INTRODUCTION

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Xanthan is an extracellular heteropolysaccharide produced by a plant-pathogenic bacterium, Xanthomonas campestris, and is composed of cellulosic backbone with linear trisaccharide side chains consisting of a mannosyl-glucuronyl-mannose sequence attached at the C-3 position on alternate glucosyl residues (16, 27). The internal and terminal mannosyl residues of the side chain are frequently acetylated and pyruvylated, respectively, depending on both the growth conditions and the bacterial strains (30). Xanthan has been widely used as a gelling and stabilizing agent in the food, pharmaceutical, and oil industries (29) because the polysaccharide shows superior rheological properties, such as pseudoplasticity, high viscosity at low concentration, and tolerance toward a wide range of pHs and temperatures (15, 19).

However, the polymer is considered to play a key role in the virulence of Xanthomonas bacterial cells against plants (5). Therefore, a biosynthetic pathway of xanthan in X. campestris has been well characterized, especially with respect to the relationship between pathogenicity and the polysaccharide (5, 18). A cluster of 12 genes has been suggested to be involved in the biosynthesis of xanthan (18). However, a depolymerization pathway of xanthan by organisms has not been elucidated. Although some microorganisms and their enzymes have been reported to participate in the depolymerization of the polysaccharide (2, 4, 22, 33, 34), enzymes responsible for the complete depolymerization of xanthan have not been identified.

In the course of a study on the assimilation of heteropolysaccharides by microbes, we have isolated a bacterium, Bacillus sp. strain GL1, that is able to depolymerize a bacterial heteropolysaccharide (gellan) (9, 12). Gellan is composed of polymerized tetrasaccharide repeating units [3)--D-Glcp-(14)--D-GlcAp-(14)--D-Glcp-(14)--L-Rhap-(1] (17, 26). Recently, Bacillus sp. strain GL1 cells have been also found to utilize xanthan for their growth, and xanthan lyase acting on the side chain of xanthan has been characterized (13). Subsequent to this study, we attempted to clarify the xanthan depolymerization pathway of the bacterium and to characterize one of the xanthan depolymerization enzymes, -D-glucanase, which hydrolyzes the main chain of xanthan.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods References

Materials. Pyruvylated xanthan (average molecular mass, 2 × 10⁶; pyruvylation of terminal mannosyl residue in the side chain, 50%) was a kind gift from The Kohjin Co., Tokyo, Japan. Modified xanthan was prepared as follows. Xanthan (0.5 g) was treated at 30°C for 90 h with 0.5 U of purified xanthan lyase, precipitated with ethanol to remove pyruvylated mannose, and dissolved in distilled water. Gellan (molecular mass, 5 × 10⁵; 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. Butyl- and DEAE-Toyopearl 650M were purchased from Tosoh Co., Tokyo, Japan, and DEAE-Sepharose CL-6B and Sephacryl S-200HR were from Pharmacia Biotech Co., Uppsala, Sweden. Bio-Gel P2 was from Bio-Rad Laboratories, Hercules, Calif. p-Nitrophenyl (pNP)-sugars, lichenan from Cetraria islandica, and -glucan from barley were from Sigma Chemical Co., St. Louis, Mo. Xylan from oat spelt was from Nacalai Tesque Co., Kyoto, Japan.

Microorganism and culture conditions. For the purification of -D-glucanase, Bacillus sp. strain GL1 cells were aerobically cultured at 30°C and at 100 rpm for 48 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). To investigate the activities of xanthan depolymerization enzymes, xanthan in the medium was replaced with gellan or pectin (0.5%).

DNA isolation and 16S rRNA analysis. A genomic DNA of Bacillus sp. strain GL1 was isolated as described previously (28). A 16S rRNA gene of the bacterium was amplified by PCR by using the genomic DNA as a template and 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGCTACCTTGTTACGACTT-3') as primers (21). The nucleotide sequence of the amplified 16S rDNA was determined by the dideoxy-chain termination method by using a model 377 automated DNA sequencer (Applied Biosystems Division of Perkin-Elmer, Foster City, Calif.) (31). Phylogenetic analysis was performed by using the FASTA and CLUSTALW programs on the DDBJ server (Shizuoka, Japan).

Assay for enzymes. -D-Glucanase was incubated at 30°C for 30 min in 0.5 ml of a reaction mixture containing 0.1% xanthan after treatment with xanthan lyase and 50 mM potassium phosphate buffer (KPB), pH 7.0, and the activity was determined by measuring the release of a reducing sugar according to the method of Lever (23). One unit of the enzyme activity was defined as the amount of enzyme required to release 1 µmol of the reducing sugar from the substrate per min. -D-Mannosidase and -D-glucosidase were assayed by using 0.4 mM pNP-sugars as substrates. One unit of the enzyme activity was defined as the amount of enzyme required to release 1 µmol of p-nitrophenol from the substrate per min. Unsaturated glucuronyl hydrolase was incubated at 30°C in 1 ml of a reaction mixture containing 0.005% tetrasaccharide from gellan by gellan lyase and 50 mM KPB, pH 7.0, and the activity was determined by monitoring the decrease of the A₂₃₅ arising from the double bond in the tetrasaccharide. One unit of the enzyme activity was defined as the amount of enzyme required to produce the decrease of 1.0 in the A₂₃₅ per min (10). The protein content was determined by the method of Lowry et al. (24), with bovine serum albumin as a standard, or by measuring the A₂₈₀, assuming that an E₂₈₀ of 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 (Insonator, Kubota model 201M; Kubota, Tokyo, Japan) 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.

TLC. Xanthan depolymerization products by extra- and intracellular enzymes were analyzed by TLC with a solvent system of 1-butanol-acetic acid-water (2:1:1 [vol/vol]). The products were visualized by heating the TLC plate at 110°C for 5 min after spraying it with 10% (vol/vol) sulfuric acid in ethanol.

Purification of xanthan depolymerization products. Xanthan depolymerization products were applied to a gel filtration of Bio-Gel P2 column (0.9 by 122 cm) previously equilibrated with distilled water. The products were eluted at room temperature with distilled water, and 0.8-ml fractions were collected every 7 min. The eluted products were detected by TLC.

Mass spectrometry. Each purified xanthan depolymerization product was analyzed by electrospray ionization-mass spectrometry (ESI-MS) by using an API III triple quadrupole mass spectrometer (Perkin-Elmer Sciex; Perkin-Elmer, Thornhill, Ontario, Canada) equipped with an atmospheric pressure ionization ion source. The mass spectrometer was operated in the positive or negative modes. The mass spectrum was scanned from 150 to 1,500 atomic mass units (amu) in 0.1-amu steps. Molecular weights were calculated on the basis of deconvolution mass spectra.

Purification of -D-glucanase. Unless otherwise specified, all operations were done at 0 to 4°C. Bacillus sp. strain GL1 cells were cultured at 30°C and 100 rpm for 48 h 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-Sepharose CL-6B column (4 by 22 cm) previously equilibrated with 20 mM KPB (pH 7.0). The enzyme was eluted with a linear gradient of NaCl (0 to 1 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, dialyzed against 20 mM KPB (pH 7.0), and applied to a DEAE-Toyopearl 650M column (4 by 8 cm) previously equilibrated with 20 mM KPB (pH 7.0). The enzyme was eluted with a linear gradient of NaCl (0 to 0.5 M) in 20 mM KPB (pH 7.0; 0.5 liters), and 4.2-ml fractions were collected every 3 min. The active fractions, which were eluted with 0.3 M NaCl, were combined and saturated with ammonium sulfate (30%), and then the enzyme solution was applied to a Butyl-Toyopearl 650M column (2.7 by 3.5 cm) previously equilibrated with 20 mM KPB (pH 7.0) and 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; 100 ml), and 0.83-ml fractions were collected every minute. The active fractions, which were eluted with 20 mM KPB (pH 7.0) and saturated with ammonium sulfate (10%), were combined, concentrated to about 3 ml by ultrafiltration with an Amicon model 8200 apparatus (Amicon Co., Beverly, Mass.) by using a membrane with a molecular-weight cutoff of >10,000, and applied to a Sephacryl S-200HR column (2.7 by 64 cm) previously equilibrated with 20 mM KPB (pH 7.0) and containing 0.15 M NaCl. The enzyme was eluted with the same buffer, and 3-ml fractions were collected every 6 min. The enzyme was eluted between fractions 51 and 58. These fractions were combined, dialyzed against 20 mM KPB (pH 7.0), and then used as the purified enzyme.

Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously (20).

Nucleotide sequence accession number. The nucleotide sequence for 16S rDNA reported here has been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. AB024598.

	RESULTS AND DISCUSSION

Phylogenetic analysis of Bacillus sp. strain GL1. Bacillus sp. strain GL1 isolated from a soil can utilize gellan and xanthan as a carbon source for its growth. The taxonomic properties of the bacterium have been described previously (12). To determine the phylogenetic position of the bacterium, the nucleotide sequence of 16S rRNA gene (1,514 bp) was determined. From the detailed taxonomical properties of Bacillus sp. strain GL1, the bacterium has been found to resemble Bacillus circulans (12). The gene of the 16S rRNA exhibited the highest identity score (91%) with that of Paenibacillus sp. (GenBank accession number AJ011322) and high similarity with those of Bacillus circulans (82%) (X60613) and Bacillus subtilis (82%) (Z99104). The phylogenetic gene tree which resulted from multiple-sequence analysis is shown in Fig. 1. The bacterium was most closely related to Brevibacillus thermoruber (Z26921) and was closer to Bacillus circulans (X60613) than to Paenibacillus sp. (AJ011322).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Phylogenetic tree based on the 16S rRNA sequences of Bacillus sp. strain GL1 and bacteria close to it. The accession numbers of bacterial 16S rRNA sequences are indicated.

Activities of xanthan depolymerization enzymes in Bacillus sp. strain GL1. Sufficient growth of Bacillus sp. strain GL1 was observed when cultured in the medium containing xanthan, gellan, or pectin as a sole carbon source, and the activities of possible xanthan depolymerization enzymes such as xanthan lyase, -D-glucanase, and exoglycosidases were determined (Table 1). Extracellular xanthan lyase (13) and -D-glucanase were produced only in the xanthan medium. The following three kinds of exoglycosidases are thought to be localized in cytosol because no signal sequence has been found in the deduced amino acid sequences from their nucleotide ones (10, 11, 25). -D-Mannosidase was specifically induced in the xanthan medium. -D-Glucosidase activity was found in all of the media tested. Unsaturated glucuronyl hydrolase, which catalyzes the hydrolysis of unsaturated oligosaccharides produced by polysaccharide lyases, was induced in the presence of gellan or xanthan (10).

Enzymatic route for xanthan depolymerization in Bacillus sp. strain GL1. When xanthan was incubated with the extracellular enzyme source of Bacillus sp. strain GL1 cells grown in the xanthan medium, three major products with R_f values from a maximum of 0.44 (P1) to 0.26 (P2) and 0.19 (P3), along with a minor product (0.14 [P4]), were observed as the final products on the TLC plate (Fig. 2). Xanthan depolymerization products produced by enzymes in the extracellular fraction were analyzed by ESI-MS (Fig. 3). The molecular weights of three major products (P1 to P3) were determined to be 250 (P1), 662 (P2), and 704 (P3), which were calculated from m/z 249, 661, and 703 ions corresponding to the deprotonated ions [M-H] in the negative mode of ESI-MS (Fig. 3), respectively, judging from the determination of the molecular weight of each purified product by ESI-MS. The product P1 was confirmed to be a pyruvylated mannose first liberated from xanthan by xanthan lyase (13). Based on the ESI-MS and information on the known structure of xanthan, the product P2 was identified as a tetrasaccharide consisting of an unsaturated glucuronic acid, an acetylated mannose, and two glucose residues. The product P3 is thought to be a deacetylated form of P2. Therefore, the products P2 and P3 were considered to be released by -D-glucanase acting on the main chain of xanthan. The product P4 was found to be a pentasaccharide, a repeating unit of xanthan, for the following two reasons. (i) The purified product (P4) was degraded to tetrasaccharides (P2 and P3) and pyruvylated mannose (P1) by purified xanthan lyase (data not shown). (ii) The minor peaks around m/z 900 in the mass spectrum (Fig. 3) are thought to be derived from pentasaccharides with or without acetylation and/or pyruvation. For example, molecular weights of 954 and 884 from m/z 953 and 883 ions corresponding to the deprotonated ions coincided with those of pentasaccharides with or without pyruvation. These results indicate that in Bacillus sp. strain GL1, extracellular xanthan lyase attacks xanthan side chains, and then extracellular -D-glucanase hydrolyzes the linkages of the main chain to give the tetrasaccharide. However, the formation of pentasaccharide was observed after incubation of xanthan with extracellular enzyme fraction, thus indicating that -D-glucanase can partly act on xanthan prior to xanthan lyase.

View larger version (65K): [in this window] [in a new window]   FIG. 2.   Degradation of xanthan by extra- and intracellular enzymes of Bacillus sp. strain GL1. Lane 1, xanthan; lane 2, xanthan depolymerization products by extracellular enzymes. The degradation of the mixture of P2 and P3 (tetrasaccharides) by intracellular enzymes of Bacillus sp. strain GL1 is also shown. The tetrasaccharides were incubated at 30°C with intracellular enzymes. Samples of 10 µl were periodically removed from the reaction mixture and analyzed by TLC. Lane 3, tetrasaccharide (mixture of P2 and P3). The reaction times were as follows: lane 4, 0 min; lane 5, 40 min; lane 6, 2 h; and lane 7, 12 h. Lanes Glc, Man, and GlcA represent authentic D-glucose, D-mannose, and D-glucuronic acid, respectively.

View larger version (31K): [in this window] [in a new window]   FIG. 3.   ESI-mass spectrum of xanthan depolymerization products by extracellular enzyme source of Bacillus sp. strain GL1.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Xanthan depolymerization pathway in Bacillus sp. strain GL1. The cleavage sites of xanthan depolymerization enzymes are indicated by thick arrows. Thin arrows show the pathway of xanthan depolymerization. The deacetylase possibly releases the acetyl group from acetylated mannose during the transformation of tri- to disaccharide.

Purification and properties of -D-glucanase. -D-Glucanase involved in the hydrolysis of the main chain of xanthan was purified 332-fold with a recovery of 9.6% from the culture fluid (Table 2). The purified enzyme was homogeneous on an SDS-PAGE gel (Fig. 5).

View this table: [in this window] [in a new window]   TABLE 2.   Purification of -D-glucanase

View larger version (38K): [in this window] [in a new window]   FIG. 5.   Electrophoretic profile of -D-glucanase. The purified -D-glucanase was subjected to SDS-PAGE. Lane 1, molecular size standards (from top): myosin (200 kDa), -galactosidase (116 kDa), bovine serum albumin (66 kDa), aldolase (42 kDa), carbonic anhydrase (30 kDa), and myoglobin (17 kDa); lane 2, purified -D-glucanase. The arrow to the right indicates the position of -D-glucanase.

(i) Molecular mass. The molecular mass of the enzyme was determined to be 355 kDa by gel permeation chromatography (Sephacryl S-200HR) (data not shown) and 173 kDa when determined by SDS-PAGE with a gel at a low concentration of 6%, indicating that the enzyme was a homodimer.

(ii) pH and temperature. The enzyme was most active at pH 6.0 in sodium acetate buffer and at 45°C (Fig. 6A and B). The enzyme was stable below 40°C (Fig. 6C). About 30% of activity was lost after incubation at 40°C and pH 6.0 for 10 min (Fig. 6C).

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Effect of pH and temperature on the activity and stability of -D-glucanase. Experiments were carried out at 30°C with 0.05% xanthan after treatment with xanthan lyase and purified -D-glucanase. (A) Effect of pH. Reactions were performed at 30°C for 30 min in the following 50 mM buffers: sodium acetate (), potassium phosphate (), sodium-HEPES (), Tris-HCl (), and glycine-NaOH (). Activity at pH 6.0 in sodium acetate buffer was relatively taken as 100%. (B) Optimal temperature. Reactions were performed for 30 min at various temperatures in 50 mM sodium acetate buffer (pH 6.0). The activity at 45°C was relatively taken as 100%. (C) Thermal stability. After preincubation of the enzyme for 10 min at various temperatures, the residual activity was measured at 30°C for 30 min in 50 mM sodium acetate buffer (pH 6.0). The activity of the enzyme preincubated at 4°C was taken as 100%.

(iii) Metal ions and others. The reaction was performed in the presence or absence of various compounds, and the residual activity was measured. Cu²⁺ inhibited the reaction by ca. 80% at 1 mM. Other divalent metal ions (Ca²⁺, Co²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, and Zn²⁺) had little effect on the enzyme activity at 1 mM. Thiol reagents (dithiothreitol, glutathione [reduced form], 2-mercaptoethanol, iodoacetamide, and N-ethylmaleimide) and a chelator (EDTA) (1 mM each) revealed no significant effect on the enzyme activity.

(iv) Substrate specificity. To examine the substrate specificity of -D-glucanase, the enzyme was incubated at 30°C for 30 min in a mixture containing 50 mM sodium acetate buffer (pH 6.0) and various substrates (0.1%). The enzyme was specific to xanthan after treatment with xanthan lyase and liberated tetrasaccharide when analyzed by TLC. Native xanthan, carboxymethyl cellulose, and -glucan were slightly degraded (Table 3). After prolonged reaction of the enzyme in the presence of native xanthan, the enzyme was confirmed to produce a small amount of the pentasaccharide (P4) but not tetrasaccharides (P2 and P3) by TLC analysis (data not shown). This result supports the appearance of a small amount of pentasaccharide in xanthan depolymerization products produced by extracellular enzymes.

View this table: [in this window] [in a new window]   TABLE 3.   Substrate specificity of -D-glucanase