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Keywords:

  • Anoxybacillus flavithermus;
  • beta-xylosidase;
  • Sulfolobus solfataricus;
  • xylanase;
  • xylan hydrolysis;
  • xylose

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Aims:  It is evaluated the effectiveness of the combined action of two highly thermostable enzymes for the hydrolysis of xylans at high temperature in order to produce D-xylose.

Methods and Results:  Xylans from different sources were hydrolyzed at high degree at 70°C by co-action of a xylanase from the thermophilic bacterium Anoxybacillus flavithermus BC and the novel β-xylosidase/α-arabinosidase from the hyperthermophilic crenarchaeon Sulfolobus solfataricus Oα. Beechwood xylan was the best substrate among the xylans tested giving, by incubation only with xylanase, 32·8 % hydrolysis after 4 h. The addition of the β-xylosidase/α-arabinosidase significantly improved the rate of hydrolysis, yielding 63·6% conversion after 4 h incubation, and the main sugar identified was xylose.

Conclusions:  This study demonstrates that a significant degree of xylan degradation was reached at high temperature by co-action of the two enzymes. Xylose was obtained as a final product in considerable yield.

Significance and Impact of the Study:  Although the xylan represents the second most abundant polysaccharide in nature, it still doesn't have significant utilization for the difficulties encountered in its hydrolysis. Its successful hydrolysis to xylose in only one stage process could make of it a cheap sugar source and could have an enormous economic potential for the conversion of plant biomass into fuels and chemicals.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Xylan, the main component of hemicellulosic fraction of terrestrial plants, is composed by a backbone of β-1,4-linked xylopyranosyl units carrying acetyl, methylglucuronosyl and arabinofuranosyl side chains (Coughlan and Hazlewood 1993). Its complete hydrolysis is important in order to obtain, in high yields, monosaccharides like d-xylose and l-arabinose which could find applications in the food and fuel industries (Kim and Oh 2003; Ryabova et al. 2003). Because of its structural complexity, complete degradation of xylan requires, besides endo-xylanase, which plays the key role in the polysaccharide hydrolysis, several additional enzymes. In this context, glycosidases such as β-xylosidase and α-arabinosidase have a crucial role in order to achieve the full and rapid hydrolysis of branched xylans up to monosaccharides (Sunna and Antranikian 1996).

Xylanolytic enzymes from thermophilic micro-organisms have great potential at industrial level for their possible utilization for xylan digestion in processes at high temperatures.

In our previous work, we reported on the production of a thermostable multienzyme xylanase complex by a thermophilic alkali-tolerant Bacillus sp., strain BC, later classified by 16S rDNA analysis as Anoxybacillus flavithermus (Dimitrov et al. 1997), and on the isolation and characterization of a xylanase from the hyperthermophilic crenarchaeon Sulfolobus solfataricus Oα grown on oat spelt xylan as sole carbon source (Cannio et al. 2004). During xylanase purification from S. solfataricus, we detected a novel activity also involved in xylan degradation. Preliminary experiments, directed to discover the nature of the unknown activity, led to the identification of a glycosyl hydrolase possessing β-xylosidase and α-arabinosidase activities (unpublished data). The enzyme was mainly cytosolic but about 20% of its activity was localized in the cell membrane.

In order to obtain a satisfactory hydrolysis of xylan to xylose, we realized a bioconversion process, at high temperature, by the combined use of the multienzyme xylanase complex from A. flavithermus BC and the β-xylosidase/α-arabinosidase from S. solfataricus Oα.

Xylanase from S. solfataricus Oα and exoenzyme activity of the multienzyme xylanase complex from A. flavithermus BC were not taken into consideration as their activity levels were low for our trials. We performed experiments with different types of xylan for variable times of incubation, and compared the yields of xylan degradation obtained by individual or combined action of both enzymes.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Growth of the micro-organisms

To investigate the inducible character of xylanase synthesis, A. flavithermus BC was grown in PY medium (0·2% w/v peptone, 0·1% w/v yeast extract), pH 8·5 supplemented with different carbohydrates as a carbon source (0·2% w/v): oat spelt xylan, larchwood xylan, birchwood xylan, xylose, sorbose, Avicel, spelt bran, corn bran. Oat spelt xylan was chosen for further recovery of the enzyme. Cultivation was carried out in a rotary shaker at 60°C for 18 h in 100-ml conical flasks containing 15 ml of the medium. Cell-free culture supernatant was recovered after centrifugation at 6000 g for 30 min at 4°C.

Sulfolobus solfataricus strain Oα was grown in Brock's basal medium containing 0·2 % (w/v) oat spelt xylan as the sole carbon source as previously described (Cannio et al. 2004).

Preparation of the enzyme fractions

The cell-free supernatant from A. flavithermus BC was concentrated sixfold by using a Millipore ultrafiltration system (membrane cut-off 10 kDa), and the thermostability of the multienzyme xylanase complex was determined at 70°C in 25 mmol l−1 Tris-HCl buffer, pH 7·0.

β-xylosidase/α-arabinosidase from S. solfataricus Oα was purified from the cell membranes as follows: 25 g of wet cells grown on oat spelt xylan were suspended in 10 ml of 50 mmol l−1 Tris-HCl, pH 7·0 and ground in a mortar with 25 g of sand (50–150 mesh) for 1 h. After centrifugation at 2000 g for 10 min in order to remove sand and unbroken cells, the supernatant was ultracentrifuged at 55 000 g for 30 min. The pellet, containing membrane fragments, was suspended in 25 ml of 50 mmol l−1 Tris-HCl pH 7·0/0·5% Triton X-100 and incubated overnight at 70°C. The suspension was ultracentrifuged as reported earlir and the supernatant was extensively dialysed against 25 mmol l−1 Tris-HCl pH 7·0. After dialysis, the supernatant (33·5 ml), exhibiting xylanolytic activities, was indicated as TX-extract.

The TX-extract was concentrated c. 15-fold by using a Millipore ultrafiltration system (membrane cut-off 10 kDa) and then dialysed against 25 mmol l−1 Tris-HCl, 200 mmol l−1 NaCl, pH 8·4. β-xylosidase and α-arabinosidase activities were separated from the xylanase activity by AKTA Fast Protein Liquid Chromatography system (GE-Healthcare Bio-Sciences, Piscataway, NJ, USA) equipped with a Superdex 200 HR 10/30 column (Amersham Pharmacia Biotech). Fractionation was obtained at a flow rate of 0·5 ml min−1 by using 25 mmol l−1 Tris-HCl, 200 mmol l−1 NaCl buffer, pH 8·4 as eluent. Fractions containing the β-xylosidase/α-arabinosidase were pooled, dialysed against 25 mmol l−1 Tris-HCl, pH 7·0 and concentrated by ultrafiltration. These fractions were utilized for the xylan hydrolysis processes.

The thermostability of the enzyme was determined at 70 and 80°C in 25 mmol l−1 Tris-HCl buffer, pH 7·0 in sealed Eppendorf tubes with mineral oil overlaid to avoid evaporation in the absence of the substrate. The residual activity was measured at specific time intervals under the standard assay conditions.

Enzyme assays and protein estimation

Xylanase activity was measured determining the amount of reducing sugars released at 70°C from 1% birchwood xylan (w/v) in 25 mmol l−1 Tris-HCl buffer, pH 7·0. The appropriate amount of sixfold concentrated culture supernatant of A. flavithermus BC, diluted with Tris-HCl buffer, was mixed with 50 μl of 1% birchwood xylan in Tris-HCl buffer (final mixture volume: 100 μl). After 5 min of incubation, the reaction was stopped by cooling on ice and the reducing sugars liberated were revealed by the Somogyi-Nelson assay (Nelson 1944). Quantification of the sugars was performed by comparing their absorbance at 520 nm in a Cary ultraviolet (UV)-visible spectrophotometer (Varian) with that of a calibration curve ranging from 0·05 to 0·30 μmol of xylose. One enzyme unit was defined as the amount of enzyme which produces 1 μmol of d-xylose per minute at 70°C and pH 7·0.

β-xylosidase and α-arabinosidase activities were measured by using p-nitrophenyl-β-d-xylopyranoside (pNPXP) and p-nitrophenyl-α-l-arabinofuranoside (pNPAF) as the substrates, respectively. The appropriately diluted enzyme was added to 450 μl of substrate (2 mmol l−1pNPXP or pNPAF in 25 mmol l−1 Tris-HCl buffer, pH 7·0).The reaction mixture (final volume: 500 μl) was incubated at 70°C for a variable time ranging from 2 to 10 min and stopped by addition of Na2CO3 (1 ml, 1 mol l−1). The amount of released p-nitrophenol was measured at 405 nm. One enzyme unit was defined as the amount of enzyme releasing 1 μmol of p-nitrophenol per minute under the described conditions.

Protein concentration was determined as described by Bradford (1976) using the BioRad protein staining assay, and bovine albumine as the standard.

Electrophoresis and zymograms

Sodium dodecyl sulfate (SDS) polyacrylamide gel electropohoresis and zymogram for xylanase characterization were performed as previously described (Dimitrov et al. 1997).

Native polyacrylamide gel electrophoresis for β-xylosidase/α-arabinosidase was performed using 4% polyacrylamide stacking gel (upper buffer pH 7·5) and 5% polyacrylamide resolving gel (lower buffer pH 8·8) in a BioRad Mini Protean II cell unit. Zymograms were prepared by overlaying a 1% (w/v) agarose solution in 50 mmol l−1 Tris-HCl, pH 7·0 containing 4-methylumbelliferyl-7-β-d-xylopyranoside (40 μg ml−1) or 4-methylumbelliferyl-α-l-arabinofuranoside (20 μg ml−1) on the native polyacrylamide gel. After agarose solidification, the adhering gels were incubated for 30 min at room temperature. Fluorescent bands because of β-xylosidase and α-arabinosidase activities were observed on exposure to UV light and photographed using the Chemi Doc EQ System (BioRad, Hercules, CA, USA).

Degradation of xylans

Xylans from three different sources (birchwood, beechwood and oat spelt) were used separately for the realization of the xylans degradation process. Samples of the chosen xylan (10 mg ml−1) in 25 mmol l−1 Tris-HCl buffer, pH 7·0 were placed in an Eppendorf tubes in the presence of xylanase ultraconcentrate (0·2 U ml−1 incubation mixture) and β-xylosidase/α-arabinosidase (1·44 and 7·2 U ml−1 incubation mixture, respectively) either alone or in combination (final mixture volume: 2 ml). Control samples were prepared with xylan without enzymes.

Samples containing xylanase alone or both enzymes were incubated at 70°C in a thermostatic water bath for 2, 3 and 4 h. Samples with β-xylosidase/α-arabinosidase alone were incubated at the same temperature for 6 h. The hydrolysis reactions were stopped by cooling the incubation mixtures on ice and then centrifuging at 16 100 g for 15 min at 4°C to remove the unhydrolysed xylan. The amount of hydrolysis products released was estimated by the Somogyi-Nelson assay. The percentage of xylan hydrolysis was calculated by considering the quantity of reducing sugars produced in comparison with the total amount of xylan added in the incubation mixture. In addition, the cleared supernatants were qualitatively analysed as described in the following paragraphs.

High-performance anionic exchange liquid chromatography

A high-performance anionic exchange liquid chromatography system (Dionex, Sunnyvale, CA, USA), equipped with a pulsed electrochemical detector (PED) was used for identification of the products released in the incubation mixtures. Separation of the carbohydrates was achieved using a Carbopac PA-100 guard and analytical columns. The elution phase was composed of 160 mmol l−1 of NaOH (Buffer A) and 160 mmol l−1 of NaOH plus 300 mmol l−1 of CH3COONa (Buffer B). d-xylose, l-arabinose and xylo-oligosaccharides were eluted with the following gradient: t = 0 min 100% Buffer A; t = 8 min 100% Buffer A; t = 28 min 65% Buffer A; t = 38 min 65% Buffer A. Their retention times were compared with that of the following standards: xylose, arabinose, xylobiose, xylotriose, xylotetratose, xylopentaose and xylohexaose. Maltotriose was used as internal standard. The flow rate was 0·25 ml min−1.

Thin-layer chromatography

Thin-layer chromatography (TLC) was developed on precoated silica gel plates (60 F254, Merck) by using acetone/isopropyl alcohol/water (6 : 3 : 1·5 by volume) as eluent. Thirty microlitres of the clarified sample were loaded onto the TLC and the separated products were detected by spraying the plate with α-naphtol (3·5% w/v in 83% ethanol and 10% sulfuric acid) followed by heating at 150°C for 10 min.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Isolation and characteristics of the enzyme fractions

Anoxybacillus flavithermus BC secreted xylanase in the culture medium during its exponential phase of growth. The enzyme was strongly inducible and its synthesis was observed only in medium containing xylan (birchwood, 0·43 U ml−1; oat spelt, 0·33 U ml−1; larchwood, 0·19 U ml−1).

Other substrates containing β-1,4 bonds like spelt bran, corn bran and Avicel supported strain's growth but were not inducers of enzyme synthesis. The xylose, which is a final product of xylan degradation, also did not induce enzyme synthesis. Oat spelt was chosen for further work because of its large natural abundance. Zymogram analysis of the culture supernatant showed one main xylanase active band corresponding to proteins of about 92·0 kDa and one minor band at 80·0 kDa (Fig. 1a).

image

Figure 1.  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis of the multienzyme xylanase complex in supernatant ultraconcentrate from Anoxybacillus flavithermus BC. Lanes: M, molecular mass markers (phosphorylase B 92·5 kDa, bovine serum albumin 66·2 kDa, egg albumin 45·0 kDa, carbonic anhydrase 31·0 kDa); 1, supernatant ultraconcentrate; 2, zymogram (a). Zymogram analysis of TX-extract from Sulfolobus solfataricus Oα. Lanes: 1, α-arabinosidase activity; 2, β-xylosidase activity (b).

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The ultraconcentrate enzyme preparation exhibited high thermostability (its activity did not change after 40 h at 70°C) in comparison with many known thermophilic bacterial xylanases. In fact, the thermophilic xylanase from Bacillus sp. lost 11% of its activity after 2 h at 70°C (Cordeiro et al. 2002). The xylanase from Bacillus sp., strain SPS-0, had 4 h stability at 70°C (Bataillon et al. 2000), while the xylanase from Bacillus flavothermus, strain LB3A, was stable for 2 h at 70°C (Sunna et al. 1997). The high thermostability of the xylanase from A. flavithermus strain BC permitted its combination in joint action with the archaeal β-xylosidase/α-arabinosidase.

The presumed β-xylosidase/α-arabinosidase was detected in TX-extract in the course of our previous experiments directed to isolate and characterize the membrane-bound xylanase from S. solfataricus Oα. An open-reading frame encoding a β-xylosidase was identified on the genome sequence of S. solfataricus P2, and the predicted gene product showed high amino-acid sequence similarity with the β-xylosidase from Thermoanaerobacter ethanolicus, later characterized as a bi-functional β-xylosidase/α-arabinosidase (Shao and Wiegel 1992; Mai et al. 2000).

In order to establish the bi-functional nature of the enzyme, the TX-extract was fractionated by gel exclusion chromatography to separate the xylanolytic enzymes and β-xylosidase and α-arabinosidase activities were co-eluted showing the same elution profile. The enzyme was highly thermostable retaining 100% of both activities at 80°C within 24 h. Zymograms containing 4-methylumbelliferyl-7-β-d-xylopyranoside and 4-methylumbelliferyl-α-l-arabinofuranoside as substrates, were performed with the aim to study the bi-functional nature of the novel enzyme. The unique activity band observed on both gels, showing the same electrophoretic mobility, added a new element to support of the double activity of the enzyme (Fig. 1b).

Degradation of xylans by xylan-degrading enzymes

Different degrees of hydrolysis for the three xylans tested were registered after incubation at 70°C with xylanase from A. flavithermus BC. After 2 h of incubation, the highest conversion, 26·1%, was observed for beechwood xylan while birchwood and oat spelt gave 10·2% and 11·6% hydrolysis, respectively (Table 1). Incubation of beechwood and oat spelt with the xylanase from A. flavithermus BC was prolonged to 4 h in order to obtain a higher percentage of hydrolysis. Beechwood was chosen because of the highest yield of hydrolysis after 2 h and oat spelt was chosen for the presence of arabinose on the xylan backbone as side chain. As expected, beechwood degradation increased, reaching 32·8% hydrolysis, while oat spelt hydrolysis decreased, probably for a reverse enzymatic reaction.

Table 1.   Hydrolysis of xylans by xylanase from Anoxybacillus flavithermus BC and β-xylosidase/α-arabinosidase from Sulfolobus solfataricus Oα
Enzyme(s)Incubation time (h)Reducing sugars yield* (mg xylose equivalents ml−1)Xylan, hydrolysis (%)
  1. *Reducing sugars released in the incubation mixtures were estimated by the Somogyi-Nelson assay.

Birchwood
 Xylanase21·0210·2
 β-xylosidase/α-arabinosidase60·323·2
 Xylanase + β-xylosidase/α-arabinosidase22·1921·9
 Control20·0020·02
Beechwood
 Xylanase22·6126·1
32·8328·3
43·2832·8
 β-xylosidase/α-arabinosidase60·606·0
 xylanase + β-xylosidase/α-arabinosidase24·7947·9
35·5855·8
46·3663·6
 Control40·0060·06
Oat spelt
 Xylanase21·1611·6
30·636·3
40·606·0
 β-xylosidase/α-arabinosidase60·151·5
 xylanase + β-xylosidase/α-arabinosidase23·4834·8
32·5925·9
41·1211·2
 Control40·0020·02

Incubation of the xylans with β-xylosidase/α-arabinosidase from S. solfataricus Oα gave poor hydrolysis even after 6 h at 70°C; while in the beechwood xylan, the best substrate for the enzyme, only 6% hydrolysis was observed.

The addition of both enzymes to the substrates greatly enhanced sugar yield. Approximately twofold increase in the production of reducing sugars was obtained after 2 h of incubation of beechwood and birchwood xylans, indicating a cooperation of the enzymes in the substrate degradation. A similar result was observed after 3 and 4 h of incubation with beechwood xylan. Threefold increase in the yield of reducing sugars was obtained by incubating oat spelt xylan with the enzyme cocktail for 2 h, but prolonging the incubation time up to 4 h, the total reducing sugars diminished as was already observed when oat spelt xylan was incubated with xylanase alone. No hydrolysis of xylans in the respective control samples (xylans in the absence of enzymes) was observed.

Identification of the degradation products

Identification of the products released from the hydrolysis of xylans by the xylanolytic enzymes was obtained by high-performance anionic exchange liquid chromatography or, when needed, by TLC. Sugars up to six xylose units were detected after 2 h of incubation in the reaction mixtures treated by xylanase. Xylotetraose was the main xylo-oligosaccharide liberated from beechwood xylan (Fig. 2a), while xylotriose was the main reducing sugar present in the hydrolysis mixtures from birchwood and oat spelt xylans.

image

Figure 2.  High-performance liquid chromatography profiles of degradation products from beechwood xylan at 70°C by: xylanase from Anoxybacillus flavithermus BC after 2 h (a); β-xylosidase/α-arabinosidase from Sulfolobus solfataricus Oα after 6 h (b); xylanase and β-xylosidase/α-arabinosidase after 2 h (c). X, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose; X6, xylohexaose; M3, maltotriose.

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A detectable amount of xylose was obtained by the action of the β-xylosidase/α-arabinosidase on xylans only after 6 h of incubation and no xylo-oligosaccharides were released (Fig. 2b). When xylanase and β-xylosidase/α-arabinosidase acted together, xylose was already detected after 2 h of incubation as the main hydrolysis product of beechwood xylan with only negligible traces of xylobiose (Fig. 2c).

Unexpectedly, prolonging the incubation to 4 h, the reaction mixture containing oat spelt xylan and xylanase showed a decrease of the reducing sugars content suggesting a possible re-synthesis of xylo-oligosaccharides with a higher molecular weight. Our hypothesis was supported by the results obtained from high-performance liquid chromatography (HPLC) and TLC analyses. A reduction of xylose, xylobiose and xylotriose from 2 to 4 h and an increase of xylotetraose and xylopentaose were observed in the incubation mixture treated with xylanase alone. In addition, traces of xylohexaose, absent after 2 h of incubation, were detected (Fig. 3). A decrease of the reducing sugars was also measured after 4 h of incubation of oat spelt xylan with both enzymes, but the HPLC analysis of the reaction mixture revealed the exclusive presence of xylose. As only xylo-oligosaccharides containing six or less xylose units could be detected with the selected chromatographic parameters, we supposed that products with a higher degree of polymerization (DP) could be produced. The TLC analysis of the hydrolysis reaction revealed that the products at the bottom of the silica gel plate intensified their brown colour after 3 and 4 h of hydrolysis (Fig. 4, lanes 2 and 3) in comparison with the products obtained after 1 h of hydrolysis (Fig. 4, lane 1), indicating an increase of products with DP of 7 or more which did not migrate in the eluent used for the development of the TLC.

image

Figure 3.  High-performance liquid chromatography profiles of degradation products from oat spelt xylan at 70°C by xylanase from Anoxybacillus flavithermus BC after 2 h (dashed line) and 4 h (solid line). X, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose; X5, xylopentaose; X6, xylohexaose; M3, maltotriose.

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image

Figure 4.  Thin-layer chromatography showing degradation products from oat spelt xylan at 70°C by xylanase from Anoxybacillus flavithermus BC and β-xylosidase/α-arabinosidase from Sulfolobus solfataricus Oα after: 2 h (lane 1), 3 h (lane 2) and 4 h (lane 3). X, xylose.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

In general, the degradation of polysaccharides is a multistage process because of the action of different enzymes, often accompanied by changes in the operative conditions for the next enzyme. The use of enzymes with complementary specificity in one stage at the same conditions would significantly simplify the industrial processes of carbohydrate utilization. Great economic benefits in direct enzyme conversion of starch to glucose in a single step have been noticed (Pandey et al. 2000), and similar results could be expected in industrial degradation processes of other widely abundant carbohydrates like xylan.

The results obtained in the experiments we performed, unequivocally revealed the significant improvement of xylan hydrolysis that was attained by the combined action of endo-xylanase and β-xylosidase/α-arabinosidase.

Xylans degradation by xylanase from A. flavithermus BC alone gave a yield ranging from 10·2% for birchwood to 26·1% for beechwood. Xylotriose was the major oligosaccharide released from oat spelt and birchwood, while xylotetraose was the main hydrolysis product from beechwood. These data were in agreement with the observations of Liab et al. (2000) regarding a different behaviour of the xylanases with respect to the xylan structures.

Xylans hydrolysis by β-xylosidase/α-arabinosidase from S. solfataricus Oα produced only low amounts of xylose, demonstrating the exo-glycosyl hydrolytic nature of the enzyme. The yield of xylose obtained from the xylans used in our trials by the β-xylosidase/α-arabinosidase was very low even prolonging the incubation till 6 h, thus indicating that the enzyme takes part in the second step of the xylan hydrolysis.

The action of both enzymes on beechwood xylan resulted in an increased yield of the reducing sugars, confirming that the β-xylosidase/α-arabinosidase degraded the xylo-oligosaccharides released by the xylanase. Almost exclusively, xylose was detected in the hydrolysis mixture of beechwood, while little amount of arabinose was also found in oat spelt incubation mixture.

Examples of a synergic action of xylanolytic enzymes in order to obtain high degradation of xylans were described for Thermotoga maritima (Xue and Shao 2004) and Bacillus thermoantarcticus (Lama et al. 2004). Xylanase and β-xylosidase from T. maritima hydrolysed corncob xylan to xylose at 90°C and the xylanolytic enzymes from B. thermoantarcticus were able to convert birchwood xylan to xylose at 70°C with a yield of 70% after 24 h.

The combined action of the xylanase from A. flavithermus BC and the β-xylosidase/α-arabinosidase from S. solfataricus Oα seemed to be more effective in hydrolysing xylan, as 63·6 % of beechwood xylan was degraded after 4 h against 30% hydrolysis of birchwood xylan obtained by the xylanolytic enzymes from B. thermoantarcticus in the same time and at the same temperature. Almost complete hydrolysis of xylan to xylose was obtained by the enzymes from T. maritima at higher temperature but in a more prolonged time (12 h).

Our trials confirm that not only significant increase in xylan degradation can be achieved by the synergic action of the enzymes, but also that it is possible to obtain xylose with low or no impurities in considerable yield. Moreover, the high thermal stability displayed by the two enzymes, make them good candidates for industrial applications, such as conversion of lignocellulosic materials at high temperature in order to produce value-added products like fuels in one step. In fact, thermostability is an important prerequisite for industrial application of biocatalysts as their prolonged recycling in biotechnological processes allows enzyme cost reduction and efficient operative regimen especially in those biotransformations that require elevated temperatures (Bragger et al. 1989).

The increase of xylo-oligosaccharides with high DP observed during the hydrolysis of oat spelt xylan suggested the occurrence of both hydrolytic and transfer reactions. Several xylanases were described to possess transglycosylation activity (Christakopoulos et al. 1996; Jiang et al. 2004), and it was also established for the xylanase from A. flavithermus BC by our investigations. One possible reason for moving the reaction to the synthetic side could be the complex structure of the oat spelt xylan; its side chains could hinder the enzyme action on the xylan backbone, while the reverse reaction could be preferred for the presence of high amounts of small sugar molecules in the reaction mixture.

At this stage, we cannot exclude the participation of the β-xylosidase/α-arabinosidase in the reverse reaction, as some β-xylosidases, such as that from Aspergillus sp., were reported to possess transglycosylation activity (Eneyskaya et al. 2003). The optimization of the reaction conditions, with the aim to move the reaction towards the hydrolytic side for the improvement of sugar yield from oat spelt xylan, is currently in progress.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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  • Cannio, R., Di Prizito, N., Rossi, M. and Morana, A. (2004) A xylan-degrading strain of Sulfolobus solfataricus: isolation and characterization of the xylanase activity. Extremophiles 8, 117124.
  • Christakopoulos, P., Kekos, D., Macris, B.J., Claeyssens, M. and Bhat, M.K. (1996) Purification and characterisation of a major xylanase with cellulase and transferase activities from Fusarium oxysporum. Carbohydr Res 289, 91104.
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