α-Glucosidase activity was detected in the crude extract of cell and envelope of Capnocytophaga ochracea ATCC 33596. The enzyme associated with the envelope was effectively dissolved by cetyltrimethylammonium bromide. The enzyme was purified from both fractions by combination of ammonium sulfate precipitation, ion-exchage chromatography, gel filtration, and isoelectric focusing. Properties of both enzymes were quite similar: they were basic proteins, metalloenzymes, and most active at pH7.0. They hydrolyzed only p-nitrophenyl-α-D-gluconopyranoside

FUJIMURA SETSUO

NAKAMURA TAKESHI

SHIBATA YUKINAGA

English

application/pdfα-Glucosidase activity was detected in the crude extract of cell and envelope of Capnocytophaga ochracea ATCC 33596. The enzyme associated with the envelope was effectively dissolved by cetyltrimethylammonium bromide. The enzyme was purified from both fractions by combination of ammonium sulfate precipitation, ion-exchage chromatography, gel filtration, and isoelectric focusing. Properties of both enzymes were quite similar: they were basic proteins, metalloenzymes, and most active at pH7.0. They hydrolyzed only p-nitrophenyl-α-D-gluconopyranoside.journal articl

FUJIMURA, SETSUO

SHIBATA, YUKINAGA

NAKAMURA, TAKESHI

Matsumoto Dental University Repository

(Original) Matsumoto S.higaku 17 :key words: Capnocutophagaev-Glucosidase of      Dissolution,285'v288, 1991- a-glucosidase - rnicrobialbiochemistrya-Glucosidase  Capnocytophaga ochracea; Its Cellular Location, Dissolution, Purification, and Partial Characterization Cellular Location,Char cterizationSETSUO FUJIMURA YUKINAGA SHIBATA and TAKESHI NAKAMURA     DePartment of Oral Microbiology, Matsumoto Dental College                  (Chief : Prof T. Nahamura)Summary       a-GIucosidase activity was detected in the crude extract of cell and envelope of   CaPnocytoPhaga ochracea ATCC 33596. The enzyme associated with the envelope was   effectively dissolved by cetyltrimethylammonium bromide. The enzyme was purified from   both fractions by combination of ammonium sulfate precipitation, ion-exchage chromato-   graphy, gel filtration, and isoelectric focusing. Properties of both enzymes were quite   similar : they were basic proteins, metalloenzymes, and most active at pH7.0. They hydro-   lyzed only P-nitrophenyl-a-D-gluconopyranoside. '                      ' Introduction   Genus CaPnocytoPhaga has been implicated as pathogenic agent of periodontal disease as wellas Porphyromonas gingivalis and Actinobacilltts actinomycetemcomitans (2, 7). However, thebiolological properties, especially production and characterization of substances which may beresponsible for pathogenic action have not sufficiently been investigated. Laughon et al reportedthat CaPnoaytoPhaga elaborated phosphatase, enzyme hydrolyzed trypsin synthetic substrate, andglucosidase (4). Recently, isolation and characterization of bacteriocin of C. ochracea, which mayplay an ecological role in oral flora were reported (5). More information would be necessary forexamination of behavior of this genus'in the oral cavity. In this report, we describe about partialbiological properties of a-glucosidase of C. ochracea.                                     Methods   CaPnoaytoPhage ochracea ATCC 33596 was cultured in General Anaerobic Medium (NissuiSeiyaku, Tokyo) in anaerobic glove box filled with gases (N2+H2+C02 ; 85 : 10 : 5) at 37"C for 4days. a-glucosidase activity was assayed using P-nitrophenyl-a-D-gluconopyranoside (PNP-a-glucoside) as substrate. The reaction mixture contained 1.0 ml of 2.5 mM PNP'a-glucoside in 50mM Tris-maleate buffer (pH7.0), O.lml of enzyme sample, and 1.9ml of 50mM Tris-maleate buffer(pH7.5). After incubation of the reaction mixture at 37thC for 30min, O.5ml of 3.0M Na2 C03 to stopthe reaction, and the released p-nitrophenol was determined by measurement of the absorbance at420nm. One unit of activity was defined as the amount that increased O.1 absorbance unit per 1 min.(accepted for publication on October 24, 1991)286 Fujimura, et aL:a-Glucosidase of Capnoc)ttophaga ochraceaSDS-polyacrylamide gel electrophoresis for examination of purity and determination of molecularweights was carried out by the method of Laemmli (3). Gels were stained with Coomassie brilliantblue R250. Marker proteins for molecular weight estimation were phosphorylase b (94 kDa), bovineserum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydlase (30 kDa), soybean trypsin inhibitor(20.1 kDa), and a-lactalbumin (14.4kDa). Cells were harvested by centrifugation at 10,OOOg for 10min, washed twice with saline, and suspended in 50 mM Tris-hydrochloride buffer (pH8.0). The cellswere disrupted by sonication at 9 kHz for 20min. After sonication, the sonicated sample wascentrifuged at 5,OOOg for 10min to remove the unbroken cells. The supernatant was centrifuged at120,OOOg for 60min and the resultant precipitate (envelope) and supematant (S120 fraction) werekept at -40"C until used.Results and Discussion    a-Glucosidase activity was detected in S120 fraction and envelope but in the culture super-natant significant amounts of the activity could not be obtained. Attempts were made to dissolvethe enzyme from the envelope. It could not be extracted by neutral saline (O.5 to 1.5M NaClsolutions). Of the detergents tested (Triton X-100, polyoxyethylene 10-tridecyl ether, cetyltrimeth-ylammonium bromide), cetyltrimethylammonium bromide was most effective for extraction.Almost all activity was dissolved with this detergent. On the other hand, about 60 to 70% theactivity could be extracted by the other two detergents. The envelope was suspended in 50mM Tris-maleate buffer (pH7.5) and cetyltrimethylammonium bromide was added to a concentration of 1%and gently stirred at 37eC for 3.5h. Then this mixture was centrifuged at 120,OOOg for 60min and thesupernatant was dialyzed against 50mM Tris-hydrochloride buffer (pH8.0).    a-Glucosidase activity was found in Sl20 fraction and dissolved envelope fraction, but nosignificant activity was found in the culture supernatant. The ratio of activity in S120 fraction andin the dissolved envelope fraction was about 3 : 1. S120 fraction was applied to a column of QAE-Sephadex A25 equilibrated with 50 mM Tris-hydrochloride buffer (pH 8.0) and washed with thisbuffer. In this step a-glucosidase activity was recovered in the washings of the column. The activefractions were combined and dialyzed against 50 mM Tris-hydrochloride buffer (pH 7.5) and appliedto a column of CM 32 equilibrated with the same buffer. Then the proteins in the column were elutedby linearly increasing concentration of NaCl (O to 500 mM). The active fractions were combined,concentrated using rotary evaporater, and dialyzed against 50 mM Tris-hydrochloride buffer (pH7.5) containing 200 mM NaCl. The dialyzed sample was applied to a column (2.6 by 95 cm) of'Sephacryl S-300 equilibrated with 50 mM Tris-hydrochloride buffer (pH 7.5) containing 200 mMNaCl and eluted with the same buffer saline. The active fractions obtained from gel filtration wasdialyzed against 150 mM glycine solution and futher purified by electrophoresis on an isoelectricfocusing column (110 ml) with pH range of 5 to 11 (Fig. Ia). The electrophoresis was carried out at600 V for 48h. The peak of the activity found at pH 8.9 was dialyzed against 50 mM Tris-maleatebuffer (pH7.5) and this was the purified enzyme from S120 fraction. Similarly, a-glucosidase in thedissolved envelope fraction was purified using the methods described above. It was obtained at pHof 9.0 in the electrophoresis on isoelectric focusing (Fig. Ib). a-glucosidase purified from S120fraction and dissolved envelope fraction were referred to as S120 enzyme and envelope enzyme,respectively.   As shown in Fig. 2, both preparations were found to be homogeneous on SDS'PAGE. Thepositions of bands were rightly the same and the molecular weight was calculated to be 70 kDa.if}JzNca\ 17(3) 1991 287PNP-P-glucoside, PNP-a-frucoside, PNP-a-xyloside, and PNP-a-galactoside were not hydro-lyzed by both purified enzyme preparation. Both enzymes were completely inhibited by 1 mM ofEDTA. EGTA also inhibited by 96% and 94% the activity of S120 enzyme and envelope enzyme,respectively. However, O-phenanthrolin did not inhibited significantly both enzymes. No effect ofCa2' or Mg2' on the activity of both enzymes was observed. S120 enzyme and envelope enzymeexhibited maximum activity at pH 7.0. Both were inactivated by heating at 50Åé for 10 min.   Purification and characterization of a-glucosidase in crude extract and in envelope from C.ochracea have revealed that both enzymes show substantially identical properties. Different fromglucosidase of Bacillus (1) and Ruminococcus (6), the pl of C. ochracea was very high, those valuesof the enzyme of the former and the latter were reported to be 4.6 and 4.4, respectively.pH40E 3o}l:' 202:iti 10ooa10     20FRACTION NO3010864A2soO.6O.4O.240opH30=E.s 20st)5:; 10;mb 10864A2soO.8O.6O.4O.2       O 10 20 30 40                         FRACTION NOFig. 1 : Isoelectric focusing of a-glucosidase of S120 fraction (a) and envelope (b).     Symbols; e : a-glucosidase activity, O : absorbance at 280 nm, A : pH288 Fujimura, et al. : a-Glucosidase of CaPnoclytoPhaga ochraceaA c        94.eK67.eKew 43.eK- 3e.oK20.IK:- -•••• 14.4KFig. 2:SDS-PAGE of purified a-glucosidase of S120 fraction (Lane A),     and envelope (Lane B). Standard proteins were run in Lane C.References1) Fogarty, W.M., Kelly, C.T. and Kadam, S.K. (1985) Separation and characterization of an a   "glucosidase and maltase from llacillzts amyloliquefaciens. Can. J. Microbiol. 31 : 670-674.2 ) Holdernan L. V., Moore W. E. C., Cato E. P., Burmeister J. A., Palcanis K. G. and Ranney R. R. (1985)   Distribution of CaPnocytophaga in periodontal microfloras. J. Periodont. Res. 20 : 475-483.3 ) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage   T4. Nature 227 : 680-685.4 ) Laughon, B. E., Syed, S. A. and Loesche, W. J. (1982) API ZYM systern for identification of Bacteroides   spp., CaPnocytoPhaga spp., and Spirochetes of oral origin. J. Clin. Microbiol. 15 : 97-102.5 ) Nakamura, T., Shibata, Y., Shimura, R. and Fujimura, S. Isolation and properties of the CaPnoclytoPhaga   ochracea bacteriocin. Oral Microbiol. Immunol. (in press).6 ) Ohyama, K., Shirai, M. S., Kurachi, Y. and Shimizu, S. (1985) Isolation and properties of P-glucosidase   from Ruminococcus albus. J. Bacteriol. 161': 432-434.7) Slots, J. and Genco, R.J. (1984) Black-pigmented Bacteroides species, CapnoaytoPhaga species, and   Actinobacillus actinomycetemcomitans in human periodontal disease : virulence factors in colonization,  survival, and tissue destruction. J. Dent. Res. 63 : 412-421.teJ"st : Capnocytophaga ochracea e a-fJv=S N-t' i ?diXEtl!t, fiTnfit` It, rsNSS iftr"eeeNanJk, ;,vaf,N$,x" , tiitEr rt (kE}lscdeJk • ovakMg)  CaPnocytoPhaga ochracea ATCC33596esl6D wats tS ma L == 7 A" p - 7" trc cr-thV - "S B" - -{t"ts#Z}rsttSLk. ;=y.x" p- 7" tcreaLte a-`7" lv-s s"--tfeX-lt eL F iJ 1flv7 s- -e =9A 7"p s F" -(i'pmNe9 trc:fiTts IL " 2"L te. di iudi f]` rb ts 6 cD a- P'• 7v = s ptL -tf ig ert :rf; '(t t`SZ va, Åq' 7h y 7/xtS ga `7 p =?p F jf i7 7 l -, ti`" 7V ts me,9E,fi.agfi}fi(itv('geReeLk. asee$2ttciffifptptoDtii)rkOtffUJ/ L'( ?sb deZt i5iggMiteiLei`Åq fi'i5', ,pt pt ew* -(i- i{5 O , ifi pH et7.0e- iE) D tc. g k-e i7 = F p 7 = ; 7v -a-D- i," 7v = 7 lt' 7 7 •s F" cD lk ig hnltÅq fige L te .

α-Glucosidase of Capnocytophaga ochracea; Its Cellular Location, Dissolution, Purification, and Partial Characterization

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capnocytophaga ochraceaのα-グルコシダーゼ;その局在性,可溶化,精製および性状

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