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Applied and Environmental Microbiology, April 2006, p. 2483-2490, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2483-2490.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Paenibacillus curdlanolyticus Strain B-6 Xylanolytic-Cellulolytic Enzyme System That Degrades Insoluble Polysaccharides

Patthra Pason, Khin Lay Kyu,* and Khanok Ratanakhanokchai

School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok, Thailand

Received 3 April 2005/ Accepted 21 January 2006


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ABSTRACT
 
A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, ß-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, ß-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.


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INTRODUCTION
 
Many cellulolytic microorganisms and their cellulase systems have been studied extensively for hydrolysis of naturally abundant lignocellulosic substances to valuable products such as fermentable sugars, chemicals, and liquid fuel (7, 13). Cellulolytic microorganisms produce cellulolytic systems consisting of various kinds of cellulases that are different for different species. In nature, cellulose and hemicelluloses are associated with plant cell walls, and microorganisms synthesize a number of different enzymes in order to grow on them since they exist as complex substrates. The following three types of enzymes are in the cellulase system: endoglucanases (endo-1,4-ß-glucanases, or 1,4-ß-glucan 4-glucanohydrolases [EC 3.2.1.4]), exoglucanases, including cellobiohydrolase (exo-1,4-ß-glucanases, or 1,4-ß-glucan cellobiohydrolases [EC 3.2.1.91]), and ß-glucosidases (ß-D-glucoside glucohydrolases [EC 3.2.1.21]). The endoglucanases cleave at random at internal amorphous sites in the cellulose glucan chains, and the exoglucanases act processively to release cellobiose primarily from the chain ends (47, 48). Exoglucanases can also act on microcrystalline cellulose (Avicel), presumably peeling cellulose chains from the microcrystalline structure (47). The compositions and structures of xylan vary according to the source. Xylanolytic enzymes such as endo-1,4-ß-xylanase (1,4-ß-D-xylan xylanohydrolase [EC 3.2.1.8]) and ß-xylosidase (EC 3.2.1.37) that cleave the backbone chain and {alpha}-L-arabinofuranosidase (EC 3.2.1.55), acetyl esterase (EC 3.1.1.6), and {alpha}-D-glucuronidase (EC 3.2.1.1) that cleave the side chain of xylan are needed to hydrolyze xylan completely (12). Multiple cellulolytic and xylanolytic enzymes are thus required to efficiently hydrolyze cellulose and xylan, which are present in lignocellulosic substances (43). Moreover, a tight interaction between the enzymes and their substrates and the cooperation of multiple enzymes are also required to enhance hydrolysis due to the complex structures of their substrates (6, 28). Various anaerobic bacteria have been reported to produce cellulases and xylanases containing cellulose-binding modules and/or xylan-binding modules which are associated with a discrete, high-molecular-weight cellulolytic or xylanolytic enzyme complex known as the cellulosome (1, 4, 8, 14, 16, 23, 41) or xylanosome (21, 31). Thus, multiple forms of cellulases and xylanases are organized into such complexes, which are dedicated to hydrolyzing lignocellulosic substances because of their ability to bind to insoluble cellulose and/or xylan via cellulose-binding modules and xylan-binding modules, respectively (4). The carbohydrate binding modules (CBMs) effect binding to the cellulose surface, presumably to facilitate cellulose hydrolysis by bringing the catalytic domain in close proximity to the substrate, insoluble cellulose (33). Thus, the arrangement of cell wall-degrading enzymes into a multienzyme complex has advantages over single-enzyme systems (45). In contrast, aerobic bacteria produce numerous individual extracellular cellulolytic enzymes with binding modules (42, 43). However, aerobic bacteria and fungi which do not adhere to cellulose produce noncomplex cellulases (33).

Previous work in our laboratory showed that the facultatively anaerobic bacterium Paenibacillus curdlanolyticus B-6 (formerly Bacillus circulans B-6), isolated from an anaerobic digester fed with pineapple wastes, is a true cellulolytic/xylanolytic organism, as it could grow on xylan, microcrystalline cellulose (49), {alpha}-cellulose, or agricultural wastes as a sole source of carbon under aerobic conditions and produced a multixylanase and -cellulase enzyme system (unpublished data). Moreover, it was also found that the bacterium could grow on xylan under anaerobic conditions and could produce a multixylanase and -cellulase enzyme system (unpublished data), but the patterns of catalytic proteins produced in cultures varied depending on the carbon source (unpublished data). Cellulolytic rumen bacteria can degrade xylan but mostly cannot grow on that substrate. P. curdlanolyticus B-6 was found to be able to adhere to xylan or cellulose during growth on xylan or cellulose under aerobic conditions. Therefore, it was of interest to undertake a study on the xylanase-cellulase enzyme system produced by P. curdlanolyticus B-6 under aerobic conditions.

In this paper, we describe the adhesion of the facultatively anaerobic bacterium Paenibacillus curdlanolyticus B-6 grown on xylan under aerobic conditions to insoluble xylan and show that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.


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MATERIALS AND METHODS
 
Bacterial strain.
The bacterial strain B-6 used in this study was isolated from an anaerobic digester fed with pineapple wastes.

Bacterial identification.
Morphological properties and taxonomic characteristics of the bacterium were studied according to the methods in Bergey's Manual of Systematic Bacteriology (46). The bacterial strain B-6 was a facultatively anaerobic, spore-forming, gram-positive, motile, rod-shaped organism and produced catalase. Thus, this bacterium was identified as belonging to the genus Bacillus according to Bergey's Manual of Systematic Bacteriology (46). The bacterium was also identified by 16S rRNA gene sequence analysis (19). The use of a specific PCR primer designed for differentiating the genus Paenibacillus from other members of the Bacillaceae showed that this strain had the same amplified 16S rRNA gene fragment as a member of the genus Paenibacillus. Based on this observation, this strain was transferred to the genus Paenibacillus (44). The 16S rRNA sequence of this strain has 1,424 bp and 97% similarity with Paenibacillus curdlanolyticus. Therefore, it was identified as Paenibacillus curdlanolyticus B-6 (formerly Bacillus circulans B-6) and is on deposit with the BIOTEC Culture Collection, the National Center for Genetic Engineering and Biotechnology (BIOTEC) Thailand, where the accession number is BCC no. 11175.

Culture medium.
The bacterium was grown in Berg's mineral salts medium, pH 7.0 (10), which contains 0.2% NaNO3, 0.05% K2HPO4, 0.02% MgSO4 · 7H2O, 0.002% MnSO4 · H2O, 0.002% FeSO4 · 7H2O, 0.002% CaCl2 · 2H2O, and 0.5% commercial oat spelt xylan (Sigma-Aldrich Chemical Co.), consisting of 60% insoluble xylan. The culture was incubated in a rotary incubator at 200 rpm and 37°C.

SEM.
The surfaces of the cells grown on xylan (referred to hereafter as xylan-grown cells), harvested at the late exponential growth phase and at the early decline phase, were analyzed by scanning electron microscopy (SEM) (3). The cell samples were filtered through a Nuclepore membrane filter (0.6-µm pore size), dehydrated by a series of graded ethanol solutions, and critical point dried with liquid CO2. The samples were then coated with gold and examined with a JEOL JSM-35 scanning electron microscope.

Preparation of insoluble xylan.
To prepare insoluble fractions of commercial oat spelt xylan, 1 gram of xylan was suspended in 20 ml of water, and the suspension was brought to pH 10 with 1 N NaOH and stirred gently at room temperature for 1 h. The xylan was centrifuged at 3,000 x g for 5 min and washed twice with water and then with 50 mM sodium acetate buffer (pH 5.5). The pellet was then suspended in 10 ml of 95% ethanol and filtered on Whatman no. 1 paper. The pellet was dried in a desiccator and ground as finely as possible with a mortar and pestle (20).

Adhesion of bacterial cells to insoluble substances.
The adhesion of P. curdlanolyticus B-6 cells to insoluble oat spelt xylan was performed during growth on xylan (41). The cells were harvested by centrifugation at 8,000 x g for 7 min at the appropriate time of growth. Cell suspensions were collected and washed three times with phosphate-buffered saline (PBS) containing 0.15 M sodium chloride and 100 mM potassium phosphate (pH 7.0). Each washed cell suspension was adjusted with PBS to an optical density at 400 nm of 0.2 and then brought to a total volume of 3 ml with 1 ml of 20% insoluble xylan and 1 ml of PBS. The suspension was mixed for 40 seconds, and insoluble xylan containing adhered bacterial cells was allowed to settle at room temperature for 30 min. The turbidity of the suspension was measured at 400 nm and compared with that of an identical cell suspension wherein PBS was substituted for the insoluble xylan suspension.

Cellulose- and xylan-binding assay.
The binding assay was conducted by adding 0.23 mg protein from the culture supernatant to 50 mg of insoluble xylan in 1.0 ml of 100 mM phosphate buffer (pH 6.0) in 1.5-ml Eppendorf tubes. Samples were shaken at intervals at 4°C for 30 min before centrifugation. The amount of activity remaining in the supernatant was determined by the standard xylanase assay method. The activity lost from the supernatant was assumed to be the bound activity (20). The cellulose-binding assay was also conducted according to the above procedure, using Avicel (Fluka).

Enzyme assays.
Xylanase, endoglucanase (carboxymethyl cellulase [CMCase]), ß-xylosidase, and arabinofuranosidase activities were determined as described previously (42). {alpha}-Amylase, mannanase, avicelase, and chitinase were assayed under the same conditions as those described above, using soluble starch (BDH), mannan (locust bean gum) (Sigma-Aldrich), Avicel, and chitin (Sigma-Aldrich) as the substrates, respectively. The concentration of reducing sugars was determined by the Somogyi-Nelson method (38). One unit of enzyme activity was defined as the amount of enzyme that liberated 1 µmol of reducing sugars per minute under the above conditions. ß-Glucosidase was determined under the same conditions as the ß-xylosidase assay, using p-nitrophenyl glucopyranoside as a substrate. Acetyl esterase activity was determined as described by Mackenzie et al. (34). Cellobiohydrolase activity was determined by the method of Kohring et al. (25). ß-Xylosidase, ß-glucosidase, arabinofuranosidase, cellobiohydrolase, and acetyl esterase activities were expressed as µmol of p-nitrophenol released per minute per milliliter of enzyme solution. Protein concentrations were measured as described by Lowry et al. (32). The protein content of the eluate from the gel filtration chromatography column was measured at 280 nm.

Isolation of multienzyme complex.
To separate the multienzyme complex from the noncomplexed enzymes, the multienzyme complex was isolated by affinity purification on cellulose. After P. curdlanolyticus B-6 was grown on xylan for 3 days, the culture was harvested by centrifugation, and the crude enzyme preparation (300 ml) was concentrated (10-fold) by using a rapid-flow filtration capsule with a 10-kDa-cutoff membrane (Minimate TFF capsule with 100ka Omega). To collect the cellulose-binding proteins, which are mainly in the multienzyme complex, the crude enzyme preparation, consisting of 229 mg protein and 20 g microcrystalline cellulose (Avicel) in 50 ml of 250 mM PBS (pH 7.0), was shaken periodically at 4°C for 30 min. After centrifugation, the cellulose-binding proteins were washed four times with 250 mM phosphate-buffered saline (pH 7.0) and then eluted with 1% triethylamine. The eluate was dialyzed and freeze-dried before being subjected to gel filtration chromatography on a Sephacryl S-300 column.

Gel filtration chromatography.
The cellulose-binding proteins were subjected to a Sephacryl S-300 high-resolution column (0.9 x 50 cm) which was equilibrated with 50 mM phosphate buffer (pH 6.0) containing 150 mM NaCl at 4°C and were eluted with the same buffer at a flow rate of 30 ml h–1. The elution of the standard proteins thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa) (high-molecular-mass standards; Amersham Pharmacia Biotech) was conducted in the same manner.

Gel electrophoresis analysis and zymograms.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 7% polyacrylamide gel by the method of Laemmli (26). After electrophoresis, the proteins were stained with Coomassie brilliant blue R-250. The molecular weight standards used were from a high-molecular-weight calibration kit (Pierce). Xylanase and CMCase zymograms were prepared from SDS-7% polyacrylamide gels containing 0.1% soluble xylan or 0.1% CMC, as described previously (42).

Hydrolysis of insoluble polysaccharides.
The insoluble lignocellulosic substance of corn hulls was ground (40-mesh) and washed several times in warm distilled water to remove the remaining free sugars. Insoluble birchwood xylan (Sigma-Aldrich), Avicel, and corn hulls (2% dry weight [each]) were incubated at 50°C with 0.2 U of the crude enzyme preparation, using 100 mM phosphate buffer, pH 6.0. At the appropriate time, the hydrolysis products were taken, and the amounts of reducing sugars released were determined by the Somogyi-Nelson method (38).

Analysis of hydrolysis products.
Analysis of hydrolysis products by high-performance liquid chromatography (HPLC) was performed using a refractive index detector. The column used for separation was a Shodex Ionpak KS-800 P column packed with strong cation-exchange resin gels. The HPLC instrument was operated at 45°C by using water at a flow rate of 0.5 ml/min as the mobile phase. Additional conditions conformed to the instructions of the manufacturer.


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RESULTS
 
Production of xylanolytic-cellulolytic enzyme system by P. curdlanolyticus B-6.
During growth of P. curdlanolyticus B-6 on xylan, the protein concentration in the medium was low up to the late stationary growth phase, although the cell mass was high (Fig. 1A). The cell adhesion test showed that the bacterial cells adhered to xylan from the exponential growth phase to the late stationary growth phase (Fig. 1A and B). The adhesion of cells to xylan reached the maximum at the late exponential growth phase and then rapidly decreased after the late stationary growth phase, while the amount of extracellular xylanase rapidly increased due to the release of enzymes from the cells into the culture medium. CMCase and xylanase activities could be detected in the culture medium after 24 and 36 h, respectively (Fig. 1B). At 30 h, the amount of CMCase was higher than that of xylanase; however, after 42 h, the amount of xylanase dramatically increased and proteins were increasingly released as well. Xylanase production correlated with the production of protein (Fig. 1A and B). The reducing sugars in the culture medium accumulated in very small amounts up to the late stationary growth phase, indicating that nearly all of the released sugar was consumed by the bacteria that adhered to xylan. However, after that phase, reducing sugars increased due to the continued action of released enzyme activities in the culture medium containing both soluble and insoluble xylan. After the bacterium was cultivated on xylan for 72 h, enzyme activities were detected in the culture medium, and the culture was harvested by centrifugation. The crude enzyme preparation consisted of xylanolytic-cellulolytic enzymes, mannanase, chitinase, and amylase, as shown in Table 1, indicating that the preparation was capable of hydrolyzing various substrates.


Figure 1
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  		FIG. 1. Cell growth and production of protein by P. curdlanolyticus B-6 cultivated on xylan (A) and production of xylanase, CMCase, and reducing sugar and adhesion of bacterial cells to xylan in the culture medium of P. curdlanolyticus B-6 grown on xylan (B).


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  		TABLE 1. Enzymatic activities of crude enzyme preparation and cellulose-binding proteins of P. curdlanolyticus B-6

Hydrolysis of insoluble polysaccharides.
The activity of the crude enzyme preparation was found to reach the maximum within the temperature range of 50 to 60°C and was stable at temperatures up to 60°C (data not shown). The reaction exhibited broad pH optima and stability in the pH range of 4 to 9 (data not shown). Since the multienzyme complex, or cellulosome, is responsible for degrading insoluble substances efficiently (1, 4, 28, 45), it is considered an important factor in the efficient degradation of insoluble cellulosic materials (39). In this study, to test the ability of the bacterium to degrade insoluble polysaccharides, the initial hydrolysis of insoluble polysaccharides with low enzyme activity was conducted. The crude enzyme preparation was incubated with insoluble birchwood xylan, Avicel (microcrystalline cellulose), and corn hulls at 50°C and pH 6.0. Since cellulose hydrolysis requires prior binding of enzymes to cellulose, the substrate-binding ability of the crude enzyme preparation was determined. In the reaction mixtures, the crude enzyme preparation bound to the substrates xylan, corn hulls, and Avicel at 57, 18, and 30%, respectively. Cellulases are known to differ in their mode of action (endo- or exo-) and also in their way of binding to the surface of the substrate (43). With short incubation times, the crude enzyme preparation showed the ability to degrade all insoluble polysaccharides tested, as shown in Fig. 2. The hydrolysis products were identified by HPLC. The hydrolysis products of insoluble birchwood xylan were xylobiose and xylose (Fig. 3A) and those of corn hulls were cellobiose, xylobiose, glucose, and xylose (Fig. 3C), while the end hydrolysis product of Avicel was glucose, within 20 min of incubation (Fig. 3B). Moreover, the enzyme preparation could hydrolyze cellulose and xylan present in lignocellulosic substances such as corn hulls to soluble sugars, exclusively glucose and xylose. As shown in Table 1, the crude enzyme preparation consisted of cellobiohydrolase, which is essential for the hydrolysis of microcrystalline cellulose (33), and avicelase activities in addition to CMCase or endoglucanase and ß-glucosidase activities. Xylanase, ß-xylosidase, and debranching enzymes such as arabinofuranosidase and acetyl esterase were also detected in the crude enzyme preparation. These results indicate that the xylanolytic-cellulolytic enzyme system produced by P. curdlanolyticus B-6 is capable of hydrolyzing not only pure insoluble polysaccharides, but also xylan and cellulose present in lignocellulosic substances such as corn hulls. The hydrolytic activities on other polysaccharides such as mannan, chitin, and starch were rather low, although manannase, chitinase, and amylase were present in the crude enzyme preparation.


Figure 2
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  		FIG. 2. Initial hydrolysis of insoluble polysaccharides by xylanolytic-cellulolytic enzyme system produced by P. curdlanolyticus B-6.


Figure 3
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  		FIG. 3. HPLC analysis of hydrolysis products released from the insoluble polysaccharides insoluble birchwood xylan (A), Avicel (B), and corn hulls (C) at 0 min and 20 min.

Adhesion of cells to xylan.
To determine the adhesion of xylan-grown cells to insoluble xylan, cells harvested at the late exponential growth phase and at the decline phase were analyzed by SEM. Figure 4A shows that the surfaces of xylan-grown cells harvested at the late exponential growth phase were coated with xylan, indicating the adhesion of cells to xylan, similar to the case of cellulosomal cells (29). However, the xylan-grown cells harvested at the early decline phase showed no adhesion to xylan (Fig. 4B).


Figure 4
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  		FIG. 4. SEM of cell surface structure of a P. curdlanolyticus B-6 cell harvested at the late exponential growth phase and coated with xylan, indicating the adhesion of the cell to xylan (A), and a cell harvested at the early decline phase, with the lack of xylan showing no adhesion of the cell to xylan (B).

Interaction of xylanase-cellulase enzyme system with insoluble polysaccharides.
Since the xylan-grown cells were found to be able to adhere to insoluble xylan, the xylanolytic-cellulolytic enzyme system in those cells must be responsible for the adhesion of the bacterium to the insoluble substrate. Thus, the binding of the crude enzyme preparation of P. curdlanolyticus B-6 to insoluble xylan and Avicel was tested. The crude enzyme preparation was found to be able to bind to both insoluble xylan and Avicel at 77% and 63%, respectively (data not shown). The crude enzyme preparation exhibited a higher affinity for insoluble xylan than for Avicel. This reflects the available exposed sites and surface areas of the substrates. The adsorption of enzymes to insoluble substrates plays an important role in the efficiency of the enzymatic hydrolysis of the substrates (11). This result shows that CBMs are possibly present either with the complexes or with single enzymes.

Isolation of multienzyme complex.
The cellulose-binding proteins, which are mainly in the multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. The crude enzyme preparation was incubated with Avicel for 30 min to allow the multienzyme complex present in the crude enzyme preparation to bind to the crystalline cellulose. The cellulose-binding proteins were separated from the unbound proteins of the crude enzyme preparation by centrifugation, eluted from the cellulose, and analyzed by SDS-PAGE and zymograms. As shown in Table 1, although the crude enzyme preparation consisted of 11 enzymatic activities, only 8 enzymatic activities were detected in the cellulose-binding protein preparation, showing that mannanase, chitinase, and amylase were unbound proteins. The concentrated cellulose-binding proteins were subjected to gel filtration chromatography on a Sephacryl S-300 column (Fig. 5). Two major fractions containing both xylanase and CMCase activities were isolated. The first peak (fractions 13 to 19) eluted closely after the void volume, and the molecular mass of the complex was estimated to be 1,450 kDa; the second peak (fractions 22 to 30) had a molecular mass of 400 kDa. These peaks formed the multienzyme complexes. The third peak (fractions 31 to 40), with only xylanase activity, was estimated to be 160 kDa, and thus it seems that this peak contained single enzymes with CBMs that could be provided from the two peaks with high-molecular-weight multienzyme complexes being dissociated during growth. The fourth peak (fractions 40 to 45) with both activities was estimated to be 40 kDa. Peak IV may contain single enzymes with substrate-binding modules and could also be made up of fragments from the high-molecular-weight multienzyme complexes. Zverlov et al. (52) reported a detailed analysis of all cellulosomal genes showing that the majority of enzymatically active cellulosomal components seemed to be well above 50 kDa and that smaller proteins found after protein preparation were obviously degradation products.


Figure 5
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  		FIG. 5. Gel filtration chromatography on Sephacryl S-300 column of the crude enzyme preparation of P. curdlanolyticus B-6 grown on xylan.

SDS-PAGE analysis and zymograms of protein components.
The protein compositions of the crude enzyme preparation and the cellulose-binding proteins were analyzed by SDS-PAGE analysis and zymograms, using CMC and soluble xylan as substrates to determine the enzyme activities of the catalytic components. When subjected to SDS-PAGE, the crude enzyme preparation showed at least 17 proteins with molecular masses in the range of 224 to 48 kDa (Fig. 6A, lane 1), of which 12 proteins had xylanase activity (Fig. 6B, lane 1) and 9 proteins had CMCase activity (Fig. 6C, lane 1) on zymograms. Among them, eight high-molecular-mass proteins, of 216, 201, 187, 157, 127, 102, 80, and 63 kDa, showed both xylanase and cellulase activities. However, the cellulose-binding proteins comprising the multienzyme complex showed at least nine proteins by SDS-PAGE (Fig. 6A, lane 2), with eight of these proteins showing xylanase activity (Fig. 6B, lane 2) and six showing CMCase activity (Fig. 6C, lane 2) on zymograms. The six CMCases also showed xylanase activity on zymograms. These multiple forms of xylanases and cellulases may act synergistically during the hydrolysis of insoluble polysaccharides.


Figure 6
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  		FIG. 6. SDS-PAGE (A) and zymogram analysis of xylanase activity (B) and CMCase activity (C) of the crude enzyme preparation (lanes 1) and cellulose-binding proteins (lanes 2). All samples contained 100 µg of protein. Molecular masses, in kilodaltons, are indicated on the right.

SDS-PAGE and zymograms for xylanase and CMCase activities of the four peaks from the gel filtration column are shown in Fig. 7A, B, and C. Multienzyme complex I (peak I) and complex II (peak II) showed eight and seven proteins by SDS-PAGE (Fig. 7A, lanes 1 and 2), with seven and six proteins showing xylanase activity (Fig. 7B, lanes 1 and 2) and five and three proteins showing CMCase activity (Fig. 7C, lanes 1 and 2), respectively, on zymograms. Peak III contained three proteins (Fig. 7A, lane 3), with all three showing xylanase activity (Fig. 7B, lane 3) and none showing CMCase activity (Fig. 7C, lane 3), while peak IV had two proteins (Fig. 7A, lane 4), with both showing xylanase activity (Fig. 7B, lane 4) and one showing CMCase activity (Fig. 7C, lane 4).


Figure 7
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  		FIG. 7. SDS-PAGE (A) and zymogram analysis of xylanase activity (B) and CMCase activity (C) in peaks from gel filtration column. Lanes 1, peak I; lanes 2, peak II; lanes 3, peak III; lanes 4, peak IV. All samples contained 100 µg of protein. Molecular masses, in kilodaltons, are indicated on the right.


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DISCUSSION
 
In this study, the xylanolytic-cellulolytic enzyme system of P. curdlanolyticus B-6 was found to be associated with cells from the early exponential growth phase to the late stationary growth phase. This result was similar to those for Clostridium thermocellum and other anaerobic bacteria (3, 17, 28). It has been reported that in the early exponential growth phase, the multienzyme complex, or cellulosome, is intimately associated with the cell surface (5, 27). The structural localization of the cellulosome on the cell surface of the anaerobic bacterium C. thermocellum has been extensively studied by SEM, and it was reported that cellulosome-producing bacterial cells adhered to the substrate due to the presence of cellulose-binding modules within the cellulosome complex (5) which are anchored to the cell at the scaffoldin protein (45). P. curdlanolyticus B-6 cells were found to adhere to xylan, but the mechanism for adherence is unknown. The results for this bacterium supported the hypothesis that the xylanolytic-cellulolytic enzymes were organized as a complex and were cell associated. It has been reported that the multienzyme complex can effectively degrade crystalline cellulosic substrates and associated plant cell wall polysaccharides (37). The crude enzyme preparation from P. curdlanolyticus B-6 exhibits activity over a broad range of pHs and has good stability across temperatures and pHs. It is capable of degrading crystalline cellulose, insoluble xylan, and agricultural waste (corn hulls) to their end product monomers within the initial period of incubation due to the ability of the enzymes to bind insoluble xylan and cellulose. Thus, the xylanolytic-cellulolytic enzyme system contains the enzymes required for complete hydrolysis of these insoluble polysaccharides, and the enzymes have activity. The hydrolysis of insoluble cellulosic substances requires the actions of multiple cellulases and xylanases with different modes of action (50). Due to the presence of cellulose-binding ability, some microorganisms, such as the cellulosome-producing strains C. thermocellum and Clostridium cellulovorans, hydrolyze lignocellulosic substances efficiently (2, 35, 37). In the multienzyme complexes produced by anaerobic bacteria, catalytic subunits such as endoglucanases, exoglucanase, and xylanases are held together into a huge complex, which serves to promote their synergistic action (1, 28).

Many investigators have reported that the components of the cellulase systems of C. thermocellum and other anaerobic bacteria are organized as a discrete complex, the cellulosome, which has been shown to be a multiprotein complex containing catalytic and noncatalytic polypeptide subunits (8, 14, 18, 30, 36, 51). In this study, the multienzyme complex produced by P. curdlanolyticus B-6 showed a molecular mass of >1,400 kDa. This mass is similar to that of the cellulosome of C. thermocellum (2,100 kDa) (3) and larger than those of other cellulosomes, e.g., 600 kDa for Clostridium cellulolyticum (9), 900 kDa for C. cellulovorans (15), 700 kDa for Clostridium josui (22), and 600 kDa for Clostridium papyrosolvens (40). The molecular masses of the two major cellulase complexes of Bacillus circulans F-2 grown on Avicel were reported to be about 669 and 443 kDa (24). The xylanolytic complex of Streptomyces olivaceoviridis E-86 was reported to exhibit a very high molecular mass of approximately 1,200 kDa (21). The extracellular complex CB, a xylanosome, was reported for Butyrivibrio fibrisolvens H17c (31). It has a molecular mass of >669 kDa and is composed of 11 proteins with xylanase activity and 3 proteins with endoglucanase activity. SDS-PAGE analysis indicated that the multienzyme complex produced by P. curdlanolyticus B-6 consists of at least eight polypeptides and that seven of them are active toward xylan and five are active toward CMC (Fig. 7A, B, and C, lanes 1).

P. curdlanolyticus B-6 possesses two multienzyme complexes, of 1,450 and 400 kDa, composed of multienzyme subunits. The results obtained in this study show that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes. This is the first report on xylanolytic-cellulolytic enzyme complexes produced by P. curdlanolyticus B-6 under aerobic conditions. Currently, we are working on the cloning and analysis of catalytic and noncatalytic genes of the multienzyme complexes of P. curdlanolyticus B-6.


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ACKNOWLEDGMENTS
 
This work was supported by a National Center for Genetic Engineering and Biotechnology (BIOTEC) grant and the Royal Golden Jubilee Ph.D. program of the Thailand Research Fund.


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FOOTNOTES
 
* Corresponding author. Mailing address: School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand. Phone: 662 470 7753. Fax: 662 452 3479. E-mail: khin.kyu{at}kmutt.ac.th. Back


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Applied and Environmental Microbiology, April 2006, p. 2483-2490, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2483-2490.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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