Purification and Characterization of Thermostable Trehalose Phosphorylase from Theymoanaeyobium Brockii

399

(J. Appl. Glycosci., Vol. 46, No. 4, p. 399-405 (1999) )

Purification and Characterization of Thermostable Trehalose Phosphorylase from Theymoanaeyobium brockii

Hiroto Chaen,* Tetsuya Nakada, Tomoyuki Nishimoto, Nobue Kuroda, Shigeharu Fukuda, Toshiyuki Sugimoto, Masashi Kurimoto and Yoshio Tsujisaka

Hayashibara Biochemical Laboratories, Inc. (7-7, Amase-minami machi , Okayama 700-0834, Japan)

A thermostable trehalose phosphorylase (EC 2.4.1.64) from a thermophilic anaerobe , Thermoanaero bium brockii ATCC 35047, was purified to an electrophoretically homogeneous state by successive

column chromatographies on DEAE-Toyopearl 6505, Butyl-Toyopearl 650 M and Ultrogel AcA 44 . The molecular weight of the enzyme was estimated to be 190,000 Da by gel filtration, and 88,000 Da by

SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity in the range of pH

7.0 to 7.5 for phosphorolysis, and pH 6.0 to 7.0 for synthesis . The optimum temperature of the enzyme

was 70•Ž in both directions. The enzyme was stable from pH 6.0 to 9.0, and up to 60•Ž . This result

showed that our trehalose phosphorylase was the most thermostable in those reported to date . Activity was inhibited by Cue, Hg2+, Mgt, Mn2+, Pb2+, and Zn2t The Kms for trehalose, Pi, D-glucose

and R-D-glucose 1-phosphate were 0.97, 0.57, 2.4 and 0.75 mM, respectively.

Trehalose phosphorylase (a, a-trehalose: a cell-free extract from a thermophilic anaer orthophosphate R-D-glucosyl transferase; EC obe, The rmoanaero bium brockii ATCC 35047, 2.4.1.64; TPase) catalyzes the reversible phos showed activity to produce a trisaccharide, phorolytic cleavage of trehalose as follows: selaginose (2-O-a-D-glucopyranosyl a-D-gluco trehalose+Pi•¬ƒÀ-G1P+D-glucose. pyranosyl a-D-glucopyranoside), from treha This enzyme was first demonstrated in lose. In the course of our investigation, it was Euglena gracilis 1,2)and later in several micro- suggested that selaginose was produced by organisms such as Bradyrhizobium japonicum,3) two reactions which were catalyzed by two Catellatospora ferruginea,4,5) Plesiomonas sp.6) thermostable phosphorylases, TPase and a and Micrococcus varians.7) Although TPase novel phosphorylase, kojibiose phosphorylase from various origins has been studied, there are (KPase) .12) In this paper, we describe the few reports of the enzyme being purified to purification and properties of thermostable electrophoretically homogeneous states. In TPase from T brockii ATCC 35047. recent years, many attempts have been made to utilize TPase to synthesize trehalose8,9) and MATERIALS AND METHODS other non-reducing trehalose-like saccharides, such as xylosylglucoside and fucosylgluco Materials. Trehalose, neotrehalose, maltose, side.10,11) However, the thermal stability of isomaltose and kojibiose were prepared in our TPases of these origins was not suitable for laboratory. ƒÀ-G1P and nigerose were purchased industrial application. Recently, we found that from Sigma Chemical Co. Laminaribiose was

purchased from Seikagaku Corporation. Selag- * Corresponding author . inose (2-O-a-glucosyl trehalose), 4-O-a-gluco Abbreviations: TPase, trehalose phosphorylase; KPase, kojibiose phosphorylase; R-G 1 P, ƒÀ-D-glu- syl trehalose and 6-O-a-glucosyl trehalose were

cose 1-phosphate; Pi, inorganic phosphate. prepared in our laboratory. 3-Morpholino 400 J. Appl. Glycosci., Vol. 46, No. 4 (1999) propane-sulfonic acid (MOPS) was obtained 280 nm was used to monitor protein in the from Dojindo Laboratories. DEAE-Toyopearl column eluate. 650S and Butyl-Toyopearl 650 M were pur Purification of TPase. chased from Tosoh Co. Ultrogel AcA 44 was Step 1. Extraction. The cells (238 g-wet) purchased from Sepracor. Other chemicals were suspended in 10 mM sodium phosphate were purchased from Wako Pure Chemicals. buffer (pH 7.0), and disrupted in an ice bath by Microorganism and cultivation. The rmo ultrasonication using a model UH-600 ho anaerobium brockii ATCC 35047 was used in mogenizer (MST Co.). Cell debris was re- this study. The medium and conditions for moved by centrifugation at 16,000•~g for 30 min. cultivation were described in our previous The supernatant (1400 mL) was used as the paper.12) After cultivation for 48 h, the cells cell-free extract. were harvested by centrifugation from 80 liters Step 2. Ammonium sulfate precipitation. of culture broth. Solid ammonium sulfate was added to the cell TPase assay. free extract to 60% saturation. After this was Phosphorolytic activity. A reaction mixture left for 16 h, the resulting precipitate was col consisting of 50 mM sodium phosphate buffer lected and dissolved in 10 mM sodium phosphate (pH 7.0), 1% trehalose, and the enzyme in a buffer (pH 7.0), and was then dialyzed against total volume of 2 mL was incubated at 60•Ž for the same buffer. 30 min. The reaction was stopped by 10 min of Step 3. DEAE-Toyopearl 650S column incubation in a boiling water bath. The glucose chromatography. The dialyzed enzyme solution released was measured by the glucose oxidase was poured into a DEAE-Toyopearl 650S peroxidase method.13) One unit of enzyme column (2.2 X 100 cm) equilibrated with the activity was defined as the amount of enzyme same buffer. After the unadsorbed protein was that liberates glucose l umol/min under these washed out from the column, the enzyme was conditions. eluted using a linear gradient from 0 to 0.5 M Synthetic activity. A reaction mixture consist- NaCI in the same buffer. The active fractions ing of 20 mM MOPS-NaOH buffer (pH 7.0), were collected and brought to a 1.5 M concentra 0.1% R-G1P, 0.1% D-glucose, and the enzyme in tion of (NH4) 2SO4 by adding solid (NH4) 2SO4. a total volume of 2.2 mL was incubated at 60•Ž Step 4. Butyl-Toyopearl 650 M column for 30 min. The reaction was stopped by chromatography. The enzyme solution was boiling for 10 min. The amount of Pi liberated poured into a Butyl-Toyopearl 650 M column from R-G1P was measured by the method of (1.6 X 48 cm) equilibrated with 10 mM sodium Fiske-Subbarow.14) One unit of enzyme activity phosphate buffer (pH 7.0) containing 1.5 M was defined as the amount of enzyme that (NH4) 2SO4. The adsorbed enzyme was liberates Pi 1 umol/min. eluted using a linear gradient from 1.5 to 0.5 M KPase assay. KPase activity was assayed in (NH4) 2SO4 in the same buffer. The active the direction of phosphorolysis. The reaction fractions were collected and concentrated to a mixture consisting of McIlvaine buffer (pH 5.5, volume of 6 mL on a OF module, Ultracent-30 Pi concentration; 102 mM), 0.1% kojibiose, and (Tosoh Co. Ltd.). the enzyme in a total volume of 2.2 mL was Step 5. Ultrogel AcA 44 column chromatog incubated at 60•Ž for 30 min. The reaction was raphy. Concentrated enzyme solution was stopped by boiling for 10 min. The glucose poured into an Ultrogel AcA 44 column (2.0 X 97 released was measured by the glucose oxidase cm) equilibrated with 10 mM sodium phosphate peroxidase method. One unit of enzyme activ buffer (pH 7.0) containing 0.2 M NaCI and was ity was defined as the amount of enzyme that eluted with the same buffer. The active frac liberates glucose l ,umol/min. tions were collected as the purified enzyme Protein assay. Protein was measured by the preparation. method of Lowry et al.15) using bovine serum Electrophoresis. Polyacrylamide gel electro albumin as a standard protein. Absorbance at phoresis (PAGE) was performed following the Thermostable Trehalose Phosphorylase from Thermoanaerobium brockii 401 method of Davis.16) The molecular weight of the subunit of enzyme was estimated by SDS- PAGE according to the method of Laemmli.17) After electrophoresis, the proteins were stained with Coomassie Brilliant Blue R-250. Molec ular weight markers (Bio-Rad Laboratories) were used as standard proteins. Estimation of the isoelectroc point of the enzyme was done by gel isoelectrof ocusing using Ampholine carrier ampholyte (Pharmacia Biotech). Molecular weight measurement. A TSKgeI Fig. 1. Separation of TPase and KPase by DEAE G4000SW column (0.75 x 60 cm) (Tosoh Co. Toyopearl 6505 column chromatography. ----- Ltd.) was used for molecular weight measure , absorbance at 280 nm; •¬, NaCI concentra ment of the purified enzyme. Analysis was tion; •›, TPase activity; and •œ KPase activity. carried out in 50 mM sodium phosphate buffer (pH 7.0) containing 0.2 M NaCI at room temper sis. TPase activity was completely separated ature at a flow rate of 0.9 mL/min. A molec from KPase activity by DEAE-Toyopearl 650S ular weight marker kit for gel filtration (Bio- column chromatography (Fig. 1). Purification Rad Laboratories) was used for standard pro of TPase from T brockii ATCC 35047 is sum teins. marized in Table 1. The enzyme was purified N- Terminal amino acid sequence analysis. about 1500-fold with a 25.8% yield from the cell- Automated Edman degradation18) and identifica free extract, and the specific activity was 78.2 tion of PTH-amino acids were carried out using U/mg of protein. The purified enzyme prepara a model 473A protein sequenator (Applied tion showed no KPase activity (less than Biosystems). 0.05%), and gave a single band by PAGE. HPLC. HPLC analysis of sugar was carried out using a CCPM pump, a RI-8012 differential Physical properties of TPase. refractive index monitor, and a SC-8010 data The molecular weight of the subunit of processor (all from Tosoh Co.) under the enzyme was estimated to be 88,000 Da by SDS- following conditions: column, TSK-GEL Amido- PAGE (Fig. 2A). The molecular weight of the 80 (250 x 4.6 mm) ; column temperature, 35t; native enzyme was estimated to be 190,000 mobile phase, acetonitrile-water (71: 29, v/v) ; Da by gel filtration on a TSKgeI G4000SW and flow rate, 1 mL/min. column (Fig. 2B). The pI of the enzyme was 5.4 by gel isoelectrof ocusing. The N-terminal RESULTS amino acid sequence up to the 15th residue was determined to be: NHZ-AIa-Asn-Lys-Thr-Lys- Purification of TPase. Lys-Pro-Ile-Tyr-Pro-Phe-Glu-Asp-Trp-Val-. Throughout the purification, TPase activity was measured in the direction of phosphoroly

Table 1. Purification of trehalose phosphorylase from T. brockii ATCC35047. 402 J. Appl. Glycosci., Vol. 46, No. 4 (1999)

Fig. 2. Homogeneity of purified trehalose phosphory lase and molecular weight measurement. Fig. 3. Effect of pH on trehalose phosphorylase activ ity during trehalose phosphorolysis and synthe (A) SDS-PAGE. Lane 1, purified trehalose phosphory sis. lase; and lane 2, standard protein mixture containing myosin (Mw 200,000), ƒÀ-galactosidase (Mw 116,250), To examine the optimum pH for phosphorolysis, the phosphorylase b (Mw 97,400), serum albumin (Mw 66,200), buffer in the standard assay system was replaced by and ovalbumin (Mw 45,000). (B) Gel filtration (TSK- citrate-NaAsO2 for pHs between 4.0 and 6.0, and by HC1 gel G4000SW). •›, purified trehalose phosphorylase; •œ NaAsO2 for pHs between 5.0 and 9.0. The enzyme 1, thyroglobulin (Mw 670,000) ; 2, gamma globulin (Mw activities were measured in 100 mM arsenate solutions. 158,000); 3, ovalbumin (Mw 44,000); 4, myoglobin (Mw To examine the optimum pH for synthesis, the buffer in 17,000) ; and 5, vitamin B12 (Mw 1350). the standard assay system was replaced by acetate for

pHs below 6.5, and standard MOPS buffer was used for pHs between 6.0 and 8.0. Optimum pH for phosphoroly Enzymatic properties of Tease. sis with citrate-NaAsO2 (ƒ¢) and HC1-NaAsO2 (•›). The effects of pH on phosphorolytic and Optimum pH for synthesis with acetate (•£) and MOPS (•œ . synthetic activity are shown in Fig. 3. The optimum pH range for phosphorolysis was pH 7.0 to 7.5, and that for synthesis was pH 6.0 to halose and 6-O-a-glucosyl trehalose were not 7.0. The enzyme was stable from pH 6.0 to 9.0 phosphorolized (data not shown). (data not shown). The optimum temperature Substrate specificity in the synthetic direction was 70•Ž in both directions, and the enzyme was was examined using ƒÀ-G1P as a glucosyl donor stable up to 60•Ž (Fig. 4, for phosphorolysis). and various mono- and oligosaccharides as The effects of metal ions on phosphorolytic acceptors. As shown in Table 3, D-xylose, D- activity are shown in Table 2. The enzyme galactose, L-arabinose, D-fucose, L-fucose, D- activity was inhibited by Cue, Hg2+, Mgt, glucosamine and 2-deoxy D-glucose can act as Mn2+, Pb2+, and Zn2t substitutes for D-glucose in the synthetic reac tion. However, little or no activity was observ Substrate specificity. ed with L-xylose, D-fructose, D-mannose, D- In order to investigate the phosphorolytic arabinose, L-sorbose, D-ribose and N-acetyl D- specificity of TPase, various disaccharides were glucosamine. No transfer product was detect- examined as substrates. TPase was specifically ed with various other di- or oligosaccharides. active on trehalose to produce ƒÀ-G1P and D- glucose, but was inactive on other saccharides Kinetic parameters. such as neotrehalose, kojibiose, nigerose, The initial velocity of the reaction was stud laminaribiose, maltose, cellobiose, isomaltose, ied in both directions. All double reciprocal sucrose, and lactose. Furthermore, selaginose plots on initial velocities versus reciprocals of (2-O-a-glucosyl trehalose), 4-O-a-glucosyl tre trehalose, Pi, l-G1P, and D-glucose showed a Thermostable Trehalose Phosphorylase from Thermoanaerobium brockii 403

Table 3. Acceptor specificity for disaccharide and oligosaccharide synthesis.

Fig. 4. Effects of temperature on trehalose phosphory lase activity and stability during trehalose phos phorolysis.

Enzyme activity was assayed at various temperatures

(50-85•Ž) according to the method described in the enzyme assay. To examine thermal stability, the enzyme was incubated at various temperatures (40- 80•Ž) for 1 h in 10 mM sodium phosphate buffer (pH 7.0), and cooled immediately. The residual activity was assayed at 60•Ž. •›, activity;•œ stability.

Table 2. Effects of metal ions on phosphorolytic activity.

The reaction mixture (1 mL) containing 1% R-G1P, 1% acceptor and the enzyme (10 U/g ƒÀ-G1P) in 25

mM MOPS buffer (pH 7.0) was incubated at 60•Ž for 24 h. The reaction products were analyzed by HPLC under the conditions described in materials and methods. + + +, transfer product greater than 40%; ++, 40 to 20%; +, 20 to 5%; -, no transfer

product.

Table 4. Kinetic parameters of trehalose phos phorylase for its substrates.

The enzyme was incubated at 50•Ž for 60 min in the

presence of 1 mM of each metal ion. The residual activity was assayed and the relative activity was expressed as a percentage of the enzyme in the absence of metal ions. 404 J. Appl. Glycosci., Vol. 46, No. 4 (1999)

series of straight lines (data not shown). The As shown in Table 3, TPase from T. brockii Km and kcat for substrates calculated from these can use D-xylose, D-fucose, D-galactose, L-arabi plots are summarized in Table 4. The Km for nose, L-fucose, D-glucosamine and 2-deoxy-D- trehalose, Pi, glucose and R-G1P were 0.94, 0.57, glucose as substitutes for D-glucose in disaccha 2.4 and 0.75 mM, respectively. ride synthesis. In contrast, Kizawa et al.7) reported that TPase from M. varians could not DISCUSSION use D-xylose, D-galactose, L-arabinose, D-glucos amine and 2-deoxy-D-glucose as glucosyl accep In our previous paper,12) we reported the for tors. Aisaka et al." reported that TPase from mation of a non-reducing trisaccharide, selagi Catellatospora ferruginea was useful in the nose, from trehalose by a cell-free extract of T synthesis of xylosylglucoside or f ucosylgluco brockii. In addition, we proposed a hypothesis side. Thus, it seems that the substrate that two phosphorylases, TPase and KPase, specificity of TPase in the synthetic direction were involved in the formation of selaginose as may be quite different depending on the enzyme follows; origin. As described above, it is considered that TPase from T brockii is useful in the synthesis of various trehalose-like disaccharides because of its high thermal stability and relatively broad In this paper, TPase was purified from T specificity. At present, however, the enzyme brockii, and some of its properties were productivity of T brockii is low. We have studied. Compared with The rmoanaero bium already cloned the TPase gene from T brockii TPase, enzymatic properties of TPases from genomic DNA. Studies on the overexpression three other sources are summarized in Table 5. of the TPase gene in an appropriate host are The The rmoanaero bium enzyme showed the now in progress. These results will be reported molecular weight of its subunit to be 88,000 Da, elsewhere. and that of the native enzyme to be 190,000 Da. As shown in Fig, 1, the KPase preparation These results indicate that the enzyme has a was also obtained by DEAE-Toyopearl 650S homodimer structure. Both molecular weights column chromatography. The purification and of this enzyme were similar to those of properties of this novel enzyme will be reported Plesiomonas TPase. The optimum pH for in our next paper. phosphorolysis was almost the same for all four, but T brockii TPase had the highest opti We thank Ms. L. Keleher for helpful comments during mum temperature and the best thermal stabil the preparation of this manuscript. ity.

Table 5. Comparison of the molecular and enzymatic properties of trehalose phosphorylase from various sources.

* Phosphorolytic activity .

•¬ Thermostable Trehalose Phosphorylase from Thermoanaerobium brockii 405

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