Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by β-xylanase, α-l -arabinofuranosidase and β-xylosidase

Carbohydrate Research 407 (2015) 79e96

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Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by b-xylanase, a-L-arabinofuranosidase and b-xylosidase Barry V. McCleary*, Vincent A. McKie, Anna Draga, Edward Rooney, David Mangan, Jennifer Larkin Megazyme International Ireland, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 November 2014 Received in revised form 21 January 2015 Accepted 23 January 2015 Available online 7 February 2015

A range of a-L-arabinofuranosyl-(1-4)-b-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 a-L-arabinofuranosidase, by several GH10 and GH11 b-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of a-L-arabinofuranosidases and b-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific a-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum b-xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 32-a-L-Araf-(1-4)-b-D-xylobiose (A3X), 23-a-L-Araf-(1-4)-b-D-xylotriose (A2XX), 33-a-L-Araf-(1-4)-b-D-xylotriose (A3XX), 22-a-L-Araf-(1-4)-b-D-xylotriose (XA2X), 32-a-L-Araf (1-4)-b-Dxylotriose (XA3X), 23-a-L-Araf-(1-4)-b-D-xylotetraose (XA2XX), 33-a-L-Araf-(1-4)-b-D-xylotetraose (XA3XX), 23,33-di-a-L-Araf-(1-4)-b-D-xylotriose (A2þ3XX), 23,33-di-a-L-Araf-(1-4)-b-D-xylotetraose (XA2þ3XX), 24,34-di-a-L-Araf-(1-4)-b-D-xylopentaose (XA2þ3XXX) and 33,34-di-a-L-Araf-(1-4)-b-D-xylopentaose (XA3A3XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the a-L-arabinofuranosidases, with specific action on AXOS, have been combined with b-xylosidase and b-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Wheat flour arabinoxylan Arabinoxylo-oligosaccharides b-Xylanase a-L-Arabinofuranosidase b-Xylosidase Enzymic hydrolysis

1. Introduction Arabinoxylan is a significant component of cereal hemicelluloses and a knowledge of the complete degradation of this polysaccharide to its component monosaccharides is essential for the efficient conversion and utilisation of most plant biomass. Wheat endosperm contains arabinoxylan at low, but significant, levels in both soluble (~0.7% w/w) and insoluble (~1.2% w/w) forms.1 Although this represents just 2.1% of flour weight, it binds up to ~20% of the water added in making pan bread.2 Significant work has been performed on the hydrolysis of wheat arabinoxylan by endo-1,4-b-xylanase (b-xylanase) and on the characterisation of b-xylanase released arabinoxylo-

* Corresponding author. E-mail address: [email protected] (B.V. McCleary). http://dx.doi.org/10.1016/j.carres.2015.01.017 0008-6215/© 2015 Elsevier Ltd. All rights reserved.

oligosaccharides (AXOS).3e10 The structural basis for substrate and inhibitor recognition in eukaryotic GH11 b-xylanases such as that from Neocallimastix patriciarum was studied using EnXyl 11A (encoded by an environmental DNA sample) that could be cocrystallised with the ligand ferulic acid-1,5-a-L-Araf-1,4-b-D-xylotriose.11 The crystal structure of the EnXyl 11A-FAX3 complex showed that the a-L-Araf can be accommodated on O-2 and O-3 at subsites 3 and þ2. a-L-Arabinofuranosidases, which hydrolyse terminal a-L-arabinofuranose from polymeric arabinoxylans are termed arabinoxylan arabinofuranosidases (AXH) and these have been divided into two groups depending on their substrate specificities. The enzyme AXH-m acts on a-L-(1-2)- and (1-3)-linked Araf units on mono-substituted Xylp residues, whereas AXH-d3 releases only a-L-(1-3)-linked Araf units from disubstituted Xylp residues.12,13 Just three examples of AXH-d3 type a-L-arabinofuranosidases have been reported to date.12e15 Little is known about the action of specific a-L-arabinofuranosidase on defined

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oligosaccharides. The crystal structures of GH62 a-L-arabinofuranosidases from Ustilago maydis (UmAbf 62A) and Podospora anserina (PaAbf 62A) were recently published. Both enzymes readily hydrolyse wheat flour arabinoxylan releasing only a-L-Araf. U. maydis a-L-arabinofuranosidase also hydrolysed sugar beet arabinan, but not debranched arabinan, indicating that the hydrolytic action was directed exclusively towards a-1,2 and a-1,3 bonds that link side chain L-Araf to the main chains of the polymer.16 Recently, Borsenberger et al.17 reported the action of several a-L-arabinofuranosidases from Bifidobacterium adolescentis and Thermobacillus xylanilyticus on 23,33-di-a-L-Araf-(1-4)-b-D-xylotriose (A2þ3XX) and three synthetic di-arabinofuranosylated substrates and demonstrated that these substrates can be used to characterise and probe selectivity of the arabinoxylan-active a-L-arabinofuranosidases. They noted that two GH51 Abfs (TxAbf from T. xylanilyticus and AbfB from B. adolescentis) act readily on the L-Araf groups on both monosubstituted D-Xylp and di-substituted D-Xylp in heteroxylans, a point that had not previously been fully appreciated. However, the ability of Aspergillus niger a-L-arabinofuranosidase (GH51 UNIPROT Accession Number B3GQR2) to remove essentially all L-Araf groups from wheat flour arabinoxylan has been reported.13 On hydrolysis of wheat flour arabinoxylan, b-xylanases from glycoside hydrolase family 10 (GH10) are able to produce shorter chain AXOS than are GH11 xylanases, consistent with work done on the hydrolysis of 4-O-methyl-D-glucurono-D-xylan and release of aldouronic acids.18 Oligosaccharides produced on hydrolysis of the arabinoxylan from the endosperm of white wheat flour by Aspergillus species b-xylanase were fractionated on Bio-Gel P-6 and analysed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and characterised by NMR studies. AXOS from degree of polymerisation (dp) 3-14 have been characterised by Hoffmann, et al.4,5 Similar studies were performed on alkali-extracted wheat flour arabinoxylan in which two different b-xylanases from Aspergillus awamori CMI 142717 (termed I and III) were employed.6,7 While b-xylanase III produced an array of AXOS very similar to those reported by Hoffmann et al.4 with an Aspergillus sp. b-xylanase, the oligosaccharides produced by b-xylanase I were quite different. Differences in the rates of hydrolysis of insoluble wheat flour arabinoxylan by GH family 10 and 11 endo-1,4-b-xylanases have been reported by Vardakou et al.8 The patterns of feruloylated AXOS also differed. Detailed HPLC-MALDI-TOF/TOF-MS/MS studies on AXOS produced on hydrolysis of water soluble wheat flour arabinoxylan by b-xylanases from Cellvibrio japonicus (Xyl IDA; GH10) and N. patriciarum (Xyl 11A, GH11) were reported by Maslen et al.10 The structures of the AXOS produced were consistent with the known specificities of the enzymes. Methods have been published for the preparation of specific AXOS. Dekker and Richards3 reported on the production of 32-a-LAraf-(1-4)-b-D-xylobiose (A3X), 32-a-L-Araf-(1-4)-b-D-xylotriose (A3XX) and 34-a-L-Araf-(1-4)-b-D-xylotetraose (A3XXX) on hydrolysis of wheat endosperm arabinoxylan by Ceratocystis paradoxa bxylanase. A procedure for the production of A3X by hydrolysis of wheat flour arabinoxylan by a GH family 10 endo-1,4-b-xylanase from Aspergillus aculeatus was more recently described by Rantanen et al.19 (Note: nomenclature rules are based on procedures described for a-gluco-oligosaccharides,20 galactomanno-oligosaccharides,21 xylogluco-oligosaccharides22 and arabino-xylo-oligosaccharides23). Pastell et al.24 reported a step-wise enzymatic preparation of AXOS substituted singly or doubly by a-L-Araf on the non-reducing D-xylose residue of xylobiose or xylotriose. Aspergillus aculeatus b-xylanase was used in combination with B. adolescentis a-L-arabinofuranosidase (AXH-d3; Megazyme cat. no. EAFAM2) or a-L-arabinofuranosidase from Novozymes (AXH-m; not commercially available). Preparation of wheat flour arabinoxylan

with just doubly substituted D-xylosyl residues, or of wheat flour arabinoxylan with just singly substituted D-xylose residues, has been described by Soerensen et al.9 Also described were procedures to prepare AXOS mixtures containing a-L-Araf groups (1-3)-linked to non-reducing end terminal D-xylosyl residues, (1-3)-linked to internal D-Xylp residues, and (1-2)-linked to internal D-Xylp residues. Qualitative studies were performed on the action of specific a-L-arabinofuranosidases on these modified polysaccharides and oligosaccharide mixtures. These authors also described a “minimal” enzyme cocktail containing b-xylosidase and novel b-xylanase and a-L-arabinofuranosidase activities for hydrolysis of wheat flour arabinoxylan. These enzymes could be useful for development of a specific assay procedure for wheat flour arabinoxylan, but several of these are covered by patent and not generally available. The ultimate aim of the current work was to develop an enzymic procedure for specific measurement of wheat flour arabinoxylan. In this paper, we describe aspects of the action of b-xylanase GH10 and GH11 on wheat flour arabinoxylan (WAX), and on acid debranched wheat arabinoxylan (ADWAX) in the absence or presence of an AXH-d3 a-L-arabinofuranosidase. Methods for the large scale production of specific AXOS and the use of these oligosaccharides in the characterisation of the action patterns of specific aL-arabinofuranosidases are also discussed. Finally, a proposed enzyme cocktail for the hydrolysis and measurement of wheat flour arabinoxylan is described. 2. Results and discussion It is well established that b-xylanases of families GH10 and GH11 have quite different action patterns on wheat flour arabinoxylan,3e12 producing different types of AXOS.3e12,19,23e25 This is confirmed in the current study using a range of each type of bxylanase. Hydrolysis curves for the action of two GH10 and two GH11 b-xylanases on WAX (Ara:Xyl¼39:61) and ADWAX (prepared by incubating WAX at low pH and high temperature under well controlled conditionsdsee Experimental Section 4.2.4) (Ara:Xyl¼24:76) are shown in Fig. 1. As expected, the more highly substituted WAX is hydrolysed to a lesser extent than the less substituted ADWAX, and the extent of hydrolysis by the GH10 bxylanases (C. japonicus and Cellvibrio mixtus) is greater than that by

Fig. 1. The rates and extents of hydrolysis of WAX and ADWAX were studied by incubating 1300 U of b-xylanase with 500 mL of WAX or ADWAX (20 mg/mL) in 10 mM sodium acetate buffer (pH 4.5) (A. niger, N. patriciarum b-xylanase) or in 10 mM sodium phosphate buffer (pH 6.3) (C. japonicus or C. mixtus b-xylanase) at 40  C. Aliquots (0.5 mL) were removed at 0, 0.5, 1, 2 and 4 h and the b-xylanase inactivated by incubating the tubes in a boiling water bath for 5 min or by adding the 0.5 mL aliquot to 9.5 mL of 20 mM acetic acid, pH 2. All volumes were adjusted to 10 mL and 0.1 mL aliquots were removed for measurement of reducing sugar level (Nelson/Somogyi21,22) and for total carbohydrate (phenol sulphuric acid method20) and the degree of hydrolysis calculated. See Materials and Methods for full incubations conditions.

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the GH11 b-xylanases (A. niger and N. patriciarum). Very similar results were obtained with the other GH10 and GH11 b-xylanases studied (as listed in Table 1). The patterns of AXOS produced on hydrolysis of ADWAX by the four GH10 b-xylanases studied were very similar, but different from the patterns obtained with the four GH11 b-xylanases. The Bio-Gel P-2 patterns for two of each of these types of b-xylanases, C. mixtus and Thermatoga maritima b-xylanases (GH10) and N. patriciarum and A. niger b-xylanases (GH11) are shown in Fig. 2. Incubations with the GH10 b-xylanases result in a higher degree of hydrolysis and thus larger proportions of low DP oligosaccharides are produced. Included in these are (1-4)-b-xylooligosaccharides substituted (either singly or doubly) on the nonreducing end D-Xylp residue with a-L-Araf. The patterns and types of AXOS produced on hydrolysis of WAX by C. mixtus and N. patriciarum b-xylanases (Fig. 3a, WAXCM and WAXNP) were in line with that previously reported for GH10 and GH11 b-xylanases.3e10 With C. mixtus xylanase, the oligosaccharide products of DP 2e5 were xylobiose, xylotriose, 32-a-L-Araf-(1-4)-b3 D-xylobiose (A X) (Table 2), very little tetrasaccharide, but significant amounts of 23,33-di-a-L-Araf-(1-4)-b-D-xylotriose (A2,3XX) together with minor amounts of an uncharacterised pentasaccharide composed of a-L-Araf linked to (1-4)-b-D-xylotetraose. Incubations employing N. patriciarum b-xylanase resulted in a lower degree of hydrolysis of WAX with low amounts of xylose, xylobiose and xylotriose and negligible amounts of tetrasaccharide in the hydrolysis mixture. The lowest DP AXOS was XA3XX (Fig. 4, spot C; Supplementary data 5). No oligosaccharides containing (12)-linked a-L-Araf residues were found in the hydrolysates of WAX,

analysis through the Complex Carbohydrate Research Centre, Athens, Georgia, USA (see Supplementary data). Acid debranched WAX (ADWAX) was prepared by incubating WAX at low pH and high temperature under well controlled conditions (see Experimental Section 4.2.5). In this process, a-L-Araf groups are released from the (1-4)-b-D-xylan backbone of WAX much more rapidly than the xylan backbone is degraded. Due to the non-selective nature of this process, the a-L-Araf depleted backbone would be expected to contain xylose residues singly substituted by (1-2)-a-L-Araf groups as well as xylose residues singly substituted with (1-3)-a-L-Araf groups. This was confirmed by identification of the types of AXOS released on hydrolysis with bxylanase. Hydrolysis of ADWAX by C. mixtus b-xylanase (Fig. 3a; ADWAXCM) produced xylotriose and A3X as the only trisaccharides. Two a-L-Araf containing tetrasaccharides, A2XX and A3XX (Fig. 5, spot E) are produced, showing that the enzyme can cleave adjacent to, and on the non-reducing side of a D-Xylp residue substituted by either a (1-2)- or a (1-3)-a-L-Araf group, i.e. the enzyme can accommodate either substitution of the D-Xylp residue at the þ1 binding subsite. The observation that A3X is the only a-LAraf containing trisaccharide produced indicates that the C. mixtus b-xylanase cannot accommodate a (1-2)-a-L-Araf group at the 2 subsite. The ability of the enzyme to accommodate both (1-3)- and (1-2)-a-L-Araf groups at the þ1 subsite is shown by the production of A2XX, A3XX and A2,3XX on hydrolysis of ADWAX. The lesser accumulation of A3XX in the hydrolysate may be due to a more rapid further hydrolysis of this to A3X than A2XX to A2X by the C. mixtus b-xylanase.

consistent with other studies15,17e19,23e26 and also consistent with conclusions that (1-2)-linked a-L-Araf only occurs on doubly substituted D-xylosyl residues in WAX. The structures of most of the oligosaccharides reported here were confirmed by NMR and MS

The smallest L-arabinose containing AXOS produced by N. patriciarum b-xylanase on hydrolysis of ADWAX were a mixture of XA3XX and XA2XX (Fig. 6, spot C). The structures of XA3XX separately and of a mixture of XA3XX and XA2XX have been

Table 1 Enzyme, source organism, pH optima, UNIPROT Accession number, CAZY number and Megazyme Catalogue Number of the enzymes used in the current study Enzyme

Organism

pH optima

UNIPROT Accession number

CAZY number

Catalogue number

b-Xylanase

Trichoderma viride Trichoderma longibrachiatum Aspergillus niger Thermotoga maritima Cellvibrio mixtus Cellvibrio japonicas Neocallimastix patriciarum Bacillus stearothermophilus

4.5 6.0 4.5 5.0 6.5 5.0 5.5e6.0 6.5

Q9UVF9 F8W669 P55329 Q60037 O68541 P14768 P29127 P40943

GH11 GH11 GH11 GH10 GH10 GH10 GH11 GH10

E-XYTR1 E-XYTR3 E-XYAN4 E-XYLATM E-XYNBCM E-XYNBCJ E-XYLNP E-XYNBS

a-L-Arabinofuranosidase

Ustilago maydis Aspergillus niger Bacteroides ovatus BACOVA_03417 Bacteroides ovatus BACOVA_03421 Bacteroides ovatus BACOVA_03425 Bifidobacterium adolescentis

4.5 4.0 6.5 7.5 6.5e7.0 5.5e6.0

Q4P6F4 B3GQR2 A7LZZ1 A7LZZ4 A7LZZ8 B3GQR2

GH62 GH51 GH43 GH43 GH43 GH43

E-ABFUM E-AFASE E-ABFBO17 E-ABFBO21 E-ABFBO25 E-AFAM2

b-Xylosidase

Selenomonas ruminantium

5.5

O52575

GH43

E-BXSR

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Fig. 2. Bio-Gel P-2 chromatographic patterns of the oligosaccharides produced on hydrolysis of ADWAX (Ara:Xyl¼24:78) by b-xylanases from a) C. mixtus, b) T. maritima, c) A. niger and d) N. patriciarum. Hydrolysis conditions are those described in Fig. 1 and Experimental. The hydrolysates (~10 g) were concentrated to 80 mL and a 20 mL aliquot applied to the Bio-Gel P2 column (595 cm) and eluted with degassed, deionised water at 60  C. Aliquots (20 mL) were collected and samples (5e10 mL) were analysed for total carbohydrate using the phenol-sulphuric acid method. The numbers in the figures are the DP of the oligosaccharide.

confirmed by NMR studies (Supplementary data 5 and 6) and by enzymatic degradation experiments with a-L-arabinofuranosidase, b-xylanase and b-xylosidase. On incubation with C. mixtus bxylanase, XA3XX (Fig. 6, spot C) was hydrolysed to XA3X (Fig. 6, spot D) and xylose. The structure of XA3X was confirmed by NMR and MS studies (Supplementary data 2). In the same way, the mixture of the two pentasaccharides (Fig. 6, spot E) were hydrolysed to a mixture of XA3X and XA2X and D-xylose (Fig. 6, spots FeH) and completely hydrolysed to a-L-Araf and (1-4)-b-D-xylotetraose on incubation with A. niger a-L-arabinofuranosidase (Fig. 6, spot L) or U. maydis a-L-arabinofuranosidase (Fig. 6, spot M). There was no hydrolysis of the oligosaccharides by Selenomonas elenomonas ruminantium b-xylosidase (Materials and Methods 4.2.8), showing that substitution by a-L-Araf on the penultimate D-xylosyl residue at the non-reducing end of an AXOS prevents cleavage of the terminal D-xylosyl residue. The smallest AXOS containing a D-Xylp residue doubly substituted with a-linked L-Araf in the N. patriciarum b-xylanase hydrolysate of WAX or ADWAX is a hexasaccharide. HPAEC analysis of this fraction using a Dionex ICS5000þ DP equipped with a Dionex CarboPac PA200 analytical column showed one major oligosaccharide peak (~95%). Incubation of this fraction with U. maydis a-L-arabinofuranosidase resulted in no hydrolysis of the major hexasaccharide fraction (Fig. 7B), while the second fraction (~5%) was completely hydrolysed to (1-4)-b-D-xylopentaose (confirmed by TLC) and L-Araf (i.e. derived from the hydrolysis of a-L-Araf-b-D-xylopentaose). The major hexasaccharide fraction was completely hydrolysed by B. adolescentis a-L-arabinofuranosidase to a pentasaccharide and L-Araf (Fig. 7C) confirming that it contains a doubly substituted D-xylosyl residue (see Table 2). Hydrolysis with B. adolescentis a-L-arabinofuranosidase at pH 6.0, followed by U. maydis a-L-arabinofuranosidase at ~pH 5.0, gave complete hydrolysis of the

oligosaccharide fractions to L-Araf and (1-4)-b-D-xylotetraose (major fraction) and (1-4)-b-D-xylopentaose (very minor fraction) (Fig. 7D). There was no hydrolysis of the oligosaccharide on incubation with either S. ruminantium b-xylosidase or C. mixtus bxylanase Materials and Methods 4.2.8). On the basis of these studies, it was concluded that the structure of the major hexasaccharide component was XA2þ3XX, and this has been confirmed by NMR studies (Supplementary data 7). The heptasaccharide fraction produced on hydrolysis of WAX by N. patriciarum b-xylanase (termed WAXNP Hepta) (Fig. 8A) contains a major component (~60%; TLC RXyl¼0.55; HPAEC retention time 12.59) and a minor component (~40%; TLC RXyl¼0.33; HPAEC retention time 10.46). Incubation of WAXNP Hepta with a mixture of U. maydis and B. adolescentis a-L-arabinofuranosidases at pH 5.0 (Materials and Methods 4.2.8) gave complete hydrolysis to L-Araf and xylopentaose. The major component (I, Fig. 8; HPAEC-retention time 12.59) was not hydrolysed by B. adolescentis a-L-arabinofuranosidase, indicating that this oligosaccharide contained only D-Xylp residues singly substituted by L-Araf residues. This was confirmed by the fact that U. maydis a-L-arabinofuranosidase gave complete hydrolysis of this component to L-Ara and (1-4)-b-D-xylopentaose (as shown by HPAEC patterns). NMR confirmed this structure as XA3A3XX (Supplementary data 8). The second heptasaccharide fraction (II, Fig. 8; HPAEC retention time also 10.46) is not hydrolysed by U. maydis a-L-arabinofuranosidase, but is hydrolysed by B. adolescentis a-L-arabinofuranosidase to LAraf and two lower DP oligosaccharides (major component ~70%; HPAEC retention time 8.77, and minor component ~30%; HPAEC retention time also 9.05); NMR (Supplementary data 8) confirmed that the major component is XA2þ3XXX, but enzymic studies (as above) indicate that a second minor component, possibly XXA2þ3XX4,7,25 was also present. The two oligosaccharides obtained on hydrolysis of WAXNP Hepta with B. adolescentis a-L-

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Fig. 3. a & b. Bio-Gel P-2 chromatographic patterns of the oligosaccharides produced on hydrolysis of WAX (39% Ara) and ADWAX (24% Ara) by C. mixtus (CM) and N. patriciarum (NP) b-xylanase in the presence (3a) or absence (3b) of B. ovatus BACOVA_03417 a-L-arabinofuranosidase. WAX or ADWAX (1 L, 10 mg/mL) in 10 mM sodium phosphate buffer (pH 6.3) was incubated with 1.0 KU of C. mixtus or 1.5 KU of N. patriciarum b-xylanase for 16 h at 40  C in the presence or absence of 2.8 KU of B. ovatus BACOVA_03417 a-L-arabinofuranosidase. Reaction was terminated by heating solutions to ~95  C in a microwave oven. Solutions were concentrated to 100 mL and aliquots (20 mL) fractionated on Bio-Gel P2 at 60  C. Aliquots (5e10 mL) were analysed for total carbohydrate using the phenol sulphuric acid method. Individual fractions were collected, concentrated and adjusted to 10 mg/ mL for analysis by HPLC, TLC and ion chromatography. The numbers in the figures are the DP of the oligosaccharide.

arabinofuranosidase (Materials and Methods 4.2.17), were recovered separately by chromatography on Bio-Gel P-2. Hydrolysis of these individual fractions (hexasaccharide, ~30% and heptasaccharide, ~70%) by C. mixtus b-xylanase (see Materials and Methods 4.2.8) is shown in Fig. 8 and the scheme below. The heptasaccharide fraction (Fig. 8D) is hydrolysed to a hexasaccharide (presumed to be XA3A3X) and free D-Xylp (Fig. 8E), consistent with the structure determined for this oligosaccharide

by NMR (Supplementary data 8), namely XA3A3XX. The b-xylanase removes the D-Xylp residue at the reducing end of the oligosaccharide. The lesser hexasaccharide fraction (Fig. 8B; ~30%) is hydrolysed by C. mixtus b-xylanase to give XA2XX and D-Xyl (presumably from XA2XXX) and xylobiose plus A2XX (presumably from XXA2XX) as shown in the scheme above and consistent with structures of AXOS, which contain D-Xylp residues that are doubly substituted by L-Araf residues.

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The above studies made it possible to produce sufficient quantities of many of the oligosaccharides listed in Table 2 to allow a comparison of the action patterns of several a-L-arabinofuranosidases on these oligosaccharides. The relative rates of hydrolysis of a range of AXOS by several a-L-arabinofuranosidases are shown in Table 3a & b. a-L-Arabinofuranosidases from B. adolescentis (GH43) and Bacteroides ovatus BACOVA_03417 (GH 43) specifically hydrolysed (1-3)-a-L-Araf groups on doubly substituted D-xylosyl residues (i.e. AXH-d3). The specific activities on the oligosaccharide A2þ3XX were greater than 200 U/mg. Interestingly, B. adolescentis (GH43) displayed significantly higher hydrolytic activity on the pentasaccharide than on the native polysaccharide while B. ovatus BACOVA_03417 (GH43) exhibited the opposite preference. Neither of these enzymes had any action on a-L-Araf groups singly substituted either (1-3)- or (1-2)- to Dxylosyl residues in (1-4)-b-D-xylo-oligosaccharides, whether within the xylo-oligosaccharide chain or at the non-reducing end. Of the a-L-arabinofuranosidases studied, the A. niger enzyme (GH51) is unique in that it had greatest activity on A3X. It also rapidly hydrolysed p-nitrophenyl a-L-arabinofuranoside and (1-3)a-L-Araf branches on sugar beet arabinan. This enzyme releases LAraf from all of the AXOS currently studied. U. maydis a-L-arabinofuranosidase has no ability to hydrolyse a-L-Araf units from doubly substituted Xylp residues. It only hydrolyses a-L-Araf groups, which are singly substituted to Xylp residues. It displays highest activity on arabinoxylan and on a-L-Araf units (1-2)-linked [and to a lesser extent, (1-3)-linked] to a Xylp residue within a (1-4)-b-Dxylo-oligosaccharide chain. The other B. ovatus a-L-arabinofuranosidases (BACOVA_03425 and BACOVA_03421; GH43) have no action on a-L-Araf on doubly substituted Xylp residue, but rapidly hydrolyse a-L-Araf (1-3)-linked and (1-2)-linked to a Xylp residue located within a xylo-oligosaccharide chain. They also rapidly hydrolyse a-L-Araf units singly linked to the non-reducing terminal Xylp residue in (1-4)-b-D-xylo-oligosaccharides where the DP of the xylo-oligosaccharide is 3 or greater. With this information available, it is possible to design experiments to optimise the production of specific AXOS. Rantanen et al.19 reported on the production of AXOS with one Xylp residue doubly substituted with a-L-arabinofuranosyl residues using an a-Larabinofuranosidase (AXH-m) from Novozymes A/S, followed by Aspergillus aculeatus b-xylanase. Presumably, the a-L-arabinofuranosidase was from Humicola insolens as described by Soerensen

et al.9 in Patent WO 2006/114095 AI. In the current experiments, we used a commercially available a-L-arabinofuranosidase from U. maydis to remove a-L-Araf groups that were singly substituted to D-Xylp residues within the xylan chain of WAX, together with hydrolysis by C. mixtus b-xylanase. The enzymes were added together into the incubation mixture because, if incubation was performed stepwise as described by Rantenan et al.,19 the arabinoxylan precipitated from solution as soon as the a-L-Araf content reduced to ~23%, making the polysaccharide less susceptible to hydrolysis by b-xylanase, and thus reducing yields. A typical oligosaccharide pattern obtained following such an incubation is shown in Fig. 9. The oligosaccharide of interest, A2þ3XX (peak 5 in Fig. 9) was obtained in ~16% yield (based on carbohydrate determination on this fraction using the phenol-sulphuric acid procedure as a percentage of the carbohydrate added to the Bio-Gel P-2 column). The monosaccharide fraction consisted of L-arabinose and D-xylose in a ratio of 4:1; the sole disaccharide was (1-4)-b-D-xylobiose; the trisaccharide fraction consisted of equal proportions of (1-4)-b-D-xylotriose and A3X; and there was essentially no tetrasaccharide. A3X presumably persisted in the hydrolysate because U. maydis a-Larabinofuranosidase (GH62) has little action on the terminally linked a-L-Araf unit in this oligosaccharide (Table 3). The level of this trisaccharide could be reduced, or removed totally, by adjusting the levels of the two enzymes used or by performing an initial incubation with excess levels of U. maydis a-L-arabinofuranosidase with just sufficient C. mixtus b-xylanase (GH10) to prevent precipitation of the polysaccharide. A3X was obtained devoid of (1-4)b-D-xylotriose by incubating the trisaccharide fraction with bxylosidase as described in Materials and Methods 4.2.15. XA3XX was obtained by hydrolysing WAX with N. patriciarum bxylanase (GH11), but yields were relatively low. Good yields of a mixture of XA3XX and XA2XX were obtained by hydrolysing ADWAX with N. patriciarum b-xylanase, and even higher levels were obtained when B. adolecentis a-L-arabinofuranosidase (GH43) was added into the incubation mixture (Fig. 3b; ADWAX NP and ADWAX B NP). The purity of the various oligosaccharides produced as described here and purified by Bio-Gel P-2 chromatography, is shown in Figs. 4e6. Single major spots were obtained on TLC for the mono-components. The pentasaccharide fraction produced on hydrolysis of ADWAX by N. patriciarum b-xylanase appears as two distinct spots. The purity of these compounds was further

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confirmed by HPAEC using a Dionex system (Fig. 10A). Structures have been confirmed by NMR and GC-MS and are consistent with literature reports7 (refer to Supplementary data 5 and 6). With the known structure of a range of AXOS, and the known action patterns of the various enzymes described here, it is possible to produce other structures that may prove valuable in characterising the action patterns of enzymes, or in studies of the binding of particular oligosaccharides into the active sites of specific enzymes during crystallisation. For example, pure A2XX has been prepared by incubation of A2þ3XX with B. adolescentis a-L-arabinofuranosidase. The a-L-Araf containing pentasaccharides produced on hydrolysis of WAX or ADWAX by N. patriciarum b-xylanase are susceptible to hydrolysis by C. mixtus b-xylanase to produce XA3X (from the WAX derived XA3XX) or a mixture of XA2X and XA3X (from the ADWAX derived mixture of XA2XX and XA3XX) (Fig. 10).

arabinofuranosidase (170 U/mL) as an ammonium sulphate suspension. Full details are given in Materials and Methods 4.2.10. In the standard assay protocol, 0.05 mL of WAX (0e1.6 mg/mL) was mixed with 0.05 mL of 100 mM sodium succinate buffer (pH 5.5) and 5 mL of enzyme mixture added, mixed and incubated at 40  C. Released L-arabinose and D-xylose were measured with mixtures of galactose dehydrogenase/galactose mutarotase and of xylose dehydrogenase/xylose mutarotase as described in Materials and Methods 4.2.4). The effect of changing the concentration of, or deleting specific a-L-arabinofuranosidases from the enzyme mixture on the rates and extents of hydrolysis of WAX is also shown. The maximum conversion of water soluble WAX to Larabinose and D-xylose using the optimal enzyme mixture is ~90%. No increase could be achieved by adding more of any one of the enzymes used. This would suggest the presence of other groups in

The structure of XA3X has been confirmed by NMR (Supplementary data 2). The scheme in Fig. 11 summarises the various hydrolytic enzymatic pathways employed to produce the compounds of interest reported herein using either WAX or ADWAX as starting materials. Described here is a tool-box of enzymes that can be used to produce AXOS of specific structures for use in further studies on the action patterns of various enzymes. Also, from these studies, an insight into the action patterns of the enzymes has been obtained, allowing their use in the development of a specific assay procedure for arabinoxylan. In the development of such a procedure using a combination of enzymes, a knowledge of the pH activity, pH stability and temperature stability is essential so that optimal incubation conditions are chosen. The pH activity of each of the enzymes considered for this assay are shown in Fig. 12, and the stabilities of these enzymes under conditions likely to be used in the assay (pH 5.5, 40  C) and at concentrations that would be used in the assay are shown in Fig. 13. Each of the enzymes, except B. ovatus BACOVA_03417 a-L-arabinofuranosidase (AXH-d3), are stable at pH 5.5 and 40  C for 2 h. The B. ovatus BACOVA_03417 a-Larabinofuranosidase is rapidly inactivated under the incubation conditions employed. Thus, B. adolescentis a-L-arabinofuranosidase, also an AXH-d3 a-L-arabinofuranosidase, was used in preference. The extent of hydrolysis of water soluble WAX by the selected mixture of enzymes is shown in Fig. 14. This mixture of b-xylanase, b-xylosidase and three a-L-arabinofuranosidases was determined empirically, and the optimal mixture is shown as “A” in Fig. 14. The enzyme suspension used to hydrolyse the arabinoxylan consisted of N. patriciarum b-xylanase (1300 U/mL), S. ruminantium b-xylosidase (200 U/mL), B. adolescentis a-L-arabinofuranosidase (300 U/ mL), U. maydis a-L-arabinofuranosidase (75 U/mL) and A. niger a-L-

the arabinoxylan, such as ferulic acid, are preventing complete hydrolysis of WAX to monosaccharides. 3. Conclusions In these studies, the action of various b-xylanases on wheat flour arabinoxylan and acid debranched arabinoxylan in the absence or presence of AHX-d3 a-L-arabinofuranosidase has been studied and the oligosaccharides produced, up to DP 7, have been characterised by both instrumental and enzymic techniques. Several a-L-arabinofuranosidases have been produced/purified and the action pattern of these on a range of AXOS have been studied. Combinations of these enzymes have been employed in the optimisation of the production of specific AXOS and in the measurement of water soluble wheat flour arabinoxylan. Research at this stage is directed towards identifying and characterising the components in water soluble WAX that resist hydrolysis, which will assist identification of additional enzymes that may be required for quantitative analysis of this polysaccharide. Application of the described arabinoxylan assay procedure to measurement of water insoluble WAX in wheat endosperm tissue is currently underway. 4. Experimental 4.1. Materials

b-Xylanase from Trichoderma viride (cat. no. E-XYTR1; UNIPROT Accession no. Q9UVF9; GH11), Trichoderma longibrachiatum (cat. no. E-XYTR3; UNIPROT Accession no. F8W669; GH11), Aspergillus niger (cat. no. E-XYAN4; UNIPROT Accession no. P55329; GH11), Thermatoga maritima (cat. no. E-XYLATM; UNIPROT Accession no.

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Table 2 Structures, nomenclature chromatographic details and key references for arabinoxylo-oligosaccharides studied in this paper Structure and name

HPAEC retention timeb (min)

Shorthand name26

Mobility on TLC (RXyl)a

A3X

0.95

8.43

2,7

A2XX

0.72

7.60

24

A3XX

0.83

8.13

2

XA3X

0.82

8.04

7,25

XA2X

0.61

8.41

A2þ3XX

0.77

10.36

7,24,27

XA3XX

0.69

8.88

4,7,25

Key references

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Table 2 (continued ) Structure and name

D-Xylose L-Arabinose

1,4-b-D-Xylobiose 1,4-b-D-Xylotriose 1,4-b-D-Xylotetraose 1,4-b-D-Xylopentaose 1,4-b-D-Xylohexaose

HPAEC retention timeb (min)

Shorthand name26

Mobility on TLC (RXyl)a

XA2XX

0.58

8.56

27

XA2þ3XX

0.58

9.99

4,7,25,27

XA3A3XX

0.55

12.59

4,7,25,27

XA2þ3XXX

0.33

10.46

4

X Ara XX XXX XXXX XXXXX XXXXXX

1.00 0.87 0.85 0.73 0.56 0.41 0.25

2.51 2.42 2.84 3.23 3.94 5.92 7.07

Key references

a TLC performed on Merck DC-Alufolien Kieselgel 60 0.2 mm pre-coated plates and the plates were developed once with 7:1:2 n-propanoleethanolewater. RXyl is the retention time of the oligosaccharide relative to D-xylose. b HPAEC was performed using a Dionex ICS5000þ DP equipped with CarboPac PA200 guard and analytical columns as described in Materials and Methods 4.2.1.

Q60037; GH10), Cellvibrio mixtus (cat. no E-XYNBCM; UNIPROT Accession no. O68541; GH10), Cellvibrio japonicus (cat. no. EXYNACJ; UNIPROT Accession no. P14768; GH10), Neocallimastix patriciarum (cat. no. E-XYLNP; UNIPROT Accession no. P29127; GH11), Bacillus stearothermophilus (cat. no. E-XYNBS; UNIPROT Accession no. P40943; GH10) plus a-L-arabinofuranosidases from Aspergillus niger (cat. no. E-AFASE; UNIPROT Accession no. B3GQR2; GH51) and Bifidobacterium adolescentis (cat. No. E-AFAM2; UNIPROT Accession no. Q5JB56; GH43), b-xylosidase from Selenomonas ruminantium (cat. no. E-BXSR; UNIPROT Accession no. O52575; GH43) and a mixture of xylose dehydrogenase/xylose mutarotase (cat. no. E-XYLMUT) were obtained from Megazyme International. Plasmid for a-L-arabinofuranosidase from Ustilago maydis UmAbf62_INRA (cat. no. E-ABFUM; UNIPROT Accession no. Q4P6F4, GH62) was obtained from Jean-Guy Berrin, (INRAdBiotechnologie des Champignons Filamenteux UMR 1163dPolytech Marseille  des Sciences de Luminy 13009 Marseille France). Plasmids Faculte

for a-L-arabinofuranosidases from Bacteroides ovatus BACOVA_03417 (cat. no. E-ABFBO17; UNIPROT Accession no. A7LZZ1, GH43), Bacteroides ovatus BACOVA_03425 (cat. no. EABFBO25; UNIPROT Accession no. A7LZZ8; GH43) and Bacteroides ovatus BACOVA_03421 (cat. no. E-ABFBO21; UNIPROT Accession no. A7LZZ4, GH43) were obtained from Dr. David Bolam, Institute for Cell and Molecular Biosciences, Medical School, Framlington Place, Newcastle University, Newcastle upon Tyne, UK) (Table 1). Wheat flour arabinoxylan medium viscosity (WAX; Lot 40601; cat. no. P-WAXYM; Ara: Xyl¼39:61), wheat flour arabinoxylan low viscosity (LVWAX; cat. no. P-WAXYL, Lot 120601; Ara: Xyl¼39:61), sugar beet arabinan (cat. no. P-ARAB; Lot 80902b), debranched arabinan (cat. no. P-DBAR; Lot 100401b) and L-Arabinose/D-Galactose Assay Kit (cat. no. K-ARGA) were obtained from Megazyme International. Oligosaccharides used in this study were A3X (see Table 2), A3XX, A2XX, XA3XX, XA2XX and A2þ3XX and were prepared as described in this paper and characterised by NMR and

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Sigma Aldrich, Lennox Laboratory Supplies or Lab unlimited (Carl Stuart Group) and were analytical reagent grade. Bio-Gel P-2, (<45 mm) was obtained from Bio-Rad, UK. 4.2. Methods

Fig. 4. Thin layer chromatography of the pentasaccharide fractions obtained on hydrolysis of WAX and ADWAX by N. patriciarum b-xylanase. A. xylose, xylotriose and xylopentaose; B. xylobiose, xylotetraose and xylohexaose; C. pentasaccharide from N. patriciarum xylanase on WAX; D. pentasaccharide mixture from N. patriciarum xylanase on ADWAX; E. xylose, xylotriose and xylopentaose; F. xylobiose, xylotetraose and xylohexaose.

enzymic hydrolysis using specific a-L-arabinofuranosidases, b-Dxylosidase from S. ruminantium and b-xylanase from C. mixtus. These oligosaccharides are also available commercially from Megazyme International. All other chemicals used were purchased from

Fig. 5. Thin layer chromatography of various oligosaccharides obtained on hydrolysis of WAX or ADWAX with N. patriciarum or C. mixtus b-xylanases. A. xylose, xylotriose and xylopentaose; B. xylobiose, xylotetraose and xylohexaose; C. A2þ3XX obtained on hydrolysis of ADWAX by C. mixtus b-xylanse; D. A2XX obtained by hydrolysis of A2þ3XX with B. adolescentis a-L-arabinofuranosidase; E. A2XX plus A3XX obtained on hydrolysis of ADWAX by C. japonicus b-xylanase; F. A3X from hydrolysis of ADWAX by C. mixtus bxylanase; G. xylose, xylotriose and xylopentaose; H. xylobiose, xylotetraose and xylohexaose.

4.2.1. General methods Carbohydrate concentrations were determined using the phenol-sulphuric acid procedure28 with appropriate D-Xylp/L-Araf standard solutions. Reducing sugar concentrations were determined using the Nelson-Somogyi29,30 procedure with either D-Xylp or L-Araf as standard. Thin layer chromatography (TLC) was performed on Merck DC-Alufolien Kieselgel 60 (0.2 mm pre-coated plates. Aliquots (10 mL) of oligosaccharides (10 mg/mL) were applied to the plates, and the plates were developed once with 7:1:2 n-propanoleethanolewater. Spots were detected by spraying with 5% sulfuric acid in ethanol and heating at 110  C in a small oven. HPLC was performed using an HPLC equipped with an oven to maintain a column temperature of 90  C and a 50 mL injection loop. The column routinely employed was a Waters Sugar-Pak 6.5 by 300 mm (part number WAT085188). Operating conditions were temperature, 90  C; mobile phase, distilled water plus EDTA (50 mg/L); and flow rate, 0.5 mL/min. Bio-Gel P-2 chromatography was performed on a column (595 cm) of Bio-Gel P-2 (<45 mm) maintained at 60  C. Samples (approx. 20 mL, 120 mg/mL) were applied via a sample loop and the column was eluted with degassed, deionised water at approximately 2.5 mL/min using a Waters 515 HPLC pump. Elution patterns were monitored by refractive index using a Knauer RI detector K2401 and fractions of 20 mL were collected using a Buchi C660 fraction collector. In general, individual peaks of carbohydrate were collected, concentrated to dryness and redissolved in deionised water to a concentration of 10 mg/mL. Oligosaccharides for IC analysis were diluted in water (>18 MU, Lennox Laboratories, cat. no. CH951L) to a concentration of approx. 10 pM and analysed by high-performance anion-exchange chromatography (HPAEC) using a Dionex ICS5000þ DP equipped with Dionex CarboPac PA200 guard and analytical columns (3250 mm) maintained at a temperature of 30  C. Appropriately diluted solutions of NaOH (50e52% in H2OdFluka, cat no 72064-500ML) and anhydrous NaOAc (electrochemical gradedThermoFisher, cat. no. 059326) were used as eluents. A stepwise linear gradient was applied over 30 min by mixing solutions of 100 mM NaOH (Solution A) and 120 mM NaOAc in 100 mM NaOH (Solution B) with a constant flow rate of 0.5 mL/ min. The gradient applied was as follows: from 100% Solution A to 45% Solution B over 5 min; from 45% to 70% Solution B over 4 min and from 70% to 100% Solution B over 1 min. The eluting mixture was maintained at 100% Solution B for an additional 15 min, before returning to 100% Solution A over 2 min 100% Solution A was maintained for an additional 3 min to allow column reconditioning (total method runtime¼30 min). Eluted oligosaccharides were monitored by PAD detection using an Au electrode (waveform: Carbohydrate, standard quad). Chromatograms were recorded and analysed using Chromeleon 7.0 and peaks were assigned by using oligosaccharide standards [(1-4)-b-Dxylo-oligosaccharides and those detailed in Table 2]. 4.2.2. Measurement of enzyme activity b-Xylanase was assayed by incubating 0.2 mL of suitably diluted enzyme with 0.5 mL of medium viscosity wheat flour arabinoxylan (Lot 40601; Ara:Xyl¼39:61, 10 mg/mL) in 100 mM sodium acetate buffer (pH 4.0, 4.5 or 5.0) or sodium phosphate buffer (pH 6 or 6.5) at 40  C and the reaction was terminated after 0, 3, 6, 9 and 12 min by adding 0.5 mL of Nelson/Somogyi29,30 reagent C with vigorous stirring of the reaction tube. Colour was developed using the

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Fig. 6. Thin layer chromatography of the DP 5 AXOS obtained on hydrolysis of WAX or ADWAX by N. patriciarum b-xylanase and further hydrolysed by C. mixtus b-xylanase. A. xylose, xylotriose and xylopentaose; B. xylobiose, xylotetraose and xylohexaose; C. XA3XX obtained on hydrolysis of WAX by N. patriciarum b-xylanase; D. XA3X; the fraction shown in “C” after incubation with C. mixtus b-xylanase and purification by Bio-Gel P2 chromatography; E. XA3XX plus XA2XX from hydrolysis of ADWAX by N. patriciarum b-xylanase; F, G & H. fraction shown in “E” after incubation with C. mixtus b-xylanase for 10, 30 and 60 min, respectively; I. xylose, xylotriose and xylopentaose; J. xylobiose, xylotetraose and xylohexaose; K. XA3XX plus XA2XX from hydrolysis of ADWAX by N. patriciarum b-xylanase; L. fraction “K” hydrolysed to the limit with A. niger a-L-arabinofuranosidase; M. fraction “K” hydrolysed to the limit with U. maydis a-L-arabinofuranosidase; N. xylose, xylotriose and xylopentaose; O. xylobiose, xylotetraose and xylohexaose P. L-arabinose. See Materials and Methods for full incubations conditions.

standard Nelson/Somogyi29,30 reducing sugar method. Xylose standards (50 mg, in the presence of 0.5 mL of arabinoxylan substrate), in quadruplicate, were developed concurrently. b-Xylosidase was routinely assayed by incubating 0.2 mL of suitably diluted enzyme with 0.2 mL of p-nitrophenyl b-D-xylopyranoside (10 mM) in sodium succinate buffer (pH 5.3) at 40  C. Reaction was

terminated by adding 3 mL of 2% tris solution (pH 11) and colour was measured at 400 nm. a-L-Arabinofuranosidase activity was assayed either on wheat flour arabinoxylan or on p-nitrophenyl a-Larabinofuranoside. In the former assay, 0.2 mL of suitably diluted enzyme was incubated with 0.5 mL of wheat flour arabinoxylan (10 mg/mL) in 100 mM sodium acetate buffer (pH 4e5.5) or sodium

Fig. 7. HPAECePAD analysis of the hexasaccharide fraction obtained on hydrolysis of wheat arabinoxylan with N. patriciarum b-xylanase. A. Hexasaccharide fraction; B. fraction “A” hydrolysed to the limit with U. maydis a-L-arabinofuranosidase; C. fraction “A” hydrolysed to the limit with B. adolescentis a-L-arabinofuranosidase; D. fraction “A” hydrolysed to the limit with B. adolescentis plus U. maydis a-L-arabinofuranosidases (see Experimental 4.2.7 for incubation details).

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phosphate buffer (pH 6e7.5) at 40  C and the reaction was terminated after 0, 3, 6, 9 and 12 min by adding 0.5 mL of Nelson/ Somogyi29,30 reagent D. Colour was developed using the standard Nelson/Somogyi29,30 reducing sugar method. L-Arabinose standards (50 mg, in the presence of 0.5 mL of arabinoxylan substrate), in quadruplicate, were developed concurrently. In the latter assay, a-L-arabinofuranosidase was assayed by incubating 0.2 mL of suitably diluted enzyme with 0.2 mL of p-nitrophenyl a-L-arabinofuranoside (5 mM) in sodium acetate buffer (pH 4.0) at 40  C. Reaction was terminated by adding 3 mL of 2% tris solution (pH 11) and colour was measured at 400 nm.

Fig. 8. Thin layer chromatography of the heptasaccharide fraction obtained on hydrolysis of WAX by N. patriciarum b-xylanase and further hydrolysed by B. adolescentis a-L-arabinofuranosidase and C. mixtus b-xylanase. A. heptasaccharide fraction obtained from Bio-Gel P-2 chromatography of the hydrolysate of WAX by N. patriciarum bxylanase; B. the hexasaccharde fraction obtained from the hydrolysis of “A” to the limit with B. adolescentis a-L-arabinofuranosidase and separated by Bio-Gel P-2 chromatography; C. “B” hydrolysed to the limit by C. mixtus b-xylanase; D. the heptasaccharde fraction obtained on hydrolysis of “A” to the limit with B. adolescentis a-L-arabinofuranosidase and separated by Bio-Gel P-2 chromatography; E. “D” hydrolysed to the limit by C. mixtus b-xylanase; F. xylose, xylotriose and xylopentaose; G. xylobiose, xylotetraose and xylohexaose. See Materials and Methods for full incubations conditions.

4.2.3. Definition of units of enzyme activity One Unit of enzyme activity is defined as the amount of enzyme required to hydrolyse one micromole of glycosidic bonds or release one micromole of product per minute from the defined substrate at a defined pH and at 40  C. One Unit of b-xylanase is the amount of enzyme required to release one micromole of D-Xylp reducingsugar equivalents from medium viscosity wheat arabinoxylan (Lot 40601; Ara:Xyl¼39:61, 10 mg/mL) per minute at 40  C and the optimal pH for activity of the enzyme (Table 1). One Unit of a-Larabinofuranosidase is routinely defined as the amount of enzyme required to release one micromole of L-Araf from medium viscosity wheat arabinoxylan (Lot 40601; 10 mg/mL) per minute at 40  C and the optimal pH for activity of the enzyme, measured either as reducing sugar using the Nelson/Somogyi29,30 reducing sugar method; or measuring L-Araf using the Megazyme L-Arabinose/DGalactose test kit as described in the kit booklet. For A. niger a-Larabinofuranosidase, one Unit of activity is defined as the amount of enzyme required to release one micromole of p-nitrophenol from p-nitrophenyl-a-L-arabinofuranoside (5 mM) per minute at pH 4.0 and 40  C. One Unit of S. ruminantium b-xylosidase is defined as the amount of enzyme required to release one micromole of p-nitrophenol from p-nitrophenyl-b-D-xylopyranoside (5 mM) per minute at pH 5.3 and 40  C. The activity of this enzyme on (1-4)-b-D xylobiose is 1.4-fold higher than that on p-nitrophenyl-b-Dxylopyranoside.

Table 3 Relative rates of hydrolysis of arabinoxylo-oligosaccharides by a-L-arabinofuranosidase enzymes. a. Activities expressed relative to a rate of 100 for the most rapidly hydrolysed substrate. b. Activities expressed as Units/mg of enzyme protein Substrate

(a) A3Xa A2XXa XA3XXa XA2XX and XA3XXa A2þ3XXa Arabinoxylan (wheat)b Arabinan (sugar beet)b Debranched Arabinan (sugar beet)b p-NP-a-L-Arafc (b) A3Xa A2XXa XA3XXa XA2XX and XA3XXa A2þ3XXa Arabinoxylan (wheat)b Arabinan (sugar beet)b Debranched Arabinan (sugar beet)b p-NP-a-L-Arafc a b c

B. adolescenti Q5JB56 (GH43)

B. ovatus BACOVA_03417 A7LZZ1 (GH43)

B. ovatus BACOVA_03421 A7LZZ4 (GH43)

B. ovatus BACOVA_03425 A7LZZ8 (GH43)

U. maydis Q4P6F4 (GH62)

A. niger B3GQR2 (GH51)

0 0 0 0 100 23 0.18 0 0.01

0 <0.50 0 0 32 100 0.24 0 0.156

9.2 100 100 100 0 56 d d d

7 90 37 100 0 83 0.04 0 0.328

2 9 21 88 0 100 0.01 0 0.012

100 16 4 4 3 0.3 7.7 0.23 22

0 0 0 0 285 67 0.54 0 0.031

0 <0.02 0 0 208 650 0.53 0 0.325

2.6 28.5 28.5 28.5 0 16 d d d

1.9 24.9 10.2 27.7 0 23 0.013 0 0.328

0.6 2.7 6.3 26.4 0 30 0.003 0 0.012

133 21 5 5 4 0.7 10.3 0.31 29

Rates of hydrolysis of AXOS were determined by measuring the rates of release of L-arabinose as described in Materials and Methods 4.2.9. Rates of hydrolysis of arabinoxylan (10 mg/mL), arabinan (10 mg/mL) and debranched arabinan (10 mg/mL) were measured as described in Materials and Methods 4.2.2. Action on p-NP-a-L-Araf was as described in Materials and Methods 4.2.2.

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and then neutralised by addition of 1.3 M NaOH and diluted to 100 mL with distilled water. Aliquots (0.2 mL) were analysed for LAraf and D-Xylp using the L-Arabinose/D-Galactose assay kit (cat. no. K-ARGA) and a mixture of Xylose dehydrogenase/xylose mutarotase (cat. no. E-XYLMUT). The procedure in the L-Arabinose/DGalactose kit booklet was followed exactly except that 0.2 mL of sample was analysed (as compared to 0.1 mL in the booklet method). After measurement of L-Araf, xylose dehydrogenase/ xylose mutarotase suspension (5 mL, E-XYLMUT) was added to each cuvette, the contents mixed thoroughly and the tubes incubated at 37  C for 10 min. The absorbance at 340 nm was again measured and the content of D-Xylp calculated.

Fig. 9. Bio-Gel P2 chromatographic pattern of the oligosaccharides produced on hydrolysis of WAX with C. mixtus b-xylanase in the presence of U. maydis a-L-arabinofuranosidase. See Materials and Methods for full incubations conditions. The numbers in the figures are the DP of the oligosaccharide.

4.2.4. Acid hydrolysis of WAX and ADWAX and determination of L -arabinose and D-xylose content Concentrated HCl (0.55 mL, 12.1 M) was added to 5 mL of ice cold WAX or ADWAX (~5 mg/mL) in water and mixed thoroughly. The tubes were capped and incubated in a boiling water bath for 1 h

Fig. 10. HPAEC-PAD analysis of the pentasaccharide fraction obtained on hydrolysis of ADWAX by N. patriciarum b-xylanase before (A) and after (B) incubation with C. mixtus b-xylanase (see Experimental 4.2.7 for incubation details).

4.2.5. Preparation of acid-debranched WAX (ADWAX) 2 kg of low viscosity WAX (Lot 70501, 7 cSt, Megazyme International cat. no. P-WAXYL) was added to 100 L of deionised water and dissolved by stirring and heating to 80  C. Concentrated formic acid (4 L) was added and the pH was adjusted to 1.45 by the addition of 5 M HCl. This solution was incubated for 1 h at 80  C. The solution was quickly transferred in 50 L lots into two 250 L polypropylene tanks containing 150 L of industrial methylated spirits (IMS) and mixed with an explosion proof Silverson homogeniser. The precipitated ADWAX was allowed to settle over 2 h and then recovered by filtration through a nylon filtration screen. The recovered polysaccharide was suspended in 25 L of IMS, homogenised and then stirred for 2 h. The arabinoxylan was again recovered on nylon screen and squeezed free of liquid. This washing process was repeated and the ADWAX was recovered by freeze-drying over 2 days. The recovered ADWAX had an arabinose:xylose ratio of ~24:76. 4.2.6. Hydrolysis of WAX and ADWAX by b-xylanase 60 g of low viscosity WAX (Ara: Xyl¼39:61; ~10 cSt) was added to 2.0 L of deionised water at ~80  C and dissolved by stirring on a magnetic stirrer for 15e20 min. To this solution, either 15 mL of 2 M sodium acetate buffer, pH 4.5 or 60 mL of 0.5 M sodium phosphate buffer, pH 6.3, was added. The volume was adjusted to 3.0 L with distilled water and the pH adjusted if necessary. This solution was mixed and lots of 500 mL were transferred into Duran® bottles and placed in a water bath set at 40  C. b-Xylanase (1300 U) was added to each bottle and the mixture incubated at 40  C for 6 h. 60 g of ADWAX (Ara:Xyl¼24:76; ~5 cSt) was added to 2.0 L of deionised water at ~80  C and stirred on a magnetic stirrer for 15e20 min, and then in microwave oven, heating to ~95  C (until the solution became clear). The beaker was immediately removed from the oven and either 15 mL of 2 M sodium acetete buffer, pH 4.5 or 60 mL of 0.5 M sodium phosphate buffer, pH 6.3, was added to give a concentration of 10 mM. The volume was immediately adjusted to 2.8 L with distilled water and the pH was adjusted if required. The volume was then quickly adjusted to 3 L and the solution mixed and poured in lots of 500 mL into Duran® bottles and cooled to 40  C in a sink full of cold water. As soon as the temperature reached 40  C, the appropriate xylanase (1300 U) was added and the bottles stirred and placed in a water bath set at 40  C and incubated for 6 h. The b-xylanases studied were from 1. T. viride; 2. A. niger; 3. T. martima; 4. C. japonicus; 5. T. longibrachiatum; 6. B. stearothermophilus; 7. C. mixtus and 8. N. patriciarum as described in materials section. b-Xylanases 1-4 were incubated with substrates at pH 4.5, and b-xylanases 5-8 were incubated with substrates at pH 6.3. The degree of hydrolysis was followed by removing samples (0.5 mL) from all incubations except those containing T. martima and B. stearothermophilus b-xylanases at 0, 0.5, 1, 2 and 4 h and incubated at 100  C for 5 min. Water (9.5 mL) was then added to

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Fig. 11. Schematic overview of the enzymatic pathways employed to produce the AXOS discussed.

each tube and the contents mixed. For the incubations containing T. martima and B. stearothermophilus b-xylanases, the 0.5 mL samples were added to 9.5 mL of 20 mM acetic acid solution, pH 3. Aliquots (0.1 mL) of all diluted solutions were analysed for reducing sugar using the Nelson/Somogyi reducing sugar procedure.29,30 DXylp (50 mg) was analysed in quadruplicate with each set of samples. The degree of hydrolysis was calculated as the reducing sugar

value (mg/mL) as a percentage of total carbohydrate determined using the phenol-sulphuric acid procedure28 with an L-Araf:D-Xylp standard (100 mL of a 40:60 arabinose:xylose solution at 0.5 mg/ mL). After incubation for 4 h with WAX or ADWAX, the reactions employing all b-xylanases except 3 and 6 (thermostable) were terminated by heating the solutions to ~90  C in a microwave oven. Those containing the thermostable b-xylanases were terminated by

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Fig. 12. Optimal pH for activity of a-L-arabinofuranosidase from A. niger, U. maydis and B. adolescentis, b-xylosidase from S. ruminatium and b-xylanase from N. patriciarum. For U. maydis a-L-arabinofuranosidase, B. adolescentis a-L-arabinofuranosidase and N. patriciarum b-xylanase assays were performed on WAX (10 mg/mL) at 40  C in citrate/phosphate buffer (pH 3-6.0). S. ruminatium b-xylosidase was assayed with pnitrophenyl b-D-xyloside (5 mM) at 40  C in citrate/phosphate buffer (pH 3-6.0) and A. niger a-L-arabinofuranosidase with p-nitrophenyl a-L-arabinofuranosidase (5 mM) at 40  C in citrate/phosphate buffer (pH 3-6.0) as described in Materials and Methods.

lowering the pH to 2 with addition of 5 M HCl and storing the solutions overnight at 4  C. These solutions (~500 mL) were concentrated by rotary evaporation to near dryness, adjusted to 80 mL with deionised water and centrifuged at 10,000 rpm for 10 min to remove insoluble material. 20 mL of this solution was applied directly to a Bio-Gel P-2 column (595 cm) and eluted with deionised water at approximately 2.5 mL/min at 60  C. TLC, HPLC and Dionex chromatography were performed as described in the general methods section. 4.2.7. Hydrolysis of WAX, ADWAX and enzyme debranched WAX (EDWAX) by b-xylanase WAX (lot 40601; Ara:Xyl¼39:41) and ADWAX (Lot 140501; Ara:Xyl¼22:78) (1 L, 10 mg/mL) in 10 mM sodium phosphate buffer, pH 6.3 were incubated with either 1.5 KU of N. patricarium or 1 KU

Fig. 13. Stability of enzymes at pH 5.5 and 40  C. The described enzymes were incubated at 40  C and pH 5.5 as described in Materials and Methods. Samples were removed at various time intervals, chilled on ice and appropriately diluted to concentrations suitable for assay. The enzymes were: A. A. niger a-L-arabinofuranosidase in BSA (0.5 mg/mL); B. U. maydis a-L-arabinofuranosidase in BSA (0.5 mg/mL); C. N. patriciarum b-xylanase in BSA (0.5 mg/mL); D. S. ruminatium b-xylosidase in BSA (0.5 mg/mL); E. B. adolescentis a-L-arabinofuranosidase in BSA (0.5 mg/mL), and F. B. ovatus BACOVA_03417 a-L-arabinofuranosidase in BSA (0.5 mg/mL).

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Fig. 14. Time course of hydrolysis of soluble wheat flour arabinoxylan (WAX) by an optimised enzyme cocktail (mixture A) and variations: A. 0.05 mL of WAX (82 mg) plus 0.05 mL of 100 mM sodium succinate buffer (pH 5.5) plus 5 mL of an enzyme mixture containing; N. patriciarum b-xylanase (1300 U/mL); S. ruminantium b-xylosidase (200 U/mL); B. adolescentis a-L-arabinofuranosidase (700 U/mL); U. maydis a-L-arabinofuranosidase (75 U/mL) and A. niger a-L-arabinofuranosidase (170 U/mL); B. mixture A but with 2.5-fold higher U. maydis a-L-arabinofuranosidase; C. mixture A but with 40% of A. niger a-L-arabinofuranosidase; D. mixture A but with no U. maydis a-L-arabinofuranosidase; E. mixture A but with no A. niger a-L-arabinofuranosidase; and F. mixture A but with no B. adolescentis a-L-arabinofuranosidase. The broken line shows the concentration of WAX in the solution as determined by acid hydrolysis followed by neutralisation and specific measurement of L-arabinose and D-xylose.

of C. mixtus b-xylanase for 16 h at 40  C. Parallel incubations were performed where 2.8 KU of B. ovatus BACOVA_03417 a-L-arabinofuranosidase was also added. All reactions were terminated by heating the solutions to ~95  C in a microwave oven. Solutions were analysed for degree of hydrolysis using the Nelson-Somogyi29,30 reducing sugar method and phenol sulphuric total carbohydrate procedure.28 The bulk solutions were concentrated to 100 mL and an aliquot was chromatographed on Bio-Gel P-2. Samples of the total hydrolysate and individual fractions recovered from Bio-Gel P2 were analysed by TLC, HPLC and HPAEC-PAD as described above. 4.2.8. Hydrolysis of xylo-oligosaccharides and AXOS by a-Larabinofuranosidases, b-xylosidase and C. mixtus b-xylanase To 0.5 mL of oligosaccharide (10 mg/mL in deionised water) was added either 0.1 mL of S. ruminantium b-xylosidase (50 U/mL on pNP-b-xylanopyranoside) in 50 mM sodium succinate buffer, pH 5.5; 0.1 mL of U. maydis a-L-arabinofuranosidase (100 U/mL on WAX) in 50 mM sodium acetate buffer, pH 4.5; A. niger a-L-arabinofuranosidase (100 U/mL on p-NP-a-L-arabinofuranoside) in 50 mM sodium acetate buffer, pH 4.0; 0.1 mL of B. ovatus BACOVA_03417 aL-arabinofuranosidase (100 U/mL on WAX) in 50 mM sodium phosphate buffer, pH 6.3; 0.1 mL of B. adolescentis a-L-arabinofuranosidase (100 U/mL on WAX) in 50 mM sodium maleate buffer, pH 5.5; or C. mixtus b-xylanase (100 U/mL on WAX) in 50 mM sodium phosphate buffer, pH 6.3, and the tubes incubated at 40  C for 20 and 60 min. Reactions were terminated by incubation of the reaction tubes at ~95  C for 2 min. Tube contents were transferred to microfuge tubes and centrifuged at 13,000 rpm for 5 min. The supernatant solutions were analysed by TLC (10 mL samples), HPLC and HPAEC-PAD. 4.2.9. Determination of the rates of hydrolysis of AXOS by a-Larabinofuranosidases To 0.05 mL of oligosaccharide (20 mg/mL) plus 0.05 mL of appropriate buffer, 0.1 mL of suitably diluted a-L-

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arabinofuranosidase was added and incubated at 40  C. The reaction was terminated at 3, 6, 9 and 12 min by heating the tubes in a boiling water bath for 3 min. The solutions were transferred to microfuge tubes and centrifuged in a microfuge (13,000 rpm, 5 min). Aliquots (0.1 mL) were removed and analysed for L-Araf using a Megazyme International L-Arabinose/D-Galactose assay kit. Zero time points were obtained by heating the enzyme solution in buffer in a boiling water bath for 3 min before adding the substrate and heating a further 2 min. The buffers employed with the different a-L-arabinofuranosidases were: for A. niger a-Larabinofuranosidased100 mM sodium acetate buffer, pH 4.0 plus 0.5 mg/mL BSA; for U. maydis a-L-arabinofuranosidased100 mM sodium acetate buffer, pH 4.5 plus 0.5 mg/mL BSA; for B. adolescentis a-L-arabinofuranosidased100 mM sodium maleate buffer, pH 5.5 plus 0.5 mg/mL BSA; for B. ovatus BACOVA_03417 a-Larabinofuranosidased100 mM sodium phosphate buffer, pH 6.5 plus 0.5 mg/mL BSA; for B. ovatus BACOVA_03421 a-L-arabinofuranosidase; 100 mM sodium phosphate buffer, pH 7.0; and for B. ovatus BACOVA_03425 a-L-arabinofuranosidase; 100 mM sodium phosphate buffer, pH 6.5 plus 0.5 mg/mL BSA. The individual a-Larabinofuranosidases were prepared by centrifuging an ammonium sulphate suspension of the specific enzyme (~10 mg) and dissolving in the appropriate buffer as mentioned above and chromatographing through a Bio-Rad PD10 desalting column in the appropriate buffer. Fractions of 1 mL were collected and those containing the enzyme were pooled (usually 3 mL) and stored on ice awaiting use. The enzymes were then used either undiluted or diluted up to 2000-fold to obtain an activity suitable for the assay. All enzymes except b-xylosidase were stable to freezing and thawing. 4.2.10. Measurement of water soluble wheat flour arabinoxyan A solution containing wheat flour arabinoxylan (0.05 mL, ~80 mg) in water and 100 mM sodium succinate buffer (0.05 mL, pH 5.5) was transferred to the bottom of several glass test tubes suitable for use in a Statfax® 4500 spectrophotometer (Megazyme cat no D-STATFAX) and the tubes placed in a dry hot-block at 40  C. Enzyme suspension (5 mL) was then transferred to the bottom of the tubes and the tubes incubated at 40  C for 10, 20, 40, 60 and 90 min and the reaction terminated by addition of 2.0 mL of buffer (pH 8.5) from the Arabinose/Galactose assay kit. A zero time point was prepared by adding the buffer (pH 8.5) to 0.05 mL of arabinoxylan solution plus 0.055 mL of sodium succinate buffer (pH 5.5). LArabinose was determined using the procedure in the L-Arabinose/ D-Galactose kit booklet. After measurement of L-Araf, xylose dehydrogenase/xylose mutarotase suspension (5 mL, E-XYLMUT) was added to each cuvette, the contents mixed thoroughly and the tubes incubated at 37  C for 10 min. The absorbance at 340 nm was again measured and the content of D-Xylp calculated. This enzyme mixture contains N. patriciarum b-xylanase (1300 U/mL on wheat arabinoxylan), S. ruminantium b-xylosidase (200 U/mL on p-nitrophenyl b-xylopyranoside), A. niger a-L-arabinofuranosidase (170 U/mL on p-nitrophenyl a-L-arabinofuranoside), U. maydis a-L-arabinofuranosidase (75 U/mL on wheat arabinoxylan) and B. adolescentis a-L-arabinofuranosidase (300 U/ mL on wheat arabinoxylan) all as an ammonium sulphate suspension. 4.2.11. Stability of enzymes used for arabinoxylan determination at 40  C, pH 5.5 over two hours Each enzyme in the mixture used to measure wheat arabinoxylan was diluted in 100 mM sodium succinate buffer (pH 5.5), or the same buffer plus BSA (0.5 mg/mL), to the same protein concentration as present in the final incubation mixture as used in arabinoxylan measurement. An aliquot (3.0 mL) of each of these

enzyme solutions were incubated at 40  C for up to 2 h. Aliquots (0.2 mL) were removed at 0, 20, 40, 60, and 120 min and transferred to 12 mL polypropylene tubes containing 2 mL of 100 mM sodium succinate buffer (pH 5.5) containing 0.02% sodium azide and BSA (0.5 mg/mL) on ice. At the end of the incubation, all of these enzyme solutions were further diluted with the appropriate volume of 100 mM sodium succinate buffer (pH 5.5) containing 0.02% sodium azide and BSA (0.5 mg/mL) to give the correct dilution for assay based on the results of the preliminary experiments. Each enzyme was assayed using the standard assay protocol described in this paper. 4.2.12. Production of doubly substituted AXOS and specifically A2,3XX 1 kg of low viscosity WAX was dissolved in 50 L of deionised water containing 1 L of 0.5 M sodium phosphate buffer (pH 6) with homogenisation at 60  C with a Silverson homogeniser. The solution was cooled to 40  C and the pH adjusted to 6.0 if necessary. U. maydis a-L-arabinofuranosidase (2.7 KU) and C. mixtus b-xylanase (38 KU) were added and the solution incubated at 40  C for 20 h. Reaction was terminated by heating the solution to 90  C and concentrated by rotary evaporation to a total carbohydrate concentration of 120 mg/mL and centrifuged at 12,000 rpm to remove insoluble material. This solution was then fractionated on Bio-Gel P-2 at 60  C with deionised water as the eluent. (Note: U. maydis a-L-arabinofuranosidase has no action on a-L-Araf residues doubly substituted to D-Xylp, but rapidly hydrolyses -a-L-Araf residues singly substituted to internal D-xylosyl residues within xylooligosaccharide chains and also hydrolyses singly substituted a-LAraf residues on non-reducing terminal D-Xylp residues in xylooligosaccharides, but more slowly (see Table 3). 4.2.13. Production of singly substituted AXOS internally linked (12)- and (1-3)- to (1-4)-b-D-xylo-oligosaccharides and specifically XA3XX and XA2XX 1 kg of ADWAX (24% arabinose) was dissolved in 25 L of 2% w/v sodium hydroxide solution at approx. 40  C with homogenisation. This was diluted to 50 L with water at approx. 40  C and adjusted to pH 6.0 by addition of solid maleic acid with stirring. N. patriciarum b-xylanase (80 KU) and B. adolescentis a-L-arabinofuranosidase (28 KU) were added and the solution incubated at 40  C for 6 h. Reaction was terminated by heating the solution to 90  C and concentrated by rotary evaporation to a total carbohydrate concentration of 120 mg/mL and centrifuged at 12,000 rpm to remove insoluble material. This solution was then fractionated on Bio-Gel P-2 at 60  C with deionised water as the eluate. (Note: B. adolescentis a-L-arabinofuranosidase has no action on a-L-Araf residues singly substituted to D-xylosyl residues, but rapidly hydrolyses (1-3)-linked -a-L-Araf residues that are doubly substituted to D-xylosyl residues in xylo-oligosaccharide chains (see Table 2). 4.2.14. Production of XA3XX 1 kg of low viscosity WAX was dissolved in 50 L of 10 mM sodium maleate buffer (pH 6.5) and homogenised at 60  C with a Silverson homogeniser. The solution was cooled to 40  C and the pH adjusted to 6.0 if necessary. N. patriciarum b-xylanase (100 KU) was added and the solution incubated at 40  C for 6 h. Reaction was terminated by heating the solution to 90  C and concentrated by rotary evaporation to 15 L. IMS (45 L) was added with continual stirring to precipitate higher molecular weight AXOS. The solution was stored at room temperature to allow the precipitate to settle. The clear supernatant solution was carefully decanted and the residue was collected on nylon filter cloth and squeezed free of liquid, which was collected and added to the supernatant solution. The solution was concentrated to a thick syrup and re-dissolved in

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water to a concentration of 120 mg/mL and fractionated by Bio-Gel P-2 chromatography to recover XA3XX and oligosaccharides of other DP. 4.2.15. Preparation of A3X The trisaccharide fraction recovered from Bio-Gel P-2 chromatography of the C. mixtus hydrolysate of ADWAX (100 mL, 5 g) in 10 mM sodium maleate buffer (pH 5.3) was incubated with 500 U of S. ruminantium b-xylosidase at 40  C for 1 h. The reaction was terminated by incubating the solution in a boiling water bath for 5 min and the solution was concentrated and chromatographed on Bio-Gel P-2 to separate A3X from D-xylose and salts. 4.2.16. Preparation of XA3X and a mixture of XA3X and XA2X A preparation of XA3XX (50 mL, 0.5 g) (recovered from N. patriciarum b-xylanase hydrolysis of WAX) in 10 mM sodium maleate buffer (pH 6.3) was incubated with 1 KU of C. mixtus bxylanase at 40  C for 1 h. The reaction was terminated by incubating the solution in a boiling water bath for 5 min and the solution was concentrated and chromatographed on Bio-Gel P-2 to recover pure XA3X. Alternatively, a mixture of XA3XX plus XA2XX (50 mL, 0.5 g) (recovered from N. patricairium b-xylanase hydrolysis of ADWAX) in 10 mM sodium maleate buffer (pH 6.3) was incubated with 1 KU of C. mixtus xylanase at 40  C for 1 h. The reaction was terminated by incubating the solution in a boiling water bath for 5 min and the solution was concentrated and chromatographed on Bio-Gel P-2 to recover a mixture of pure XA3X plus XA2X. 4.2.17. Hydrolysis with B. adolescentis a-L-Arabinofurnosidase of the heptasaccharide fraction obtained on hydrolysis of wheat flour arabinoxylan with N. patriciarum b-xylanase A preparation of the heptasaccharide fraction (50 mL, 0.4 g) (recovered from N. patriciarum b-xylanase hydrolysis of WAX) in 10 mM sodium acetate buffer (pH 5.5) was incubated with 200 U of B. adolescentis a-L-arabinofurnosidase at 40  C for 1 h. The reaction was terminated by incubating the solution in a boiling water bath for 5 min and the solution was concentrated by rotary evaporation and chromatographed on Bio-Gel P-2 to recover a heptasaccharide fraction (~60%) and a hexacaccharide fraction (~35%) and free L-Araf. The heptasaccharide and heptasaccharide fractions were concentrated and adjusted to 10 mg/mL. 4.2.18. NMR spectroscopy (performed at the Complex Carbohydrate Research Center, Athens Georgia, USA) Each sample (~10 mg) was deutero-exchanged twice by lyophilisation in D2O (99.9% D, Aldrich). The sample was dissolved in 0.3 mL D2O (99.96% D, Cambridge Isotope), 10 mL 20% acetone in D2O was added, and the sample was placed into 3-mm NMR tube. NMR spectra were acquired at 600 (1H) or 150 MHz (13C) on a Varian Inova-600 instrument at 25  C. One-dimensional proton and two-dimensional gCOSY, zTOCSY, ROESY, gHSQC and gHMBC spectra were acquired. Chemical shifts were referenced relative to DSS (0.000 ppm) by setting the chemical shifts of acetone to 2.218 ppm in the proton dimension and 33.0 ppm in the carbon dimension. 1-D proton spectrum was signal-averaged from 32 scans. The 2-D gCOSY, zTOCSY spectra were acquired in 16 scans per increment and 128 increments, the ROESY and gHSQC spectra in 32 scans and 128 increments, and the gHMBC spectrum in 64 scans and 96 increments. Spectral width was 3600 Hz for the proton spectrum, 11,000 Hz for the carbon spectrum. Acquisition time was 2.5 s for the 1-D proton spectrum and 150 ms for the 2-D spectra. Mixing time for the TOCSY experiment was 80 ms.

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4.2.19. Linkage analysis (performed at the Complex Carbohydrate Research Center, Athens Georgia, USA) For glycosyl linkage analysis, the sample was permethylated, depolymerised, reduced, and acetylated; and the resultant partially methylated alditol acetates (PMAAs) analysed by gas chromatography-mass spectrometry (GC-MS) as described by York et al.31 Initially, 500 mg of the sample was suspended in about 200 mL of dimethyl sulfoxide and placed on a magnetic stirrer for 2 days. Each sample was then permethylated by the method of Ciukanu and Kerek32 (treatment with sodium hydroxide and methyl iodide in dry DMSO). The sample was subjected to the NaOH base for 15 min then methyl iodide was added and left for 40 min. The base was then added for 15 min again and finally more methyl iodide was added for 40 min. This addition of more methyl iodide and NaOH base was to insure complete methylation of the sample. Following sample workup, the permethylated material was hydrolysed using 2 M trifluoroacetic acid (2 h in sealed tube at 121  C), reduced with NaBD4, and acetylated using acetic anhydride/trifluoroacetic acid. The resulting PMAAs were analysed on an Agilent 7890 A GC interfaced to a 5975 C MSD (mass selective detector, electron impact ionisation mode); separation was performed on a 30 m Supelco 2330 bonded phase fused silica capillary column. 4.2.20. MALDI-TOF mass spectrometry (performed at the Complex Carbohydrate Research Center, Athens Georgia, USA) MALDI-TOF MS was obtained on Bruker ultrafleXtreme MALDITOF MS instrument with FlexControl software. 2% DHBA in 50% aqueous methanol was used as a matrix. Samples were de-salted prior to MS analysis by treating samples with DOWEX 50Wx8 in  NHþ 4 form overnight at 4 C. The matrix solution (1 mL) was deposited first on the target, then an equal volume of the sample (1 mg/mL in pure water) was deposited. The MS was obtained in reflector positive mode at 60% of the full laser power. Data was processed by Bruker FlexAnalysis software. Acknowledgements The authors thank Dr. David Bolam, Institute for Cell and Molecular Biosciences, Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH for supplying the plasmids for the a-L-arabinofuranosidases from B. ovatus. The authors also thank Dr. Jean-Guy Berrin, INRA, Laboratoire de Biotechnologie des Champignons Filamenteux, Marseilles, France for supplying the plasmid for the a-L-arabinofuranosidase from U. maydis. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.carres.2015.01.017. References 1. 2. 3. 4. 5. 6. 7. 8. 9.

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