Purification and Characterization of a Novel Phosphorylase, Kojibiose
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423 (J. Appl. Glycosci., Vol. 46, No. 4, p. 423-429 (1999)) Purification and Characterization of a Novel Phosphorylase , Kojibiose Phosphorylase, from Thermoanaerobium brockii Hiroto Chaen,* Takuo Yamamoto, Tomoyuki Nishimoto, Tetsuya Nakada, Shigeharu Fukuda, Toshiyuki Sugimoto , Masashi Kurimoto and Yoshio Tsujisaka Hayashibara Biochemical Laboratories, Inc. (7-7 , Amase-minami machi, Okayama 700-0834, Japan) The thermophilic anaerobe Thermoanaerobium brockii ATCC 35047 produces a novel phosphorylase , k ojibiose phosphorylase, which catalyzes the reversible phosphorolysis of kojibiose to form ƒÀ-glucose 1-phosphate and D-glucose. The enzyme was purified from a cell-free extract to an electrophoretically homogeneous state by successive column chromatography on DEAE-Toyopearl 650S , CM-Toyopearl 6505, Hydroxyapatite, Ultrogel AcA44, Mono Q, and Butyl-Toyopearl 650 M . The enzyme had a molecular weight of 83,000 by SDS-polyacrylamide gel electrophoresis and a pl of 4 .3 to 4.4 by gel isoelectrofocusing. The enzyme showed the highest activity at pH 5 .5 and 65•Ž, and was stable from pH 5.5 to 9.7 and up to 65•Ž. The enzyme activity was inhibited by Hg2+ and Pb2+. The Km values for kojibiose, Pi, glucose, and ƒÀ-glucose 1-phosphate were 0 .77, 0.85, 3.52, and 0.77 mM, respectively. Kojibiose (2-O-a -D-glucopyranosyl a-D-gluco- novel phosphorylase catalyzed the reversible pyranose) is a disaccharide that has been iso phosphorolysis of kojibiose as follows: lated from koji-extract,1) beer,2) honey,3) and ƒÀ-G1P+D-glucose •¬ kojibiose+Pi starch hydrol.4) In general, this disaccharide is We proposed that the novel enzyme was koji prepared from a partial acetolyzate of Leuco biose phosphorylase (KPase). At present, five nostoc mesenteroides NRRL B-1299 dextran.5) enzymes, sucrose phosphorylase (EC 2.4.1.7), Chiba et al.6) reported the enzymatic synthesis maltose Phosphorylase (EC 2.4.1.8), cellobiose of kojibiose and nigerose (3-O-a-D-glucopyrano phosphorylase (EC 2.4.1.20), laminaribiose syl a-D-glucopyranose) from starch by the phosphorylase (EC 2.4.1.31) and trehalose phos transglucosylation of buckwheat a-glucosidase. phorylase (EC 2.4.1.64), are classified into dis As reported in our previous paper,1) we found accharide phosphorylase.8-12) However, no that a cell-free extract from a thermophilic report has been observed on the phosphorylase anaerobe, The rmoanaero bium brockii ATCC that acts on the a-1,2 glucosidic linkage. 35047, showed the activity to produce a tris In this paper, we describe the purification and accharide, selaginose (2-O-a-D-glucopyranosyl properties of a novel enzyme, KPase, from a a-D-glucopyranosyl a-D-glucopyranoside) , from thermophilic anaerobe, Thermoanaerobium bro trehalose. In addition, it was suggested that ckii ATCC 35047. selaginose was produced by two reactions which catalyzed by two thermostable phosphorylases, MATERIALS AND METHODS trehalose Phosphorylase (TPase) and a novel phosphorylase. It was considered that the Materials. CM-Toyopearl 650S was purchas * Corresponding author ed from Tosoh Co., Ltd. Hydroxyapatite was . Abbreviations: KPase, kojibiose phosphorylase; TP purchased from Wako Pure Chemical Indus ase, trehalose phosphorylase; Q-G 1 P, a-D-glucose 1- tries. Mono Q was purchased from Pharmacia phosphate; Pi, inorganic phosphate. Biotech. 424 J Appl. Glycosci., Vol. 46, No. 4 (1999) Microorganism and cultivation. The rmoana Step 5. Hydroxyapatite column chromatogra erobium brockii ATCC 35047 was used in this phy. The dialyzate was put on a column (1.6 •~ study. Medium and conditions for cultivation 22 cm) of Hydroxyapatite equilibrated with 10 were described in our previous paper.7) mM sodium phosphate buffer (pH 7.0). The Enzyme assay. enzyme was eluted with a linear gradient from Phosphorolytic activity. The reaction mixture 0.01 to 0.2 M sodium phosphate. The active for kojibiose phosphorolysis contained 0.1% fractions were combined and concentrated to a kojibiose, McIlvaine buffer (pH 5.5 Pi concen volume of 3 mL on an OF module, Ultracent-30 tration; 102 mM), and the enzyme in a total (Tosoh Co.). volume of 2.2 mL. After incubation at 60•Ž for Step 6. Ultrogel AcA44 column chromatogra 30 min, the reaction was stopped by boiling for phy. The concentrated enzyme solution was 10 min. Released glucose was measured by the put on a column (2.0 •~ 97 cm) of Ultrogel AcA44 glucose oxidase-peroxidase method.13) One unit equilibrated with 10 mM sodium phosphate of enzyme activity was defined as the amount of buffer (pH 7.0) containing 0.2 M NaCI and elut the enzyme that liberates glucose at 1umol/ ed with the same buffer. The active fractions min under the above-mentioned conditions. were combined and dialyzed against 10 mM Synthetic activity. The reaction mixture for sodium phosphate buffer (pH 7.0) containing kojibiose synthesis contained 0.1% glucose, 0.1 M NaCI. 0.1% fl-glucose 1-phosphate, 50 mM acetate Step 7. Mono Q column chromatography. buffer (pH 5.5), and the enzyme in a total The dialyzate was put on a column (0.5 •~ 5 cm) volume of 1.1 mL. After incubation at 60•Ž for of Mono Q equilibrated with 10 mM sodium various times, each reaction was stopped by phosphate buffer (pH 7.0) containing 0.1 M boiling for 10 min. Released Pi was measured NaCI. The enzyme was eluted with a linear by the method of Fiske and Subbarow.14) gradient from 0.1 to 0.4 M NaCI in the same Purification of KPase. buffer. The active fractions were combined Step 1. Extraction. The cell-free extract of and brought to a 1.2-M concentration of The rmoanaero bium brockii ATCC 35047 was (NH4) 2504 by adding solid (NH4) 2504. obtained as described in our accompanying Step 8. Butyl-Toyopearl 650 M column chro paper (H. Chaen, accompanying paper). matography. The enzyme solution was put on a Step 2. Ammonium sulfate precipitation. column (1.0 •~ 11 cm) of Butyl-Toyopearl 650 M Ammonium sulfate precipitation was done as equilibrated with 10 mM sodium phosphate described in our accompanying paper (H. buffer (pH 7.0) containing 1.2 M (NH4) 2504. It Chaen, accompanying paper). was eluted with a linear gradient from 1.2 to Step 3. DEAE- Toyopearl 6505 column chro 0 M (NH4) 2SO4 in the same buffer. The active matography. Chromatography on a DEAE fractions were pooled as the purified enzyme Toyopearl 650S column was done as described preparation. in our accompanying paper (H. Chaen, ac Substrate specificity. Substrate specificities of companying paper). The active fractions of the enzyme for phosphorolysis were tested using KPase were collected and dialyzed against 10 various disaccharides. Enzyme activity was mM citrate-Na2HPO4 buffer (pH 5.2). measured by the glucose liberated. The sub Step 4. CM- Toyopearl 6505 column chroma strate concentration was 0.1% in 0.1 M sodium tography. The dialyzate was put on a column arsenate-citrate buffer (pH 5.5). (1.6 •~ 22 cm) of CM-Toyopearl 6505 equilibrat Acceptor specificity. Acceptor specificities of ed with 10 mM citrate-Na2HPO4 buffer (pH the enzyme were tested according to the synthe 5.2). The enzyme was eluted with a linear sis, substituting various mono-, di-, and oligo gradient from 0 to 0.4 M NaCI in the same saccharides as acceptors for glucose. The reac buffer. The active fractions were combined tion mixture was analyzed by thin-layer chro and dialyzed against 10 mM sodium phosphate matography (TLC) and gas-liquid chromatog buffer (pH 7.0). raphy (GLC). Novel Phosphorylase from Thermoanaerobium brockii 425 HPLC. HPLC analysis of sugar was carried out using a CCPM pump, a RI-8012 differential refractive index monitor, and SC-8010 data processor (all from Tosoh Co.) under the following conditions: column, YMC-pak ODS AQ-303 (250 x 4.6 mm, YMC Co.) ; column tem perature, 35•Ž; mobile phase, water; and flow rate, 0.4 mL/min. Unless otherwise described above, all mate rials and methods used for this study were the same as those described in our accompanying paper (H. Chaen, accompanying paper). RESULTS Fig. 1. Homogeneity of purified KPase and molecular Purification of KPase. weight measurement. Table 1 shows a summary of the purification (A) SDS-PAGE. Lane 1, purified KPase; lane 2, stan of KPase from T. brockii ATCC 35047. The dard protein mixture containing myosin (Mw 200,000), enzyme was purified about 5500-fold with a ,O-galactosidase (116,250) , phosphorylase (97,000) , serum albumin (66,200), and ovalbumin (45,000). (B) Gel 10.6% yield from the cell-free extract. The filtration (TSKge1 G4000SW).•›, purified KPase;•œ 1-5, specific activity of the purified enzyme pre respectively, correspond to thyroglobulin (Mw 670,000) , paration obtained by Butyl-Toyopearl 650 M gamma globulin (158,000), ovalbumin (44,000), myoglo column chromatography was 71.4 U/mg pro bin (17,000), and vitamin B-12 (1350). tein. The purified enzyme preparation showed no TPase activity (less than 0.05%) and gave a Val-Lys-His-Met- Phe-Leu-Glu-Asp-V al-Asn- single band by PAGE. Asn-Leu-Ile-Ser. Physical properties of KPase. Enzymatic properties of KPase. The molecular weight of the enzyme was The effects of pH and temperature on the estimated to be 83,000 by SDS-PAGE (Fig. IA). phosphorolytic activity and stability are shown The molecular weight of the native enzyme was in Fig. 2A and B, respectively. The optimum estimated to be 500,000 by gel filtlation on a pH was 5.5, and the enzyme was stable from pH TSKge1 G4000SW column (Fig. 113). The pI 5.5 to 9.7. The optimum pH in the synthetic was from 4.3 to 4.4 by gel isoelectrofocusing .