Commercial cellulolytic enzyme mixtures, Celluclast 1-5L and Cellic Ctc2, were inhibited by the wheat straw PL and its’s fractions produced via XAD fractionation and enzymatic pre-hydrolysis. Comparison of different types of cellulases here showed that the CBHs in particular were sensitive to the inhibition. CBHs are key enzymes in the hydrolysis of crystalline cellulose , and thus their inhibition is expected to have major overall net effect on the cellulose saccharification by the cellulolytic enzyme mixtures.
Strong inhibition of CBHs by hemicellulosic oligosaccharides is well known  and observed also in this study. This is caused by binding of the xylooligosaccharides the tunnel-shaped active site of CBHs . However, this type of inhibition can be relieved by oligosaccharide-degrading enzymes present in cellulolytic cocktails. Interestingly, we observed that a substantial fraction of xylooligosaccharides was bound to the hydrophobic XAD resin, suggesting that they were interlinked with phenolics. The Phe-Fr fraction, containing these phenolics-associated oligosaccharides, was clearly more inhibitory to the CBHs than Ctec-PL fraction, where PL was pretreatead and partially hydrolysed by Cellic Ctec 2 enzymes.
XAD-resins have been previously used in binding of lignin-fragments and other aromatic compounds from lignocellulose extracts and hydrolysates [39, 40]. Fractions isolated with XAD resins from pretreated hardwood, softwood and sugarcane baggase have also been reported to contain lignin-carbohydrate complexes (LCCs) [39, 41]. On the other hand, wheat straw LCCs are known to contain lignin bound to glucan and xylan moieties via ferulic acid and phenyl glycosidic linkages, respectively  and feruloylated oligosaccharides have also previously been identified in wheat straw hydrothermal pretreatment liquor . The LCC type of compounds can thus be the source of oligosaccharides in the XAD bound fraction of wheat straw PL. This was supported by the GC-MS analysis, which showed that 4-hydroxycinnamic and ferulic acid esters with pentoses were present in the XAD bound Phe-Fr.
The effect of LCC type of compounds on cellulolytic enzymes is not well known, though the inhibition of cellulases by softwood LCCs was reported . The study showed clear difference in inhibition by ethanol organosolv dissolved lignin and its intensively hydrolysed fraction (enzymatic residual lignin), which correlates well with difference of Phe-Fr and Ctec-PL inhibition in our study. Proposed inhibition mechanism involved interactions of enzymes with the minor solubilized low-molecular lignin component, along with binding to lignin .
Inhibition of CBHs was relieved by degradation of oligosaccharides by other enzymes present in cellulolytic cocktails. Endoglucanases and xylanases from T. reesei were also able to release sugars from the oligosaccharides in Phe-Fr, suggesting that they can contribute to relieving the inhibitory effect of LCCs. Ferulic acid derivatives have been reported to bind in xylanase active site . One can speculate that the open-cleft architecture in the active site of EGs allows similar binding of these type of compounds. EG TrCel7B is known to be able to hydrolyse a wide range of substrates, including celluloses, xyloglucan, arylated disaccharides and xylans . The TrCel12A also hydrolyses, besides cellulose at least, β-glucan, glucomannan and xyloglucan [21, 46]. However, it is noteworthy that despite high enzyme load (16.5 mg protein/g non-monomeric xylan), all the bound saccharides in PL were not hydrolysed to monosaccharides during pretreatment by Cellic Ctec2.
The inhibition of enzymes by Ctec-PL, i.e. PL that was pre-hydrolysed by Cellic Ctec2, represents an inhibitory power of PL that cannot be mitigated by the enzymes present in this state-of-the-art enzyme cocktail. Combating with this remaining inhibitory power, may pave a way for mitigating the enzymes inhibition and improving their performance on lignocellulose degradation.
The composition of the soluble Phe-Fr in the wheat straw PL was found to be very complex. Numerous monomeric and dimeric compounds were detected using the GC-MS analysis, but their proportion of the total amount of phenolics was low (0.4 g/L of the 4.5 g/L). The effects of pure phenolic monomers on enzymes have been widely studied [18, 47]. The reported inhibitory/deactivating concentrations of the phenolic monomers are typically in the order of 1–10 g/L, which is much higher than the concentrations detected in the PL studied in present work. This suggests that the phenolic monomers may not necessarily be the major drivers of the inhibition of enzymes.
A vast majority of the phenolic compounds in the PL of wheat straw had Mw characteristic to oligophenolic compounds (between 450 and 1700 Da). It was proposed that wheat straw derived oligophenolic compounds are responsible for phenolic inhibition of commercial enzyme cocktail, Cellic Ctec3 via non-specific binding to the enzymes . The wheat straw LCCs should also fall into this Mw fraction. However, the analytical fractionation of the PL showed that the carbohydrates bound to phenolics were not the only cause of enzyme inhibition, since purer phenolic fractions, eluted with higher ethanol concentrations, were more inhibitory to the enzymatic cellulose hydrolysis, than carbohydrate containing fractions. Tannic acid was used as model compound to represent HMW phenolics in the inhibition study. Tannic acid was reported to be a strong inhibitor for T. reesei CBHs (TrCel7A and TrCel6A) and EGs (TrCel7B and TrCel5A) . However, here the inhibition by tannic acid had only a moderate effect on purified enzymes and Cellic CTec2 in concentration mimicking the amount of HMWs in the Phe-Fr.
An activating effect of phenolic compounds was steadily observed with Cellic Ctec2 in hydrolysis of BMCC and Avicel, while not present with Celluclast 1.5L. Our recent study has revealed that the phenolic compounds in PL of wheat straw can support LPMOs with both electrons and H2O2 co-substrate . The major difference between Celluclast 1.5L and Cellic Ctec2 is that the latter is rich in LPMOs . Thus, the overall effect of PL/Phe-Fr/Ctec-PL on cellulose degradation by Cellic Ctec2 may reflect the balance between LPMO supporting and enzymes inhibiting effects.