INTRODUCTION

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Xanthan is an exopolysaccharide produced by the plant pathogenic bacterium Xanthomonas campestris and consists of a main cellulosic chain with trisaccharide side chains, each of which is composed of a glucuronyl and two mannosyl residues attached at the C-3 position on an alternate glucosyl residue (11, 16) (Fig. 1). Each of the internal and terminal mannosyl residues of the side chain has an O-acetyl group at the C-6 position and a pyruvate ketal at the C-4 and C-6 positions, respectively, depending on the growth conditions and bacterial strains (18).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Structure of xanthan. An arrow indicates the cleavage site for the xanthan lyase analyzed in this article. Glc, glucose; GlcA, glucuronic acid; Man, mannose.

Xanthan has peculiar rheological properties of pseudoplasticity, high viscosity at low concentration, and tolerance for a wide range of pHs and temperatures (10, 12) and is widely utilized as a gelling and stabilizing agent in the food and pharmaceutical industries (17).

Two types of xanthan-degrading enzymes are known to exist in microbes. One is xanthanase (endo-1,4--D-glucanase) catalyzing the hydrolysis of the main chain of xanthan. A number of xanthanases have been identified, and some of them are categorized as cellulase family members (9, 20). The other type is a xanthan lyase (4,5-transeliminase), which cleaves the linkage between the terminal mannosyl and glucuronyl residues of the side chain of xanthan (1, 21). Although xanthanase has been well documented, xanthan lyase has rarely been characterized. The lyase-type enzyme acting on the side chain of xanthan is unique because, as seen for the lyases for alginate, pectin, and chondroitin, almost all the polysaccharide lyases so far elucidated can act on the main chains of polysaccharides. Although xanthan variants produced by mutant cells of X. campestris have been well studied (7, 19), the characterization of the lyase-treated xanthan with an unsaturated uronic acid at the terminals of the side chains has seldom been reported (1). Therefore, the preparation of the modified xanthan by treatment with the lyase may result in new applications of xanthan in biopolymer-based industries.

We have isolated the bacterium Bacillus sp. strain GL1, which degrades gellan, a polysaccharide in the sphingan family, and have shown that gellan lyase is one of the enzymes involved in the degradation of gellan (4, 6). Recently, Bacillus sp. strain GL1 cells were found to utilize xanthan for their growth and thereby produced gellan lyase in addition to xanthan lyase. Although Kennedy and Sutherland (13) have reported that the bacterium producing xanthan lyase is able to grow in the gellan medium, the involvement of gellan lyase in gellan degradation is still controversial. In order to clarify the difference between the two lyases and to investigate the possibility of preparing modified xanthan having novel rheological properties, we attempted to purify and characterize xanthan lyase produced by Bacillus sp. strain GL1.

	MATERIALS AND METHODS

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Materials. Pyruvylated xanthan (average molecular mass, 2 × 10⁶ Da; percent pyruvylation of the terminal mannosyl residue in the side chain, 50%) was a kind gift from Kohjin Co., Tokyo, Japan. Gellan (molecular mass, 5 × 10⁵ Da; deacetylated) and pectin were purchased from Wako Pure Chemicals Co., Osaka, Japan. Silica gel 60/Kieselguhr F254 thin-layer chromatography (TLC) plates were obtained from E. Merck, Darmstadt, Germany. DEAE-cellulose and sodium alginate were purchased from Nacalai Tesque Co., Kyoto, Japan; butyl-Toyopearl 650M was purchased from Tosoh Co., Tokyo, Japan; and Sephacryl S-200HR, Sephadex G-15, and QAE-Sephadex A-25 were purchased from Pharmacia Biotech. Co., Uppsala, Sweden. Fucoidan was from Sigma Chemical Co., St. Louis, Mo.

Microorganisms and culture conditions. For the production of xanthan lyase, Bacillus sp. strain GL1 was aerobically cultured at 30°C and 100 rpm for 36 h in a liquid xanthan medium consisting of 0.1% (NH₄)₂SO₄, 0.1% KH₂PO₄, 0.1% Na₂HPO₄, 0.01% MgSO₄ · 7H₂O, 0.01% yeast extract, and 0.5% xanthan (pH 7.2). For the production of gellan lyase, xanthan was replaced with gellan (0.5%).

Assay for enzymes. Xanthan lyase was incubated in 1 ml of the reaction mixture containing 0.05% xanthan and 50 mM potassium phosphate buffer (KPB), pH 7.0, and the activity was determined by monitoring the increase of the absorbance at 235 nm arising from the double bond in the reaction product. One unit of enzyme activity was defined as the amount of enzyme required to produce an increase of 1.0 in the absorbance at 235 nm per min. Gellan lyase was assayed as described previously (4). Protein content was determined by the method of Lowry et al. (15), with bovine serum albumin as the standard, or by measuring the absorbance at 280 nm assuming that E₂₈₀ = 1.0 corresponds to 1 mg/ml.

Preparation of extra- and intracellular fractions. Cells grown at 30°C for 48 h were harvested by centrifugation at 13,000 × g at 4°C for 10 min, and the resulting culture fluid was used as an extracellular enzyme source. Collected cells were washed in 20 mM KPB, pH 7.0, and then resuspended in the same buffer. The cells were ultrasonically disrupted at 0°C and 9 kHz for 5 min, and the clear solution obtained after centrifugation at 13,000 × g at 4°C for 20 min was dialyzed against 20 mM KPB, pH 7.0, overnight. The dialysate was used as an intracellular enzyme source.

Viscosity of xanthan. The viscosity of xanthan was determined at 25°C with a concentric cylinder viscometer (Rotovisco RV-11; Haake Co., Karlsruhe, Germany) as described previously (4).

Purification of xanthan lyase. Unless otherwise specified, all operations were done at 0 to 4°C. Bacillus sp. strain GL1 was cultured for 70 h at 30°C in 10 liters of xanthan medium (1 liter/flask). After cultivation, the cells were removed by centrifugation at 13,000 × g at 4°C for 10 min. The fluid (crude enzyme solution; 8.4 liters) was applied to a DEAE-cellulose column (4.7 by 41 cm) previously equilibrated with 20 mM KPB, pH 7.0. The enzyme was eluted with a linear gradient of NaCl (0 to 0.7 M) in 20 mM KPB, pH 7.0 (2 liters), and 18-ml fractions were collected every 9 min. The active fractions, which were eluted with 0.5 M NaCl, were combined and fractionated with ammonium sulfate, and the precipitate (50 to 90% saturation) was collected by centrifugation at 13,000 × g at 4°C for 15 min. The enzyme was dissolved in 2 ml of 20 mM KPB, pH 7.0, and applied to a Sephacryl S-200HR column (2.7 by 64 cm) previously equilibrated with 20 mM KPB, pH 7.0, containing 0.15 M NaCl. The enzyme was eluted with the same buffer, and 3-ml fractions were collected every 3 min. The enzyme eluted between fractions 63 and 74; these fractions were combined and saturated with ammonium sulfate (30%), and then the enzyme solution (38 ml) was applied to a butyl-Toyopearl 650M column (2.7 by 17 cm) previously equilibrated with 20 mM KPB, pH 7.0, saturated with ammonium sulfate (30%). The enzyme was eluted with a linear gradient of ammonium sulfate (30 to 0%) in 20 mM KPB, pH 7.0 (500 ml). Four-milliliter fractions were collected every 3 min. The active fractions, which were eluted with 20 mM KPB, pH 7.0, saturated with ammonium sulfate (9%), were combined and dialyzed against 20 mM KPB, pH 7.0, and then the enzyme solution (19 ml) was applied to a QAE-Sephadex A-25 column (0.9 by 20 cm) previously equilibrated with 20 mM KPB, pH 7.0. The enzyme was eluted with a linear gradient of NaCl (0 to 0.3 M) in 20 mM KPB, pH 7.0 (100 ml), and 0.8-ml fractions were collected every 3 min. The active fractions, which were eluted with 0.24 M NaCl, were combined and used as the purified enzyme.

Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE were performed as described previously (3, 14).

TLC. Products of the degradation of xanthan by xanthan lyase were analyzed by TLC with a solvent system of 1-butanol-acetic acid-water (3:2:2 [vol/vol]) as described previously (8). The products were visualized by heating the plate at 110°C for 5 min after spraying it with 10% (vol/vol) sulfuric acid in ethanol.

Preparation of the xanthan derivative. The removal of pyruvyl or acetyl groups from xanthan was performed as described by Bradshaw et al. (2).

Purification of the xanthan degradation product. The product was separated from xanthan and xanthan lyase by ultrafiltration using Ultrafree C3LGC (Japan Millipore Co., Tokyo, Japan) and then subjected to gel filtration with a Sephadex G-15 column (1.0 by 50 cm) equilibrated with distilled water. The product was eluted with distilled water, and an 0.67-ml fraction was collected every 3 min.

Mass spectrometry. The purified product was analyzed by electrospray ionization-mass spectrometry (ESI-MS) using an API III triple-quadrupole mass spectrometer (Perkin-Elmer Sciex, Thornhill, Ontario, Canada) equipped with an atmospheric pressure ionization ion source. The mass spectrometer was operated in the negative mode. The ion spray voltage was 4,000 V, and the orifice voltage was 30 to 50 V. During the analysis, the mass spectrum was scanned from 150 to 400 atomic mass units (AMU) in 0.1-AMU steps. Molecular weights were calculated on the basis of deconvolution mass spectra.

Acid hydrolysis and pyruvate assay. The product was hydrolyzed in 2.5 M trifluoroacetic acid at 100°C for 6 h. After hydrolysis, trifluoroacetic acid was evaporated under vacuum. The resultant product was analyzed by TLC and pyruvate assay. The product after acid hydrolysis was incubated in 50 mM KPB, pH 7.0, containing 0.1 mM NADH and 7 mU of lactate dehydrogenase per ml. Pyruvate was determined by measuring the decrease in absorbance at 340 nm.

	RESULTS AND DISCUSSION

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Assimilation of xanthan by Bacillus sp. strain GL1. When Bacillus sp. strain GL1 cells were cultivated in the medium containing xanthan as the sole carbon source, the viscosity of the culture decreased with increasing cell growth and reached the level of water when cell growth approached a plateau (Fig. 2). No change in viscosity was observed in the medium without cells. These results indicated that xanthan was depolymerized by certain degrading enzymes and that the products were utilized for cell growth. The culture fluids were analyzed by TLC. Several products with low molecular masses were found in the culture fluid (Fig. 3), although such products were not detected in the fluid without cells. The indicated product (Fig. 3, arrow) seems to be pyruvylated mannose, as described below. The bacterium grown in the presence of xanthan produced both xanthan and gellan lyases extracellularly. On the other hand, in the presence of gellan, the bacterium produced only gellan lyase, thus suggesting that xanthan can affect promoters for both xanthan and gellan lyases, although the structures of the two biopolymers are quite different. We have already cloned the gellan lyase gene from Bacillus sp. strain GL1 (5), and we are analyzing the expression of the lyase gene in the presence of xanthan and other biopolymers.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Effect of cell growth on the viscosity of xanthan. The culture was performed in 200 ml of 0.5% xanthan medium at 30°C. The same medium without cells was used as a control. For the measurement of cell growth (absorbance [OD] at 600 nm) and viscosity, samples of 10 ml were periodically removed from both media with (open symbols) and without (solid symbols) cells. , , absorbance at 600 nm; , , viscosity of the medium.

View larger version (73K): [in this window] [in a new window]   FIG. 3.   Degradation of xanthan by Bacillus sp. strain GL1. The culture was performed in 200 ml of 0.5% xanthan medium at 30°C. Samples of 10 µl were periodically (see lane identifications) removed from the medium and analyzed by TLC. Lane 1, 0 h; lane 2, 16 h; lane 3, 28 h; lane 4, 60 h. Lanes 5, 6, and 7 represent authentic D-glucose, D-mannose, and D-glucuronic acid, respectively. The product indicated by the arrow was later found to be pyruvylated mannose.

Purification and properties of xanthan lyase. Xanthan lyase was purified 46.9-fold with a recovery of 0.85% from the culture fluid (Table 1). The low level of recovery was presumably due to the overestimation of the enzyme in a crude solution containing xanthan-degrading products. The purified enzyme was homogeneous as determined by both SDS-PAGE and native PAGE (Fig. 4).

View larger version (46K): [in this window] [in a new window]   FIG. 4.   Electrophoresis profile of xanthan lyase. The purified xanthan lyase (5 µg) was subjected to SDS-PAGE (A) and native PAGE (B). Proteins were stained with Coomassie brilliant blue R-250. (A) Lane 1, purified xanthan lyase, lane 2, molecular size standards (from the top) myosin (200 kDa), -galactosidase (116 kDa), bovine serum albumin (66 kDa), aldolase (42 kDa), carbonic anhydrase (30 kDa), and myoglobin (17 kDa). Arrows indicate xanthan lyase.

(i) Molecular mass. The enzyme was a monomer with a molecular mass of 75 kDa as determined by SDS-PAGE (Fig. 4A) and by gel permeation chromatography (Sephacryl S-200HR) (data not shown).

(ii) pH and temperature. The enzyme was most active at pH 5.5 in sodium acetate buffer and at 50°C. The enzyme activity was inhibited by KPB, and this inhibition is thought to be caused by potassium ions, because the enzyme activity was not affected by sodium phosphate buffer (data not shown). The enzyme was stable between pH 6.5 and 9 at 4°C, and below 37°C. About 60% activity was lost after incubation at 45°C, pH 7.0, for 10 min.

(iii) Metal ions and others. The reaction was performed in the presence or absence of various compounds, and residual activity was measured (Table 2). Co²⁺ slightly enhanced the activity of the enzyme at 1 mM. Hg²⁺ almost completely inhibited the reaction at 1 mM. Other divalent metal ions had no effect on the enzyme activity at 1 mM. The activity of the enzyme was intensely inhibited by NaCl and KCl at 150 mM. This suggests that Na⁺ and K⁺ are responsible for this inhibition since, as seen from the effects of various metals, Cl had no appreciable effects on enzyme activity. Dithiothreitol, glutathione (reduced form), and 2-mercaptoethanol (1 mM each) had no significant effect on enzyme activity. On the other hand, iodoacetamide and N-ethylmaleimide partially inhibited the reaction at 1 mM, suggesting the participation of the thiol moiety in the lyase reaction.

(iv) Substrate specificity. To examine the substrate specificity of xanthan lyase, the lyase was incubated at 30°C for 10 min in a mixture containing 50 mM sodium acetate buffer (pH 5.5) and various substrates (0.05%). The enzyme was highly specific for xanthan, and other polysaccharides such as alginate, fucoidan, gellan, pectin, and gellan-related polysaccharides (S-88, welan, rhamsan, and S-198; gifts from T. J. Pollock, Shin-Etsu Bio, Inc., San Diego, Calif.) were inert as substrates. The effects of the pyruvyl and acetyl groups in xanthan on enzyme activity were investigated. The enzyme appeared to be specific for pyruvylated xanthan, because the activity of the enzyme toward native xanthan (100%) was much higher than that toward modified xanthan without pyruvate residues (69%). Furthermore, the enzyme released only pyruvylated mannose, not mannose from half-pyruvylated xanthan as described below. The observed activity with the modified xanthan was due to the pyruvylated mannose remaining after the removal of pyruvyl group. On the other hand, the presence or absence of the acetyl group in xanthan had no effects on enzyme activity.

Product of the xanthan lyase reaction. The reaction product of the xanthan lyase reaction was analyzed by TLC. The formation of only one reaction product was confirmed by TLC, and the product migrated just between the positions for mannose and glucose (Fig. 5A). The brown color of the product was similar to that of mannose produced by TLC. The purified product had a molecular weight of 250, calculated from the m/z-249 ion corresponding to deprotonated ion [M-H] in the negative mode of ESI-MS (Fig. 5B). The value matched the theoretical one for pyruvylated mannose. The purified product was subjected to acid hydrolysis. The hydrolysate contained a sugar with the same mobility as mannose, as determined by TLC, and pyruvate, as determined with lactate dehydrogenase (data not shown). These results indicate that xanthan lyase acts on the linkage between the terminal mannosyl and glucuronyl residues of the side chains of xanthan and releases the pyruvylated mannose.

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Degradation of xanthan by xanthan lyase. (A) Reaction was performed at 30°C in 1 ml of mixture containing 1.3% xanthan, 50 mM KPB (pH 7.0) and xanthan lyase (5 mU). An aliquot (5 µl) of the reaction mixture was analyzed by TLC at the times indicated below. Lane 1, 0 h; lane 2, 2 h; lane 3, 4 h; lane 4, 6 h; lane 5, 8 h. Lanes 6, 7, and 8 represent authentic D-glucose, D-mannose, and D-glucuronic acid, respectively. (B) ESI-MS of the xanthan-degrading product produced by xanthan lyase.