Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans

Enzyme and Microbial Technology 49 (2011) 312–320

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Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans L. Pouvreau a,1 , M.C. Jonathan a , M.A. Kabel c , S.W.A. Hinz b , H. Gruppen a , H.A. Schols a,∗ a b c

Laboratory of Food Chemistry, Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands Dyadic Nederland B.V., Nieuwe Kanaal 7-S, 6709 PA Wageningen, The Netherlands Royal Nedalco, PO Box 6, 4600 AA Bergen op Zoom, The Netherlands

a r t i c l e

i n f o

Article history: Received 13 December 2010 Received in revised form 10 May 2011 Accepted 16 May 2011 Keywords: Acetyl xylan esterase Chrysosporium lucknowense Xylan Acetylated xylooligosaccharides Eucalyptus

a b s t r a c t Two novel acetyl xylan esterases, Axe2 and Axe3, from Chrysosporium lucknowense (C1), belonging to the carbohydrate esterase families 5 and 1, respectively, were purified and biochemically characterized. Axe2 and Axe3 are able to hydrolyze acetyl groups both from simple acetylated xylo-oligosaccharides and complex non-soluble acetylglucuronoxylan. Both enzymes performed optimally at pH 7.0 and 40 ◦ C. Axe2 has a clear preference for acetylated xylo-oligosaccharides (AcXOS) with a high degree of substitution and Axe3 does not show such preference. Axe3 has a preference for large AcXOS (DP 9–12) when compared to smaller AcXOS (especially DP 4–7) while for Axe2 the size of the oligomer is irrelevant. Even though there is difference in substrate affinity towards acetylated xylooligosaccharides from Eucalyptus wood, the final hydrolysis products are the same for Axe2 and Axe3: xylo-oligosaccharides containing one acetyl group located at the non-reducing xylose residue remain as examined using MALDI-TOF MS, CE-LIF and the application of an endo-xylanase (GH 10). © 2011 Elsevier Inc. All rights reserved.

1. Introduction An increasing demand for energy, soaring prices of oil and environmental pollution associated with the use of fossil fuel have resulted in an increased worldwide interest for the production and use of biofuels [1,2]. If ethanol is to be used as an alternative source of energy, the production will only be economically feasible if from lignocellulosic material both the cellulose and hemicellulose are used as source for monosaccharides. Cellulose is the most abundant polymer in lignocellulosic material, which can make up to 40% of the total material [3]. The chemical structure of cellulose is relatively simple, and only three enzyme classes are needed for complete hydrolysis [4]: endo-glucanases, exoglucanases (including cellobiohydrolases) and ␤-glucosidases. On the other hand, hemicellulose which makes up to approximately 30% of the lignocellulosic material [1], has a very complex chemical structure. The main hemicellulosic polymer xylan [5,6] consists of

Abbreviations: Axe, acetyl xylan esterase; AcXOS, acetylated xylooligosaccharides; Ac-MeGlcAXylan, xylan polymer containing 4-O-methyl-d-glucopyranosyl uronic acid and acetyl groups; CE-LIF, capillary electrophoresis with laser induced fluorescence detection. ∗ Corresponding author. Tel.: +31 317 482239; fax: +31 317 484893. E-mail address: [email protected] (H.A. Schols). 1 Present address: NIZO Food Research B.V., PO Box 20, 6710 BA Ede, The Netherlands. 0141-0229/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2011.05.010

␤-d-(1,4)-linked xylopyranosyl backbone, which can be substituted with l-arabinosyl, acetyl, 4-O-methyl-d-glucopyranosyl uronic acid and feruloyl residues [7–9]. It can form strong networks through feruloyl residues that can be linked to hydroxyl groups of other feruloyl residues or lignin [6,10–12]. Besides that xylan will form hydrogen bonds with cellulose [13]. Complete hydrolysis of xylans requires an array of accessory enzymes, including arabinofuranosidases [14,15], ␣-glucuronidases [5,16–19], feruloyl esterases [20–22] and acetyl xylan esterases [23,24], in addition to backbone depolymerising enzymes, like endo-(1,4)-␤-xylanases [25] and ␤-xylosidases [25]. The structure of xylan differs highly for different plant materials. Cereal xylans are mainly substituted with arabinosyl residues, whereas hard wood xylans are substituted with 4-O-methyl-d-glucopyranosyl uronic acid residues [26,27] and acetyl residues [27,28]. Arabinosyl and feruloyl residues are not present in hard wood xylans. Xylosyl residues of Eucalyptus wood xylan are highly substituted: approximately 60% of the total xylosyl residues are acetylated (assuming that only one acetyl group is present per xylosyl residue) and 30% are substituted with 4O-methyl-d-glucopyranosyl uronic acid. In comparison, corn cob xylans are only substituted approximately 35 and 10% with acetyl and 4-O-methyl-d-glucopyranosyl uronic acid residues, respectively [28]. Enzymatic removal of these acetyl residues can be performed by acetyl xylan esterases, which are produced by fungi such as Aspergillus niger, Trichoderma reesei and Penicillium purpurogenum [14,16,29,30], and bacteria such as Streptomyces lividans

L. Pouvreau et al. / Enzyme and Microbial Technology 49 (2011) 312–320

[31] and Bifidobacterium animalis subsp. lactis [32]. Acetyl xylan esterases cleave the ester bonds between the acetyl residue and the xylopyranosyl residue. Herewith the enzymes create more cleavage sites for endo-(1,4)-␤-xylanases. In general, fungi seem to express several acetyl xylan esterases belonging to different carbohydrate esterase (CE) families (CAZy classification [33]). It is expected that these different acetyl xylan esterases differ in substrate specificity as a consequence of the large substrate heterogeneity [31,34]. However, so far the CAZy classification is not a prediction for the substrate specificity. Acetyl xylan esterase from T. reesei, which is classified as CE5 enzyme has a higher affinity towards the acetyl group located at position O-2 [16,35], which is also shown for the acetyl xylan esterases from S. lividans, which has been classified as CE4 enzyme [31,35] and for the acetyl xylan esterase from Schizophyllum commune, which has been classified as a CE1 enzyme [23,35]. A more recent discovered class of acetyl esterases, CE16 [24] shows more preference for acetyl residues at positions O-3 and O-4 [35]. The ascomycete Chrysosporium lucknowense C1 is an industrial strain optimized in cellulase and hemicellulase production. The C1 genome has been fully sequenced and annotated. Of more than 200 candidate genes that have high similarities to carbohydrate active enzymes, about 50 glucuro(arabino)xylan active enzymes have been putatively identified, among them fourteen esterases, classified in the family’s CE1, CE4, CE5 and CE16 [36,37]. Based on these genomic data, it can be concluded that C1 seems to be a good platform for the degradation of glucurono(arabino)xylanrich biomass. Two of the putative esterases have been selected for expression, purification and characterization, Axe2 (classified as CE5 enzyme) and Axe3 (classified as CE1 enzyme). Here we report the mode of action of the two acetyl xylan esterases from C. lucknowense C1 towards acetylated Eucalyptus xylan and xylo-oligosaccharides. 2. Materials and methods 2.1. Materials Eucalyptus alcohol insoluble solids (AIS) and a purified xylan polymer containing 4-O-methyl-d-glucopyranosyl uronic acid and acetyl groups (Ac-MeGlcAXylan) were obtained as described previously [28]. Eucalyptus wood AIS consist mainly of cellulose and heteroxylan, which are entangled in a complex and insoluble cell wall network. The purified polymer Ac-MeGlcAXylan from Eucalyptus wood, is a high molecular weight and partly insoluble xylan, decorated with acetyl groups at positions O-2 and/or O-3 and with 4-O-methyl-d-glucopyranosyl uronic acid (MeGlcA) at position O-2 of xylosyl residues [23]. Neutral acetylated xylo-oligosaccharides (AcXOS; with a DP of 4–12 xylosyl residues) were obtained from thermally processed Eucalyptus wood [13]. These AcXOS are purified xylo-oligosaccharides, in which xylosyl residues are solely substituted by acetyl groups [38]. The monosaccharide compositions of these substrates are presented in Table 1. Methyl ferulate, sinapate and caffeate were purchased from Apin Chemicals Ltd. (UK) and p-nitrophenyl-acetate was obtained from Fluka (Sigma–Aldrich, Germany).

313

2.3. SDS-PAGE SDS-PAGE, under reducing conditions, was performed with a Pharmacia PhastSystem (GE Healthcare) according to the instructions of the manufacturer using Gradient 8–25% Phastgels (GE Healthcare, Uppsala, Sweden). Gels were stained according to the Coomassie blue staining procedure provided by the manufacturer. 2.4. Purification of Axe2 and Axe3 An Akta purifier chromatography system and column used for the protein purification were obtained from GE Healthcare. The eluates were monitored at 215, 280 and 320 nm. The fermentation supernatants containing Axe2 or Axe3 were applied to a Superdex 75 gel filtration column (10 mm × 300 mm), equilibrated with 50 mM sodium acetate buffer, pH 5.0, containing 50 mM NaCl. The proteins were eluted using the same buffer at a flow rate of 0.8 mL/min. Two protein peaks were eluted from the column for both enzyme samples: the activity assays used showed that the first peak did not contain any esterase activity, while the second peak showed high activity towards AcXOS in both cases. Fractions (0.5 mL), containing esterase activity, were collected, pooled and were subsequently stored in small aliquots at −20 ◦ C. SDS-PAGE of peak 1 showed that the proteins represented the host proteins (based on a background control), while hardly any protein was detected in peak 2 using SDS-PAGE. The detection limit of proteins using Coomassie blue staining is very low and the dye binding a certain amount of proteins differs highly from one protein to another [40]. MALDI-TOF MS was performed to determine the Mw of the enzymes. The enzymes were found to have a molecular mass of 23.6 and 33.6 kDa for Axe2 and Axe3, respectively, which are very similar to the sequencebased molecular masses (22.3 and 31.5 kDa, respectively). 2.5. Enzyme assays The esterase activity of the acetyl xylan esterase towards AIS, Ac-MeGlcAXylan and AcXOS from Eucalyptus wood dispersed in millipore water was measured using a pH stat. The reaction mixture (5 mL) consisted of AIS (2 mg), Ac-MeGlcAXylan (1 mg) or AcXOS (1 mg) and an enzyme dose of 5 ␮g protein per mg of substrate. The acetic acid released was continuously back-titrated with 1 mM NaOH to the desired pH (pH range: 4.5–7.5), employing a thermostated autotitration system (719 S Titrino, Metrohm Ion analysis, Metrohm Ltd., Herisau, Switzerland). Temperature was controlled at the desired level (35–60 ◦ C). The reactions were continued until the end point was reached, resulting in typical incubation times of 4–6 h, depending on the substrate used. Temperature and pH optima were determined using the pHstat and AcXOS as the substrate. The specific activity was defined as 1 ␮mol of acetic acid released per min per mg protein. The pH optimum was determined within the pH range 4.0–7.5 at 35 ◦ C. pHs higher than 7.5 were not tested due to autohydrolysis of the ester linkage. The temperature profile was determined within the temperature range 35–60 ◦ C at the optimum pH determined. The specific activities were determined after 5 min of incubation. Hydrolysis products of Axe2 and Axe3 were monitored using MALDI-TOF MS and CE-LIF. In these experiments, substrate (10 mg/mL) and enzyme (5 ␮g enzyme per mg substrate) were incubated in a 50 mM sodium phosphate buffer pH 7.0 using a micro mixer (700 rpm) for a defined time at 40 ◦ C and the reaction was successively stopped by boiling (10 min). For CE analysis, the oligosaccharides were derivatized with the fluorescent APTS before incubation with the enzyme. The samples were stored at −20 ◦ C until the analysis took place. 2.6. MALDI-TOF MS The hydrolysis products of AcXOS were analyzed using MALDI-TOF MS, as described previously [41]. The molecular masses of the purified enzymes were determined using MALDI-TOF MS, as described by Pouvreau et al. [42]. 2.7. CE-LIF

2.2. Cloning of acetyl xylan esterase encoding genes The ascomycetous fungus C. lucknowense C1 (accession number: VKM F-3500D of the All-Russian Collection of Microorganisms of Russian Academy of Sciences) was used for the production of hemicellulases. The full genome of C1 has been sequenced and revealed many genes encoding for cellulases, hemicellulases and accessory enzymes [36]. Annotated genes, encoding for the putative acetyl xylan esterases Axe2 and Axe3, have been cloned into a specially designed C1-expression host [39] that allows production of relatively pure enzyme. The amino acid sequences have been deposited at GenBank with the accession numbers HQ324256 (Axe2) and HQ324257 (Axe3). The strains, containing extra copies of the axe2 or axe3 gene were grown aerobically in 2 L fermentors in mineral medium, containing glucose as carbon source, ammonium sulfate as nitrogen source and trace elements for the essential salts. Growth is performed under glucose limitation at pH 6.0 and 32 ◦ C [39]. After fermentation, the supernatants containing the enzymes were tested on acetylated substrates and both Axe2 and Axe3 showed activity towards acetylated xylan releasing acetic acid.

Samples for CE-LIF were prepared using the ProteomeLab Carbohydrate Labeling and Analysis Kit (Beckman Coulter, Fullerton, CA). The samples were derivatized with the fluorescent APTS overnight at room temperature and analyzed as described previously [43]. The enzyme incubation (40 ◦ C and pH 7.0) took place after the oligosaccharides were labeled. CE–MS was performed as described previously [41].

3. Results and discussion Two putative acetyl xylan esterase genes, axe2 and axe3, were annotated manually in the C. lucknowense C1. The sequence of axe2 encodes a 228 amino acid sequence, a signal sequence is predicted at positions 1–17 and the mature protein starts from residue 18 onwards. Calculated from the amino acid sequence, the molecular mass is 22.3 kDa and the iso-electric point (pI) 3.7. Based on homology, the enzyme Axe2 can be assigned to CAZy family CE5 [33].

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Table 1 Sugar composition (mol%) of Eucalyptus wood AIS, Ac-MeGlcAXylan and AcXOS. Total sugara

AIS Ac-MeGlcAXylan AcXOS a b

73 61 75

Molar composition Rha

Ara

Xyl

Man

Gal

Glc

MeGlcA

2 0 3

1 0 0

24 36 85

1 1 5

2 1 7

63 1 3

7 10 1

MeGlcA/Xylb

Ac/Xylb

0.29 0.27 0.01

0.64 0.70 0.60

Neutral sugars + uronic acids (UA) expressed as weight percentage of each fraction (dm). Ratio mol/mol.

Axe2 showed 68% homology with an acetyl xylan esterase from Neurospora crassa (GenBank accession number: EAA29494 [44]). The sequence of axe3 encodes a 313 amino acid sequence; a signal sequence is predicted at positions 1–21 and the mature protein starts from position 22 onwards. Calculated from the amino acid sequence, the molecular mass is 31.5 kDa and the iso-electric point (pI) 5.4. Based on homology, the enzyme Axe3 can be assigned to CAZy family CE1 [33]. Axe3 showed 69% homology with acetyl xylan esterase (AxeI) from P. purpurogenum (GenBank accession number: AAM93261 [30,45]). 3.1. pH and temperature profiles 3.1.1. pH profile Axe2 and Axe3 were tested on AcXOS in the pH range 4.0–7.5 using the pH stat method. Results showed that Axe2 and Axe3 have a very similar pH profile (Fig. 1A). Both enzymes did not show any activity towards AcXOS at pH 4.0 and both showed two distinct maxima at pH 6.0 and 7.0, respectively, and a dip in activity at pH 6.5. Measurements at pH 6.0, 6.5 and 7.0 were repeated several times and the results confirmed the current data. We have no explanation for this phenomenon. The specific activity of Axe3 was generally 2–3 times lower than the specific activity of Axe2 on the substrate used. Besides specific activity, we have also measured the amount of acetic acid (expressed as a percentage of the total amount of acetyl group present in AcXOS) released after a prolonged incubation period (80 min). The results showed that both Axe2 and Axe3 released more acetic acid at pH 7.0 than at pH 6.0 (15 and 7% more, respectively, data not shown). Combining both results, it was concluded that both Axe2 and Axe3 have their optimal pH at 7.0. 3.1.2. Temperature profile The temperature profiles of Axe2 and Axe3 were determined in the temperature range 35–60 ◦ C at pH 7.0. The results showed that Axe2 has clearly an optimal temperature at 40 ◦ C, whereas Axe3 does not display a very clear optimum (Fig. 1B). This experiment confirmed that the specific activity of Axe2 is higher than that of Axe3. The amount of acetic acid (expressed as a percentage of the total amount of acetyl groups present in AcXOS) released after a prolonged period (80 min) was determined. The results confirmed the previous findings: Axe2 has a clear optimal temperature at 40 ◦ C and Axe3 has a more broad “optimum” temperature range between 35 and 45 ◦ C (data not shown). In order to compare Axe2 and Axe3, it was decided to use a temperature of 40 ◦ C in all further experiments. 3.2. Substrate specificity Axe2 and Axe3 were tested for their activity towards different substrates in order to determine their substrate specificity. Results shown in Table 2 indicate that Axe2 was not active towards the synthetic substrate p-nitrophenyl-acetate. On the counterpart, Axe3 showed activity towards this substrate. Tenkanen et al. [46] showed that, so far only CE4 acetyl xylan esterases were found to lack the

ability of degrading artificial substrates as ␣-naphthyl-acetate and p-nitrophenyl-acetate. Since the CAZy esterase families include a large number of different types of esterases, such as cinnamoyl and feruloyl esterases, Axe2 and Axe3 were tested for activity towards methyl ferulate, methyl sinapate and methyl caffeate. Both Axe2 and Axe3 did not show any activity towards these 3 synthetic phenolic substrates (Table 2). Based on these results, it was concluded that both enzymes do not contain cinnamoyl or feruloyl esterase activity. Three xylan or xylo-oligosaccharide substrates were used to determine the substrate specificity of Axe2 and Axe3 towards natural substrates. These three Eucalyptus wood xylans and xylooligosaccharides differ in solubility, homogeneity, chain length and substitution (Table 1). The activity towards the substrates is shown in Table 2 and Fig. 2. Fig. 2A shows the specific activity of Axe2 and Axe3 towards the substrates, whereas Fig. 2B shows the amount of acetate released by the enzymes. As described earlier, it was shown that Axe2 has a higher specific activity than Axe3 on AcXOS, this was also found for Eucalyptus wood AIS. The specific activity towards Ac-MeGlcAXylan was equal for both enzymes. Both Axe2 and Axe3 have the highest specific activity towards oligomers (AcXOS), followed by the polymer (Ac-MeGlcAXylan) and the insoluble material (Eucalyptus wood AIS). The relative specific activity for the different substrates differs for both enzymes. These results suggest that Axe2 and Axe3 are differently influenced by the size, accessibility and/or the heterogeneous substitution of their substrates. From literature it is known that acetyl xylan esterases may have different specific activities towards xylo-oligosaccharides and polymers. For example, the esterase from S. commune acts preferentially on acetylated xylo-oligomers [47], whereas Axe from T. reesei has a high affinity for polymeric substrate and is hindered by high levels of acetylation [16,48]. Despite differences in the velocity of the reactions, the total amount of acetic acid released at the end point was very similar for both enzymes and for all substrates tested (Fig. 2B). In one experiment extra AcXOS were added to the digest after 50 min of incubation. The initial velocities of both enzymes were exactly the same as the ones observed at the starting point of the experiments (no further data shown). This suggested that the enzymes still have the same activity at the end point of the reactions. Although Axe2 and Axe3 show a difference in velocity, they remove acetyl groups efficiently from both oligomers and polymers. Approximately 80–90% of the total acetyl groups were

Table 2 Substrate specificity of Axe2 and Axe3; +, enzyme is active towards this substrate; −, enzyme is not active towards this substrate. Substrate

Axe2

Axe3

p-Nitrophenyl-acetate Methyl ferulate Methyl sinapate Methyl caffeate Eucalyptus wood AIS Ac-MeGlcAXylan AcXOS

− − − − +/− + +

+ − − − +/− + +

L. Pouvreau et al. / Enzyme and Microbial Technology 49 (2011) 312–320

16

315

A

14

activity ( mol/min/mg)

12

10

8

6

4

2

0 4

4.5

5

5.5

6

6.5

7

7.5

8

pH 25

B

Activity ( mol/min/mg)

20

15

10

5

0 30

35

40

45

50

55

60

65

Temperature (°C) Fig. 1. (A) pH optima of Axe2 (square) and Axe3 (triangle) towards AcXOS from Eucalyptus wood. Experiments at various pH were performed at 35 ◦ C, an AcXOS concentration of 0.2 mg/mL and an enzyme loading of 5 ␮g/mg substrate. (B) Temperature profile of Axe 2 (square) and Axe 3 (triangle) towards AcXOS from Eucalyptus wood. Experiments at various temperatures were performed at pH 7.0, an AcXOS concentration of 0.2 mg/mL and an enzyme loading of 5 ␮g/mg substrate.

released from AcXOS and Ac-MeGlcAXylan. Both substitutions of acetyl groups to position O-2 or O-3 are equally present in acetylated Eucalyptus oligosaccharides [38]. Since most of the acetyl groups are removed, it is suggested that Axe2 and Axe3 can remove acetyl groups from both positions O-2 and O-3. This observation seems to be in contrast with data presented by Biely et al. [35] stating that five acetyl xylan esterases tested from families CE1, CE4 and CE5 showed a strong preference for acetyl group at the O-2 position. The only type of esterase that seems to act differently is a CE16 enzyme from T. reesei, which has a preference for acetyl groups substituted to the O-3 and O-4 positions. Regarding the insoluble AIS substrate, both enzymes were only able to release approximately 20% of the total amount of the acetyl

groups present. These results indicated that both Axe2 and Axe3 are hindered by the more complex structure of the acetylated glucuronoxylan within the cell wall and/or by the interactions between xylan and cellulose, making the acetyl groups inaccessible. 3.3. End products of Axe2 and Axe3 The deacetylation of AcXOS by Axe2 and Axe3 was followed in time. Products were analyzed by MALDI-TOF MS and the results are depicted in Fig. 3. Fig. 3A shows the MALDI-TOF mass spectrum of the substrate AcXOS as an example. The result of deacetylation of the substrate by the enzymes is shown in Fig. 3B and C. The figure clearly shows that AcXOS have a high degree of

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Activity (µmol/min/mg protein)

25

A

20

15

10

5

0

100

AcXOS

AcGlcAmeXylan

AIS

AcXOS

AcGlcAmeXylan

AIS

B

Acetic acid released (%)

90 80 70 60 50 40 30 20 10 0

Fig. 2. (A) Specific activity of Axe2 (grey) and Axe3 (white) on various substrates from Eucalyptus wood (5 min incubation) and (B) amount of acetic acid released (as percentage of the total amount of acetic acid present in each substrate) by Axe2 (grey) and Axe3 (white) from different substrates (4–6 h depending of the substrate). Experiments were performed with a substrate concentration of 0.4 mg/mL (AIS) or 0.2 mg/mL (Ac-MeGlcAXylan and AcXOS) and an enzyme loading of 5 ␮g/mg substrate at pH 7.0 and 40 ◦ C.

substitution by acetyl groups (Fig. 3A). Although non-acetylated oligosaccharides are also present in the substrate as well as in the enzyme digests, the presence of these oligomers is not indicated in Fig. 3, since only the highest level of acetylation per oligomer is presented. The fast deacetylation of Axe2 is again shown in this figure. Already after 30 min of incubation, all oligosaccharides have been deacetylated to a maximum of two acetyl groups being substituted (Fig. 3B), which are mainly the highly substituted XOS. The XOS containing only 2 acetyl groups accumulate. These results indicate that Axe2 has a clear preference for AcXOS with a high degree of substitution. In this first 30 min the enzyme is still in the linear part of the deacetylation reaction (as determined by pH-stat analysis). Only after 5 h of incubation all oligosaccharides have been deacetylated to a maximum of one acetyl substitution per oligomer. In contrast, Axe3 deacetylated the XOS much slower than Axe2. The xylo-oligomers remain highly acetylated after 30 min of incubation with Axe3 (Fig. 3C). The number of acetyl groups per xylooligomers declines gradually with increasing incubation time to finally reach the same end-point as Axe2 after 5 h of incubation. The

degree of substitution decreases faster for XOS with a DP of 9–12. This is especially visible within the first 30 min of incubation (which is still in the linear range of the reaction). These results indicate that Axe3 has a small preference for larger AcXOS (DP 9–12). As clearly shown in Fig. 3, XOS substituted with only one acetyl group accumulate in the final digest of both enzymes. This result supports the hypothesis that an acetyl group present at a specific position was resistant for enzymatic deacetylation by both Axe2 and Axe3. It is expected that this residue is located at or close to the reducing or non-reducing end of the oligomer. 3.4. Monitoring acetyl release by CE-LIF Since the MALDI-TOF MS method is only able to differentiate AcXOS based on their masses, CE-LIF was used to differentiate the XOS in the enzyme digests based both on their mass and the position of the acetylated xylose residue within the oligomer [43]. Digestion of AcXOS by Axe2 was followed in time and analyzed using CE-LIF (Fig. 4). A small amount of AcXOS was saponified and used as a reference for a 100% de-acetylated substrate. The

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317

Fig. 3. MALDI-TOF MS spectra of (A) the initial substrate (AcXOS); (B) and (C) show the remaining acetyl groups per oligomer in the hydrolysis products of Axe2 and Axe3, respectively, at different incubation times: 0, 0.5, 1, 2 and 5 h. The Y-axis indicates the highest level of acetylation present in each XOS DP, as based on MALDI-TOF MS. Experiments were performed with a substrate concentration of 10 mg/mL and an enzyme loading of 5 ␮g/mg substrate at pH 7.0 and 40 ◦ C.

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X3

5h

X4

30 min

X5 X2

AcXOS

X6

Fluorescence

10

X7

11

12

X8

X9

X10

Saponified AcXOS.

X11

X12

5h 30 min

10 min AcXOS 4.00

5.00

6.00

7.00

8.00

9.00

10.00

11.00

12.00

Time (min) Fig. 4. CE electropherograms of saponified AcXOS and AcXOS digested by Axe2, at different incubation times. Experiments were performed with a substrate concentration of 10 mg/mL and an enzyme loading of 5 ␮g/mg substrate at pH 7.0 and 40 ◦ C. The inset shows a magnification of the graph between 10 and 12 min. The arrows point to the remaining acetylated oligomer for every oligomer size. Xn indicates a xylo-oligomer containing n xylosyl residues.

3.5. Position of xylose residue carrying the remaining acetyl group To determine the precise location of the remaining acetyl group, the APTS-labeled hydrolysis products from the 5 h digest with Axe2 was hydrolyzed using an endo-(1,4)-␤-xylanase (GH 10) from Aspergillus awamori (Fig. 5). In this digest only the oligosaccharides with one substituted acetyl group were present. Comparing the digests before and after the hydrolysis with endo-(1,4)-␤-xylanase will give a clear indication on the xylose residue carrying the remaining acetyl groups. The APTS group is located at the reducing end of the oligosaccharides, therefore only two possible results can be expected from this experiment: the remaining acetyl group is located at or close to the reducing end. This will result in an increase in smaller APTS-labeled XOS still carrying an acetyl group.

Alternatively the remaining acetyl group is located at or close to the non-reducing end. This will result in an increase in non-acetylated, APTS-labeled xylobiose and xylotriose. In this latter case, products released from the non-reducing end containing an acetyl group will not be visible in the electropherograms, since they are not labeled by APTS. Fig. 5 shows that only APTS-labeled xylobiose and xylotriose are formed by the action of xylanase. No acetylated, APTS-labeled oligomers are formed. For Axe3, similar results were obtained (data not shown). Based on these results and on our knowledge on the activity of an endo-(1,4)-␤-xylanase (GH 10) from A. awamori [25], we can conclude that the remaining acetyl group is located at or close to the non-reducing end. CE–MS2 experiments [41] for mono-acetylated

X3 X2

Fluorescence

CE electropherogram of the initial substrate (AcXOS in Fig. 4) gives an insufficient resolution to separate all acetylated XOS molecules due to the high degree of acetylation and the high number of possible acetylated isomers for each xylo-oligosaccharide. This is especially due to large oligomers where the curve has a wavy shape (see zoom in Fig. 4). Nevertheless, at increasing incubation times, more and sharper peaks become visible in the electropherograms representing less complex and less acetylated xylo-oligosaccharides. Axe3 digestion showed very similar trends and is therefore not shown. The CE-LIF results support the MALDI-TOF MS data: Axe2 has a higher initial activity than Axe3 (data not shown) and the final hydrolysis products are similar for both enzymes. Incubation with both enzymes resulted in fully deacetylated XOS of different DP, next to minor levels of acetylated XOS of different DP. From the MALDI-TOF MS data it is known that these substituted oligosaccharides are only substituted with one acetyl group. Since the remaining single acetylated XOS eluted exactly at the same relative time for both enzyme digests, we can anticipate that the position of the remaining acetyl group is the same for both enzymes and for each DP [30].

Digested by Axe2, followed by endo-xylanase GH10

X1

X4

X5

Digested by Axe2

3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0

Time(min) Fig. 5. CE eletropherograms of AcXOS digested by Axe2 (incubation conditions: 5 h, pH 7.0 and 40 ◦ C, substrate concentration of 10 mg/mL, enzyme loading 5 ␮g/mg substrate) (A) without and (B) with subsequent hydrolysis by a GH 10 endo-(1,4)-␤xylanase from A. awamori (incubation conditions: 16 h, pH 5.0 and 40 ◦ C). Arrows ↑↓ indicate when a peak increases or decreases in relative abundance after incubation of the Axe2 digest with endo-(1,4)-␤-xylanase. Xn indicates a xylo-oligomer containing n xylosyl residues.

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xylo-triose and -tetraose confirmed that the remaining acetyl group was located on the xylosyl residues located at the non-reducing end of the xylo-triose and -tetraose (data not shown). Literature data show that acetyl groups located at the non-reducing end of an oligomer can migrate over the positions O-2, O-3 and O-4. This occurs in aqueous systems and is dependent on the pH [49]. It is anticipated that under the conditions used equilibrium of all three forms might be present. As indicated earlier both Axe2 and Axe3 are expected to remove acetyl groups when substituted to the O-2 and O-3 positions, but it is not expected that the enzymes remove the acetyl group when it is located at the O-4 position. So far, only CE16 acetyl esterases are described to deacetylate oligosaccharides at the O-4 position [35].

4. Conclusions The acetyl xylan esterases, Axe2 and Axe3 from C. lucknowense have been purified and characterized. Both enzymes performed optimally at pH 7.0. Axe2 showed a clear optimal temperature at 40 ◦ C, while Axe3 showed a much broader optimal temperature range from 34 to 45 ◦ C. Axe2 and Axe3 are able to hydrolyze acetyl groups from acetylated xylo-oligosaccharides and complex insoluble polymeric substrates. Both Axe2 and Axe3 have a preference for xylooligosaccharides. Axe2 has a higher specific activity than Axe3, especially towards oligosaccharides. Axe2 showed a higher affinity towards oligosaccharides substituted by more than 3 acetyl groups. Axe3 showed a somewhat higher affinity for larger oligosaccharides. Both enzymes seem to efficiently remove acetyl groups when they are substituted to the O-2 and O-3 positions of xylo-oligosaccharides. They were unable to cleave acetyl groups located at the non-reducing end of a xylooligosaccharide, which could possibly be located at the O-4 position of the xylose residue as a result of transmigration.

Acknowledgement This research has been financed by the Dutch Ministry of Economic Affairs through an EOS/ES grant.

References ˜ [1] Pérez J, Munoz-Dorado J, de la Rubia T, Martínez J. Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview. Int Microbiol 2002;5:53–63. [2] Zaldivar J, Nielsen J, Olsson L. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl Microbiol Biotechnol 2001;56:17–34. [3] Timell TE. Recent progress in the chemistry of wood hemicellulose. Wood Sci Technol 1967;1:45–70. [4] Ingram LO, Doran JB. Conversion of cellulosic materials to ethanol. FEMS Microbiol Rev 1995;16:235–41. [5] de Vries RP, Poulsen CH, Madrid S, Visser J. aguA, the gene encoding an extracellular alpha-glucuronidase from Aspergillus tubingensis, is specifically induced on xylose and not on glucuronic acid. J Bacteriol 1998;180:243–9. [6] Saha B. Hemicellulose bioconversion. J Ind Microbiol Biotechnol 2003;30:279–91. [7] Schoonveld-Bergmans MEF, Beldman G, Voragen AGJ. Structural features of (glucurono)arabinoxylans extracted from wheat bran by barium hydroxide. J Cereal Sci 1999;29:63–75. [8] Shibuya N, Iwasaki T. Structural features of rice bran hemicellulose. Phytochemistry 1985;30:488–94. [9] Wende G, Fry SC. O-ferulated, O-acteylated oligosaccharides as side chains of grass xylans. Phytochemistry 1991;44:1019–30. [10] Carpita N, McCann M. The functions of cell wall polysaccharides in composition and architecture revealed through mutations. Plant Soil 2002;247:71–80. [11] Das NN, Das SC, Mukherjee AK. On the ester linkage between lignin and 4-Omethyl-d-glucurono-d-xylan in jute fiber (Corchorus capsularis). Carbohydr Res 1984;127:345–8. [12] Tabahashi N, Koshijima T. Ester linkages between lignin and glucuronoxylan in lignin–carbohydrate complex from beech (Fagus crenata) wood. Wood Sci Technol 1988;22:231–41.

319

[13] Kabel MA, van den Borne H, Vincken JP, Voragen AGJ, Schols HA. Structural differences of xylans affect their interaction with cellulose. Carbohydr Polym 2007;69:94–105. [14] Kormelink FJM, Gruppen H, Voragen AGJ. Mode of action of (1→4)-beta-d-arabinoxylan arabinofuranohydrolase (AXH) and alphal-arabinofuranosidases on alkali-extractable wheat-flour arabinoxylan. Carbohydr Res 1993;249:345–53. [15] Sørensen HR, Jørgensen CT, Hansen CH, Jørgensen CI, Pedersen S, Meyer AS. A novel GH43 ␣-l-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 ␣-l-arabinofuranosidases on wheat arabinoxylan. Appl Microbiol Biotechnol 2006;73:850–61. [16] Poutanen K, Sundberg M, Korte H, Puls J. Deacetylation of xylans by acetyl esterases of Trichoderma reesei. Appl Microbiol Biotechnol 1990;33: 506–10. [17] Heneghan MN, McLoughlin L, Murray PG, Tuohy MG. Cloning, characterisation and expression analysis of ␣-glucuronidase from the thermophilic fungus Talaromyces emersonii. Enzyme Microb Technol 2007;41: 677–82. [18] Tenkanen M, Siika-aho M. An ␣-glucuronidase of Schizophyllum commune acting on polymeric xylan. J Biotechnol 2000;78:149–61. [19] Ryabova O, Vrˇsanská M, Kaneko S, Van Zyl WH, Biely P. A novel family of hemicellulolytic ␣-glucuronidase. FEBS Lett 2009;583:1457–62. [20] Shin HD, Chen R. A type B feruloyl esterase from Aspergillus nidulans with broad pH applicability. Appl Microbiol Biotechnol 2007;73:1323–30. [21] de Vries RP, van Kuyk PA, Kester HCM, Visser J. The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds. Biochem J 2002;363:377–86. [22] Faulds CB, Williamson G. Release of ferulic acid from wheat bran by a ferulic acid esterase (FAE-III) from Aspergillus niger. Appl Microbiol Biotechnol 1995;43:1082–7. [23] Biely P, MacKenzie CR, Schneider H. Acetylxylan esterase of Schizophyllum commune. Methods Enzymol 1988;160:700–7. [24] Li X-L, Skory CD, Cotta MA, Puchart V, Biely P. Novel family of carbohydrate esterases, based on identification of the Hypocrea jecorina acetyl esterase gene. Appl Environ Microbiol 2008;74:7482–9. [25] Kormelink FJM, Searle-van Leeuwen MJF, Wood TM, Voragen AGJ. Purification and characterization of three endo-(1,4)-␤-xylanases and ␤-xylosidase from Aspergillus awamori. J Biotechnol 1993;27:249–65. [26] Togashi H, Kato A, Shimizu K. Enzymatically derived aldouronic acids from Eucalyptus globulus glucuronoxylan. Carbohydr Polym 2009;78: 247–52. [27] Evtuguin DV, Tomás JL, Silva AMS, Neto CP. Characterization of an acetylated heteroxylan from Eucalyptus globulus Labill. Carbohydr Res 2003;338: 597–604. [28] Kabel MA, Carvalheiro F, Garrote G, Avgerinos E, Koukios E, Parajó JC, et al. Hydrothermally treated xylan rich by-products yield different classes of xylooligosaccharides. Carbohydr Polym 2002;50:47–56. ˜ L, Gutiérrez R, Caputo V, Peirano A, Steiner J, Eyzaguirre J. Purification and [29] Egana characterization of two acetyl xylan esterases from Penicillium purpurogenum. Biotechnol Appl Biochem 1996;24:33–9. [30] Gordillo F, Caputo V, Peirano A, Chávez R, Van Beeumen J, Vandenberghe I, et al. Penicillium purpurogenum produces a family 1 acetyl xylan esterase containing a carbohydrate-binding module: characterization of the protein and its gene. Mycol Res 2006;110:1129–39. [31] Biely P, Puchart V. Recent progress in the assays of xylanolytic enzymes. J Sci Food Agric 2006;86:1636–47. [32] Kim JF, Jeong H, Yu DS, Choi S-H, Hur C-G, Park M-S, et al. Genome sequence of the probiotic bacterium Bifidobacterium animalis subsp. lactis AD011. J Bacteriol 2009;191:678–9. [33] Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 2009;37:D233–8. [34] Hakulinen N, Tenkanen M, Rouvinen J. Three-dimensional structure of the catalytic core of acetylxylan esterase from Trichoderma reesei: insights into the deacetylation mechanism. J Struct Biol 2000;132:180–90. [35] Biely P, Mastihubova M, Tenkanen M, Eyzaguirre J, Li X-L, Vrˆsanská M. Action of xylan deacetylating enzymes on monoacetyl derivatives of 4-nitrophenyl glycosides of ␤-d-xylopyranose and ␣-l-arabinofuranose. J Biotechnol 2011;151:137–42. [36] Hinz SWA, Pouvreau L, Joosten R, Bartels J, Jonathan MC, Wery J, et al. Hemicellulase production in Chrysosporium lucknowense C1. J Cereal Sci 2009;50:318–23. [37] Verdoes JC, Punt PJ, Burlingame R, Bartels J, Van Dijk R, Slump E, et al. A dedicated vector for efficient library construction and high throughput screening in the hyphal fungus Chrysosporium lucknowense. Ind Biotechnol 2007;3: 48–57. [38] Kabel MA, de Waard P, Schols HA, Voragen AGJ. Location of O-acetyl substituents in xylo-oligosaccharides obtained from hydrothermally treated Eucalyptus wood. Carbohydr Res 2003;338:69–77. [39] Verdoes JC, Punt PJ, Burlingame RP, Pynnonen CM, Olson PT, Wery J, et al. New fungal production system. Int Patent WO/2010/107303; 2010. [40] Neuhoff V, Stamm R, Pardowitz I, Arold N, Ehrhardt W, Taube D. Essential probelms in quantification of proteins following colloidal staining with coomassie brilliant blue dyes in polyacrylamide gels, and their solution. Electrophoresis 1990;11:101–17.

320

L. Pouvreau et al. / Enzyme and Microbial Technology 49 (2011) 312–320

[41] Albrecht S, Van Muiswinkel GCJ, Schols HA, Voragen AGJ, Gruppen H. Introducing capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) for the characterization of konjac glucomannan oligosaccharides and their in vitro fermentation behavior. J Agric Food Chem 2009;57:3867–76. [42] Pouvreau L, Gruppen H, Piersma SR, Van den Broek LAM, Van Koningsveld GA, Voragen AGJ. Relative abundance and inhibitory distribution of protease inhibitors in potato juice from cv. Elkana. J Agric Food Chem 2001;49: 2864–74. [43] Albrecht S, Schols HA, van den Heuvel EGHM, Voragen AGJ, Gruppen H. CELIF–MSn profiling of oligosaccharides in human milk and feces of breast-fed babies. Electrophoresis 2010;31:1–10. [44] Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, et al. The genome sequence of the filamentous fungus Neurospora crassa. Nature 2003;422:859–68.

[45] Chávez R, Bull P, Eyzaguirre J. The xylanolytic enzyme system from the genus Penicillium. J Biotechnol 2006;123:413–33. [46] Tenkanen M, Eyzaguirre J, Isoniemi R, Faulds CB, Biely P. Comparison of catalytic properties of acetyl xylan esterases from three carbohydrate esterase families. ACS Symp Ser 2003;855: 211–29. [47] Biely P, MacKenzie CR, Puls J, Schneider H. Cooperativity of esterases and xylanases in the enzymatic degradation of acetyl xylan. Biotechnology 1986;4:731–3. [48] Mitchell DJ, Grohmann K, Himmel ME, Dale DE, Schroeder HA. Effect of the degree of acetylation on the enzymatic digestion of acetylated xylans. J Wood Chem Technol 1990;10:111–21. [49] Mastihubová M, Biely P. Lipase-catalysed preparation of acetates of 4nitrophenyl ␤-d-xylopyranoside and their use in kinetic studies of acetyl migration. Carbohydr Res 2004;339:1353–60.