Simultaneous production of endo-β-1,4-xylanase and branched xylooligosaccharides by Thermomyces lanuginosus

Journal of Biotechnology 137 (2008) 34–43

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Simultaneous production of endo-␤-1,4-xylanase and branched xylooligosaccharides by Thermomyces lanuginosus Vladimír Puchart, Peter Biely ∗ Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava 45, Slovak Republic

a r t i c l e

i n f o

Article history: Received 6 May 2008 Received in revised form 25 June 2008 Accepted 7 July 2008 Keywords: endo-␤-1,4-Xylanase Glycoside hydrolase family 11 Thermomyces lanuginosus Aldopentaouronic acid Arabinoxylooligosaccharides

a b s t r a c t When grown on beech-wood glucuronoxylan, two strains of the thermophilic fungus Thermomyces lanuginosius, IMI 84400 and IMI 96213, secreted endo-␤-1,4-xylanase of glycoside hydrolase family 11 and simultaneously accumulated an acidic pentasaccharide in the medium. The aldopentaouronic acid was purified and its structure was established by a combination of NMR spectroscopy and enzyme digestion with glycosidases as MeGlcA3 Xyl4 . Both strains showed limited growth on wheat arabinoxylan as a carbon source. An essential part of the polysaccharide was not utilized, and it was converted to a series of arabinoxylooligosaccharides differing in the degree of polymerization. The structure of the shorter arabinoxylooligosaccharides remaining in the wheat arabinoxylan-spent medium was established using mass spectrometry and digestion with glycosidases. Xylose and linear ␤-1,4-xylooligosaccharides generated extracellularly during growth on either hardwood or cereal xylan were efficiently taken up by the cells and metabolized intracellularly. The data suggest that due to a lack of extracellular ␤-xylosidase, ␣-glucuronidase, and ␣-l-arabinofuranosidase, the widely used T. lanuginosus strains might become efficient producers of branched xylooligosaccharides from both types of xylans. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Microorganisms produce a set of enzymes for decomposition of plant xylan, the second most abundant polysaccharide in nature. A crucial enzyme for xylan depolymerization is endo-␤-1,4xylanase (E.C. 3.2.1.8), which acts in the regions of low substitution. The debranching of the polymer and its fragments is performed by the concerted action of the so-called auxiliary enzymes—␣glucuronidases (E.C. 3.2.1.139), acetylxylan esterases (E.C. 3.1.1.72), feruloyl esterases (E.C. 3.1.1.73) and ␣-l-arabinofuranosidases (E.C. 3.2.1.55). The resulting ␤-1,4-xylooligosaccharides serve as substrates for ␤-xylosidases (E.C. 3.2.1.37), which act from the nonreducing end. Deacetylated glucuronoxylans are efficiently hydrolyzed by xylanases, affording a mixture of linear and branched xylooligosaccharides carrying usually a single 4-O-methyl-␣-d-glucuronic acid

Abbreviations: AnArf, Aspergillus niger ␣-l-arabinofuranosidase; BspArf, Bifidobacterium sp. ␣-l-arabinofuranosidase; GH, glycoside hydrolase; MeGlcA, 4O-methyl-d-glucuronic acid or 4-O-methyl-␣-d-glucopyranuronosyl; MeGlcA3 Xyl4 , ␤-d-xylopyranosyl-1,4-[2-O-(4-O-methyl-␣-d-glucopyranuronosyl)]-␤-dxylopyranosyl-1,4-␤-d-xylopyranosyl-d-xylopyranose; XlnD, Aspergillus niger ␤-xylosidase heterologously expressed and secreted by Saccharomyces cerevisiae. ∗ Corresponding author. Tel.: +421 2 59410275; fax: +421 2 59410222. E-mail address: [email protected] (P. Biely). 0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2008.07.1789

residue. Their decomposition requires ␣-glucuronidase. With one exception (Tenkanen and Siika-aho, 2000), all ␣-glucuronidases described thus far belong to glycoside hydrolase family 67 (Coutinho and Henrissat, 1999), which act solely on aldouronic acids in which the uronic acid is linked to the non-reducing terminal xylosyl residue (Nurizzo et al., 2002; Vrˇsanská et al., 2007). l-Arabinose side chains strongly impede the xylanase-catalyzed depolymerization of arabinoxylans. The fragments released by xylanase represent usually a complex mixture of arabinoxylooligosaccharides. Their xylosyl residues may be non-substituted, singly substituted at position 2 or 3, or disubstituted at both positions. Studies of mode of action of the ␣-l-arabinofuranosidases on arabinoxylooligosaccharides suggest that the enzymes belonging to glycoside hydrolase family 51 and 54 act on both types of monoarabinosylated xylosyl residues (Sørensen et al., 2006; de Wet et al., 2008). In contrast, all enzymes active on doubly arabinosylated residues belong to glycoside hydrolase family 43 (van den Broek et al., 2005; Sørensen et al., 2006). These enzymes remove only the arabinose residues linked to position 3. The second arabinosyl residues linked to position 2 can be removed by ␣-l-arabinofuranosidases of GH51 or GH54. The distinct substrate specificities of GH43 versus GH51 or GH54 ␣l-arabinofuranosidases was exploited for preparation of singly and doubly substituted arabinoxylan (Sørensen et al., 2006).

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In nature, microorganisms in a given habitat usually cooperate in the utilization of a complex carbon source. As a result individual microorganisms may not possess all of the enzymes needed for complete plant cell wall degradation. This seems to be the case in the widely studied fungus Thermomyces lanuginosus. This efficient producer of a cellulase-free xylanase previously was shown to leave some fragments of glucuronoxylan in the medium (Puchart et al., 1999). These fragments have been isolated and identified in this work. We also report here that the fungus utilizes neither branched xylooligosaccharides formed during growth on arabinoxylan. The arabinose-substituted xylooligosaccharides were also isolated and their tentative structure determined by a combination of mass spectrometry and hydrolysis with three different glycosidases. Thus an example of a simultaneous production of endoxylanase and oligosaccharides is presented. 2. Materials and methods 2.1. Chemicals Partially debranched beechwood xylan was donated by Dr. G. Gamerith (Lenzing AG, Lenzing, Austria). Deacetylated beechwood 4-O-methyl-␣-d-glucurono-␤-1,4-d-xylan was prepared from beech sawdust by alkaline extraction as described by Ebringerová et al. (1967). The glucuronoxylan hydrolyzate, containing a mixture of singly substituted aldouronic acids was generated by an application of Erwinia chrysanthemi xylanase A as reported recently (Vrˇsanská et al., 2007). 4-O-Methyl-d-glucuronic acid (MeGlcA) was prepared by enzymatic deesterification of its ˇ methyl ester according to Spániková et al. (2007). Corn fiber xylan was provided by the late Dr. R.B. Hespell (Agricultural Research Service, USDA, Peoria, IL, USA). Wheat arabinoxylan of medium viscosity and linear ␤-1,4-xylooligosaccharides (xylobiose through xylohexaose) were purchased from Megazyme (Bray, Ireland). Arabinoxylobiose (␣-l-arabinofuranosyl-1,3␤-d-xylopyranosyl-1,4-d-xylopyranose) and arabinoxylotriose [␤-d-xylopyranosyl-1,4-(2-O-␣-l-arabinofuranosyl)-␤-dxylopyranosyl-1,4-d-xylopyranose] were generous gifts of Dr. S. Kaneko (National Food Research Institute, Tsukuba, Japan). 2.2. Enzymes Aspergillus niger ␤-xylosidase XlnD (GH3) was heterologously expressed in Saccharomyces cerevisiae as reported (Biely et al., 2000a). E. chrysanthemi xylanase A (GH5) was a kind gift of Prof. James F. Preston (University of Florida, Gainesville, FL, USA). Aspergillus tubingensis ␣-glucuronidase (GH67) was obtained from Drs. Jaap Visser and Ronald P. de Vries (Wageningen Agricultural University, Wageningen, The Netherlands). ␣-l-Arabinofuranosidases from Aspergillus niger (AnArf) and Bifidobacterium sp. (BspArf) were obtained from Megazyme. AnArf has not yet been classified and its positional specificity on arabinoxylan or the corresponding oligosaccharides is unknown. BspArf is classified to GH43 and is highly specific for a removal of arabinose from position 3 of doubly arabinosylated xylosyl residues (van Laere et al., 1997; van den Broek et al., 2005).

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and the liberated 4-nitrophenol was quantified spectrophotometrically at 410 nm. Beechwood glucuronoxylan (0.2% (w/v)) was used as a substrate for xylanases. Reducing sugars were quantified by the 3,5-dinitrosalicylic acid method (Miller, 1959). One unit of ␤-xylosidase, ␣-l-arabinofuranosidase, or xylanase activity corresponds to the amount of enzyme liberating 1 ␮mol of 4-nitrophenol or xylose equivalents in 1 min. A ␤-xylosidase (XlnD)-coupled assay using 4-nitrophenyl 4-O-methyl-␣-d-glucopyranuronosyl-1,2-␤d-xylopyranoside was used for determination of ␣-glucuronidase activity (Biely et al., 2000a). 2.4. Cultivation Thermomyces lanuginosus strains IMI 84400 (ATCC 22070) and IMI 96213 were obtained from International Mycological Institute (at present part of CAB International, Egham, Surrey, UK). They were grown in a medium composed of (in 1-l): l-asparagine, 4 g; KH2 PO4 , 3 g; K2 HPO4 , 2 g; MgSO4 ·7H2 O, 0.5 g, CoCl2 ·6H2 O, 2 mg; FeSO4 ·7H2 O, 5 mg; MnSO4 ·H2 O, 2 mg; ZnCl2 , 1.6 mg; and the main carbon source, 20 g. Mycelium grown on glucose for 4 days at 50 ◦ C and 200 rpm was used as inoculum for the media containing Lenzing xylan, deacetylated beechwood glucuronoxylan, corn fiber xylan or wheat arabinoxylan as a carbon source. Aliquots of the cultures were taken daily, centrifuged and the supernatants analyzed for the presence of xylanolytic enzymes and for the unconsumed sugars. Larger volumes of the cultures (20 ml) were used for determination of biomass dry weight (105 ◦ C). 2.5. Isolation of major non-utilized oligosaccharide from glucuronoxylan-spent medium Cultures obtained after an 8-day cultivation of the fungus on deacetylated beechwood glucuronoxylan were centrifuged (30 min, 14,000 × g) and the supernatants were subjected to ultrafiltration through a 10 kDa cut-off polyethersulfone membrane (Millipore, Bedford, MA, USA). After dilution with water the retentate was desalted by repeated ultrafiltration and stored at 4 ◦ C. The filtrate was subjected to anion exchange chromatography on a sinter funnel (9 cm × 3 cm) filled with Dowex 1 × 8 (Serva, Heidelberg, Germany) in the OH− form. Substances that passed through were combined with those eluted with water. Adsorbed compounds were liberated by elution with 4 M acetic acid, evaporated to dryness, dissolved in a minimal volume of water, loaded onto a column of Biogel P-2 (200 cm × 1.8 cm; Biorad, Hercules, CA, USA) and eluted with water at a flow rate of 16 ml h−1 . 4-ml (15 min) fractions were collected and analyzed by TLC. Pure carbohydrates of distinct TLC mobilities were separately pooled and freeze-dried. 2.6. Isolation of non-utilized oligosaccharide fragments after growth on wheat arabinoxylan Culture media obtained after 8-day cultivation of the fungus on wheat arabinoxylan were concentrated ca. 10-fold by vacuum evaporation and subjected to preparative TLC. After detection of the guide strips, the corresponding bands were eluted with methanol and the recovered saccharides were dried under vacuum.

2.3. Enzyme assays

2.7. Thin layer chromatography

All enzyme assays were done in 50 mM sodium acetate buffer, pH 5.0, at 40 ◦ C. ␤-Xylosidase and ␣-l-arabinofuranosidase activities were determined on the corresponding 4-nitrophenyl glycosides (1 mM). The reactions were terminated by the addition of two volumes of a saturated solution of sodium tetraborate,

Analytical TLC was performed on aluminum-coated silica gel 60 plates (Merck, Darmstadt, Germany) in ethyl acetate/acetic acid/1-propanol/formic acid/water (25:10:5:1:15, by vol.; two developments). The sugars were visualized with 0.2% (w/v) orcinol solution in ethanol/sulfuric acid (1:9, v/v) and quantified by

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densitometry (UN-SCAN-IT; Silk Scientific, Orem, UT, USA) using calibration curves of xylose, arabinose and MeGlcA. 2.8. NMR spectroscopy For NMR spectroscopy all oligosaccharides were freeze-dried twice from D2 O. 1 H and 13 C NMR spectra were recorded in D2 O at 25 ◦ C on a Bruker instrument operating at 300 and 75 MHz, respectively. Chemical shifts are referred to d6 -acetone at 2.225 ppm (1 H) and 31.07 ppm (13 C) as an external standard and are reported relative to 3-(trimethylsilyl)-propionic acid-d4 , sodium salt (TSP). 2.9. Nanospray mass spectrometry Isolated compounds dissolved in a mixture of methanol/water (1:1, v/v) were analyzed by electrospray ionisationquadrupole/time-of-flight mass spectrometry using a PicoTip® emitter (New Objective, Woburn, MA, USA) mounted onto nanospray of Q/ToF Premier (Waters, Milford, MA, USA). The mass spectrometer was operated in the positive V mode. Prior to direct infusion at a flow rate of 1–5 ␮l min−1 , the sample solutions were diluted to an approximate concentration of 50 ␮M. The ESI conditions were optimized for the highest sensitivity detection. The capillary and cone voltages were maintained at 3.5 kV and 45 V, respectively. The source temperature was set to 80 ◦ C. Nitrogen (30 l h−1 ) and argon were applied as the cone and collision gases, respectively. The ESI-Q/ToF mass spectrometer was calibrated with a 500 fmol ␮l−1 solution of [Glu-1]-fibrinopeptide delivered through a reference sprayer of the NanoLock Spray (Waters) source at a flow rate of 300 nL min−1 . 2.10. Enzyme treatment of purified carbohydrates Glucuronoxylan fragments accumulated in the culture fluid were dissolved in 50 mM sodium acetate buffer, pH 5.0 (1%, w/v), and treated with ␤-xylosidase XlnD (0.014 U ml−1 ) and A. tubingensis ␣-glucuronidase (0.16 U ml−1 ) at 30 ◦ C for 16 h. The enzymes were added separately or sequentially after thermal inactivation (100 ◦ C, 5 min) of the first catalyst. The acidic oligosaccharides were also treated with Erwinia chrysanthemi xylanase A (16.5 mU ml−1 ) under identical conditions. Oligosaccharides isolated from the wheat arabinoxylan medium were treated with XlnD (0.12 U ml−1 ) for 2 h, and for 24 h with ␣-l-arabinofuranosidase from A. niger (0.4 U ml−1 ) or with ␣-larabinofuranosidase from Bifidobacterium sp. (0.001 U ml−1 ). The ␣-l-arabinofuranosidases were added either sequentially or simultaneously. After their inactivation (100 ◦ C, 5 min) XlnD was added. The enzymatic processing of arabinoxylooligosaccharides was analyzed by TLC. 3. Results 3.1. Incomplete utilization of glucuronoxylan by T. lanuginosus strains Both strains of T. lanuginosus, IMI 84400 and IMI 96213, grew well in a synthetic medium containing alkali-extracted beechwood glucuronoxylan or Lenzing beechwood glucuronoxylan as the main carbon source. Biomass dry weight in all cases was in the range of 13–16 mg/ml, which is significantly lower than that on glucose (20–21 mg/ml). Extracellular ␤-xylosidase and ␣glucuronidase were absent, although both strains secreted xylanase which reached a maximum level of 226 and 133 U ml−1 , respectively. On day 2 the following oligosaccharides were detected by TLC: xylose, ␤-1,4-xylobiose, traces of ␤-1,4-xylotriose, and four

acidic oligosaccharides having chromatographic mobility similar to that of aldotetrao- (trace amount), aldopentao-, aldoheptao- and aldooctao-uronic acids. All neutral sugars disappeared on day 3. In regards to uronic acid content, both strains converted the polymers almost quantitatively to an aldopentaouronic acid in approximately 35% yield which corresponds to the substitution of the main chain with MeGlcA (xylose to uronic acid ratio approximately 10:1). 3.2. Limited arabinoxylan utilization by T. lanuginosus strains Corn fiber xylan (arabinose:xylose:galactose:uronic acid ratio 33.8:51.9:6.5:7.4) did not serve as a carbon source for the microorganisms. This polysaccharide was also found to be completely resistant to the action of various purified endoxylanases. Wheat arabinoxylan (arabinose to xylose ratio 41:59) was utilized by both T. lanuginosus strains, but to a very limited extent despite higher xylanase production (>500 U ml−1 ) than that on glucuronoxylan. Biomass dry weight on wheat arabinoxylan was in the range of 6–8 mg/ml, which is 2–3 times lower than on beechwood glucuronoxylan. In contrast to essentially a single product remaining in the medium with the acidic polysaccharide, the incomplete utilization of wheat arabinoxylan led to the accumulation of a complex mixture of branched oligosaccharides accounting more than 90% of the starting polysaccharide. From the wheat arabinoxylan-spent media of both strains we were able to isolate eight oligosaccharide fractions by preparative TLC. 3.3. Isolation and structural determination of the major acidic xylooligosaccharide The oligosaccharides remaining in the glucuronoxylan-spent medium were adsorbed to anion exchange resin and liberated with acetic acid. The major acidic oligosaccharide, migrating as aldopentaouronic acid, was purified by gel permeation chromatography. The structure of the isolated compound was established by NMR spectroscopy and by glycosidase treatment. The most intensive signal in one-dimensional 1 H NMR spectrum (Fig. 1) was observed at 3.497 ppm (s) and corresponds to the methyl group linked to position O-4 of the glucuronic acid residue, demonstrating that uronic acid present in the isolated fragment is in the form of 4O-methyl-d-glucuronic acid. The most downfield shifted signal in the spectrum at 5.323 ppm (J = 3.7 Hz, relative intensity 1.0) corresponds to the anomeric H-1 atom of 4-O-methyl-d-glucuronic acid. The presence of a single doublet at 5.323 ppm suggests that the MeGlcA is linked neither to the reducing end terminal nor to the adjacent xylosyl moiety. In such cases the anomeric signal of the MeGlcA residue would be split into two adjacent doublets (at approximately 5.3 ppm), differing considerably in their intensities (Cavagna et al., 1984; Excoffier et al., 1986; St John et al., 2006). The reported equilibrium ratio of the ␣ and ␤ anomer of the reducing end xylose is approximately 0.3:0.7 (St John et al., 2006). The anomeric region of the spectrum also contains five other doublets corresponding to the non-reducing end ␤-xylosyl residue, 2 internal ␤-xylosyl residues and to the two anomers of the reducing end terminal xylose. Relative intensities of these resonances, 1.0:1.0:0.9:0.6:0.3, are consistent with the presence of four xyloses linked by ␤-1,4-glycosidic linkages. Assignment of the signals in the individual 1 H and 13 C NMR spectra (Fig. 1) was performed with the aid of two-dimensional 1 H–1 H homocorrelated and 1 H–13 C heterocorrelated NMR spectra. In contrast to earlier papers (Biely et al., 1997; Nacos et al., 2006), our signal assignment is complete (Table 1) and corresponds to the structure MeGlcA3 Xyl4 . The assignment is in full agreement with the signal assignment of glucuronoxylans (Kardoˇsová et al., 1998; Moine et al., 2007) and of shorter aldouronic acids synthesized

V. Puchart, P. Biely / Journal of Biotechnology 137 (2008) 34–43

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non-reducing end. In accord with this observation, ␤-xylosidase shortened the substance by one xylosyl unit (Fig. 2). After this step the oligosaccharide became susceptible to ␣-glucuronidase, which resulted in the generation of MeGlcA and ␤-1,4-xylotriose (Fig. 2). This was another proof that the isolated acidic fragment carries a MeGlcA substituent at the penultimate xylosyl residue counting from the non-reducing end. The structure of the major xylan fragment remaining in the culture fluid of T. lanuginosus strains grown on glucuronoxylan was further confirmed by its hydrolysis with Erwinia chrysanthemi xylanase A to give the aldotetraouronic acid, MeGlcA2 Xyl3 , and xylose (Fig. 2). 3.4. Structure of oligosaccharides remaining in the wheat arabinoxylan-spent medium

Fig. 1. 1 H (part A) and 13 C (part B) NMR spectra of purified aldopentaouronic acid MeGlcA3 Xyl4 accumulated in the culture medium after 8-day growth of T. lanuginosus IMI 96213 on beechwood glucuronoxylan as a sole carbon source.

chemically (Kováˇc et al., 1980; Hirsch et al., 1981; Cavagna et al., 1984; Excoffier et al., 1986). The structure of the isolated acidic fragment, MeGlcA3 Xyl4 , was confirmed by enzymatic treatment. The compound was resistant to the action of GH67 ␣-glucuronidase, which removes only d-glucuronic acid or its 4-O-methyl ether attached to the nonreducing end xylosyl residue (Biely et al., 2000a,b). This excludes the possibility that the acidic oligosaccharide is substituted at the

Eight different fractions of oligosaccharides were isolated by preparative TLC from the medium (Fig. 3). They were analyzed by nanospray MS and digested with various glycosidases. Their tentative structures are shown in Table 2. Treatment of the culture medium or the isolated fractions with ␤-xylosidase XlnD did not result in xylose liberation, which implies that the culture fluid was free of linear ␤-1,4-xylooligosaccharides. This result pointed also to the fact that all non-utilized arabinoxylooligosaccharides were singly or doubly substituted at the non-reducing terminal xylosyl residue, or singly O-3 substituted or O-3 and O-2 doubly substituted at the penultimate xylosyl residue. It is known that certain types of ␤-xylosidases are unable to cleave substituted non-reducing-end terminal xylosyl residues or non-substituted non-reducing-end terminal xylosyl residues linked to O-3 substituted xylosyl residues (Kormelink et al., 1993a; Tenkanen et al., 1996; Herrmann et al., 1997; Smaali et al., 2006). However, such enzymes do recognize penultimate xylosyl residues that carry the substituent at position O-2, as in the case of MeGlcA3 Xyl4 (this study; Tenkanen et al., 1996; Herrmann et al., 1997). The structures of arabinoxylooligosaccharides shown in Table 2 were established by an application of the following rules: (1) oligosaccharide degree of polymerization was deduced from its MS signal; (2) simultaneous or sequential action of BspArf and AnArf (in this order) yielded fully debranched oligosaccharides, thus unraveling a length of the xylooligosaccharide backbone; (3) complete hydrolysis of the product to xylose and arabinose by sequential action of AnArf and XlnD revealed the presence

Table 1 Assignment of chemical shifts (in ppm) in the 1 H and 13 C NMR spectra of the isolated aldopentaouronic acid MeGlcA3 Xyl4

Atom

Xyl-I

Xyl-II

H

1 2 3 4 5 6 OMe

C

␣

␤

␣

␤

5.220 (3.6) 3.57 3.63 3.63 3.52eq, 3.83ax

4.617 (7.9) 3.27 3.59 3.82 3.40eq, 4.09ax

92.89 72.23 71.82 77.42 59.70

97.38 74.85 74.78 77.27 63.74

Xyl-III

Xyl-IV

MeGlcA

H

C

H

C

H

C

H

C

4.502 (7.6) 3.32 3.61 3.82 3.43eq, 4.13ax

102.55 73.53 74.55 77.27 63.74

4.660 (7.5) 3.51 3.67 3.87 3.47eq, 4.17ax

102.27 77.69 72.12 76.85 63.74

4.493 (7.7) 3.32 3.47 3.67 3.32eq, 4.02ax

102.86 73.70 76.48 70.08 66.09

5.323 (3.7) 3.62 3.795 3.253 4.364 (10.1)

98.38 72.12 73.09 83.34 73.24 177.71 60.76

The values in parentheses are coupling constants J in Hz. Sugar rings are marked according to the compound formula.

3.497

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Fig. 2. Schematic presentation of the action of GH5 xylanase A from Erwinia chrysanthemi, GH3 XlnD, and GH67 ␣-glucuronidase from Aspergillus tubingensis on aldopentaouronic acid isolated from the beechwood glucuronoxylan-spent medium after 8-day growth of T. lanuginosus IMI 96213.

of solely singly substituted compounds; (4) liberation of arabinose accompanied with a change of TLC mobility after digestion with BspArf reliably identified a doubly substituted compound; (5) partial dexylosylation with XlnD alone or after prior debranching with AnArf or BspArf occurred only when the non-reducing terminal xylosyl residue is not substituted and the penultimate xylosyl residue is not substituted at position O-3 (this is a simple approach to establish the decoration pattern of the first substituted xylosyl residue from the non-reducing terminus); (6) the precise location of several substitutents is based on the assumption that the products generated with GH11

xylanase (discussed below) contain at least two consecutive nonsubstituted xylosyl residues at the reducing terminus (Kormelink et al., 1993a; Ordaz-Ortiz et al., 2004). This approach to unraveling the structure of arabinoxylooligosaccharides is illustrated in Table 3. 4. Discussion There are several reports on xylanase production by T. lanuginosus strains on glucuronoxylan and arabinoxylan-containing agricultural residues (Puchart et al., 1999). However, to the best

Fig. 3. TLC analysis of arabinoxylooligosaccharides remaining in the wheat arabinoxylan-spent medium after 8-day growth of T. lanuginosus IMI 96213. Panel A: lane M, crude culture medium; lanes 1–8, fractions 1–8 isolated from this medium by preparative TLC. S1—standards of xylose and ␤-1,4-xylobiose through ␤-1,4-xylohexaose; S2—standards of l-arabinose, arabinoxylobiose and arabinoxylotriose. Panel B: densitogram of lane M from panel A. Position of the isolated fractions within the densitogram are marked with the corresponding numbers.

Table 2 Tentative structure of arabinoxylooligosaccharide products obtained after cultivation of T. lanuginosus on wheat arabinoxylan and their separation by preparative TLC Fraction no. (relative abundance)

Major product(s)

Minor product(s)

1 RXyl = 0.72 (11.5%)

2 RXyl = 0.64 (26.0%)

4 RXyl = 0.54 (10.6%)

Structure not established

V. Puchart, P. Biely / Journal of Biotechnology 137 (2008) 34–43

3 RXyl = 0.57 (14.0%)

5 RXyl = 0.49 (10.0%)

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Table 2 (Continued) 40

Fraction no. (relative abundance)

Major product(s)

Minor product(s)

6 RXyl = 0.47 (6.9%)

8 RXyl = 0.32 (12.0%)

Retention factor (RXyl ) is chromatographic mobility after two subsequent developments relative to that of xylose (RXyl = 1.00). Hexagonals and pentagonals stand for d-xylopyranosyl residues joined by ␤-1,4-linkages (horizontal bars, –) and l-arabinofuranosyl residues connected to the xylooligosaccharide backbone by ␣-1,2- (vertical bars, |) and ␣-1,3-linkages (slashes, /).

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7 RXyl = 0.40 (9.0%)

Table 3 Schematic presentation of the action of A. niger ␤-xylosidase XlnD, A. niger ␣-l-arabinofuranosidase AnArf and Bifidobacterium sp. ␣-l-arabinofuranosidase BspArf on low degree of polymerization arabinoxylooligosaccharides, generated by T. lanuginosus xylanase, having singly arabinosylated xylosyl residue(s), doubly arabinosylated xylosyl residue and those consisting of xylosyl residues of both types of substitution Substrate

First enzyme

Product structure after the first treatment

BspArf

Second enzyme

Product structure after the second treatment

XlnD

AnArf N. c.

AnArf

XlnD

XlnD

N. c.

AnArf

BspArf

BspArf

AnArf

V. Puchart, P. Biely / Journal of Biotechnology 137 (2008) 34–43

XlnD or BspArf

XlnD Hexagonals and pentagonals stand for d-xylopyranosyl residues joined by ␤-1,4-linkages (horizontal bars, –) and l-arabinofuranosyl residues connected to the xylooligosaccharide backbone by ␣-1,2- (vertical bars, |) and ␣-1,3-linkages (slashes, /). N.c. – no change.

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of our knowledge, this is the first report on the accumulation of branched oligosaccharides by this thermophilic microorganism in growth media containing the polysaccharides as carbon sources. Regardless of the type of xylan, the fungus utilized only xylose and linear xylan fragments as carbon sources. These fragments were observed in the media only at early stages of cultivation. Since ␤xylosidase was not detected in the medium, the disappearance of linear oligosaccharides could be due to their internalization, presumably by membrane-located transporters as previously shown in other microorganisms (Krátky´ and Biely, 1980; Kremnicky´ and Biely, 1998; Shulami et al., 1999). This work confirms that xylanase of glycoside hydrolase family 11 is the only xylanolytic enzyme secreted by T. lanuginosus strains. The extracellular activities of ␤-xylosidase, ␣-glucuronidase and ␣-l-arabinofuranosidase were in all cases extremely low, close to the limits of their detection. The absence of the three auxiliary enzymes is an explanation for the observed accumulation of branched oligosaccharides in the growth media. Trace amounts of aldotetraouronic acid of a putative structure of MeGlcA2 Xyl3 , accompanying the major acidic product, aldopentaouronic acid MeGlcA3 Xyl4 , originate most probably from the reducing end of individual glucuronoxylan molecules. The main fragment, whose yield is approximately 35%, is known as the shortest final acidic fragment of glucuronoxylan degradation with GH11 endoxylanases (Biely et al., 1997; Bennett et al., 1998; Christakopoulos et al., 2003; Katapodis et al., 2003; Kolenová et al., 2006; Vardakou et al., 2008). This agrees well with the production of a single xylanase, belonging to GH11 family, as evidenced by IEF and native PAGE analysis of the culture media from various T. lanuginosus strains (Purkarthofer et al., 1993; Singh et al., 2000, 2003). The structure of arabinoxylooligosaccharides also corresponds to exclusive secretion of a GH11 xylanase in culture filtrate of T. lanuginosus. The shortest fragment identified in the medium is arabinoxylotriose with arabinose linked to the internal xylosyl residue. This compound is known as the shortest fragment generated from arabinoxylans by an action of GH11 xylanases (Kormelink et al., 1993a,b; Vardakou et al., 2003). On the contrary, family 10 xylanases produce arabinoxylobiose as the shortest fragment (Gruppen et al., 1992; Kormelink et al., 1993a; Vardakou et al., 2003; Pell et al., 2004; Rantanen et al., 2007). Extremely low yields of arabinoxylotriose are commonly reported for arabinoxylan hydrolyzates generated with family 11 xylanases. We hypothesize that this singly substituted xylotriose, as well as the doubly arabinosylated analogue found also in trace amounts in fraction 2, originates from the reducing end of the polysaccharide. Consistent with previous reports (Kormelink et al., 1993a; Quéméner et al., 2006), all other oligosaccharides remaining in the culture fluid possess non-substituted xylobiose residue at their reducing end. The structures of longer arabinoxylooligosaccharides determined in this study are identical with those generated by different GH11 endoxylananases and established by other methodologies (Kormelink et al., 1993a; Quéméner et al., 2006; Maslen et al., 2007). Our data support the conclusion drawn by Kormelink et al. (1993a) that family 11 xylanases require as a rule 3 penultimate non-substituted xylosyl residues to cleave the xylan backbone, leaving two of them at a newly formed reducing end. That is, these enzymes accommodate substituted xylosyl residues at subsites −3 and +2 but not at subsites −2, −1 or +1. The data presented in this study suggest that three glycosidases, BspArf, AnArf and XlnD, supplemented with an endoxylanase, could bring about a complete hydrolysis of cereal arabinoxylan to pentoses. Exceptions could include highly substituted arabinoxylans, e.g., those in corn fiber (Hespell, 1998) as well as arabinoxylans with a higher content of uronic acids.

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