The cellulose binding region in Trichoderma reesei cellobiohydrolase I has a higher capacity in improving crystalline cellulose degradation than that of Penicillium oxalicum

Bioresource Technology 266 (2018) 19–25

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The cellulose binding region in Trichoderma reesei cellobiohydrolase I has a higher capacity in improving crystalline cellulose degradation than that of Penicillium oxalicum

T

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Jian Dua, Xiu Zhanga, Xuezhi Lia, Jian Zhaoa, Guodong Liua,b, , Baoyu Gaob, Yinbo Qua,c a

State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, Shandong, PR China Shandong Key Laboratory of Water Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Shandong University, Qingdao 266237, Shandong, PR China c National Glycoengineering Research Center, Shandong University, Qingdao 266237, Shandong, PR China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Cellulase Cellobiohydrolase Crystalline cellulose Trichoderma reesei Domain exchange

Commercial cellulase preparations for lignocellulose bioconversion are mainly produced by the fungus Trichoderma reesei. The maximum cellulose conversion of T. reesei cellulase mixture was 15%–20% higher than that of Penicillium oxalicum in the hydrolysis of corncob residue and Avicel. Nevertheless, both preparations hydrolyzed more than 92% of cellulose in NaOH-mercerized Avicel. When added to Avicel hydrolysis residue that was less reactive to P. oxalicum cellulases, cellobiohydrolase I (CBH I) from T. reesei resulted in a higher cellulose conversion than its homologous proteins from P. oxalicum and Aspergillus niger at the same protein loadings. Further domain exchange experiment attributed the high hydrolytic efficiency of T. reesei CBH I to its inter-domain linker and cellulose-binding domain. The results in part explained the superior performance of T. reesei cellulases on the degradation of native crystalline cellulose, and highlighted the important role of cellulose-binding region in determining the degree of hydrolysis by cellulases.

1. Introduction Bioconversion of lignocellulosic materials to fuels and chemicals is a sustainable route to solve the energy and resource problems of human society (Dutta and Wu, 2014). The low efficiency of cellulolytic enzymes has been a significant barrier to the industrialization of the processes developed in this area. Therefore, improvement of the performance of cellulase preparations has been a major task to lower the cost of cellulose saccharification in the past decades (Harris et al., 2014). Cellulase preparations are generally enzyme mixtures including three classes of glycoside hydrolases, i.e. cellobiohydrolase (EC 3.2.1.91), endo-1,4-β-glucanase (EC 3.2.1.4) and β-glucosidase (EC 3.2.1.21), which act on cellulose chain ends, internal cellulose chains and cello-oligosaccharides, respectively (Lynd et al., 2002). Each class of cellulase contains members in different glycoside hydrolase families according to the classification in CAZy database (Lombard et al., 2014). In addition, the enzyme mixtures often contain accessory proteins (e.g. swollenin and lytic polysaccharide monooxygenase (Harris et al., 2014) that facilitate the degradation of cellulose. For example, the exoproteome of Trichoderma reesei, the widely used cellulase producer in

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industry, contains two cellobiohydrolases (accounting for about 70% of total proteins), at least three kinds of endo-1,4-β-glucanases, one βglucosidase, and one lytic polysaccharide monooxygenase (Bischof et al., 2016; Herpoel-Gimbert et al., 2008). It has been extensively suggested that cellobiohydrolases are necessary for the efficient degradation of type I crystalline cellulose (Gusakov et al., 2007; Morozova et al., 2010; Szijarto et al., 2008), which is the major allomorph of crystalline cellulose in plants. If the structure of crystalline cellulose I was disrupted or transformed to other crystalline allomorphs, the rate of enzymatic saccharification could be greatly improved (Chundawat et al., 2011; Igarashi et al., 2007; Szijarto et al., 2008). Adsorption of cellulases to the surface of insoluble cellulose is a prerequisite for hydrolysis. For multi-domain cellulases, the adsorption is mainly mediated by the cellulose binding domain (Linder and Teeri, 1997). In fungal cellulases, the cellulose binding domain is often a module consisting of about 40 amino acids, which is classified into carbohydrate-binding module family 1 (CBM 1) in CAZy database (Boraston et al., 2004). Removal or inactivation of CBM 1 in T. reesei cellobiohydrolase I (CBH I) greatly reduced its binding ability and hydrolytic activity on crystalline cellulose (Amore et al., 2017; CruysBagger et al., 2013; Reinikainen et al., 1992), while fusion of CBM 1 to

Corresponding author at: State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, Shandong, PR China. E-mail address: [email protected] (G. Liu).

https://doi.org/10.1016/j.biortech.2018.06.050 Received 22 April 2018; Received in revised form 16 June 2018; Accepted 18 June 2018 Available online 19 June 2018 0960-8524/ © 2018 Elsevier Ltd. All rights reserved.

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restriction enzymes and ligated to a plasmid containing the promoter of P. oxalicum amylase gene amy15A, the terminator of Aspergillus nidulans trpC, and the Escherichia coli hygromycin B phosphotransferase gene hph. The sequences of CBH I-coding regions were the same with those in the above-mentioned GenBank records, as confirmed by Sanger sequencing. Then, the expression cassettes were amplified by PCR and transformed into P. oxalicum A11Δ, a mutant with low extracellular protein background on starch (Hu et al., 2015). The transformants were identified by PCR. Correct transformants were cultivated in Vogel’s salt solution (Vogel, 1956) supplemented with 15 g l−1 starch and 5 g l−1 glucose for 3 days for induced production of CBH I proteins. Additionally, strain A11Δ and the recombinant strain expressing T. reesei CBH I were cultivated in the previously optimized medium for cellulase production (Han et al., 2017) at 30 °C for 5 days. The supernatants of fermentation broths were concentrated with centrifugal ultrafiltration concentrators with a molecular cut-off of 3 kDa (Pall Corporation, USA) and used for saccharification. FPase and cellobiohydrolase activities were measured with filter paper and pNPC as the substrate, respectively, as previously described (Deshpande et al., 1984; Ghose, 1987). Protein concentration was measured using the Bradford method (Bradford, 1976).

CBM 1-lacking cellulases could significantly improve their hydrolytic activities on crystalline cellulose (Szijarto et al., 2008; Thongekkaew et al., 2013). In addition, CBH I with CBM 1 exhibited higher cellulose conversions at the plateau of hydrolysis (i.e. maximum conversion) than the catalytic domain alone (Amore et al., 2017; Voutilainen et al., 2008), implying that CBM 1 is critical for the degradation of the highly recalcitrant fraction in the heterogeneous cellulosic materials. The cellulase mixture produced by T. reesei was identified as an outstanding degrader of native crystalline cellulose by screening 14,000 moulds more than 60 years ago (Allen et al., 2009). Compared with enzymes of many other sources (e.g. Aspergillus, Penicillium and Fusarium species), T. reesei cellulases showed a much higher conversion of cotton after long-time hydrolysis, while its efficiency on carboxymethylcellulose as a soluble amorphous substrate was undistinguished (Mandels and Weber, 1969). This difference raised the question of why the cellulase mixtures had similar compositions but quite different efficiencies in the hydrolysis of insoluble crystalline cellulose. In this study, the hydrolysis performances of cellulases produced by T. reesei and another cellulolytic fungus Penicillium oxalicum (Liu et al., 2013a) were compared. The difference of the two enzyme mixtures in final cellulose conversion was found to be dependent on the structure of cellulose. Subsequently, the different degrees of cellulose hydrolysis between the two mixtures were attributed to the efficiency of CBH I, and further its cellulose binding region.

2.4. Enzymatic hydrolysis Enzymatic hydrolysis was performed in 0.2 M Na-acetate buffer (pH 4.8) containing 1 g l−1 sodium azide. The loadings of substrates and cellulases were indicated in the text. The ingredients were mixed in 125 ml Erlenmeyer flasks with a reaction volume of 40 ml, and incubated at 48 °C using a thermostated air bath shaker set at 150 rpm. Samples were taken for sugar analysis by HPLC, and cellulose conversion was calculated as previously described (Han et al., 2017).

2. Material and methods 2.1. Cellulosic materials Avicel PH-101, a nearly pure cellulose, was purchased from Aladdin, China. Corncob residue (CCR) as a byproduct of xylose industry with diluted acid pretreatment of corncob was provided by Shandong Longlive Biotechnology Co., Ltd. (Shandong, China). The composition of CCR was determined according to the NREL method (Sluiter et al., 2008).

2.5. X-ray diffraction analysis of cellulosic materials X-ray diffraction (XRD) was performed with a Bruker D8-Advance instrument (Bruker, Germany). Samples were dried, ground, and scanned from 2θ = 8° to 80°. The data of XRD were analyzed by software MDI Jade 5.0.

2.2. NaOH pretreatment of Avicel Avicel was treated with 20% (w/v) NaOH with a solid to liquid ratio of 1:5 for 1 h at 45 °C. After the treatment, the solid was collected with centrifugation, neutrilized by 6 M H2SO4, and washed with distilled water. These NaOH-treated cellulose was then dried with lyophilization in vacuum.

2.6. Determination of enzyme adsorption To determine the adsorption isotherms of CBH I proteins, Avicel at 10% DM was added with CBH I solutions ranging from 1 to 15 mg g−1 DM. The ingredients were mixed in 10 ml centrifuge tubes with a reaction volume of 6 ml, and horizontally placed in a thermostated air bath shaker set at 150 rpm and 48 °C for 3 h. Then, samples were centrifuged at 9500g for 10 min, and the concentration of free enzymes in the supernatant was determined. The concentration of bound enzymes (total enzyme minus bound enzyme) was plotted against that of free enzyme, which was fitted with the equation as below:

2.3. Cellulase production Cellulase mixtures were produced by cultivating previously engineered strains in 500-mL Erlenmeyer flasks at 30 °C for 7 days. T. reesei SCB18 was obtained by genetic modification of strain SN1 (Gao et al., 2017), and P. oxalicum I1-13 was obtained by engineering strain 114-2, as previously described (Yao et al., 2016). Unless specifically indicated, the T medium and P medium were used for cellulase production by SCB18 and I1-13, respectively. The T medium contained 20 g l−1 microcrystalline cellulose, 2.5 g l−1 (NH4)2SO4, 5 g l−1 KH2PO4, 0.6 g l−1 MgSO4·7H2O, 1 g l−1 CaCl2·2H2O, and 20 g l−1 corn steep liquor (Qian et al., 2016). The P medium contained 36 g l−1 microcrystalline cellulose, 21 g l−1 soybean cake powder, 36 g l−1 wheat bran, 0.5 g l−1 KH2PO4 and 0.5 g l−1 MgSO4·7H2O. For the production of CBH I proteins, the coding sequences of native enzymes were amplified from the genomic DNA of corresponding fungal strains. The gene encoding CBH I in Aspergillus niger was cloned from strain N593 (ATCC number 64973). GenBank accession numbers of CBH I homologs from T. reesei, P. oxalicum and A. niger are EGR44817.1, EPS32984.1 and AAF04492.1, respectively. The sequences encoding catalytic domain and linker-CBM 1 from different species were fused via PCR. The obtained fragments were cut by

B=

nF Kd−1 + F

B and F are the concentrations of bound enzyme (μmol g−1 cellulose) and free enzyme in the solution (μM), respectively. n and Kd, obtained by regression analysis, are the maximum adsorption (μmol g−1 cellulose) and the Langmuir adsorption equilibrium constant (μM−1), respectively. In addition, for the adsorption of 1 mg·g−1 DM of CBH I on Avicel or NaOH-treated Avicel at 10% DM, the supernatant and precipitate after centrifugation were analyzed by SDS-PAGE. 2.7. Sequence analysis and structural modelling For sequence analysis, the linker regions between catalytic domain and CBM 1 in CBH I homologs were extracted from the amino acid after 20

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the conserved P-I-G-S-T sequence to the amino acid six residues ahead of Q-C-G-G sequence (Sammond et al., 2012). The 36 amino acid-sequence at the C-terminus of CBH I was extracted as CBM 1. The structure homology models of CBM 1 domains were built on the SWISSMODEL server (Biasini et al., 2014) using the determined structure of CBM 1 in T. reesei CBH I (Kraulis et al., 1989) as the template. The PyMol software (http://www.pymol.org/) was used for the visualization of structures and the creation of images. 3. Results and discussion 3.1. T. reesei cellulases efficiently degraded the cellulose in corncob residue that was recalcitrant to P. oxalicum cellulases In our previous study (Song et al., 2016), the performances of three commercial cellulase preparations were compared on the hydrolysis of corncob residue (CCR), which contained 56.7% cellulose, 5.0% hemicellulose and 23.2% lignin. The two preparations produced by T. reesei resulted in higher cellulose conversions than that produced by P. oxalicum at the same protein loadings. This disparity, however, was not observed during the hydrolysis of delignified CCR (Song et al., 2016). To further explore this phenomenon, the performances of cellulase mixtures produced in-house by T. reesei and P. oxalicum strains with clear genetic backgrounds (Gao et al., 2017; Yao et al., 2016) were compared. The two cellulase preparations were named CT and CP, respectively. Both strains were engineered for the over-expression of extracellular β-glucosidases, which eliminated the inhibition of accumulated cellobiose on cellulose hydrolysis (Song et al., 2016; Yao et al., 2016). Indeed, no cellobiose was detected by high performance liquid chromatography (HPLC) in the hydrolysates in this study. CT showed a faster hydrolysis of CCR during the first 12 h of saccharification than CP of the same filter paper enzyme (FPase) activity (Fig. 1A). In addition, CT reached a final cellulose conversion of 66.1% at 72 h, higher than that of CP (49.5%). Actually, only a small increase in cellulose conversion was observed for CP after 24 h of saccharification. Why did CP show a poor degradation of cellulose in the later stage of CCR saccharification? It was first hypothesized that CP might lose its hydrolyzing activity quickly during the saccharification. Heat, glucose, xylo-oligosaccharides and vanillin were all known to diminish cellulase activities during lignocellulose saccharification. However, CT and CP were similarly affected by these factors. Another possibility was that there might be some cellulose in CCR resistant to the degradation by CP but not CT. To test this hypothesis, the solid residue of CCR after 72 h of saccharification by CP was boiled and washed to remove glucose and active CP, and then hydrolyzed by freshly added enzymes. The residue was efficiently hydrolyzed by fresh CT but poorly by fresh CP (Fig. 1B), suggesting that the cellulose contained in this residue was indeed hard to degrade for CP. The characteristics of CP-resistant cellulose in CCR were studied by comparing the residues obtained from different hydrolysis setups. XRD spectra of CCR indicated it mainly contained cellulose in crystalline allomorph I (Garvey et al., 2005; Park et al., 2010). The cellulose Icharacteristic diffraction peaks at around 16°, 22.5° and 35° significantly decreased in the residues after CT hydrolysis, but remained similar in the residue of CP.

Fig. 1. Enzymatic hydrolysis of corncob residue by crude cellulase preparations. (A) Time course of hydrolysis with enzyme dosage of 10 FPU g−1 DM. (B) Sequential hydrolysis. The residual solids after 72 h of hydrolysis with enzyme dosage of 20 FPU g−1 DM were boiled for 10 min to inactivate enzymes, and washed with 0.2 M Na-acetate buffer. Then, fresh enzymes of 20 FPU g−1 DM were added for another 72 h of hydrolysis. The enzymes used in the two stages are indicated in the figure (e.g. “CP + CT” means hydrolysis by CP during the first 72 h and by CT during the second 72 h). In both panels, the substrates were loaded at 5% DM, and the hydrolyses were performed in triplicate. Data represent mean ± SD.

(Fig. 2A). This disparity was also observed when using cotton fiber, containing mainly cellulose Iβ (Zhang et al., 2006), as the substrate (data not shown). The P. oxalicum strain was also cultivated with the T medium used for CT production (see Section 2), and the obtained cellulase showed a cellulose conversion of 16.8% after 72 h of Avicel hydrolysis. Thus, the different hydrolytic capacities between CT and CP were not due to the use of different media. The maximum cellulose conversions by the two preparations in Avicel hydrolysis were measured by using a low substrate loading (1% dry matter, DM) and a series of enzyme dosages. At the dosage of 50 FPase units (FPU) g−1 DM, both CT and CP reached their maximum cellulose conversion, with 68.6% and 44.0% of Avicel degraded respectively (Fig. 2B). Supplementation of fresh enzymes after 72 h of Avicel hydrolysis also confirmed that the residual cellulose of CP hydrolysis could be more efficiently degraded by fresh CT than fresh CP (Fig. 2C). These results, if connected to those of CCR hydrolysis, suggested that the low degradability of cellulose in CCR for CP was more likely due to the structure of cellulose itself rather than the blocking effect of non-cellulose components (e.g. lignin). The crystalline form of cellulose has a remarkable effect on the hydrolytic efficiency of cellulases (Igarashi et al., 2007). The performances of CT and CP were compared on treated Avicel with a different crystalline structure. Mercerization of Avicel with NaOH swelled cellulose fibers, and XRD analysis revealed a typical spectrum of cellulose II (Ishikawa et al., 1997) after the treatment. Strikingly, cellulose conversion was similar between CT and CP throughout the hydrolysis of NaOH-pretreated Avicel, with more than 92% of the substrate hydrolyzed to glucose by both preparations after 72 h (Fig. 2D). Consistent with the higher conversions, both reaction mixtures became transparent after the saccharification. Taken together, the higher hydrolytic

3.2. The superior degrading ability of T. reesei cellulases was dependent on the structure of cellulose The above results suggested that CT had a distinctive capability in the hydrolysis of crystalline cellulose I contained in CCR, which was less reactive to CP. To validate this hypothesis, Avicel consisting of mainly cellulose I and amorphous cellulose was used for comparing the efficiencies of the cellulases. Again, CT resulted in significantly higher cellulose conversion (40.5%) than CP (22.8%) after 72 h of hydrolysis 21

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Fig. 2. Enzymatic hydrolysis of nearly pure cellulose. (A) Hydrolysis of Avicel at 5% DM with enzyme dosage of 10 FPU g−1 DM. (B) Hydrolysis of Avicel at 1% DM for 72 h with various cellulase dosages. (C) Two-stage hydrolysis of Avicel. Avicel at 5% DM was first hydrolyzed with enzyme dosage of 20 FPU g−1 DM for 72 h, and then fresh cellulases of 20 FPU g−1 DM were added to the mixture for another 72 h of hydrolysis. The enzymes used in the two stages are indicated in the figure (e.g. “CP + CT” means hydrolysis by CP during the first 72 h and addition of CT at 72 h). (D) Hydrolysis of NaOH-pretreated Avicel at 5% DM with enzyme dosage of 10 FPU g−1 DM. The hydrolyses were performed in triplicate. Data represent mean ± SD.

CT and CP were likely due to the different hydrolytic efficiencies of CBH I proteins they contained. The addition of T. reesei CBH I resulted in a greater increase in cellulose conversion than that of crude cellulase preparation CT added at the same protein loading (Fig. 4). This demonstrated that CBH I was more important in hydrolyzing the residual Avicel than the other components in CT. When the dosage of TCBH was doubled, further improvement in cellulose conversion was quite limited (from 23.8% to 25.9%), suggesting the improving effect of TCBH was close to saturation.

efficiency of CT than CP is likely to be specific on the cellulose of crystalline allomorph I.

3.3. CBH I contributed to the superior Avicel-degrading ability of T. reesei cellulase mixture Considering the important role of CBH I in the hydrolysis of crystalline cellulose by fungal cellulase mixtures, this enzyme was hypothesized to be associated with the difference in cellulose I-degrading ability between CT and CP. Previous proteomic studies showed that CBH I was the most abundant component in the exoproteome of both species (Herpoel-Gimbert et al., 2008; Liu et al., 2013b). To compare the properties of these two CBH I homologs (named TCBH and PCBH for those from T. reesei and P. oxalicum, respectively), they were expressed in P. oxalicum mutant strain A11Δ with a low extracellular-protein background on starch (Hu et al., 2015). For comparison, the ortholog of these two enzymes in A. niger (named ACBH) was also expressed. All the three enzymes have a N-terminal catalytic domain and a C-terminal CBM 1 connected by a linker peptide. In addition, two chimeric enzymes with mutual exchange of the region for cellulose binding (linker region plus CBM 1) between TCBH and PCBH were expressed and named PTCBH and TPCBH, respectively (Fig. 3A). The extracellular proteins of recombinant strains cultivated on starch contained mainly the expressed CBH I proteins (Fig. 3B), which were used for the subsequent experiments. When pNPC was used as a substrate, the raw CBH I proteins showed different specific activities, with the highest values detected for PCBH and PTCBH (Fig. 3C). The CBH I proteins were supplemented to the hydrolysis system of Avicel. The substrate was first hydrolyzed by preparation CP for 72 h, and then CBH I was added for another 72 h of hydrolysis. With the addition of Na-acetate buffer at 72 h (as a control), cellulose conversion only showed a minor increase from 72 to 144 h (from 15.2% to 16.0%), suggesting that the rest of the Avicel was difficult to hydrolyze for the residual CP in the system (Fig. 4). As expected, addition of fresh CT led to a greater increase of cellulose conversion (22.6% at 144 h) than fresh CP (18.0%). Notably, the addition of TCBH at 72 h was sufficient to enable continuous hydrolysis of Avicel, with a cellulose conversion of 23.8% reached at 144 h. Addition of PCBH showed moderate increase, while ACBH showed the least increase, in cellulose conversion. These results suggested that the different Avicel-degrading abilities between

3.4. The linker-CBM 1 in T. reesei CBH I conferred its high efficiency in crystalline cellulose degradation A recent domain exchange study between CBH I homologs from T. reesei and Penicillium funiculosum indicated that catalytic domain, linker and CBM 1 were all responsible for the different efficiencies between these two enzymes (Taylor et al., 2018). Here, the addition of the chimeric enzyme PTCBH facilitated the degradation of Avicel residue to a similar degree with that of TCBH, while the effect of TPCBH addition was similar to that of PCBH (Fig. 4). Thus, the linker-CBM 1 region is the main determinant of the degrading ability of the two CBH I proteins on Avicel residue. The five CBH I proteins also showed different adsorption behaviors on Avicel, as revealed by SDS-PAGE and calculation of adsorption-related parameters (Fig. 5). TCBH and PTCBH were efficiently adsorbed to Avicel, with negligible protein found in the supernatant. In contrast, ACBH of the same concentration only showed very weak adsorption on Avicel. Moderate adsorption was observed for PCBH and TPCBH. That is to say, the linker-CBM 1 region in TCBH has a higher binding ability on Avicel than that in PCBH. This might explain the higher hydrolytic efficiencies of TCBH and TPCBH on Avicel residue (Fig. 4). Interestingly, both TCBH and PCBH were efficiently adsorbed to NaOH-pretreated Avicel when the same enzyme-to-substrate ratio was applied (Fig. 5B), suggesting the inefficient adsorption of PCBH on Avicel was also related to the structure of cellulose. The linker-CBM 1 regions in CBH I homologs from different species were then compared from a structural perspective. The linker sequences in TCBH, PCBH and ACBH contain 32, 50 and 41 amino acids, respectively, of which 10, 27 and 32 are serine or threonine as putative O22

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linked glycosylation sites (Amore et al., 2017). The differences in the length and glycosylation of linker peptides might affect the distance between the two domains and the cellulose-binding affinity of CBH I (Amore et al., 2017). For the CBM 1 domain, sequence variation among the CBH I homologs was mainly observed for the amino acid residues from position 22 to 30. Among the five residues important for cellulose binding on the flat surface of TCBH CBM 1 (Linder et al., 1995b), Y32, Y33, N29 and Q34 are identical between the two CBM 1 sequences, and Y5 is substituted by a tryptophan in PCBH CBM 1. This substitution has been proved to increase the affinity of CBM 1 on cellulose (Linder et al., 1995a), which should therfore not account for the lower affinity of PCBH CBM 1 than TCBH CBM 1. Three other amino acid variations, L28Q, V27K and P30E, could increase the hydrophilicity of the cellulose-binding surface, the opposite surface and the tip of PCBH CBM 1, respectively. These three amino acids were also substituted in ACBH. In future study, detailed mutations of these varied amino acids are expected to be conducted to reveal their functions in the binding and disrupting (Mello and Polikarpov, 2014; Wang et al., 2008) abilities of CBM 1 and hydrolytic efficiency of CBH I on crystalline cellulose. The role of cellulose-binding region in determining the degree of crystalline cellulose degradation was observed but not explored in previous studies. The addition of linker-CBM 1 from Chaetomium thermophilum CBH I to a CBM 1-lacking CBH I enabled higher cellulose conversion in prolonged hydrolysis of Avicel than the addition of that from T. reesei CBH I, which was consistent with the higher degradation degree of native C. thermophilum CBH I than T. reesei CBH I (Voutilainen et al., 2008). In another study, CBM 1 in T. reesei CBH I was shown to enhance the binding but lower the specific activity of bound enzyme on crystalline cellulose in the first 2 h of hydrolysis (Nakamura et al., 2013). The authors speculated that CBM 1 might lead to nonproductive binding of CBH I on cellulose surface. However, the original hydrolysis curves in their study showed that CBM 1 was essential for maintaining the hydrolysis rate at later stage of reaction. Therefore, the binding of CBM 1 to the highly-recalcitrant cellulose components in the heterogeneous substrate might have opposite effects on hydrolysis between the initial and prolonged stages of saccharification.

Fig. 3. Expression of CBH I proteins of difference domain architectures. (A) Schematic representation of the domain composition. PCBH, P. oxalicum CBH I; TCBH, T. reesei CBH I; ACBH, A. niger CBH I; PTCBH, chimeric construct containing the catalytic domain of PCBH and linker-CBM 1 of TCBH; TPCBH, chimeric construct containing the catalytic domain of TCBH and linker-CBM 1 of PCBH. (B) SDS-PAGE analysis of extracellular proteins produced by different P. oxalicum strains on starch. The extracellular proteins of parent strain A11Δ were loaded as a control. (C) Specific activities of raw CBH I proteins on pNPC. Data represent mean ± SD of duplicate measurements. Prefixes of CBH I names are shown in panels B and C.

3.5. Supplementation of T. reesei CBH I enhanced the performance of P. oxalicum exoproteome on the saccharification of crystalline cellulose Since the addition of TCBH “restarted” the efficient hydrolysis of Avicel by CP that was almost aborted after 72 h (Fig. 4), the possibility of engineering the efficiency of CP by supplementing TCBH at the start of hydrolysis was explored. First, the in vitro mixture of CP and TCBH with a mass ratio of 5:1 was used for the hydrolysis of Avicel, cotton and CCR. For all three materials, cellulose conversion was significantly enhanced by adding TCBH into CP (Fig. 6A–C). However, the efficiency of the artificial cellulase mixture was still lower than that of CT with a 20% less protein dosage. Because the expression of TCBH in P. oxalicum was driven by the starch-inducible promoter amy15A(p) (Hu et al., 2015), mixture of TCBH with P. oxalicum cellulases could be obtained by cultivating the recombinant strain in the medium containing both cellulose and starch. As shown in Fig. 6D, the band of TCBH was observed in the SDS-PAGE analysis of extracellular proteins produced by the TCBH-expressing P. oxalicum strain. The specific FPase activity of the recombinant enzyme mixture was slightly higher than that of the parent strain A11Δ with the standard one-hour assay (Fig. 6E). Nevertheless, the inclusion of TCBH improved cellulose conversion in the hydrolysis of Avicel, cotton and CCR up to 1.5–1.7 fold as compared with the parent enzyme mixture at the same dosage (Fig. 6F).

Fig. 4. Hydrolysis of Avicel by CP and different CBH I proteins. Avicel at 5% DM was first hydrolyzed by CP of 5 mg g−1 DM for 72 h, and then CBH I of 1 mg g−1 DM or 2 mg g−1 DM were added to the mixture for another 72 h of hydrolysis. Prefixes of CBH I names are shown. Cellulase mixture CP or CT of 1 mg g−1 DM, or Na-acetate buffer of the same volume, was also added at 72 h as control. The hydrolyses were performed in duplicate. Data represent mean ± SD.

4. Conclusions In this study, the cellulase mixture produced by Trichoderma reesei was found to efficiently degrade crystalline allomorph I cellulose that 23

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Fig. 5. Adsorption of CBH I proteins on cellulose. (A) Adsorption of CBH I on Avicel at 10% DM. Ctrl, CBH I preparation as the control; S, supernatant at 3 h; P, precipitate at 3 h. (B) Adsorption of PCBH and TCBH on NaOH-pretreated Avicel at 10% DM. (C) and (D), the maximum adsorption and the Langmuir adsorption equilibrium constant according to adsorption isotherm, respectively.

Fig. 6. Hydrolytic efficiency of the mixture of T. reesei CBH I and P. oxalicum cellulases. For the hydrolysis of Avicel (A), cotton fiber (B) and CCR (C), substrates at 5% DM was hydrolyzed by CP or CT of 5 mg g−1 DM, or CP of 5 mg g−1 DM plus TCBH of 1 mg g−1 DM (CP + TCBH) for 72 h. (D) SDS-PAGE analysis of the extracellular proteins of the parent P. oxalicum strain A11Δ and the recombinant strain A11Δ-TCBH expressing TCBH. The band of TCBH was indicated by an arrow. (E) Specific activities of crude enzymes on filter paper. (F) Hydrolysis of cellulosic substrates at 5% DM by crude enzymes of 10 mg g−1 DM for 72 h. The hydrolyses were performed in duplicate. Data represent mean ± SD.

was less reactive to the cellulases of Penicillium oxalicum. Cellobiohydrolase I as a major component in cellulase mixture, and further its cellulose-binding region, was identified to be a determinant factor for the hydrolysis degree. Given the relationship between crystalline form of cellulose and the performance of cellulases of different origins, the effect of pretreatment method on the structure of cellulose should be seriously considered in the development of cellulase preparations for lignocellulose saccharification.

Competing interests

Acknowledgements

References

This work was supported by the National Natural Science Foundation of China – China (grant number 31700062), The Funding for Shandong Postdoctoral Innovation Project – China (201701008), and State Key Laboratory of Microbial Technology, Shandong University.

Allen, F., Andreotti, R., Eveleigh, D.E., Nystrom, J., 2009. Mary Elizabeth Hickox Mandels, 90, bioenergy leader. Biotechnol. Biofuels 2, 22. Amore, A., Knott, B.C., Supekar, N.T., Shajahan, A., Azadi, P., Zhao, P., Wells, L., Linger, J.G., Hobdey, S.E., Vander Wall, T.A., Shollenberger, T., Yarbrough, J.M., Tan, Z., Crowley, M.F., Himmel, M.E., Decker, S.R., Beckham, G.T., Taylor 2nd, L.E., 2017. Distinct roles of N- and O-glycans in cellulase activity and stability. Proc. Natl. Acad.

The authors have no competing interests to declare. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.biortech.2018.06.050.

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Penicillium: from strain screening to systems biology. Biofuels 4 (5), 523–534. Liu, G., Zhang, L., Qin, Y., Zou, G., Li, Z., Yan, X., Wei, X., Chen, M., Chen, L., Zheng, K., Zhang, J., Ma, L., Li, J., Liu, R., Xu, H., Bao, X., Fang, X., Wang, L., Zhong, Y., Liu, W., Zheng, H., Wang, S., Wang, C., Xun, L., Zhao, G.P., Wang, T., Zhou, Z., Qu, Y., 2013b. Long-term strain improvements accumulate mutations in regulatory elements responsible for hyper-production of cellulolytic enzymes. Sci. Rep. 3, 1569. Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P.M., Henrissat, B., 2014. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res. 42 (Database issue), D490–D495. Lynd, L.R., Weimer, P.J., van Zyl, W.H., Pretorius, I.S., 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66 (3), 506–577. Mandels, M., Weber, J., 1969. The production of cellulases. In: Cellulases and Their Applications, vol. 95. American Chemical Society, pp. 391–414. Mello, B.L., Polikarpov, I., 2014. Family 1 carbohydrate binding-modules enhance saccharification rates. AMB Express 4, 36. Morozova, V.V., Gusakov, A.V., Andrianov, R.M., Pravilnikov, A.G., Osipov, D.O., Sinitsyn, A.P., 2010. Cellulases of Penicillium verruculosum. Biotechnol. J. 5 (8), 871–880. Nakamura, A., Tsukada, T., Auer, S., Furuta, T., Wada, M., Koivula, A., Igarashi, K., Samejima, M., 2013. The tryptophan residue at the active site tunnel entrance of Trichoderma reesei cellobiohydrolase Cel7A is important for initiation of degradation of crystalline cellulose. J. Biol. Chem. 288 (19), 13503–13510. Park, S., Baker, J.O., Himmel, M.E., Parilla, P.A., Johnson, D.K., 2010. Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance. Biotechnol. Biofuels 3, 10. Qian, Y.C., Zhong, L.X., Hou, Y.H., Qu, Y.B., Zhong, Y.H., 2016. Characterization and strain improvement of a hypercellulytic variant, Trichoderma reesei SN1, by genetic engineering for optimized cellulase production in biomass conversion improvement. Front. Microbiol. 7, 1349. Reinikainen, T., Ruohonen, L., Nevanen, T., Laaksonen, L., Kraulis, P., Jones, T.A., Knowles, J.K., Teeri, T.T., 1992. Investigation of the function of mutated cellulosebinding domains of Trichoderma reesei cellobiohydrolase I. Proteins 14 (4), 475–482. Sammond, D.W., Payne, C.M., Brunecky, R., Himmel, M.E., Crowley, M.F., Beckham, G.T., 2012. Cellulase linkers are optimized based on domain type and function: insights from sequence analysis, biophysical measurements, and molecular simulation. PLoS One 7 (11), e48615. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008. Determination of Structural Carbohydrates and Lignin in Biomass. NREL. Song, W., Han, X., Qian, Y., Liu, G., Yao, G., Zhong, Y., Qu, Y., 2016. Proteomic analysis of the biomass hydrolytic potentials of Penicillium oxalicum lignocellulolytic enzyme system. Biotechnol. Biofuels 9, 68. Szijarto, N., Siika-Aho, M., Tenkanen, M., Alapuranen, M., Vehmaanpera, J., Reczey, K., Viikari, L., 2008. Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces. J. Biotechnol. 136 (3–4), 140–147. Taylor 2nd, L.E., Knott, B.C., Baker, J.O., Alahuhta, P.M., Hobdey, S.E., Linger, J.G., Lunin, V.V., Amore, A., Subramanian, V., Podkaminer, K., Xu, Q., VanderWall, T.A., Schuster, L.A., Chaudhari, Y.B., Adney, W.S., Crowley, M.F., Himmel, M.E., Decker, S.R., Beckham, G.T., 2018. Engineering enhanced cellobiohydrolase activity. Nat. Commun. 9 (1), 1186. Thongekkaew, J., Ikeda, H., Masaki, K., Iefuji, H., 2013. Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp. S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates. Enzyme Microb. Technol. 52 (4–5), 241–246. Vogel, H.J.A., 1956. A convenient growth medium for Neurospora (Medium V). Microb. Genet. Bull. 13, 42–43. Voutilainen, S.P., Puranen, T., Siika-Aho, M., Lappalainen, A., Alapuranen, M., Kallio, J., Hooman, S., Viikari, L., Vehmaanpera, J., Koivula, A., 2008. Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases. Biotechnol. Bioeng. 101 (3), 515–528. Wang, L., Zhang, Y., Gao, P., 2008. A novel function for the cellulose binding module of cellobiohydrolase I. Sci. China C Life Sci. 51 (7), 620–629. Yao, G., Wu, R., Kan, Q., Gao, L., Liu, M., Yang, P., Du, J., Li, Z., Qu, Y., 2016. Production of a high-efficiency cellulase complex via β-glucosidase engineering in Penicillium oxalicum. Biotechnol. Biofuels 9, 78. Zhang, Y.H., Himmel, M.E., Mielenz, J.R., 2006. Outlook for cellulase improvement: screening and selection strategies. Biotechnol. Adv. 24 (5), 452–481.

Sci. U.S.A. 114 (52), 13667–13672. Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Gallo Cassarino, T., Bertoni, M., Bordoli, L., Schwede, T., 2014. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42 (Web Server issue), W252–W258. Bischof, R.H., Ramoni, J., Seiboth, B., 2016. Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei. Microb. Cell Fact. 15 (1), 106. Boraston, A.B., Bolam, D.N., Gilbert, H.J., Davies, G.J., 2004. Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem. J. 382 (Pt. 3), 769–781. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Chundawat, S.P., Bellesia, G., Uppugundla, N., da Costa Sousa, L., Gao, D., Cheh, A.M., Agarwal, U.P., Bianchetti, C.M., Phillips Jr., G.N., Langan, P., Balan, V., Gnanakaran, S., Dale, B.E., 2011. Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate. J. Am. Chem. Soc. 133 (29), 11163–11174. Cruys-Bagger, N., Tatsumi, H., Ren, G.R., Borch, K., Westh, P., 2013. Transient kinetics and rate-limiting steps for the processive cellobiohydrolase Cel7A: effects of substrate structure and carbohydrate binding domain. Biochemistry 52 (49), 8938–8948. Deshpande, M.V., Eriksson, K.E., Pettersson, L.G., 1984. An assay for selective determination of exo-1,4,-β-glucanases in a mixture of cellulolytic enzymes. Anal. Biochem. 138 (2), 481–487. Dutta, S., Wu, K.C.W., 2014. Enzymatic breakdown of biomass: enzyme active sites, immobilization, and biofuel production. Green Chem. 16 (11), 4615–4626. Gao, J., Qian, Y., Wang, Y., Qu, Y., Zhong, Y., 2017. Production of the versatile cellulase for cellulose bioconversion and cellulase inducer synthesis by genetic improvement of Trichoderma reesei. Biotechnol. Biofuels 10, 272. Garvey, C.J., Parker, I.H., Simon, G.P., 2005. On the interpretation of X-ray diffraction powder patterns in terms of the nanostructure of cellulose I fibres. Macromol. Chem. Phys. 206 (15), 1568–1575. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59 (2), 257–268. Gusakov, A.V., Salanovich, T.N., Antonov, A.I., Ustinov, B.B., Okunev, O.N., Burlingame, R., Emalfarb, M., Baez, M., Sinitsyn, A.P., 2007. Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 97 (5), 1028–1038. Han, X., Song, W., Liu, G., Li, Z., Yang, P., Qu, Y., 2017. Improving cellulase productivity of Penicillium oxalicum RE-10 by repeated fed-batch fermentation strategy. Bioresour. Technol. 227, 155–163. Harris, P.V., Xu, F., Kreel, N.E., Kang, C., Fukuyama, S., 2014. New enzyme insights drive advances in commercial ethanol production. Curr. Opin. Chem. Biol. 19, 162–170. Herpoel-Gimbert, I., Margeot, A., Dolla, A., Jan, G., Molle, D., Lignon, S., Mathis, H., Sigoillot, J.C., Monot, F., Asther, M., 2008. Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains. Biotechnol. Biofuels 1 (1), 18. Hu, Y., Xue, H., Liu, G., Song, X., Qu, Y., 2015. Efficient production and evaluation of lignocellulolytic enzymes using a constitutive protein expression system in Penicillium oxalicum. J. Ind. Microbiol. Biotechnol. 42 (6), 877–887. Igarashi, K., Wada, M., Samejima, M., 2007. Activation of crystalline cellulose to cellulose III(I) results in efficient hydrolysis by cellobiohydrolase. FEBS J. 274 (7), 1785–1792. Ishikawa, A., Okano, T., Sugiyama, J., 1997. Fine structure and tensile properties of ramie fibres in the crystalline form of cellulose I, II, IIII and IVI. Polymer 38 (2), 463–468. Kraulis, J., Clore, G.M., Nilges, M., Jones, T.A., Pettersson, G., Knowles, J., Gronenborn, A.M., 1989. Determination of the three-dimensional solution structure of the Cterminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing. Biochemistry 28 (18), 7241–7257. Linder, M., Teeri, T.T., 1997. The roles and function of cellulose-binding domains. J. Biotechnol. 57 (1), 15–28. Linder, M., Lindeberg, G., Reinikainen, T., Teeri, T.T., Pettersson, G., 1995a. The difference in affinity between two fungal cellulose-binding domains is dominated by a single amino acid substitution. FEBS Lett. 372 (1), 96–98. Linder, M., Mattinen, M.L., Kontteli, M., Lindeberg, G., Stahlberg, J., Drakenberg, T., Reinikainen, T., Pettersson, G., Annila, A., 1995b. Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I. Protein Sci. 4 (6), 1056–1064. Liu, G., Qin, Y., Li, Z., Qu, Y., 2013a. Improving lignocellulolytic enzyme production with

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