# A new synergistic relationship between xylan-active LPMO and xylobiohydrolase to tackle recalcitrant xylan

Anastasia Zerva, Christina Pentari, Sacha Grisel, Jean-Guy Berrin, Evangelos Topakas

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Anastasia Zerva, Christina Pentari, Sacha Grisel, Jean-Guy Berrin, Evangelos Topakas. A new synergistic relationship between xylan-active LPMO and xylobiohydrolase to tackle recalcitrant xylan. Biotechnology for Biofuels, 2020, 13 (1), 10.1186/s13068-020-01777-x. http://dx.doi.org/10.1186/s13068-020-01777-x

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# A new synergistic relationship between xylan-active LPMO and xylobiohydrolase to tackle recalcitrant xylan

Anastasia Zerva<fnref n="1" />,<fnref n="2" />, Christina Pentari<fnref n="1" />, Sacha Grisel<fnref n="2" />, Jean-Guy Berrin<fnref n="2" />* and Evangelos Topakas<fnref n="1" />* ID

**Abstract**

**Background:** Hemicellulose accounts for a significant part of plant biomass, and still poses a barrier to the efficient saccharification of lignocellulose. The recalcitrant part of hemicellulose is a serious impediment to the action of cellulases, despite the use of xylanases in the cellulolytic cocktail mixtures. However, the complexity and variety of hemicelluloses in different plant materials require the use of highly specific enzymes for a complete breakdown. Over the last few years, new fungal enzymes with novel activities on hemicelluloses have emerged. In the present study, we explored the synergistic relationships of the xylan-active AA14 lytic polysaccharide monooxygenase (LPMO), *PcAA14B*, with the recently discovered glucuronoxylan-specific xylanase *TtXyn30A*, of the (sub)family GH30_7, displaying xylobiohydrolase activity, and with commercial cellobiohydrolases, on pretreated natural lignocellulosic substrates.

**Results:** *PcAA14B* and *TtXyn30A* showed a strong synergistic interaction on the degradation of the recalcitrant part of xylan. *PcAA14B* was able to increase the release of xylobiose from *TtXyn30A*, showing a degree of synergism (DS) of 3.8 on birchwood cellulosic fibers, and up to 5.7 on pretreated beechwood substrates. The increase in activity was dose- and time- dependent. A screening study on beechwood materials pretreated with different methods showed that the effect of the *PcAA14B-TtXyn30A* synergism was more prominent on substrates with low hemicellulose content, indicating that *PcAA14B* is mainly active on the recalcitrant part of xylan, which is in close proximity to the underlying cellulose fibers. Simultaneous addition of both enzymes resulted in higher DS than sequential addition. Moreover, *PcAA14B* was found to enhance cellobiose release from cellobiohydrolases during hydrolysis of pretreated lignocellulosic substrates, as well as microcrystalline cellulose.

**Conclusions:** The results of the present study revealed a new synergistic relationship not only among two recently discovered xylan-active enzymes, the LPMO *PcAA14B*, and the GH30_7 glucuronoxylan-active xylobiohydrolase *TtXyn30A*, but also among *PcAA14B* and cellobiohydrolases. We hypothesize that *PcAA14B* creates free ends in the xylan polymer, which can be used as targets for the action of *TtXyn30A*. The results are of special importance for the design of next-generation enzymatic cocktails, able to efficiently remove hemicelluloses, allowing complete saccharification of cellulose in plant biomass.

**Keywords:** Synergism, AA14, GH30, Xylanase, Xylobiohydrolase, Lignocellulose

**Background**

Recently, accumulating data on the discovery of novel enzyme activities tend to revolutionize the way microbial lignocellulose degradation was perceived during the last 30 years. Major breakthroughs in this field, such as the discovery of lytic polysaccharide monooxygenases

<footnote>*Correspondence: jean-guy.berrin@inrae.fr; vtopakas@chemeng.ntua.gr</footnote>

<footnote n="1">¹ Industrial Biotechnology and Biocatalysis Group, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, 15780 Athens, Greece</footnote>

<footnote n="2">² INRAE, Aix Marseille University, Biodiversité Et Biotechnologie Fongiques (BBF), UMR1163, 13009 Marseille, France</footnote>
(LPMOs, [1]) and more recently enzymes with xylobio-
hydrolase activity [2, 3] pave the way for major changes
in the established theories regarding lignocellulose deg-
radation. More specifically, for hemicellulose, the dis-
covery of xylan-active AA14 LPMOs [4], as well as the
conformations of xylan polymers interacting with cellu-
lose [5–7], shed new light on the hemicelluloses structure
and degradation pathways. According to a recent study,
lytic xylan oxidases belonging to AA14 family selectively
oxidize xylan polymers that cover cellulose fibrils, while
no activity is detected on soluble xylan polymers, or
underlying cellulose fibrils [4]. The explanation for this
preference was based on recent discoveries regarding
the conformations xylan polymers can receive when in
contact with cellulose [6]. The authors hypothesized that
AA14 LPMOs are active only on the twofold screw xylan
polymers, acting as a shield of cellulose against micro-
bial attack, in contrast with threefold screw soluble xylan
polymers. The flat twofold screw conformation of xylan
is recalcitrant to degradation by xylanase and displays a
certain degree of ‘crystallinity’, which might be necessary
for LPMO activity. This hypothesis is in line with earlier
reports on the activity of cellulose-acting AA9 LPMOs
on cellulose-associated xylan [8]. At the same time,
Couturier et al. found that AA14 LPMOs produce only
traces of C1-oxidized products, and therefore, the AA14
LPMO activity is detected only in synergism with a GH11
xylanase [4]. AA14 LPMOs also significantly boost the
hydrolytic effect of cellulolytic cocktails of standard com-
position, on woody substrates.

Aside from AA14 LPMOs, which selectively oxidize recalcitrant xylan, AA9 LPMOs have been reported to target xylan associated to cellulose [8, 9]. A single LPMO belonging to AA9 family has been reported to oxidize isolated xylans (PMO9A_MALCI, [10]), together with a bacterial AA10 LPMO (*Kp*LPMO10A, [11]), although with different regioselectivity; *Kp*LPMO10A released exclusively C4-oxidized sugars, while PMO9A_MALCI was shown to produce both C1- and C4-oxidized products.

On the other hand, the recent discovery of the first
fungal enzymes with xylobiohydrolase activity [2, 3]
opens up new pathways for xylan degradation. GH30_7
xylanases not only act specifically on glucuronoxylan in
an endo-manner, releasing uronic acid-decorated xylo-
oligosaccharides (UXOS), but they can also act in an exo-
manner, releasing xylobiose [2, 3] from the non-reducing
end, or xylose from the reducing end [12] of xylan poly-
mers, or xylo-oligosaccharides (XOS). The recent discov-
ery of such novel enzymatic activities raised numerous
questions regarding the role and significance of such
xylobiohydrolases in the decomposition of lignocellulose
in natural ecosystems, and also regarding the potential

of such enzymes in the design of biomass-degrading
enzyme mixtures. Moreover, the activity of GH30_7 xyla-
nases in natural woody substrates could shed light on the
substrate preference and biological role of such novel
enzymes.

Aside from the obvious significance of experimental
data on single enzymes, the recalcitrant and complex
structure of plant cell walls requires the coordinate action
of a broad range of enzymes with varied specificities, act-
ing synergistically, for the complete degradation of bio-
mass. Therefore, the focus on enzyme combinations and
their effects on lignocellulose is of equal importance.
Enzyme synergism is the enhancement of substrate con-
version observed through the combined action of two or
more enzymes. Some enzyme combinations with strong
synergistic effects are well established in the literature,
such as the cellulases–xylanases synergism and the syn-
ergism among the cellulose-acting enzymes, such as
CBHs and EGs [13, 14]. Thus, it is widely accepted that
xylan-degrading enzymes are indispensable in LCB deg-
radation, because the removal of xylan increases the
accessibility of cellulose-binding sites for cellulases [15,
16]. On the other hand, the synergism between cellulose-
acting LPMOs and cellulases is well documented [13, 17,
18].

In the present work, the role of a xylan-active LPMO
from *Pycnoporus coccineus* (*PcAA14B*) in the degrada-
tion of (hemi)cellulose was investigated through explo-
ration of its synergistic relationships with a xylanase of
the GH30_7 family (*TtXyn30A*) from *Thermothelomyces
thermophila* (previously named *Myceliophthora ther-
mophila* and *Sporotrichum thermophile*), and commercial
cellobiohydrolases, on natural lignocellulosic substrates.

**Results**

Synergism between PcAA14B LPMO and TtXyn30A
xylanase

As previously reported, PcAA14B LPMO acts synergis-
tically with a family GH11 endoxylanase (AnXyn11) in
the degradation of xylan-containing substrates [4]. In
the present work, the synergism between PcAA14B and
TtXyn30A was first studied on wood cellulosic fibers,
containing 20% xylan, similarly to Couturier et al. [4].
The cellulosic fibers' substrate and the AnXyn11 xyla-
nase were used as previously [4], to obtain comparable
results. As shown in Fig. 1, the increase in XOS release
was much higher in the case of TtXyn30A, compared to
the synergistic action of AnXyn11 xylanase. More specifi-
cally, the increase in XOS release in the case of TtXyn30A
was 338.6 ± 13.7%, corresponding to a degree of syner-
gism (DS) 3.83 ± 0.08, while in the case of AnXyn11, the
increase was only 62.8 ± 2.4%, corresponding to a DS of
1.60 ± 0.02. However, the release of total oligosaccharides
<figure><img src="image_3.png" /></figure>

was much higher with *AnXyn11* (up to 264.5 µM, Additional file 1. Table S1) than with *TtXyn30A* (up to 88.6 µM, Additional file 1. Table S1), since the only xylo-oligosaccharide detected in the supernatant of *TtXyn30A* reactions was xylobiose.

To establish that the observed increase was indeed the result of a true synergistic relationship between *PcAA14B* and *TtXyn30A*, the next step was to investigate the release of soluble XOS over time. Of note, as the major product of *TtXyn30A* is xylobiose, all quantifications were based on xylobiose concentration. As shown in Fig. 2a, the release of xylobiose reaches a plateau already after 4 h of reaction with the use of

*TtXyn30A* alone, while with the addition of *PcAA14B* the release of xylobiose is constantly increasing up to 24 h. This result is a strong indication that the action of *PcAA14B* is constantly creating new initiation sites for *TtXyn30A*. From the above results, it is strongly suggested that *TtXyn30A* alone is not capable of hydrolyzing the recalcitrant part of xylan.

In an attempt to gain further insight on the synergistic relationship between these two enzymes, we tested the effect of *PcAA14B* loading on the xylobiose release from *TtXyn30A* on cellulosic fibers. As shown in Fig. 2b, the addition of equimolar amount of *PcAA14B* to *TtXyn30A* increased xylobiose release almost
<figure><img src="image_4.png" /><figcaption>**Fig. 2** a Time course of the xylobiose release by *TtXyn30A* (filled circles) and *TtXyn30A* with *PcAA14B* (open circles). **b** The effect of molar ratio of *PcAA14B* to *TtXyn30A* on xylobiose release. *TtXyn30A* was added at a final concentration of 0.1 µM. Error bars represent the standard deviation from three independent experiments</figcaption></figure>

threefold, while increasing the molar ratio more than fivefold does not seem to increase further the synergy.

## Synergistic effects of PcAA14B LPMO and TtXyn30A xylanase on lignocellulosic substrates

The synergism between *PcAA14B* LPMO and *TtXyn30A* xylanase was tested on different pretreated beechwood substrates. The substrates used in this study were selected based on their hemicellulose content, to gain better insights of the synergy of *PcAA14B* and *TtXyn30A* in the degradation of cellulose-associated xylan. Milox process (substrates 3, 5 and 7) leads to the delignification of the material, with the concomitant production of lignin degradation products [19]. On the other hand, acetone/water oxidation (substrates 1, 2, 4 and 6) is a recently developed methodology, offering the advantage of higher delignification, while producing fewer degradation products [19]. Moreover, the parameters, including acetone to water ratio, can be optimized for the removal of hemicelluloses and lignin. For example, higher acetone content led to substrates with higher hemicellulose content (substrates 4 and 6), in contrast with high water content, leading to substrates (1 and 2), where most of the hemicellulose is removed. Overall, the selected substrates were considered representative of a wide variety of compositions in terms of cellulose, hemicelluloses, and lignin content. The properties of the substrates and the pretreatment conditions are shown in Table 1, together with the release of xylobiose and the degree of synergism. Examples of the analysis of soluble XOS released from lignocellulosic substrates, after treatment with *PcAA14B* and *TtXyn30A*, are shown in Additional file 1: Fig. S1.

lignocellulosic substrates hydrolysis, xylobiose was the only product detected from the action of *TtXyn30A*, with HPAEC-PAD analysis. Therefore, all quantifications were made based on the exo-action of *TtXyn30A*, since uronic XOS were not detected in any case.

As shown in Table 1 and Additional file 1: Fig. S1, the increase in xylobiose release as a result of the synergism between *PcAA14B* and *TtXyn30A* varies a lot among the different substrates. The highest release of xylobiose by *TtXyn30A* was observed on a substrate with relatively high hemicellulose content (substrate 4, Table 1), but, at the same time, the enhancement of *TtXyn30A* activity by *PcAA14B* was low in this case. On the contrary, the lowest xylobiose release was observed in the substrates 1, 2 and 6. The low hemicellulose content might explain the low concentration of released xylobiose from *TtXyn30A* on substrates 1 and 2. Despite the low activity of *TtXyn30A* on both substrates 1 and 2, the relative increase in xylobiose release was the highest observed (Table 1). Therefore, it seems that the hemicellulose remaining after pretreatment is the most recalcitrant to degradation, since the activity of *TtXyn30A* on this substrate is the lowest observed. On the other hand, the high increase in xylobiose release could be the result of a high LPMO activity, and therefore, this recalcitrant part of hemicellulose seems to be an ideal substrate for *PcAA14B*.

The synergistic activity of *PcAA14B* and *TtXyn30A* seems to be interrupted in the case of substrates 6 and 7. Those are the substrates with the highest hemicellulose content tested in this study. The apparent loss of *PcAA14B* activity on these substrates is a further indication of the substrate preference of the enzyme; high content of residual hemicellulose indicates that possibly the
<table><caption>Table 1 Results on the synergism between PcAA14B and TtXyn30A on different lignocellulosic substrates</caption><thead><tr><th>Substrate #</th><th>Pretreatment conditions</th><th>Cellulose (% w/w)</th><th>Acid-insoluble lignin (% w/w)</th><th>Acid-soluble lignin (% w/w)</th><th>Hemicellulose (% w/w)</th><th>TtXyn30A (µM Xylobiose)</th><th>PcAA14B + TtXyn30A (µM xylobiose)</th><th>Degree of synergism</th></tr></thead><tbody><tr><td>1</td><td>d. H<sub>2</sub>O/acetone (50/50%), Air (40% O<sub>2</sub>), 8.5 bar (175 °C, 2 h)</td><td>79.47</td><td>8.4</td><td>0.5</td><td>4.01</td><td>1.23 ± 0.00</td><td>7.00 ± 0.30 (***)</td><td>5.70 ± 0.20</td></tr><tr><td>2</td><td>d. H<sub>2</sub>O/acetone (75/25%), Air (40% O<sub>2</sub>), 40 bar, 175 °C</td><td>84.10</td><td>11.5</td><td>1.0</td><td>5.18</td><td>0.98 ± 0.06</td><td>2.30 ± 0.07 (***)</td><td>2.35 ± 0.07</td></tr><tr><td>3</td><td>Milox 80 °C, 1 h, formic acid</td><td>80.30</td><td>6.3</td><td>1.0</td><td>12.35</td><td>26.7 ± 1.80</td><td>32.3 ± 1.3 (*)</td><td>1.21 ± 0.03</td></tr><tr><td>4</td><td>d. H<sub>2</sub>O/acetone (25/75%), Air (40% O<sub>2</sub>), 40 bar, 175 °C</td><td>75.36</td><td>2.1</td><td>1.6</td><td>17.93</td><td>36.1 ± 1.20</td><td>46.1 ± 2.3 (**)</td><td>1.28 ± 0.10</td></tr><tr><td>5</td><td>Milox 80 °C, 1 h, formic acid, 1 bar</td><td>75.31</td><td>2.1</td><td>1.2</td><td>19.21</td><td>13.9 ± 1.10</td><td>17.2 ± 1.1 (*)</td><td>1.24 ± 0.04</td></tr><tr><td>6</td><td>d. H<sub>2</sub>O/acetone (25/75%), Air, 20 bar</td><td>50.22</td><td>15.7</td><td>3.0</td><td>19.71</td><td>3.10 ± 0.25</td><td>2.20 ± 0.00 (*)</td><td>–</td></tr><tr><td>7</td><td>Milox 80 °C, 3 h, acetic acid</td><td>51.84</td><td>4.6</td><td>3.0</td><td>29.06</td><td>20.1 ± 1.00</td><td>18.0 ± 0.8 (ns)</td><td>–</td></tr></tbody></table>

The data on the pretreatment conditions and compositional analyses have been transferred by [19]. Error bars represent the standard deviation from three independent experiments. Asterisks (*p < 0.1; **p < 0.01; ***p < 0.001) indicate significance based on Student's t test.

ns not significant

recalcitrant part of xylan, which is the assumed preferred substrate for PcAA14B, is not accessible by the enzyme. Substrate 6 contains much more residual lignin, which may inhibit the performance of TtXyn30A due to its adsorption. To test this hypothesis, we performed a laccase treatment in substrate 6, aiming at the removal of residual lignin. The results showed that laccase treatment of substrate 6 increased xylobiose release from TtXyn30A by 19.7 ± 2.9%, indicating that residual lignin is responsible, at least to a certain extent, for TtXyn30A inhibition.

Products of the endoxylanase activity of TtXyn30A, such as 4-O-MeGlcA decorated XOS, were not detected in the above conditions. This could not only be attributed to their low concentration in the experimental setup of the present study, or to their low abundance in the beechwood substrates, but it could also indicate that the endo-action of TtXyn30A is inhibited by the structure of the recalcitrant twofold xylan that is bound with the underlying cellulose fibrils. In this case, the role of PcAA14B could be much more crucial. To elucidate this, hydrolysis of substrates 1 and 4 was performed with increased concentration of TtXyn30A, in the absence of PcAA14B. Positive controls with soluble beechwood xylan were also included. The samples were properly diluted and analyzed with HPAEC-PAD, and the chromatograms are shown in Additional file 1: Fig. S2.

In the samples of substrate 1, no UXOS were detected, which can be explained by the low hemicellulose content of this substrate. On the other hand, in soluble beechwood substrate, UXOS were detected, and one of them was identified as 2,3-(4-O-methyl-α-D-glucuronyl)-xylotriose, against a suitable standard. In samples of substrate 4, one small peak appeared, with retention time corresponding to a possible UXOS product. The same peak was also detected in soluble beechwood xylan samples (Additional file 1: Fig. S2). This result corroborates with the higher hemicellulose content of substrate 4 and the higher activity of TtXyn30A, compared with the other tested substrates.

### Effect of sequential or simultaneous addition of enzymes on the hydrolytic reactions

Synergistic relationships among lignocellulose-acting enzymes are often time-dependent, and the order of enzyme addition can offer valuable insights regarding their mode of action on their polymer substrate. The effect of sequential or simultaneous addition of PcAA14B and TtXyn30A was examined, and the results are shown in Fig. 3. The maximum release of xylobiose was shown when both enzymes were added simultaneously. In this case, the DS was found 1.96 ± 0.30 for the first 24 h, and 2.70 ± 0.09 when the reaction was prolonged for
<figure><img src="image_5.png" /><figcaption>**Fig. 3** Time dependence of the synergism between *PcAA14A* and *TtXyn30A*. The enzymes and their combinations were added at the specified time points, after boiling the reaction mixture to deactivate the first enzyme. The reaction was performed with substrate 1, and it was stopped at 48 h, except X, where the reaction was stopped at 24 h. Error bars represent the standard deviation from three independent experiments</figcaption></figure>

48 h. When *PcAA14B* was added first for 24 h prior to the addition of *TtXyn30A*, the DS was 1.93±0.02, similarly to the case where both enzymes were added together and kept for 24 h. When *TtXyn30A* was added first for 24 h prior to the addition of *PcAA14B*, no synergism was observed, and the xylobiose release reached 2.70±0.00 µM (Fig. 3). This result is expected, since *PcAA14B* does not release soluble products in detectable quantities. Therefore, an additional experiment was performed, where the substrate was pretreated with *TtXyn30A* and after 24 h, *PcAA14B* was added, without deactivating the xylanase. The results were surprising, since the concentration of xylobiose release (2.80±0.24 µM) was similar to the reaction when the xylanase was deactivated before LPMO addition. To determine the effect of enzyme stability to the above results, stability tests were performed for both enzymes under the same conditions. Both enzymes were found to be stable at 4 °C for 48 h, while *TtXyn30A* was also found stable at 45 °C during 48 h in the absence of substrate. *PcAA14b* was found to maintain its activity fully for the first 24 h, but up to 48 h the residual activity drops to 55.9±1.4%. The stability of *TtXyn30A* was also tested in the presence of substrate 1. Surprisingly, after 24 h at 45 °C, the residual activity of *TtXyn30A* dropped to 9.00±0.57% of the initial, while after 48 h only traces of the initial activity were detected in the supernatant. This result could be due to unproductive binding of *TtXyn30A* to the substrate and specifically to the lignin part, which in substrate 1 amounts to 8.9% (w/w). This finding is in accordance with our results

regarding the diminished activity of *TtXyn30A* in substrate 6 due to its high lignin content. Therefore, when *TtXyn30A* is added first, it is possible that the enzyme is already “deactivated” due to unproductive binding on lignin prior to *PcAA14B* addition.

## Synergistic effects of PcAA14B and cellobiohydrolases on the hydrolysis of lignocellulosic substrates

The next step was to explore the synergistic relationships of *PcAA14B* with single cellulose-acting enzymes, since in our previous study, the increase of released glucose by a complex (hemi)cellulolytic cocktail by the addition of *PcAA14B* was documented [4]. The synergism between *PcAA14B* and cellobiohydrolases CBHI and CBHII was tested on substrate 1 (Table 1), since maximum *PcAA14B* activity was observed using this substrate, in the previous experiments with *TtXyn30A*. The quantification of the results was based on cellobiose release, and the results are shown in Fig. 4a. *PcAA14B*, although it is not active on cellulose, was shown to greatly increase the release of cellobiose by CBH from substrate 1, even though peaks corresponding to oxidized sugars, or xylo-oligomers, were not detected. Although the release of cellobiose was more prominent in the reaction of *PcAA14B* with CBHI, the DS was slightly higher in combination with CBHII (1.58±0.15 and 1.87±0.01, respectively). The synergism between *PcAA14B* and CBHII was further tested on microcrystalline cellulose (Avicel), with or without cysteine as electron donor, and the results are shown in Fig. 4b. The addition of *PcAA14B* enhanced the production of cellobiose from CBHII, with or without the addition of electron donor, corresponding to DS of 3.70±0.35 and 3.35±0.40, respectively.

## Discussion

Regarding the enzymatic degradation of lignocellulosic materials, the ‘blocking effect’ of xylan on the accessibility of the underlying cellulose to cellulases is widely established in literature [20]. This is further supported by the number of studies on the synergistic effects of xylanases on cellulose hydrolysis by cellulases [15, 16, 21, 22]. However, a large part of xylan is still recalcitrant to degradation by most known enzymes, leading research not only toward the discovery of new enzymatic activities, but also on the exploration of new synergistic interactions among known enzymes [20]. In the present work, not only the synergistic interactions between the xylan-active LPMO *PcAA14B* and the glucuronoxylan-specific xylobiohydrolase *TtXyn30A*, but also cellobiohydrolases, fill a significant knowledge gap on our understanding of the microbial degradation of (hemi)cellulose.

Both AA14 and GH30_7 are enzyme classes found in fungal saprotrophs [2, 4]. Bioinformatics analyses
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 4** Synergism between *PcAA14B* and cellobiohydrolases. **a** Synergistic effect of *PcAA14B* supplementation on CBH-mediated hydrolysis of substrate 1. **b** Synergistic effect of *PcAA14B* supplementation, with and without electron donor, on CBHII-mediated hydrolysis of microcrystalline cellulose (Avicel). Error bars represent the standard deviation from three independent experiments. Asterisks (\*\*p < 0.01; \*\*\*p < 0.001) indicate significance based on Student's t test</figcaption></figure>

among filamentous fungi revealed that AA14 and GH30_7 genes can both be found at the genomic level in some well-known fungal saprotrophs like *Trichoderma reesei*, *Podospora anserina* and *Fusarium oxysporum* (Fig. 5), suggesting that such synergistic activities could occur in nature.

The synergistic effects observed between *PcAA14B* and *TtXyn30A* on birchwood cellulosic fibers were

<figure><img src="image_8.png" /><figcaption>**Fig. 5** Number of genes belonging to either GH30 (light grey bars), GH30_7 (black bars) or AA14 (dark grey bars) CAZy (sub)family, in fungal genomes</figcaption></figure>
significantly higher than those reported for *PcAA14B* and a GH11 xylanase *AnXyn11*, as described by Couturier et al. [4] and repeated here for comparative purposes. The synergistic interaction between *PcAA14B* and *TtXyn30A* was found to be dose-dependent, while the addition of *PcAA14B* led to ongoing xylobiose release by *TtXyn30A* over a 24-h period, in contrast to the plateau reached by *TtXyn30A* only after 8 h, during hydrolysis of birch-wood cellulosic fibers. Altogether these results led to the hypothesis that the action of *PcAA14B* is constantly cre-ating substrate sites available for *TtXyn30A* on the xylan polymer, as shown in Fig. 6.

The activity of *TtXyn30A* was tested in pretreated natural lignocellulosic substrates for the first time in this study. *TtXyn30A* also hydrolyzed the glucuronoxylan of most tested beechwood substrates with hemicellulose content >10% (w/w), releasing xylobiose. A single exception to this was observed during hydrolysis of substrate 6, which contained high levels of residual lignin. Residual lignin was found to inhibit *TtXyn30A*. Although cellulases inhibition by the presence of lignin, due to enzyme absorption on the surface of the polymer [23], is much more prominent, xylanase activity was also reported to be negatively affected [24, 25], probably due to ionic

<figure><img src="image_9.png" /></figure>
interactions between the charged moieties of the enzyme surface and lignin -COOH and -CO groups.

Regarding the activity of *PcAA14B*, Couturier et al. proposed a specific hypothesis for the substrate preference of this LPMO [4]; *PcAA14B* would be only active on the twofold screw xylan polymer that is attached to cellulose. Simmons et al. showed that the attachment of xylan polymers to underlying cellulose fibrils induces the twofold screw conformation on the xylan molecule, in contrast with the threefold screw conformation of soluble xylan [26]. Twofold screw xylan is a more rigid structure, resembling the cellulose fibrils, and therefore shows a certain degree of ‘crystallinity’ [6]. The results of our study strengthen the initial hypothesis, revealing a relationship between the hemicellulose content of the substrate, and the degree of synergism between the two enzymes; the lower the hemicellulose content, the higher the degree of synergism observed, at least for beechwood substrates, rich in glucuronoxylan. Moreover, the synergism among the two enzymes is less obvious in substrates with high hemicellulose content (e.g., 29% w/w), where the recalcitrant part of xylan is perhaps not accessible to *PcAA14B*.

The high degree of synergism observed for cellulosic fibers and some of the tested beechwood substrates among *PcAA14B* and *TtXyn30A*, especially when compared to the results with *PcAA14B* and *AnXyn11* xylanase, can also be justified by previous findings on the conformation of xylan in plant cell walls. Grantham et al. previously showed that xylan decorations, such as acetyl, arabinosyl and GlcA or 4-O-MeGlcA groups, are essential for the proper attachment of xylan to the underlying cellulose fibrils, and therefore, for the formation of the twofold xylan structure [6]. The oxidative cleavage of recalcitrant xylan by *PcAA14B* created free ends, which in the case of glucuronoxylan are evenly decorated with 4-O-MeGlcA moieties. The newly created free ends function as substrate sites for the xylobiohydrolase activity of *TtXyn30A*, which ultimately releases xylobiose. The synergistic effect is further supported by the complementary action of both enzymes: while *PcAA14B* oxidizes at the C1 position of the xylan backbone, *TtXyn30A* releases xylose dimers from the non-reducing end. This synergistic relationship is similar to the synergism observed between CBHs and cellulose-active LPMOs during cellulose hydrolysis [13]. Products of the endoxylanase activity of *TtXyn30A*, such as 4-O-MeGlcA decorated XOS, were detected in higher enzyme loadings, and in hemicellulose-rich substrates. In regard to the endoxylanase activity, *TtXyn30A* has been shown to attack the second glycosidic bond from the 4-O-MeGlcA position, while GH11 xylanases are well known to act several xylose monomers away from the substituent position,

due to the architecture of the binding site cleft, which is not large enough to accommodate decorated xylans [27, 28]. This is also showed by Couturier et al., where the non-oxidized products found by the combined action of *PcAA14B* and *AnXyn11* were X₃MeGlcA, X₄MeGlcA and X₅MeGlcA [4].

The synergism observed between *TtXyn30A* and *PcAA14B* was found to be more prominent when both enzymes were added simultaneously rather than sequentially. This is not usual for enzymes acting on the same polymer, due to competition for binding sites. However, there are some reports showing a similar trend [29]. Simultaneous addition of enzymes with complementary activities usually is more favorable when the enzymes act on different polymers (e.g., cellulases and xylanases) [30]. Malgas et al. proposed an ‘alternating effect’ for the synergism displayed among cellulases and xylanases, where each enzyme hydrolyzes the cellulose and xylan moieties, covering each other in the complex biomass structure [31]. This seems to be the case for the synergism among *TtXyn30A* and *PcAA14B*, as shown in Fig. 6. Time dependence experiments provide valuable insights regarding the mode of action of *PcAA14B* and its synergism with *TtXyn30A*. When the substrate was pretreated with the LPMO and xylanase was added, after LPMO deactivation, for 24 h, the xylobiose release was the same as in the case where both enzymes worked together for 24 h. These data indicate that *PcAA14B* action modifies the substrate of *TtXyn30A*, making it more susceptible to hydrolysis, similarly to cellulose-acting LPMOs and cellobiohydrolases [13]. According to our hypothesis, although both enzymes act on the same polymer, its conformation is not the same: *PcAA14B* acts on the twofold helical screw xylan, creating free ends for *TtXyn30A* action, while *TtXyn30A* acts on the free non-reducing ends of the xylan polymer, due to its xylobiohydrolase activity. This synergistic relationship, aside from its obvious significance in the design of next-generation (hemi) cellulolytic cocktails, could also be employed for the production of oligosaccharides from residual biomass, which are shown to have significant prebiotic activity [32].

In the present work, the synergistic action of *PcAA14B* and cellobiohydrolases CBHI and CBHII was demonstrated for the first time. As previously shown, *PcAA14B* was found to enhance the glucose release from a (hemi) cellulolytic cocktail from *T. reesei*, but the synergism was not further studied with single enzymes [4]. Cellobiose release was found to be enhanced by the addition of *PcAA14B* on substrate hydrolysis by CBHs, and this finding is in accordance with the previous results, where the disruption of wood fibers was observed by the action of *PcAA14B* [4]. The authors hypothesized that the disruption of wood fibers revealed more substrate sites
for cellulases, and the results of this work support this hypothesis. Moreover, the synergism between PcAA14B and CBHII was shown for Avicel degradation. This synergistic effect on Avicel can also be attributed to traces of recalcitrant xylan [33], the removal of which may facilitate cellulose hydrolysis by cellobiohydrolases, as proposed by Igarashi et al. [34].

## Conclusions

In the present study, new synergistic relationships were established: the xylan-active LPMO, represented in this study by PcAA14B, and a novel GH30_7 glucuronoxylan-active xylanase with xylobiohydrolase activity, represented by TtXyn30A, were found to act synergistically on cellulosic substrates. Also, the activity of TtXyn30A was documented against pretreated lignocellulosic substrates for the first time. The addition of PcAA14B in the reaction mixture together with TtXyn30A increased greatly the xylobiose release from pretreated lignocellulosic substrates. The synergistic effects were most prominent in substrates of relatively low hemicellulose content, indicating a preference of PcAA14B for recalcitrant xylan. We hypothesized that the action of PcAA14B on recalcitrant xylan creates new free ends in the polymer, which can be used as substrate for the action of TtXyn30A. PcAA14B was also found to act synergistically with cellobiohydrolases CBHI and CBHII. Overall, the results of the present study provide additional insights into the mode of action of the PcAA14B LPMO and will contribute to the design of next-generation enzymatic cocktails for the saccharification of woody lignocellulosic substrates with residual recalcitrant xylan.

## Methods

### Enzymes and substrates

Beechwood xylan, xylo-oligosaccharides (XOS), CBHI and CBHII cellobiohydrolases from *Trichoderma longibrachiatum* and a microbial source, respectively, and GH11 endo-1,4-β- xylanase M4 from *Aspergillus niger* (*AnXyn11*), were purchased from Megazyme (Bray, Co. Wicklow, Ireland). Kraft birchwood cellulosic fibers (79% (w/w) cellulose, 21% (w/w) xylan, [4]) were kindly provided by Sandra Tapin (FCBA, Grenoble, France). Aqueous dispersions of Kraft birchwood cellulosic fibers were adjusted to pH 5.2 with acetate buffer (50 mM) in a final reaction volume of 0.5 mL. Different beechwood lignocellulosic substrates were produced from commercially available beechwood (Lignocel® HBS 150–500), pretreated, as previously described [19]. TtXyn30A was produced and purified as described in [2]. PcAA14B was produced and purified, as previously described [4]. In brief, *P. pastoris* clones bearing the recombinant proteins in frame with (His)₆-tag sequences were grown overnight

at 30 °C in BMGY medium. The cells were then transferred to BMMY medium supplemented with methanol, as described in EasySelect™ Pichia Expression kit, and incubated 3–6 days for enzyme production. Then, the supernatants were collected by centrifugation, and they were filtered, concentrated and dialyzed. Both enzymes were purified with immobilized metal ion affinity chromatography (IMAC), to homogeneity.

### Bioinformatics analyses

Bioinformatics analyses were performed on sequenced fungal genomes publicly available (NCBI non-redundant sequence database and JGI). The co-occurrence of GH30_7 and AA14 modules in fungal genomes was searched using CAZy tools (www.cazy.org).

### Synergism assays

Cellulosic fibers, lignocellulosic substrates, or Avicel were added to 50 mM sodium acetate buffer, pH 5.2 at a final concentration of 0.5% (w/v). The enzymes were added at a final concentration of 2 µM for PcAA14B and 0.1 µM for TtXyn30A, AnXyn11, CBHI or CBHII, unless stated otherwise. Cysteine was used as an electron donor on the assays using birchwood cellulosic fibers and Avicel, at a final concentration of 1 mM. The reactions (0.5 mL) were incubated for 16 h in 45 °C, at 850 rpm, in a thermomixer (Eppendorf, Montesson, France). The reaction was terminated with 10-min incubation in 100 °C, and the reactions were centrifuged to remove the insoluble fraction.

The hydrolysis products were analyzed with high-performance anion-exchange chromatography (HPAEC), using a CarboPac PA-1 (4 × 250 mm, Dionex) column with a pulsed amperometric detector (PAD) equipped with a gold electrode. The solvents used were NaOH 100 mM (solvent A) and NaOAc (1 M) in 100 mM NaOH (solvent B) at a flow rate of 1 mL min⁻¹. For the analysis of oligosaccharides, the following gradient was applied: 0–10 min 0–10% B, 10–35 min 10–30% B, 35–40 min 30–100% B 40–41 min 100–0% B, 41–50 min isocratic step, 100% A. Xylobiose, XOS and cellobiose were quantified against suitable standard curves.

The degree of synergism was calculated according to the following equation:

$$DS = \frac{X_{A,B}}{X_A + X_B}$$

where $X_{A,B}$ is the µM of product from the action of both enzymes on the substrate, $X_A$ the µM of product from the action of the LPMO and $X_B$ the µM of product from the action of xylanases TtXyn30A or AnXyn11, or cellobiohydrolases CBHI and CBHII.

The time dependence of the synergism between TtXyn30A and PcAA14B was tested, as mentioned above.
First, a pretreatment of 10 mg mL⁻¹ of substrate 1 was performed with either *TtXyn30A* (0.1 µM) or *PcAA14B* (2 µM) at 45 °C for 24 h. Then, the first enzyme was deactivated by boiling the reaction mixture, the second enzyme was added (*PcAA14B* or *TtXyn30A*), and the reaction was incubated for a further 24 h. The effect of *TtXyn30A* alone and together with *PcAA14B* added together from the start of the reaction and incubated for 24 or 48 h were used as controls. Xylobiose concentrations were determined with HPAEC-PAD, as mentioned above.

The synergism of *PcAA14B* with cellobiohydrolases CBHI and CBHII was studied, as mentioned above. In brief, CBHI and CBHII were added at a final concentration of 0.1 µM, while *PcAA14B* was added at a final concentration of 1 µM, to 15 mg mL⁻¹ of substrate 1. Cysteine (1 mM) was added as electron donor, as indicated. The reaction was incubated at 45 °C for 16 h, and then the samples were boiled and filtered before HPAEC-PAD analysis. Cellobiose quantification was performed against a suitable calibration curve.

## Detection of UXOS in the reaction supernatants of *TtXyn30A*

For the detection of UXOS products of *TtXyn30A*, 50 mg mL⁻¹ substrate 1 and 4 were treated with 0.25 mg mL⁻¹ *TtXyn30A* for 24 h in 50 °C and 1100 rpm. Beechwood xylan (5 mg mL⁻¹) was hydrolyzed with 0.02 mg mL⁻¹ *TtXyn30A* in the same conditions. The reactions were terminated by boiling the samples. The samples from hydrolysis of substrates 1 and 4 were diluted 2 times before HPAEC-PAD analysis, while the samples from beechwood xylan hydrolysis were diluted 10 times before analysis. HPAEC-PAD analysis was performed with a different elution program [2]. The solvents used were NaOH 100 mM (solvent A) and NaOAc (0.5 M) in 100 mM NaOH (solvent B) at a flow rate of 1 mL min⁻¹. For the analysis of oligosaccharides, the following gradient was applied: 0–5 min A, 5.1–30 min 0–20% B, 30.1–35 min 100% B, 35.1–50 min 100% A.

## Stability measurements

The stability of *PcAA14B* and *TtXyn30A* was determined as follows: both enzymes were incubated in sodium acetate buffer, 50 mM, pH 5.2 for 48 h either in 4 or 45 °C. The final concentrations of enzymes were 2 µM for *PcAA14B* and 0.1 µM for *TtXyn30A*. Samples were withdrawn in time points 0, 24 and 48 h, and the activity was determined. For *TtXyn30A*, the activity was measured with beechwood xylan as substrate, at a final concentration of 0.5% (w/v), for 24 h in 45 °C and 1200 rpm. The reducing sugars were quantified with the dinitrosalicylic

acid method (DNS, [35]). In addition, the stability of *TtXyn30A* was measured also in the presence of 10 mg mL⁻¹ substrate 1.

For *PcAA14B*, due to the lack of soluble products, the activity was determined by measuring the degree of synergism with *TtXyn30A*. In the *PcAA14B*-containing samples, substrate 1 was added at a final concentration of 10 mg mL⁻¹ and fresh *TtXyn30A* at a final concentration of 0.1 µM. The mixture was incubated at 45 °C, and 1200 rpm for 24 h, and the xylobiose concentration was quantified with HPAEC-PAD, as described previously.

## Laccase treatment

A treatment with laccase enzyme was performed in substrate 6 (Table 1). Laccase Novozym® 51003 was used at 10 U g⁻¹ substrate, which was added at a final concentration of 10% (w/v). The mixture was incubated for 24 h in 40 °C, pH 6, 950 rpm, and then the supernatant was removed. The laccase-treated substrate was washed three times with ultrapure water, to remove low-MW phenolic compounds released by the action of the enzyme, and freeze-dried. Finally, it was hydrolyzed by *TtXyn30A*, as described previously. *TtXyn30A* was added at a final concentration of 1 µM.

## Statistical analysis

All experiments were performed in triplicate, and the results are expressed as mean value ± standard deviation. The statistical analysis was performed with SigmaPlot 12.5 software. Student's *t* test was applied to experiment subsets, with the use of GraphPad Prism 5 software. *P* values less than 0.05 were considered statistically significant.

## Supplementary information

**Supplementary information** accompanies this paper at https://doi.org/10.1186/s13068-020-01777-x.

**Additional file 1: Table S1.** Xylo-oligosaccharide release from cellulosic fibers, after hydrolysis with *PcAA14B*, *TtXyn30A*, *AnXyn11* and their combinations, as shown in Fig. 1. Error bars represent the standard deviation from three independent experiments. **Figure S1.** Analysis of XOS release by *PcAA14B* and *TtXyn30A*, in different pretreated lignocellulosic substrates, as shown in Table 1; (a) Substrate 5, (b) substrate 2, (c) substrate 4. Green line: substrate only, black line: *PcAA14B*, red line: *TtXyn30A*, blue line: *PcAA14B* and *TtXyn30A*. DP2: xylobiose. **Figure S2.** Analysis of XOS release by *TtXyn30A*, in beechwood xylan (10X diluted, blue line: reaction, light blue line: substrate blank) and substrate 4 (2X diluted, green line: reaction, yellow line: substrate blank); red line: 23-(4-O-Methyl-α-D-Glucuronyl)-xylotriose, black line: XOS standards (DP 1-5).

## Acknowledgements

The authors are grateful to K. G. Kalogiannis and A. A. Lappas from CPERI (Thessaloniki, Greece) who kindly provided the pretreated biomass samples and to Novozymes A/S for the generous gift of Novozym® 51003. The CAZy team, most especially Elodie Drula (AFMB, Marseille) are thanked for
the functional annotation of fungal genomes. A.Z. thanks the French Embassy in Greece, together with the French Ministry of Higher Education, Research and Innovation, for the scholarship 'Séjours scientifiques de haut niveau' (SSHN).

## Authors' contributions

AZ designed and performed experiments, analyzed data and wrote the original draft, with input from all authors; CP performed experiments and analyses; SG produced and purified enzymes and performed analyses; JGB and ET designed and supervised the study, and wrote the manuscript. All authors read and approved the final manuscript.

## Funding

This study did not receive any particular funding.

## Ethics approval and consent to participate

Not applicable.

## Consent for publication

Not applicable.

## Competing interests

The authors declare that they have no competing interests.

Received: 11 May 2020 Accepted: 29 July 2020

Published online: 10 August 2020

## References

1. Vaaje-Kolstad G, Westereng B, Horn SJ, Liu Z, Zhai H, Sorlie M, Eijsink VG. An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science. 2010;330(6001):219-22. https://doi.org/10.1126/science.1192231.

2. Katsimpouras C, Deedes G, Thomaidis NS, Topakas E. A novel fungal GH30 xylanase with xylobiohydrolase auxiliary activity. Biotechnol Biofuels. 2019;12(1):120. https://doi.org/10.1186/s13068-019-1455-2.

3. Nakamichi Y, Fouquet T, Ito S, Watanabe M, Matsushika A, Inoue H. Structural and functional characterization of a bifunctional GH30-7 xylanase B from the filamentous fungus *Talaromyces cellulolyticus*. J Biol Chem. 2019;294(11):4065-78. https://doi.org/10.1074/jbc.RA118.007207.

4. Couturier M, Ladevèze S, Sulzenbacher G, Ciano L, Fanuel M, Moreau C, Villares A, Cathala B, Chaspoul F, Frandsen KE, Labourel A, Herpoël-Gimbert I, Grisel S, Haon M, Lenfant N, Rogniaux H, Ropartz D, Davies GJ, Rosso MN, Walton PH, Henrissat B, Berrin JG. Lytic xylan oxidases from wood-decay fungi unlock biomass degradation. Nat Chem Biol. 2018;14:306. https://doi.org/10.1038/nchembio.2558.

5. Busse-Wicher M, Grantham NJ, Lyczakowski JJ, Nikolovski N, Dupree P. Xylan decoration patterns and the plant secondary cell wall molecular architecture. Biochem Soc Trans. 2016;44:1. https://doi.org/10.1042/BST20150183.

6. Grantham NJ, Wurman-Rodrich J, Terrett OM, Lyczakowski JJ, Stott K, Iuga D, Simmons TJ, Durand-Tardif M, Brown SP, Dupree R, Busse-Wicher M, Dupree P. An even pattern of xylan substitution is critical for interaction with cellulose in plant cell walls. Nat Plants. 2017;3:11. https://doi.org/10.1038/s41477-017-0030-8.

7. Terrett OM, Lyczakowski JJ, Yu L, Iuga D, Franks WT, Brown SP, Dupree R, Dupree P. Molecular architecture of softwood revealed by solid-state NMR. Nat Commun. 2019;10:4978. https://doi.org/10.1038/s41467-019-12979-9.

8. Frommhagen M, Sforza S, Westphal AH, Visser J, Hinz SWA, Koetsier MJ, van Berkel WJH, Gruppen H, Kabel MA. Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase. Biotechnol Biofuels. 2015;8:101. https://doi.org/10.1186/s13068-015-0284-1.

9. Hüttner S, Várna I, Petrović DM, Bach CX, Kim Anh DT, Thanh VN, Eijsink VGH, Larsbrink J, Olsson L. Specific xylan activity revealed for AA9 lytic polysaccharide monooxygenases of the thermophilic fungus *Malbranchea cinnamomea* by functional characterization. Appl Environ Microbiol. 2019;85:e01408-01419. https://doi.org/10.1128/aem.01408-19.

10. Basotra N, Dhiman SS, Agrawal D, Sani RK, Tsang A, Chadha BS. Characterization of a novel Lytic Polysaccharide Monooxygenase from Malbranchea cinnamomea exhibiting dual catalytic behavior. Carbohydr Res. 2019;478:46-53. https://doi.org/10.1016/j.carres.2019.04.006.

11. Corrèa TLR, Júnior AT, Wolf LD, Buckeridge MS, dos Santos LV, Murakami MT. An actinobacteria lytic polysaccharide monooxygenase acts on both cellulose and xylan to boost biomass saccharification. Biotechnol Biofuels. 2019;12:117. https://doi.org/10.1186/s13068-019-1449-0.

12. Nakamichi Y, Fouquet T, Ito S, Matsushika A, Inoue H. Mode of action of GH30-7 reducing-end xylose-releasing exo-xylanase A (Xyn30A) from the filamentous fungus *Talaromyces cellulolyticus*. Appl Environ Microbiol. 2019. https://doi.org/10.1128/AEM.00552-19.

13. Song B, Li B, Wang X, Shen W, Park S, Collings C, Feng A, Smith SJ, Walton JD, Ding SY. Real-time imaging reveals that lytic polysaccharide monooxygenase promotes cellulase activity by increasing cellulose accessibility. Biotechnol Biofuels. 2018;11:41-41. https://doi.org/10.1186/s13068-018-1023-1.

14. Olsen JP, Borch K, Westh P. Endo/exo-synergism of cellulases increases with substrate conversion. Biotechnol Bioeng. 2017;114(3):696-700. https://doi.org/10.1002/bit.26179.

15. Song HT, Gao Y, Yang YM, Xiao WJ, Liu SH, Xia WC, Liu ZL, Yi L, Jiang ZB. Synergistic effect of cellulase and xylanase during hydrolysis of natural lignocellulosic substrates. Bioresour Technol. 2016;219:710-5. https://doi.org/10.1016/j.biortech.2016.08.035.

16. Hu J, Arantes V, Pribowo A, Saddler JN. The synergistic action of accessory enzymes enhances the hydrolytic potential of a "cellulase mixture" but is highly substrate specific. Biotechnol Biofuels. 2013;6(1):112. https://doi.org/10.1186/1754-6834-6-112.

17. Harris PV, Welner D, McFarland KC, Re E, Navarro Poulsen JC, Brown K, Salbo R, Ding H, Vlasenko E, Merino S, Xu F, Cherry J, Larsen S, Leggio LL. Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family. Biochemistry. 2010;49:15. https://doi.org/10.1021/bi100009p.

18. Karnaouri A, Muraleedharan MN, Dimarogona M, Topakas E, Rova U, Sandgren M, Christakopoulos P. Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from *Myceliophthora thermophila* during the hydrolysis of lignocellulosic substrates. Biotechnol Biofuels. 2017;10:126. https://doi.org/10.1186/s13068-017-0813-1.

19. Katsimpouras C, Kalogiannis KG, Kalogianni A, Lappas AA, Topakas E. Production of high concentrated cellulosic ethanol by acetone/water oxidized pretreated beech wood. Biotechnol Biofuels. 2017;10(1):54. https://doi.org/10.1186/s13068-017-0737-9.

20. Biely P, Singh S, Puchart V. Towards enzymatic breakdown of complex plant xylan structures: state of the art. Biotechnol Adv. 2016;34:7. https://doi.org/10.1016/j.biotechadv.2016.09.001.

21. Hu J, Arantes V, Saddler JN. The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect? Biotechnol Biofuels. 2011;4:36. https://doi.org/10.1186/1754-6834-4-36.

22. Couturier M, Haon M, Coutinho PM, Henrissat B, Lesage-Meessen L, Berrin JG. *Podospora anserina* hemicellulases potentiate the *Trichoderma reesei* secretome for saccharification of lignocellulosic biomass. Appl Environ Microbiol. 2011;77:1. https://doi.org/10.1128/AEM.01761-10.

23. Fong M, Berrin JG, Paës G. Investigation of the binding properties of a multi-modular GH45 cellulase using bioinspired model assemblies. Biotechnol Biofuels. 2016;9:1. https://doi.org/10.1186/s13068-016-0428-y.

24. Berlin A, Balakshin M, Gilkes N, Kadla J, Maximenko V, Kubo S, Saddler J. Inhibition of cellulase, xylanase and β-glucosidase activities by softwood lignin preparations. J Biotechnol. 2006;125:2. https://doi.org/10.1016/j.jbiotec.2006.02.021.

25. Kellock M, Rahikainen J, Marjamaa K, Kruus K. Lignin-derived inhibition of monocomponent cellulases and a xylanase in the hydrolysis of lignocellulosics. Bioresour Technol. 2017;232:183-91. https://doi.org/10.1016/j.biortech.2017.01.072.

26. Simmons TJ, Mortimer JC, Bernardinelli OD, Pöppler AC, Brown SP, deAzevedo ER, Dupree R, Dupree P. Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR. Nat Commun. 2016;7:13902. https://doi.org/10.1038/ncomms13902.
27. Paës G, Berrin JG, Beaugrand J. GH11 xylanases: structure/function/properties relationships and applications. Biotechnol Adv. 2012;30(3):564–92. https://doi.org/10.1016/j.biotechadv.2011.10.003.

28. Berrin JG, Juge N. Factors affecting xylanase functionality in the degradation of arabinoxylans. Biotechnol Lett. 2008;30:7. https://doi.org/10.1007/s10529-008-9669-6.

29. Väljamäe P, Sild V, Nutt A, Pettersson G, Johansson G. Acid hydrolysis of bacterial cellulose reveals different modes of synergistic action between cellobiohydrolase I and endoglucanase I. Eur J Biochem. 1999;266(2):327–34. https://doi.org/10.1046/j.1432-1327.1999.00853.x.

30. Murashima K, Kosugi A, Doi RH. Synergistic effects of cellulosomal xylanase and cellulases from *Clostridium cellulovorans* on plant cell wall degradation. J Bacteriol. 2003;185(5):1518–24. https://doi.org/10.1128/JB.185.5.1518-1524.2003.

31. Malgas S, Thoresen M, van Dyk JS, Pletschke BI. Time dependence of enzyme synergism during the degradation of model and natural lignocellulosic substrates. Enzyme and Microbial Technol. 2017;103:1–11. https://doi.org/10.1016/j.enzmictec.2017.04.007.

32. Yu X, Yin J, Li L, Luan C, Zhang J, Zhao C, Li S. Prebiotic potential of xylooligosaccharides derived from corn cobs and their in vitro antioxidant activity when combined with lactobacillus. J Microbiol Biotechnol. 2015;25:1084–92. https://doi.org/10.4014/jmb.1501.01022.

33. Värnai A, Siika-Aho M, Viikari L. Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs. Biotechnol Biofuels. 2013;6:30. https://doi.org/10.1186/1754-6834-6-30.

34. Igarashi K, Uchihashi T, Koivula A, Wada M, Kimura S, Okamoto T, Penttilä M, Ando T, Samejima M. Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface. Science. 2011;333:1279–82. https://doi.org/10.1126/science.1208386.

35. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem. 1959;31(3):426–8. https://doi.org/10.1021/ac60147a030.

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