Xylanase (GH11) from Acremonium cellulolyticus: homologous expression and characterization

Lignocellulosic biomass can be converted into biofuel or bio-based materials (Deutschmann and Dekker [2012]; Kumar et al. [2008]) but must be converted into fermentable sugars by saccharification. Xylan is one of the major structural components of plant cell walls and is the second most abundant renewable biomass resource (York and O’Neill [2008]). Xylan consists of a backbone of β-1,4-D-xylan with short side chains of O-acetyl, β-L-arabinofuranosyl, D-α-glucuronic acid and phenolic acid (Coughlan and Hazlewood [1993]). Xylanases (endo-1,4-β-xylanases; EC 3.2.1.8) catalyze the hydrolysis of the β-1,4 bonds of xylan and thus are important enzymes for the degradation of hemicellulosic polysaccharides (Collins et al. [2005]; Prade [1996]). Based on their amino acid sequence similarities, xylanases are mainly classified into families 10 and 11 of the glycoside hydrolases (GH; http://www.cazy.org/Glycoside-Hydrolases.html; (Coutinho and Henrissat [1999]). GH10 xylanases generally have a molecular weight ≥30 kDa and a low pI, while GH11 xylanases are generally smaller (approximately 20 kDa) and have a high pI (Beaugrand et al. [2004]). The crystal structures of xylanases show that GH10 enzymes fold into a (β/α)₈-barrel (Dimarogona et al. [2012]; Harris et al. [1994]; Lo Leggio et al. [1999]), whereas family 11 enzymes have a β-jellyroll structure (Paës et al. [2012]; Sidhu et al. [1999]). Cellulose and xylan are closely linked together in plant cell walls (Carpita and Gibeaut [1993]). Thus, cellulases and hemicellulases work coordinately in the enzymatic degradation of these polysaccharides.

Filamentous fungi produce a wide spectrum of degradation enzymes for cellulose and xylan (van den Brink and de Vries [2011]). Acremonium cellulolyticus, isolated by Yamanobe et al., is a high cellulolytic enzyme-producing fungus (Yamanobe et al. [1987]). Fujii et al. reported that the culture supernatant from A. cellulolyticus has a higher cellulase specific activity and yields more glucose from lignocellulosic materials than the culture supernatant from Trichoderma reesei (Fujii et al. [2009]). Thermostable xylanase from A. cellulolyticus was found by Mitsuishi et al. (Mitsuishi et al. [1987]), but this enzyme has not been identified. Furthermore, there is no report of the expression and characterization of the xylanases (GH11) from A. cellulolyticus. The cellulose-induced xylanases of A. cellulolyticus play an important role in the cellulolytic biomass degradation process. In this study, we searched the xylanase coding regions from the draft genome sequence of A. cellulolyticus (unpublished data) and attempted to clarify the characteristics of the all xylanases (GH11).

Analysis of the A. cellulolyticus genome identified seven ORFs exhibiting homology to the xylanase (GH11) genes: xylA, xylB, xylC, xylD, xylE, xylE and xylG, which encode XylA (209 aa), XylB (282 aa), XylC (223 aa), XylD (190 aa), XylE (234 aa), XylF (276 aa) and XylG (233 aa), respectively. Each gene also encodes the signal peptide at the N-terminus. N-terminal sequence analysis showed that each signal peptide is cleaved by an aminopeptidase N-terminal to the arginine residue in this homologous expression system (Inoue et al. [2013]). In addition, Blast alignments indicated that only XylB and XylF have a carbohydrate-binding module (CBM-1) at their C-terminus, suggesting that XylB and XylF bind more strongly to their substrate than the other xylanases. Figure 1 shows the sequence alignment of the active domain in the xylanases. Six xylanases (XylA, XylB, XylC, XylE, XylF and XylG) exhibit amino acid sequence homology of around 11 ~ 52% and strictly conserve the two catalytic glutamate residues, and also strongly conserve other resides located in the speculative catalytic cleft (Paës et al. [2012]). Although the structure of XylC or any of the other xylanases has not been determined, it is expected to have a β-jellyroll structure based on multiple sequence alignment and molecular weight analyses. This protein folding motif is extremely common in small molecular weight (<30 kDa) proteins like the GH11 family (Paës et al. [2012]). Analysis of the A. cellulolyticus genome showed the xylD gene to be smaller than the other family members (Figure 1), and that it lacks some of the regions conserved in the other xylanases, including one of the catalytic glutamic acids. It seems that the xylD gene cannot translate normal xylanase protein (XylD) in A. cellulolyticus. Amino acid sequence alignments suggest that the active domains of six xylanases (Xyl11A, B, C, E, F, and G) in A. cellulolyticus have common activity and structure, and share high homology with the GH11 family of Trichoderma reesei, which is well known for its hyper-productivity (Zou et al. [2012]). There is over 50% sequence identity between XylC and T. reesei GH11.

Six of the seven xylanases (Xyl11A, B, C, E, F, and G) were expressed and purified successfully in A. cellulolyticus transformant strains. SDS-PAGE analysis shows that the purified XylB, XylE, XylF and XylG are accompanied by one or two smaller, faint bands (Figure 2). However, these proteins exhibited the significant xylanase activity toward the birch-wood xylan (Bailey et al. [1992]). Therefore, their faint bands suggest the nick regions of the recombinant proteins expressed. The nick regions seem to be located somewhere in these folded proteins (unclear). The N-terminus of the expressed protein giving rise to each major band was also determined (Table 2). These data indicate that A. cellulolyticus efficiently translates and expresses these xylanase genes using this homologous expression system (Inoue et al. [2013]).

Activity assays conducted at various pH values showed that all the xylanases have their optimum pH between pH 4.0 – 4.5, although XylA and XylC retain activity between pH 4.0 – 6.0 and pH 4.0 – 7.0, respectively (Figure 3). In addition, we examined the specific activity of all the xylanases against xylan and found that XylC has much higher specific activity than the other xylanases (Table 3). XylC exhibited between 4.5 to 90-fold higher activity at pH 5.5 than the other xylanases, and twofold higher activity at its optimum pH (pH 4.0) than at pH 5.5. These results clearly show that XylC plays the important role as a glycoside hydrolase 11 (GH11) of all the xylanases in A. cellulolyticus. It is possible that XylC uses a different mechanism for substrate recognition and catalysis compared to other xylanases. Detailed structural studies will be required to clarify this issue.

The levels of expression of the seven xylanase genes in Y-94 cultured for 24, 72, and 120 h were measured by real-time quantitative PCR (Figure 4). No expression of any xylanase gene was observed after 24 h (data not shown). A higher expression level of xylA and xylB was observed compared to the other xylanases, indicating that these genes contribute to xylan degradation by A. cellulolyticus after 72 h. XylC, which showed the highest xylanase activity, was expressed slightly less than xylA and xylB. This result suggests that expression of xylA and xylB is important for the saccharification of biomass, and that the detailed substrate specificity of xylA and xylB may be different from that of xylC. The expression of xylD, xylE, xylF and xylG was not detected under any of the conditions tested, with the exception of xylG at 120 h, xylan cultivation (Figure 4).

Thermal shift assays conducted using differential scanning fluorimetry showed that the xylanases have a T_m of around 60°C at pH 5.5, and that their thermostability increases by up to 10°C at pH 4.0 (Table 3). These results show that all GH11s in A. cellulolyticus are most stable and active at pH 4.0. Industrial use of this enzyme will require thermostability at least up to 70°C. XylC plays an important role in degrading xylan and is a good candidate for industrial use in food, paper and chemical production (Deutschmann and Dekker [2012]; Polizeli et al. [2005]). We are currently attempting to crystallize XylC to determine its atomic-level structure by X-ray crystallography.