INTRODUCTION

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Many compounds with interesting physiological or organoleptic properties occur in nature as glycosides. Therefore, methods of glycosylating compounds which would otherwise be too volatile or have low solubility in aqueous systems are of interest to the pharmaceutical, cosmetics, and food industries.

There are two distinct methods of enzymatic glycosylation: reversed hydrolysis and transglycosylation. Because of the hydrolytic activity of the glycosidases catalyzing reversed hydrolysis, final glycoside yields are generally low. Use of high substrate concentrations or heterogeneous catalysis in organic solvents with low water activity may result in a reaction equilibrium shift toward reasonable glycoside synthesis (34). These complex reaction conditions can be avoided by using enzymes with transglycosylating activity and which cannot hydrolyze the glycosides formed.

Here we describe in detail the transglucosylating activity of glucosyltransferase-D (GTF-D) (EC 2.4.1.5) from Streptococcus mutans GS-5 toward the model acceptor compound (+)-catechin. Catechin is a polyphenol with a broad range of functions in medicinal and food applications (15). Most studies on transglycosylation have focused on saccharides as acceptor molecules, resulting in the formation of oligosaccharides (27). However, there have also been reports of various enzymes capable of transglycosylating (phenolic) alcohols: Bacillus subtilis X-23 -amylase (EC 3.2.1.1) (23-25), Bacillus macerans cyclodextrin glucanotransferase (EC 2.4.1.19) (8), and Leuconostoc mesenteroides sucrose phosphorylase (EC 2.4.1.7) (15-18) and dextransucrase (EC 2.4.1.5) (27).

GTFs from the cariogenic streptococci are believed to play an important role in the formation of dental caries due to the production of glucans from dietary sucrose (12, 19). The extracellular, constitutively produced GTFs are classified according to the primer requirement to start glucan synthesis and to the type of glucan formed. The two main categories of glucans are the water-insoluble glucans, predominantly containing -1,3-linked glucose, and the soluble glucans, rich in -1,6-linked glucose (27). Extracellular streptococcal fluids regularly contain more than one enzymatic activity, often occurring in high-molecular-weight aggregates. Genes encoding various GTFs have been isolated. S. mutans expresses four different genes, gtfA, gtfB, gtfC, and gtfD (1, 13, 14, 26). The latter gene encodes GTF-D, which produces water-soluble glucan in a primer-stimulated manner (14). Other Streptococcus species, such as S. sobrinus and S. downei, also contain multiple gtf genes encoding distinct GTFs (11, 36).

For GTFs closely related to L. mesenteroides dextransucrase, synthesis of glucan is proposed to proceed according to a two-site insertion mechanism, resulting in addition of glucose to the reducing end of the glucan (27, 29, 31). Leucrose (5-O--D-glucopyranosyl-D-fructose) and glucose (arising from an acceptor reaction with water) are formed as minor products. Besides the transfer of glucose from sucrose to glucan, other carbohydrates, such as monosaccharides (D-mannose, D-galactose), disaccharides (maltose, lactose), or oligosaccharides (maltotriose), can act as acceptors for transglucosylation by dextransucrase, resulting in a homologous series of oligosaccharide products (27, 28).

In this study, we compared the transglucosylating activities of several S. mutans and S. sobrinus GTFs toward (+)-catechin as a model acceptor. The highly efficient catechin transglucosylation by GTF-D and especially the influence of fructose on GTF-D transglucosylating activity are described in detail.

	MATERIALS AND METHODS

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Growth conditions of S. sobrinus SL-1 and partial purification of GTF-T. S. sobrinus SL-1 was kindly provided by R. R. B. Russell (Department of Oral Biology, University of Newcastle) and stored in glycerol (45%, vol/vol) at 80°C. A single colony of S. sobrinus SL-1 grown on an MRS plate at 37°C was used to inoculate 25 ml of MRS medium supplemented with cysteine (0.5 g/liter) and was grown statically at 37°C. Overnight cultures (1 ml) were used to inoculate 250 ml of modified Terleckyj et al. (32) medium and were incubated statically at 37°C. After reaching the stationary phase at an optical density at 600 nm (OD₆₀₀) of 1.5 to 2.0, the culture supernatant was obtained by centrifugation (16,300 × g, 15 min, 4°C). A mixture of four glucosyltransferases (GTF-T) was partially purified and concentrated by ammonium sulfate precipitation (7). The supernatant was brought to 55% (326 g/liter) saturation with ammonium sulfate. The precipitate was collected by centrifugation (39,100 × g, 15 min, 4°C), dissolved in potassium phosphate buffer (25 mM, pH 6.0), and dialyzed against the same buffer.

Cultivation of Escherichia coli expressing recombinant GTF-B and GTF-D and preparation of cell extracts. Escherichia coli containing S. mutans GS-5 gtfB(pYNB13) (33) and E. coli containing S. mutans GS-5 gtfD (pYND72) (30) were kindly provided by H. K. Kuramitsu (Department of Oral Biology, State University of New York at Buffalo). The E. coli strains were stored in glycerol (14%, vol/vol) at 80°C. Both strains were grown aerobically at 30°C in TY medium (1% NaCl, 3.2% tryptone, and 2% yeast extract) with 0.2 mM isopropyl--D-thiogalactoside. pYNB13 was maintained in medium supplemented with 50 µg of ampicillin per ml. pYND72 was maintained in medium supplemented with 68 µg of chloramphenicol per ml. Overnight cultures were harvested by centrifugation (16,300 × g, 15 min, 4°C). The cells were washed with Tris-hydrochloride buffer (20 mM, pH 7.5) and resuspended in the same buffer at a concentration of 0.2 ± 0.1 g of wet biomass/ml. After sonication (Sonifier 250; Branson [duty cycle, 30%; output control, 3]), 0.1 mM phenylmethylsulfonyl fluoride and 0.5 mg of DNase were added to the lysate. After centrifugation (39,100 × g, 15 min, 4°C), the supernatant was dialyzed against Tris-hydrochloride buffer (20 mM, pH 7.5) and used as a crude enzyme solution.

Partial purification of GTF-B. GTF-B produced with E. coli was partially purified by ammonium sulfate precipitation. The crude enzyme solution was brought to 10% (50 g/liter) saturation with ammonium sulfate. The supernatant with GTF activity obtained after centrifugation (39,100 × g, 15 min, 4°C) was brought to 40% (169 g/liter) saturation and allowed to stand for 30 min. After centrifugation (39,100 × g, 15 min, 4°C), the precipitate was dissolved in potassium phosphate buffer (25 mM, pH 6.0) and dialyzed against the same buffer.

Partial purification of GTF-D. GTF-D was initially partially purified by applying the cell extract on a DEAE-Sepharose CL-6B (Pharmacia) column (2.5 by 30 cm) previously equilibrated with Tris-hydrochloride buffer (20 mM, pH 7.5). Proteins were eluted by using a linear gradient of 0 to 0.7 M NaCl in the same buffer. Fractions containing GTF activity were concentrated by ultrafiltration (YM30; Amicon Corporation). The concentrated solution was dialyzed against potassium phosphate buffer (25 mM, pH 6.0). Under the same experimental conditions, GTF-D was also purified with a DE-52 cellulose anion-exchange column (Whatman).

-Glucosidase activity. Enzyme solutions were incubated with p-nitrophenyl--D-glucopyranoside (10 mM) and potassium phosphate buffer (125 mM, pH 6.0) in a volume of 1 ml. -Glucosidase activity present in the crude enzyme solution was measured by monitoring the increase of p-nitrophenolate at 405 nm at 30°C. The molar extinction coefficient for p-nitrophenolate at pH 6.0 was 2,300 M¹ cm¹.

Glucosyltransferase transglucosylating activity. GTF transglucosylating activity was quantified by two different methods: measurement of the formation of reducing sugars (RS activity) and of catechin glucosides (CG activity).

Catechin transglucosylation conditions. Catechin transglucosylation studies were performed with a reaction solution containing potassium phosphate buffer (125 mM, pH 6.0), sucrose, (+)-catechin (Sigma), and water. After addition of GTF, the reaction mixture was incubated statically at 37°C.

Separation of catechin transfer products. GTF-D catechin transfer products for structure elucidation were separated with a two-step approach. First, 200 µl of reaction mixture was put on a silica column (10 by 1.5 cm; Silica Gel 60 [Merck]; particle size, 0.063 to 0.200 mm). Two fractions (A and B) were collected by eluting the silica column with 23 ml of 50% ethanol-H₂O (vol/vol) and 18 ml of 50% ethanol-H₂O (vol/vol) with 5% acetic acid (vol/vol). Both fractions were concentrated by vacuum distillation and dissolved in 125 mM potassium phosphate, pH 6.0. Further purification of catechin transfer products was done by collecting the individual peaks with the HPLC-UV method described above.

NMR spectroscopic analysis. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were obtained with a 200 MHz AC200 spectrometer at room temperature. All nuclear Overhauser effect (NOE)-difference and two-dimensional NMR spectra measured were collected with a 400 MHz Bruker DPX 400 spectrometer.

CO₂ measurements. CO₂ concentrations were determined by analyzing 100-µl gas phase samples on a Hewlett Packard HP 6890 gas chromatography system.

Growth conditions of Saccharomyces cerevisiae T2-3D and Pichia pastoris. The mutant yeast S. cerevisiae T2-3D (35) was kindly provided by J. T. Pronk (Department of Microbiology, University of Delft). S. cerevisiae T2-3D was stored in glycerol (20%, wt/vol) at 80°C. P. pastoris (CBS 704) was stored in glycerol (50%, vol/vol) at 80°C. Both yeasts were grown aerobically in 1 liter of mineral salts medium with glucose (2%, wt/vol) and yeast extract (Difco) (0.2%, wt/vol). P. pastoris and S. cerevisiae were harvested by centrifugation (39,100 × g, 15 min, 4°C) at OD₆₆₀s of 10 and 15, respectively. After washing of the cells, P. pastoris and S. cerevisiae were stored (20°C) at concentrations of, respectively, 66 and 70 mg (dry weight) ml¹.

	RESULTS

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Partial purification of GTFs. The culture supernatant from S. sobrinus SL-1 was harvested during the stationary phase. The initial RS activity of the mixture of four glucosyltransferases (GTF-T), 0.16 U/ml (DNS method), which was comparable with other reported activities for S. mutans strains (9, 10), was purified 13-fold by ammonium sulfate precipitation with 83% recovery (0.86 U/mg). After dialysis, no -glucosidase activity was detected. This GTF-T enzyme preparation was used in the initial experiments, as shown in Table 1.

Catechin transglucosylation with different GTFs. The partially purified GTF preparations (0.3 U/ml) were compared for their transglucosylating activities toward 5 mM catechin as a model acceptor. Both the RS activity (expressed as reducing sugars released in the presence of catechin [DNS method]) and the CG activity (expressed as glucose incorporated into catechin [HPLC method]) were determined (Table 1).

Characteristics of GTF-D transfer products. HPLC analysis showed that during the incubation of GTF-D with catechin and sucrose, three distinguishable peaks, hereafter labeled peaks 1 (t_r, 7.63), 2 (t_r, 7.85), and 3 (t_r, 8.37) in order of increasing HPLC retention times (t_r), were detected (Fig. 1A). While the total area of peaks 1, 2, and 3 increased, the catechin peak area decreased (peak 4; t_r, 8.80 [Fig. 1A and B]). Catechin polymerization was not observed under these conditions. Incubation of the reaction solution with Aspergillus niger amyloglucosidase (Sigma) (EC 3.2.1.3) resulted in a decrease in the three product peaks and an increase in catechin (data not shown).

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Formation of GTF-D catechin transfer products. (A) HPLC chromatogram showing the three catechin transfer products (1, 2, and 3) and catechin (4) after 24 h of incubation with 10 mM catechin, 60 mM sucrose, and 37 mU of GTF-D per ml at 37°C. (B) Formation of the three catechin transfer products (peak 1 [], peak 2 [], peak 3 []) and catechin (×) with 10 mM catechin, 60 mM sucrose, and 37 mU of GTF-D per ml at 37°C.

Identification of catechin transfer products. The exact identity of the GTF-D catechin transfer products was established by a comprehensive NMR spectroscopic study. To obtain the purified catechin transfer products, the mixture of GTF-D catechin transfer products was separated in two steps as described in Materials and Methods. After the first step (silica column chromatography), two fractions, labeled A and B, were collected. Fraction A contained peaks 1 and 3, hereafter labeled products 1 and 3. Fraction B contained peak 2, hereafter labeled product 2, and catechin. After purification, we obtained 6.7, 3.2, and 16.1 µmol of products 1, 2, and 3, respectively, from the initial 250 µmol of catechin (25-ml reaction volume containing 60 mM sucrose and 37 mU of GTF-D/ml). Product 2 was not chemically stable upon prolonged storage, so no further attempts were made to elucidate its structure.

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Structure and atom numbering of catechin.

Hydrolysis of catechin glucosides by amyloglucosidase. The identification of product 3 as catechin-4'-O--D-glucoside and product 1 as catechin-4'-7-O--di-D-glucoside made it tempting to assume that product 2 was catechin-7-O--glucoside. Hydrolysis of the identified catechin glucosides by A. niger amyloglucosidase was used in an attempt to confirm this assumption.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Hydrolysis of the three catechin transfer products (1 [], 2 [], 3 []) and formation of catechin (×), with 140 U of A. niger amyloglucosidase per ml at 55°C.

Kinetics of GTF-D catechin transglucosylation. Catechin transglucosylation assays were performed with 125 mM potassium phosphate buffer at pH 6.0. It appeared that under these reaction conditions the reaction rate was more or less constant during the first 60 min (Fig. 1B). Subsequently initial CG activities were determined based on the amount of catechin glucoside formed after 60 min of incubation. Variations in potassium phosphate concentration (25 to 250 mM) and pH (5.0 to 7.5) had very little effect on the initial CG activity (with 60 mM sucrose and 10 mM catechin).

Fructose inhibition of GTF-D activity. The total catechin glucoside formation rate, as shown in Fig. 1B, gradually decreased as the reaction proceeded. The decrease in catechin concentration during the reaction was simulated, assuming first-order kinetics with respect to the catechin concentration and zero-order kinetics with respect to the sucrose concentration. Comparison of the experimentally determined data (Fig. 1B) with the simulated curve (data not shown) showed that the decrease in the reaction rate was stronger than would be expected based solely on the decrease in the catechin concentration. In other studies with glucosyltransferases, the accumulation of fructose was shown to competitively inhibit GTF enzyme activity (4). We therefore studied the inhibitory effect of fructose on catechin transglucosylation by GTF-D.

Glucose and fructose consumption by P. pastoris and S. cerevisiae T2-3D. To overcome the inhibitory influence of fructose accumulation on catechin transglucosylation, we studied the possibility of using yeasts to remove fructose. Two yeasts, the methylotrophic yeast P. pastoris and a mutant, S. cerevisiae T2-3D, were selected. Both strains are incapable of fermenting sucrose. This was confirmed by measuring saccharide-dependent carbon dioxide formation. No additional formation of CO₂ was observed after addition of sucrose in contrast to addition of either glucose or fructose. Furthermore, the sucrose concentration, monitored by HPLC-RI, did not change during incubation of P. pastoris or S. cerevisiae with sucrose.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   (A) Catechin glucoside formation by S. mutans GS-5 GTF-D in the absence () or presence of P. pastoris () (66 mg [dry weight]/ml) or S. cerevisiae T2-3D () (70 mg [dry weight]/ml). (B) Saccharide concentrations (, sucrose; , fructose; , glucose) in the absence (closed symbols) and in the presence (open symbols) of P. pastoris. The saccharide profile with S. cerevisiae T2-3D was very similar to the saccharide profile with P. pastoris. The reaction solutions (5 ml) contained 125 mM potassium phosphate buffer (pH 6.0), 60 mM sucrose, 10 mM catechin, and 37 mU of GTF-D per ml.

	DISCUSSION

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In this study we have compared the transglucosylating activity toward catechin of several GTFs from the genus Streptococcus. The transglucosylation characteristics of S. mutans GS-5 GTF-D were studied in more detail. Although catechin has a complex structure, containing five hydroxyl groups, this compound was used as a model acceptor to allow comparison with literature data on other catechin-transglucosylating enzymes (8, 15, 22).

The different GTF preparations tested were a mixture of the four GTFs from S. sobrinus SL-1 (GTF-T) and the heterologously produced GTF-B and GTF-D from S. mutans GS-5. Note that the three GTF preparations were not purified to homogeneity.

All GTF preparations were capable of catechin transglucosylation (Table 1), although at different rates. The CG activity of GTF-D was almost three times higher than the CG activity of GTF-B and eight times higher than the CG activity of GTF-T. Considering the ratio between the RS and CG activity of the three GTF preparations, we decided to study the transglucosylation of catechin in more detail using GTF-D from S. mutans GS-5.

Besides catechin transglucosylating activity, all three GTF preparations showed an increase in the formation rate of reducing sugars in the presence of catechin. Especially in the case of GTF-T, this increase can hardly be explained by assuming that catechin is a better glucosyl acceptor than dextran, because almost no catechin glucoside formation was observed. An explanation could be that upon dextran chain elongation the glucosyl transfer rate decreases. If we assume that the presence of catechin results in premature displacement of the growing dextran chain from the active site, catechin addition would result in an enhanced rate of (short-chain) dextran formation and hence also in an increase in reducing sugar formation.

Based on HPLC analysis, glucosylation of catechin resulted in the formation of (at least) three catechin transfer products. Two catechin transfer products were characterized in more detail. Although catechin glucoside separation was not optimized, sufficient catechin glucoside was isolated to perform ¹H and ¹³C NMR spectroscopy. The structures of two catechin transfer products were identified as (+)-catechin-4'-O--D-glucoside and (+)-catechin-4',7-O--di-D-glucoside. In the literature, 4'-O- (22) and 3'-O- (8, 15) glucosylated catechins have been described. The catechin diglucoside characterized in this study is to our knowledge the first catechin diglucoside described. The structure of the third catechin transfer product (labeled product 2) could not be spectroscopically elucidated. However, the deglucosylation study with A. niger amyloglucosidase suggests that this compound could be 7-O- monoglucosylated catechin. Note that the A. niger amyloglucosidase preferentially deglucosylates the 7-O- glucose of the diglucoside. The fast hydrolysis of product 1 into product 3 and the fast hydrolysis of product 2 into catechin are therefore indicative of glucosylation of C-7 rather than C-5, C-3, or C-3' in product 2.

In comparison with literature data concerning catechin transglucosylation (15, 22), the catechin transglucosylation efficiency of GTF-D appears to be very high. Based on the data in Table 1, a minimal glucosyltransfer efficiency of 58% can be calculated assuming that all the reducing sugars formed are fructose. In a number of studies, the transfer ratio (or yield) has been used to quantify the transglucosylating potential of enzyme preparations (15, 22). The transfer ratio is expressed as the ratio between the moles of catechin glucoside formed and moles of catechin initially added. Depending on the sucrose concentration, the transfer ratio for catechin glucosylation with GTF-D varied between 55 and 90%. Sucrose phosphorylase (15) was reported to have a similar transfer ratio, but a much higher sucrose concentration (30%, wt/vol) was used. The mixture of GTFs from S. sobrinus (22) also transglucosylates catechin, but the reported transfer ratio is lower than the transfer ratio of the partially purified GTF-D that we studied. The data reported for catechin transglucosylation with cyclodextrin glucanotransferase are difficult to compare because of the use of soluble starch as the glucosyl donor (8).

Detailed analysis of catechin glucosylation kinetics made it clear that the decrease in the catechin concentration during the reaction could not solely explain the observed decrease in GTF-D activity. We showed that fructose, which can be regarded as a side product of the transglucosylation reaction, competitively inhibits GTF-D. The observed decrease in GTF-D activity as the reaction proceeds is therefore most likely the result of a combination of the decrease in catechin concentration and fructose inhibition, although product inhibition by catechin glucosides cannot be ruled out.

To demonstrate and minimize the influence of fructose on catechin transglucosylation, the effect of fructose removal from the reaction mixture was studied. To achieve this, two yeasts, P. pastoris and S. cerevisiae T2-3D, incapable of sucrose consumption, were used. Addition of P. pastoris or S. cerevisiae to the catechin transglucosylation mixture resulted in a twofold increase of the duration of the maximal GTF-D activity, an increase of catechin glucoside production (maximum, 24%), and complete sucrose utilization (Fig. 4). The decrease in catechin glucoside formation rate after 500 min of incubation was most likely due to sucrose depletion and very low catechin concentrations. Interestingly, the removal of fructose and, hence, fructose inhibition by fermentation also resulted in complete sucrose utilization. As sucrose consumption by P. pastoris and S. cerevisiae was ruled out, the complete sucrose utilization by GTF-D is due to the removal of fructose.

The transglucosylation efficiency based on sucrose utilization by GTF-D can be very high. However, the use of yeasts to enhance the GTF-D transglucosylation rate resulted in drastically decreased efficiency of sucrose utilization. Therefore, depending on the optimization objective, yeasts should be added or omitted. It would be interesting to study whether dextran formation can also be stimulated by fructose removal, as has been shown here for catechin transglucosylation.