Expression and purification of TtGH74 and TtGH74ΔCBM
The codon-optimized genes encoding GH74 xyloglucanase from T. terrestris (TtGH74) and CBM-deleted variant TtGH74ΔCBM were successfully expressed in P. pastoris KM71H. The purified enzymes were analyzed by SDS-PAGE with molecular masses of about 90 kDa and 85 kDa, respectively. After digestion with N-glycosidase Endo H, SDS-PAGE analysis showed that the molecular masses of TtGH74 and TtGH74ΔCBM decreased closely to their theoretical values of 87.89 kDa and 83.09 kDa, respectively (Fig. 1) The analysis of amino sequence of TtGH74 by NetNGlyc 1.0 Server (https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0) confirmed that it has three putative N-glycosylation sites (N212, N325 and N409), indicating that the recombinant TtGH74 and TtGH74ΔCBM could partially be N-glycosylated.
Optimization of TtGH74 expression conditions
The protein expression level of TtGH74 was only 60% of that of TtGH74ΔCBM, therefore, the expression conditions of TtGH74 were optimized. Methanol-regulated promoter of the alcohol oxidase 1 gene (AOX1) is the key to the high expression levels of recombinant protein in P. pastoris (35), which was significantly influenced by methanol concentration. In addition, the expression level was also affected by medium pH, induction temperature and time.
The optimum culture temperature for production of TtGH74 was 28 ℃ (Fig. S4 a). As shown in Fig. S4 b, the optimal methanol concentration was observed at 1.5% (vol/vol), excessive methanol decreased the expression of TtGH74 in P. pastoris. The optimum induction pH was pH 6.0 (Fig. S4 c). Under the optimized condition, the enzyme activity of TtGH74 secreted in the supernatant reached to 95 U/mL after 5 days of induction with 1.5% methanol at pH 6.0 and 28 ℃ (Fig. S4 d).
Properties of recombinant TtGH74 and TtGH74ΔCBM from P. pastoris
Among all tested substrates, TtGH74 showed an ultra-high activity against tamarind seed XG. TtGH74 also showed activities toward barley β-glucan, konjac glucomannan, lichenan, and PASC, but the corresponding activities were only approximately 5% and 1% of that on xyloglucan. No activities were detected towards other polysaccharides such as laminarin, starch, xylan, pectin, and chitin, etc (Table 1).
Table 1
Substrate specificity of TtGH74
Substrate | Glycosidic bond | TtGH74 activity |
β-Glucan (barley) | β-1,3 and β-1,4 | ++ |
Glucomannan (konjac) | β-1,4 | ++ |
Lichenan (lichen of iceland) | β-1,3 and β-1,4 | + |
Xyloglucan (tamarind seed) | β-1,4 α-1,6 and β-1,6 | +++++ |
Laminarin (laminaria) | β-1,6-endo-β-1,3 | |
Soluble starch (potato) | α-1,4 | |
Xylan (birch) | β-1,4 | |
Arabinoxylan (wheat) | β-1,4 | |
Pectin | α-1,4 | |
Chitin | β-1,4 | |
CMC-Na | β-1,4 | |
Avicel | β-1,4 | |
Phosphoric acid swelling cellulose (PASC) | β-1,4 | + |
TtGH74 and TtGH74ΔCBM displayed the maximum activity toward tamarind seed XG at 75 ℃ and pH 5.5 (Fig. 2a, b). However, the enzymes were unstable at 70 ℃, after pre-incubation with 70 ℃ for 20 min, their residual activity was dropped to below 20% (Fig. 2c). This result indicated that the enzymes had more thermostability at presence of the substrate, which may be attributed to the stabilizing effects of substrate binding (36). Interestingly, the deletion of CBM1 significantly improved the thermostability of TtGH74ΔCBM when pre-incubation of enzymes at temperature below 70 ℃. When preincubation of enzymes at 55 ℃ to 65 ℃ for 4 h, the residual activity of TtGH74ΔCBM was 10–20% higher than that of TtGH74. When the pre-incubation of enzymes at 50 ℃, the activity of TtGH74 remained above 80%, while the activity of TtGH74ΔCBM remained almost unchanged after 4 h of pre-incubation (Fig. 2c). Conversely, the thermostability of some glycoside hydrolases from fungi were decreased after the removal CBM1 (37, 38). The inconsistent observation about CBM1 suggested that its role in the thermostability of the enzyme might be related to the kind of enzymes. The enzyme was stable in the pH range from 3 to 8 (Fig. 2d).
The effect of metal ions (1 and 5 mM) on the enzymes activities was investigated and results are shown in Fig. S5. The activities of TtGH74 and TtGH74ΔCBM were increased by 2–25% in the presence of 5 mM K+, Ca2+, Ba2+ and Ni2+ ions. At the same time, the enzymes activities were decreased by 5%-75% in the presence of EDTA, NH4+, Li+, Mg2+, Pb2+, Zn2+, Cu2+, Mn2+, Co2+, Fe3+ and Al3+ (1 and 5 mM). 5 mM Pb2+ and Fe3+ could dramatically reduce the enzyme activity, and the activities of TtGH74 and TtGH74ΔCBM were decreased by 90%.
The kinetic values were determined in the concentration ranges of 0.2-6 mg/mL of tamarind seed XG (Table 2). It can be seen from Table 2 that the catalytic activity of TtGH74 is much higher than the previous characterized GH74 xyloglucanases. TtGH74ΔCBM has slightly smaller Km and Kcat value than TtGH74 (Table 2). It was reported that the Kcat of GH74 xyloglucanase from Phanerochaete chrysosporium was increased slightly after deletion of CBM1 (39). These results suggested that CBM1 in GH74 xyloglucanases did not significantly affect its catalytic activity for the soluble XG.
Table 2
Comparison of biochemical properties of TtGH74 and TtGH74ΔCBM with other GH74 xyloglucanases
Entry name | Strain | Temperature (℃) | pH | Vmax (U/mg) | Km (mg/mL) | Kcat (s− 1) | References |
TtGH74 | Thielavia terrestris | 75 | 5.5 | 193.2 | 0.3225 | 283.36 | This paper |
TtGH74ΔCBM | 168.5 | 0.2671 | 233.09 |
PcGH74 | Phanerochaete chrysosporium | 55 | 6.0 | | 0.25 | 28.1 | (39) |
PcGH74ΔCBM | | 0.28 | 31.9 |
MtGH74 | Myceliophthora thermophila VKPM | 70–75 | 6.5 | | 0.57 | | (40) |
AfGH74 | Aspergillus fumigatus | 50 | 5.5 | 11.9 | 1.5 | 16.4 | (41) |
XEG74 | Paenibacillus sp KM21 | 45 | 6.0 | 36.8 | 0.96 | 49.2 | (42) |
PoGH74 | Paenibacillus odorifer | 50 | 6.0 | | 0.05 | 39.8 | (43) |
*Kcat: The number of moles of substrate that can be catalyzed per mole of enzyme per second. |
The end-products generated from the tamarind seed XG after TtGH74 hydrolysis were identified using matrix assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-TOF MS). A series of sodium adducts of hydrolyze products were detected. The m/z values of the major peaks were 1085, 1247 and 1409 corresponding to molecular masses of the reduced oligosaccharide ions [XXXG + Na]+, [XXLG + Na]+, and [XLLG + Na]+, respectively. Besides the main XG building blocks, various low molecular products were generated during the hydrolysis, whose m/z values detected by mass spectrometry may potentially correspond to XX, XXG, GXX, XGX, XL, XLG, GXL, LL, LG, XXL, LLG, GXXXG, GXLLG, XLLGX oligosaccharides, respectively (Fig. 3). TtGH74 released oligo-xyloglucans such as XXXG, XXLG, and XLLG which was the typical final products of endo-type xyloglucanases. In addition, the sequence alignments of TtGH74 with other fungal GH74 xyloglucanases (Fig. S1) revealed that TtGH74 have four conserved tryptophan residues which was regarded as the key amino acid residues for the endo-processive activity (26). Thus, indicated that TtGH74 is endo-processive-type xyloglucanase. |
Adsorption of TtGH74 and TtGH74ΔCBM on different substrates
The binding affinity of TtGH74 and TtGH74ΔCBM on different substrates (10 mg in 0.5 mL) was compared by measuring the unbound protein in the supernatant, the results are shown in Fig. 4a, all TtGH74 protein bound onto PASC, while only approximately 35% and 23% of TtGH74 bound onto Avicel and Whatman filter paper, respectively. For pretreated lignocellulosic substrates, the amount of bound protein in ascending order is 15%, 20% and 30% for deep eutectic solvents (DES) pretreated-corn bran, sulfuric acid pretreated-corn bran, and DES pretreated-apple pomace, respectively, directly proportional to the galactose content in the substrates (Table 3). Without CBM1, the adsorption capacity of TtGH74ΔCBM for insoluble carbohydrates is almost completely lost. We further measured the adsorption of TtGH74 under the condition of low cellulose content (1 mg in 0.5 mL) with additional different proportions of XG (Fig. 4b). Due to the relative excess amount of enzyme, the unbound protein was detected in supernatant, but as the increase of XG proportion, the amount of the unbound protein gradually decreased, which implied that the TtGH74 could bind onto XG. To further verify the adsorption of CBM1 on XG, we compared the mobility of CBM-deleted variant TtGH74ΔCBM with the TtGH74 in xyloglucan-containing native gels. It was observed that the mobility of TtGH74ΔCBM was increased significantly compared to TtGH74, which further clarified the binding affinity of CBM1 in TtGH74 onto XG (Fig. 4c, d). The binding affinity of CBM1 on cellulose and role of CBM1 in cellulase functionality were well demonstrated in literatures (44, 45), CBM1 is type A CBM, and was previously recognized as cellulose-binding domain due to first discovery from fungal cellulases (3, 46). However, the effects of CBM1 on XG binding and activity of fungal GH74 xyloglucanases were few reported. Our results suggested that CBM1 in TtGH74 displayed high binding affinity for both of cellulose and XG.
Hydrolysis action of TtGH74 and TtGH74ΔCBM on XG and XG-coated PASC
Hydrolysis action of TtGH74 and TtGH74ΔCBM on free XG was performed in the concentration ranges of 25–600 µg XG (Fig. 5a). When the amount of XG is low (less than 250 µg), TtGH74ΔCBM exhibited a slightly lower hydrolysis yield than the intact enzyme due to its low affinity to the substrate. However, as the amount of XG increased, the yield of reducing sugars by TtGH74ΔCBM hydrolysis increased significantly, and surpassed the yield by TtGH74 as the content of XG was over 250 µg.
XG-coated PASC with different proportions of XG were used as substrate to investigate the influence of CBM1 in enzyme functionality. XG can coat onto the surface of PASC once mixture, however, the association pattern between XG with PASC relies on the proportion ratio of XG/PASC. The previous researches revealed that at low XG/cellulose concentration ratio (25 µg/mg), all XG are tightly bound to the cellulose surface, and XG is not easy to be hydrolyzed by enzyme. However, with the increase of XG/cellulose ratio, XG forms accessible “loops” and “tails” on the cellulose surface, and the accessible XG gradually increases to a constant value (47). The schematic diagram of the association pattern of XG and PASC is shown in Fig. 5b.
When XG-coated PASC was reacted with TtGH74 or TtGH74ΔCBM (Fig. 5a), XG was more difficult to be hydrolyzed than free XG by TtGH74 and TtGH74ΔCBM because of the close association of XG with PASC. Similar as free XG, once the proportion of XG increased over 400 µg/mg, TtGH74ΔCBM released more reducing sugars from XG-coated PASC than TtGH74. The free XG was detected in the supernatant of XG/PASC solution once the ratio was greater than 400 µg/mg, so the higher catalytic performance of TtGH74ΔCBM than TtGH74 in the hydrolysis of XG-coated PASC solution as the proportion of XG over 400 µg/mg might be due to the presence of free XG in solution. However, it was worth noted that a relative higher reducing sugars were released from XG-coated PASC by TtGH74 than TtGH74ΔCBM as the proportion ratio of XG/PASC below 300 µg/mg. These results indicated that CBM1 might not be essential for hydrolysis of free XG, but effective for the associated XG to some degree. The coverage of XG on PASC even at low concentration also resulted in a significant decrease in the catalytic activity of EG1, in comparison, a less decrease in CBH1 activity was observed. These differences might be attributed to the different catalytic pattern between EG1 and CBH1. EG1 is a GH5 endo-glucanase which randomly attacks on internal sites in the cellulose chain (48), while CBH1 is an exo-cellulase which processively hydrolyzes the cellulose chain from reducing end (49).
Hydrolysis action of TtGH74 or TtGH74ΔCBM on pretreated corn bran and apple pomace
Corn bran and apple pomace were chosen as enzymatic substrates. The product yield was expressed in grams of reducing sugar produced by per kilogram of dry material (Reducing Sugars g/kg Dry Material, RS g/kg DM). Two substrates were pre-treated at 90 ℃ by 1% sulfuric acid or DES for different times (3, 6, 9, 12 h). Interestingly, no matter what kind of pretreatment methods and times used, TtGH74 produced more reducing sugars than TtGH74ΔCBM from pretreated corn bran after four days of reaction, while TtGH74ΔCBM has better hydrolysis performance on DES-pretreated apple pomace than TtGH74 (Fig. 6). Finally, corn bran pretreated with sulfuric acid for 6 h, corn bran and apple pomace pretreated with DES for 9 h were used as substrates for further exploration. As shown in Table 3, three pretreated substrates differed in chemical compositions due to different sources and pretreatment methods. DES pretreatment was more effective in removal of lignin and hemicelluloses than sulfuric acid pretreatment for corn bran. DES pretreated-apple pomace had the highest content of galactose among three substrates. Galactose is a specific substituent in the side branch of XG. Galactose may also come from the main chain of residual pectin, but galactose in pectin of apple accounts for approximately 1% of total pectin according to the literature (50), so the galactose in pretreated apple pomace mainly comes from the side chain of XG. Therefore, the higher content of galactose represents the higher amount of XG in lignocelluloses. This may explain the higher adsorption capacity of TtGH74 on DES pretreated-apple pomace than DES pretreated- and sulfuric acid pretreated-corn bran (Fig. 4a). This may also explain the much higher reducing sugars released from DES pretreated-apple pomace by either TtGH74 or TtGH74ΔCBM (Fig. 6c). It was worthy noted that the content of lignin in DES pretreated-corn bran is much lower than sulfuric acid pretreated-corn bran. This might contribute to the more reducing sugars generated from DES pretreated-corn bran than sulfuric acid pretreated-corn bran, although the content of XG is relatively lower in DES pretreated-corn bran (Fig. 6a, b).
Table 3
Chemical composition of pretreated corn bran and apple pomace substrates
| Lignin | Glucose | Xylose | Galactose | Mannose | Arabinose |
Sulfuric acid pretreated-corn bran | 20.73% | 66.04% | 8.10% | 0.18% | 1.82% | 0.74% |
DES pretreated-corn bran | 11.10% | 70.04% | 7.78% | 0.09% | 0.98% | 0.43% |
DES pretreated-apple pomace | 21.69% | 43.22% | 8.43% | 2.59% | 2.34% | 0.22% |
The presence of XG in three pretreated substrates was also confirmed by analyzing the TtGH74 hydrolysis end-products using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF MS. The various sodium adducts of oligosaccharides were released from sulfuric acid pretreated-corn bran, DES pretreated-corn bran and DES pretreated-apple pomace after TtGH74 hydrolysis (Fig. 7). Some of the detected m/z values may correspond to XG, XX, LG, XXG, XGX, XL, GGL, XLG, GXL, XXL, XXXG, LLG, XXLG, GXXXG oligosaccharides, respectively. However, different pretreatment methods might have various fractionalization and modification effects on XG in lignocellulosic biomass. Therefore, the content change and structural modification of XG in different pretreated lignocellulosic biomasses resulted in different end product profiles in hydrolysates. The more amount of GGL, XLG and GXL oligosaccharides were released from sulfuric acid pretreated-corn bran, while more XX, LG, XL, XXG, XGX oligosaccharides were released from either DES pretreated corn bran or apple pomace.
Time course of enzymatic hydrolysis of three pretreated substrates in different concentrations (from 10 to 80 mg) was performed and results are shown in Fig. 8. In general, TtGH74 produced more reducing sugars than TtGH74ΔCBM from sulfuric acid-pretreated corn bran and DES-pretreated corn bran after four days of hydrolysis, but the increase ranges varied depended on the concentration of substrates. For the two pretreated corn brans, at low substrate concentration (10 mg), the yield of reducing sugar by TtGH74 was 1.23 and 1.39 times higher than TtGH74ΔCBM. The yield of reducing sugar by TtGH74 was 1.36 and 1.13 times higher than TtGH74ΔCBM when the substrate concentration was 20 mg. While at concentrations of 40–80 mg, the yield of reducing sugar by TtGH74 was about 1.12 times higher than that of TtGH74ΔCBM. On the contrary, TtGH74ΔCBM produced more reducing sugars than TtGH74 from DES-pretreated apple pomace, no matter what concentration of substrate was used. The yield of reducing sugar by TtGH74ΔCBM was 1.68–2.06 times higher than that of TtGH74 at concentrations of 10–80 mg. From the time course experiment, it could be concluded that the presence of CBM1 is conducive to enzymatic hydrolysis, but its role and significance are substrate-specific because of the differences in the contents and structure of XG in different biomasses.
Synergistic action of TtGH74 or TtGH74ΔCBM with CBH1/EG1 mixture and xylanase on pretreated corn bran and apple pomace
As specific xyloglucan degrading enzymes, GH74 xyloglucanases were widely distributed in bacteria and fungi, and usually were co-expressed with cellulase and xylanase. In view of the presence of XG in pretreated corn bran and apple pomace (Table 3) and its blocking effect on EG1 and CBH1 activity towards XG-coated PASC (Fig. 5), it is reasonable to believe that xyloglucanases may be necessary for efficient enzymatic saccharification of XG-rich biomasses. So, we further investigated the synergy between xyloglucanase, xylanase and cellulase in hydrolysis of pretreated corn bran and apple pomace. As shown in Fig. 9 (a, b), the reducing sugar yields of TtGH74 hydrolysis were 13.88, 17.74, 13.97 (RS g/kg DM) for sulfuric acid pretreated-corn bran, DES pretreated-corn bran and DES pretreated-apple pomace, respectively, while the reducing sugar yield of GH10 xylanase hydrolysis were 16.54, 10.94, 5.22, respectively, and the reducing sugar yield of CBH1/EG1 mixture hydrolysis were 81.41, 70.26, 23.94, respectively. Correspondingly, when the enzymes acting synergistically, the yield of the combined action of GH10 xylanase with CBH1/EG1 mixture were 131.38, 96.70, 27.72, respectively, and the degree of synergy was 1.34, 1.19 and 0.95, respectively. The yield of the combined action of TtGH74 with CBH1/EG1 mixture were 192.30, 119.84, 47.08, and the degree of synergy reached up to 2.02, 1.36 and 1.24, respectively. Furthermore, the yield of the combined action of TtGH74, GH10 xylanase and CBH1/EG1 mixture were 241.25, 152.83, 77.19, and the degree of synergy was 2.16, 1.54 and 1.79 for sulfuric acid pretreated-corn bran, DES pretreated-corn bran and DES pretreated-apple pomace, respectively. The above results indicated that either TtGH74 or GH10 xylanase showed boosting effect on the hydrolysis efficiency of CBH1/EG1 mixture, in comparison, the synergistic action between TtGH74 and the CBH1/EG1 mixture was significantly higher than that of GH 10 xylanase and CBH1/EG1 mixture. Interesting, the degree of synergy between GH10 xylanase and TtGH74 was 0.62, 0.88 and 0.69, respectively, indicating that GH10 xylanase and TtGH74 not only had no synergy, but also had inhibition effect on each other due to the close spatial location of xyloglucan and xylan. However, the quaternary mixture of TtGH74, GH10 xylanase and CBH1/EG1 resulted in more synergistic action than ternary mixture of TtGH74 with CBH1/EG1.
For three pretreated substrates, when replacing TtGH74 with TtGH74ΔCBM in the enzymatic hydrolysis, the reducing sugar yield of TtGH74ΔCBM hydrolysis were 9.54, 16.00, 23.30 (RS g/kg DM) for sulfuric acid pretreated-corn bran, DES pretreated-corn bran and DES pretreated-apple pomace, respectively. The yield of the combined action of TtGH74ΔCBM with CBH1/EG1 mixture were 121.20, 105.39, 65.06, and the degree of synergy was 1.33, 1.22 and 1.38, respectively. The yield of the combined action of TtGH74ΔCBM with GH10 xylanase and CBH1/EG1 mixture were 195.42, 145.68, 98.80, and the degree of synergy reached up to 1.82, 1.50, 1.88, respectively. Similarly, no synergy but inhibition effect on each other was observed for TtGH74ΔCBM and GH10 xylanase, and the degree of synergy was 0.60, 0.82 and 0.44, respectively.
It can be inferred from the above results, the addition of TtGH74 or TtGH74ΔCBM into the CBH1/EG1 mixture or quaternary mixture of CBH1/EG and GH10 xylanase facilitated the overall hydrolysis of three pretreated substrates. The boosting effect of TtGH74 in CBH1/EG1 mixture or quaternary mixture of CBH1/EG and GH10 xylanase was relatively higher than TtGH74ΔCBM in terms of the degrees of synergy for sulfuric acid pretreated-corn bran (2.02 and 1.36 vs 1.33 and 1.22) and DES pretreated-corn bran (2.16 and 1.54 vs 1.82 and 1.50). In contrast, the boosting effect of TtGH74ΔCBM was much higher than TtGH74 in terms of the degree of synergy for pretreated apple pomace (1.38 and 1.88 vs 1.24 and 1.79). This is consistent with the bias of their activities when acted on three pretreated substrates alone, i.e. TtGH74 showed higher activity towards pretreated corn bran than TtGH74ΔCBM, conversely, TtGH74ΔCBM had much higher activity than TtGH74 towards pretreated apple pomace (Fig. 8).
In order to verify whether the above synergistic action between xyloglucanase and other glycoside hydrolases was attributed to the presence of XG in the substrates, the associated XG was extracted by strong alkali treatment and XG-free residuals were used as substrates for synergistic experiments. As shown in Fig. S6, no oligosaccharides could be released from strong alkali treated-residual by TtGH74 hydrolysis based on the analysis of HPAEC-PAD, indicating that that strong alkali treated-residual did not contain XG. In addition, almost no reducing sugar was produced from strong alkali treated-residual by GH10 xylanase hydrolysis (Fig. 9c), indicating that the strong alkali treatment also removed xylan from pretreated-lignocellulose. When XG-free substrates were hydrolyzed by the mixture of CBH1/EG1 and GH10 xylanase, CBH1/EG1 and TtGH74, and the mixture of TtGH74, CBH1/EG1 and GH10 xylanase, no synergistic action was observed. Their corresponding reducing sugar yields were almost as same as that of CBH1/EG1 mixture alone, and all degrees of synergy were close to 1, (Fig. 9c, d). However, we found that the hydrolysis efficiency of CBH1/EG1 mixture on the strong alkali treated residues was greatly improved. The reducing sugar yields reached up to 192.29, 114.81 and 93.54 (RS g/kg DM), respectively, which were 136.20%, 63.41% and 290.72% higher than the corresponding reducing sugar yields of CBH1/EG1 mixture on sulfuric acid pretreated-corn bran, DES pretreated-corn bran and DES pretreated-apple pomace, respectively (Fig. 9a). Thus, further confirmed that the blocks of XG is one of great significance factors impeding the degradation of xyloglucan-rich lignocellulose.
The structures, activity modes and enzymatic properties of GH74 xyloglucanases have been widely studied in literatures (26, 27, 39–43), but their roles in lignocellulose biomass degradation has been rarely reported, Benko et al.(18) studied the contribution of the added GH74 xyloglucanase from Trichoderma reesei (TrGH74) in the cellulase mixture to degradation of willow, barley straw, wheat straw, reed canary grass, corn stover and Solka Floc. It was found that the degree of synergy of TrGH74 with the cellulase mixture reached to 1.22 for barley straw, but only 1.07 and 1.10 for corn stover, and reed canary grass and willow, respectively. No synergistic effect was observed for wheat straw and Solka Floc. It was suggested that the degree of synergy was positively correlated with the content of XG in the substrate. Corn bran (or corn fibre) is an agricultural by-product obtained from corn processing. It was well recognized that corn bran polysaccharides were very difficult to be decomposed by enzymatic hydrolysis (51–54). Its recalcitrance was regarded due to the rich content of glucuronoarabinoxylan which was extensively decorated with variations of both monomeric and oligomeric substitutions (55). Apple pomace is the by-product from apple processing rich in pectin, cellulose and hemicelluloses (56), and could be a raw material for biofuel and biochemical production (57); however, the enzymatic saccharification of apple pomace has not well investigated. In this study, we demonstrated that both of them have rich in xyloglucan which was closely associated with other polysaccharides and still remained in solid residues after pretreatment. The associated XG significantly hindered the enzymatic hydrolysis efficiency. Our results demonstrated that the hydrolysis performance of cellulase mixture on pretreated XG-rich biomasses could be greatly improved by adding the so-called accessory enzyme xyloglucanase to enzyme mixture.