the text. Symbols: (A) 0, GP-V; *, GP-IV; O, GP-IIImixture; and *, GP-II mixture; (B) 0, GP-IIIB; 0, GP-IIIA; and 0,

GP-IIIC. In panel A, GP-I gave the same results as the GP-IImixture.

(A)

(B)

..~~~~

APPL. ENVIRON. MICROBIOL.

ENDO-P-N-ACETYLGLUCOSAMINIDASE FROM A. PROTOPHORMIAE 3111

concerning the induction of glycosidases that act on polysac-charides such as starch, cellulose, mannan, glucan, andchitin (19, 33). But there are no reports of the induction ofendoglycosidases that act on asparagine-linked oligosaccha-rides of glycoproteins.When A. protophormiae was grown in medium containing

ovalbumin, which is a typical glycoprotein having aspar-

agine-linked oligosaccharides, we found that ovalbumin oli-gosaccharides accumulated in the culture fluid. This factshowed that this bacterium produced endo-,-GlcNAc-asewhen grown in medium containing ovalbumin. As describedin the text, A. protophormiae does not produce a-mannosi-dase or a-mannanase at all in culture fluid. Therefore, thisbacterium does not completely degrade asparagine-linkedoligosaccharides. This result suggests that the production ofendo-p-GlcNAc-ase is not dependent upon carbon sources.

As shown in Fig. 1, the pH of the culture fluid was approx-imately 9.0, and the endo-p-GlcNAc-ase of A. proto-phormiae, unlike enzymes from other sources, showed highactivity at alkaline pHs. We also found that this bacteriumproduced alkaline protease with a high activity in culturefluid (manuscript in preparation). We already reported thatmicrobial glycoproteins such as yeast invertase and Rhizo-pus glucoamylase become sensitive to proteinases after thedeglycosylation of asparagine-linked oligosaccharides byendo-j3-GlcNAc-ase (31, 35, 36). Therefore, concerning theaction of endo-,-GlcNAc-ase, this alkaline protease of A.protophormiae may play an important role in the degrada-tion of glycoproteins.The A. protophormiae endo-p-GlcNAc-ase may specifi-

cally act on high-mannose type oligosaccharides. The sub-strate specificity of the A. protophormiae enzyme was

similar to that of Endo-C,, purified from Clostridium perfrin-gens (29). Most endo-,B-GlcNAc-ases from microorganismsare highly specific for high-mannose type oligosaccharides.Also, all microbial glycoproteins reported to date havehigh-mannose type oligosaccharides (7, 9, 22, 32). There-fore, it is reasonable that these enzymes show substratespecificities toward the degradation of microbial glycopro-teins. On the contrary, endo-p-GlcNAc-ases from patho-genic bacteria, such as Diplococcus pneumoniae, Flavobac-terium meningosepticum, and C. perfringens (C, enzyme),can act on the core structure or native form of complex typeoligosaccharides. Such oligosaccharides are widely distrib-uted in the animal kingdom. Thus, the substrate specificitiesof the enzymes seem to be influenced by the structures ofoligosaccharides encountered in different environments. A.protophormiae induced the enzyme when grown in a me-dium containing glycoproteins which the bacterium was ableto degrade. The induction mechanism of the enzyme iscurrently being investigated. Further studies on the action ofA. protophormiae endo-p-GlcNAc-ase toward various gly-coproteins are in progress.

LITERATURE CITED

1. Andrews, P. 1965. The gel filtration behavior of proteins relatedto their molecular weights over a wide range. Biochem. J.96:595-606.

2. Berger, L. R., and D. M. Reynolds. 1958. The chitinase systemof a strain of Streptomyces griseus. Biochim. Biophys. Acta29:522-534.

3. Davis, B. J. 1964. Disc electrophoresis. II. Method and applica-tion to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427.

4. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F.

Smith. 1956. Colorimetric method for determination of sugarsand related substances. Anal. Chem. 28:350-356.

5. Elder, J. H., and S. Alexander. 1982. Endo-p-N-acetylglu-cosaminidase F: endoglycosidase from Flavobacterium menin-gosepticum that cleaves both high-mannose and complex gly-coproteins. Proc. Natl. Acad. Sci. USA 79:4540-4544.

6. Gray, W. R. 1967. Dansyl chloride procedure. Methods En-zymol. 11:139-151.

7. Hashimoto, C., R. E. Cohen, W.-J. Zhang, and C. E. Ballou.1981. Carbohydrate chains on yeast carboxypeptidase Y arephosphorylated. Proc. Natl. Acad. Sci. USA 78:2244-2248.

8. Huang, C.-C., H. E. Mayer, Jr., and R. Montgomery. 1970.Microheterogeneity and paucidispersity of glycoproteins. Part I.The carbohydrate of chitin ovalbumin. Carbohydr. Res. 13:127-137.

9. Ito, S., T. Muramatsu, and A. Kobata. 1975. Endo-p-N-acetyl-glucosaminidase acting on carbohydrate moieties of glycopro-teins: purification and properties of the two enzymes withdifferent specificities from Clostridium perfringens. Arch. Bio-chem. Biophys. 171:78-86.

10. Iwase, H., S.-C. Li, and Y.-T. Li. 1983. Fractionation ofDns-glycopeptides by reversed-phase high-performance liquidchromatography. J. Chromatogr. 267:238-241.

11. Iwase, H., T. Morinaga, Y.-T. Li, and S.-C. Li. 1981. Analysis ofendo-p-N-acetylglucosaminidase activity by high-pressure liq-uid chromatography on a silica-based chemically bonded octa-decyl column. Anal. Biochem. 113:93-95.

12. Kadowaki, S., K. Takegawa, K. Yamamoto, H. Kumagai, and T.Tochikura. 1988. Effect of yeast extract on endo-,-N-acetyl-glucosaminidase production by a Flavobacterium sp. Agric.Biol. Chem. 52:2105-2106.

13. Koide, N., and T. Muramatsu. 1974. Endo-p-N-acetylglucos-aminidase acting on carbohydrate moieties of glycoproteins. J.Biol. Chem. 249:4897-4904.

14. Krauspe, R., and A. Scheer. 1986. Coomassie brilliant blueG-250 dye-binding technique for determination of autolyticprotein breakdown in Euglena gracilis and comparison to othermethods of autolysis measurement. Anal. Biochem. 153:242-250.

15. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

16. Lehle, L., R. E. Cohen, and C. E. Ballou. 1979. Carbohydratestructure of yeast invertase. Demonstration of a form with onlycore oligosaccharides and a form with completed polysaccha-ride chains. J. Biol. Chem. 254:12209-12218.

17. Liang, C.-J., K. Yamashita, and A. Kobata. 1980. Structuralstudy of the carbohydrate moiety of bovine ribonuclease B. J.Biochem. (Tokyo) 88:51-58.

18. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

19. Mann, J. W., T. W. Jeffries, and J. D. Macmillan. 1978.Production and ecological significance of yeast cell wall-degrad-ing enzymes from Oerskovia. Appl. Environ. Microbiol. 36:594-605.

20. Mescher, M. F., and J. L. Strominger. 1976. Purification andcharacterization of a prokaryotic glycoprotein from the cellenvelope of Halobacterium salinarium. J. Biol. Chem. 251:2005-2014.

21. Minobe, S., H. Nakajima, N. Itoh, I. Funakoshi, and I. Yamash-ina. 1979. Structure of a major oligosaccharide of Taka-amylaseA. J. Biochem. (Tokyo) 86:1851-1854.

22. Nakao, Y., Y. Kozutsumi, I. Funakoshi, T. Kawasaki, I. Ya-mashina, J. H. G. M. Mutsaers, H. Van Halbeek, and J. F. G.Vliegenthart. 1987. Structures of oligosaccharides on P-galac-tosidase from Aspergillus oryzae J. Biochem. (Tokyo) 102:171-179.

23. Nelson, N. 1944. A photometric adaptation of the Somogyimethod for the determination of glucose. J. Biol. Chem. 153:375-380.

24. Rhodes, M. B., P. R. Azari, and R. E. Feeney. 1958. Analysis,fractionation, and purification of egg white proteins with cellu-

VOL. 55, 1989

3112 TAKEGAWA ET AL.

lose-cation exchanger. J. Biol. Chem. 230:399-408.25. Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem.

195:19-23.26. Spik, G., B. Bayard, B. Fournet, G. Strecker, S. Bouquelet, and

J. Montreuil. 1975. Studies on glycoconjugates. LXIV. Com-plete structure of two carbohydrate units of human serotrans-ferrin. FEBS Lett. 50:296-299.

27. Stackebrandt, E., U. J. Fowler, F. Fiedler, and H. Seiler. 1983.Taxonomic studies on Arthrobacter nicotianae and related taxa.Description of Arthrobacter uratoxydans sp. nov. and Arthro-bacter sulfureus sp. nov. and reclassification of Brevibacteriumprotophormiae as Arthrobacter protophormiae comb. nov.Syst. Appl. Microbiol. 4:470-486.

28. Tachibana, Y., K. Yamashita, and A. Kobata. 1982. Substratespecificity of mammalian endo-,-N-acetylglucosaminidase:study with the enzyme of rat liver. Arch. Biochem. Biophys.214:199-210.

29. Tai, T., K. Yamashita, S. Ito, and A. Kobata. 1977. Structure ofthe carbohydrate moiety of ovalbumin glycopeptide III and thedifference in specificity of endo-o-N-acetylglucosaminidase CIIand H. J. Biol. Chem. 252:6687-6694.

30. Tai, T., K. Yamashita, M. Ogata-Arakawa, N. Koide, T. Mura-matsu, S. Iwashita, Y. Inoue, and A. Kobata. 1975. Structuralstudies of two ovalbumin glycopeptides in relation to theendo-,-N-acetylglucosaminidase specificity. J. Biol. Chem.250:8569-8575.

31. Takegawa, K., M. Inami, K. Yamamoto, H. Kumagai, T.

Tochikura, B. Mikami, and Y. Morita. 1988. Elucidation of therole of sugar chains in glucoamylase using endo-3-N-acetylglu-cosaminidase from Flavobacterium sp. Biochim. Biophys. Acta955:187-193.

32. Takegawa, K., N. Kawasaki, S. Iwahara, K. Yamamoto, T.Tochikura, B. Mikami, and Y. Morita. 1989. Primary structureof an N-linked sugar chain derived from glucoamylase ofRhizopus niveus. Biochim. Biophys. Acta 990:98-100.

33. Tanaka, H., K. Itakura, and K. Toda. 1978. Concerted inductionof P-glucanases of Bacillus circulans WL12 in response tovarious yeast glucans. Agric. Biol. Chem. 42:1631-1636.

34. Tarentino, A. L., and F. Maley. 1974. Purification and propertiesof an endo-p-N-acetylglucosaminidase from Streptomyces gri-seus. J. Biol. Chem. 249:811-817.

35. Yamamoto, K., K. Takegawa, H. Kumagai, and T. Tochikura.1986. Elucidation of the role of sugar chains in glycosylated-enzymes using endo-,-N-acetylglucosaminidase from Flavo-bacterium sp. Agric. Biol. Chem. 50:2167-2169.

36. Yamamoto, K., K. Takegawa, H. Kumagai, and T. Tochikura.1987. Effect of protease digestion on the activity of sugar-depleted enzymes prepared with endo-,-N-acetylglucos-aminidase from Flavobacterium sp. Agric. Biol. Chem. 51:1481-1487.

37. Yamashita, K., Y. Tachibana, and A. Kobata. 1978. The struc-tures of the galactose-containing sugar chains of ovalbumin. J.Biol. Chem. 253:3862-3869.

APPL. ENVIRON. MICROBIOL.