AFEX pretreatment

AFEX pretreatment of corn stover was carried out as described in our previous work [8]. A hybrid seed variety (33A14) based stover, provided by the National Renewable Energy Laboratory (USA), was harvested in 2002 from the Kramer farm in Wray (CO, USA) with 60% moisture (kg water/kg dry biomass) and transferred to a high pressure Parr reactor. Heated liquid ammonia (1 kg of ammonia/kg of dry weight biomass) was charged to the reactor vessel resulting in an immediate rise in temperature to 130°C. The reactor was maintained at 130°C for 15 min through an external heating mantle (within ± 10°C). At the end of 15 min, the pressure was reduced to an atmospheric level resulting in a precipitous drop in temperature of the reactor contents. The instantaneous pressure drop in the vessel caused the ammonia to vaporize, cooling the biomass to below 30°C. The pretreated material was left under the hood overnight to ensure the complete removal of any residual ammonia. AFEX pretreated corn stover was milled (Centrifugal mill ZM 200, Retsch, PA, USA) using a 0.1 mm sieve attachment as described elsewhere [17]. The composition of both the untreated and AFEX treated stover was approximately 34.4% glucan, 22.4% xylan and 11% lignin. Except for the addition of a small amount of ammonia and formation of certain decomposition products [15], gross biomass composition does not change due to AFEX.

Discovery and cloning of endoxylanases (LX1 and LX2) and beta-xylosidase (LβX)

The details of discovery and cloning of LβX, LX1 and LX2 was based on previous work [9].

Discovery and cloning of L-arabinofuranosidase (LArb)

Enrichment cultures were started from a grass compost and were grown in YTP-2 medium containing (per litre) 2.0 g yeast extract, 2.0 g tryptone, 2.0 g sodium pyruvate, 1.0 g KCl, 2.0 g KNO₃, 2.0 g Na₂HPO₄.7H₂O, 0.1 g MgSO₄, 0.03 g CaCl₂, and 2.0 mL clarified tomato juice. Enrichments were performed at 70°C in flasks agitated at 200 rpm. A number of aerobic cultures were purified by selection of individual colonies on plates containing the above medium and 16.0 g/L agar. For the preparation of genomic DNA, litre cultures were grown from a single colony in YTP-2 medium and collected by centrifugation. The cell was lysed using a combination of sodium dodecyl sulphate (SDS) and proteinase K and genomic DNA was isolated using a phenol/chloroform extraction [18]. The genomic DNA was precipitated, treated with RNase to remove residual contaminating RNA and fragmented by hydrodynamic shearing (HydroShear apparatus, Genomic Solutions, MI, USA) to generate fragments of either 3-5 kb or 10 kb. The fragments were purified on an agarose gel, end repaired and ligated into a high-stability, low copy vector (pSMART-LCKan, Lucigen, WI, USA). The recombinant plasmids were then used to transform Escherichia coli 10G ELITE electrocompetent (Lucigen) cells and screened on plates containing 4-methylumbelliferyl-α-D-arabinopyranoside. DNA inserts of the positive clones were sequenced. The enzymes of interest were subcloned into pET28a and the resulting vectors used to transform BL21 (DE3) chemically competent cells. Amino acid sequences were deduced from the DNA sequences of the clones using ExPaSy translate tool (http://expasy.org/tools/dna.html) and confirmed by N-terminal sequencing/sodium SDS polyacrylamide gel electrophoresis (SDS-PAGE).

Discovery and cloning of LX3, LX4, LX5, LX6 and LαGl

D. turgidum strain 6724T (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; German Collection of Microorganisms and Cell Cultures) bacterial cell concentrate was a kind gift of Dr Frank T Robb, Center of Marine Biotechnology, University of Maryland Biotechnology Institute, USA. C. thermocellum bacterial cell concentrate was a kind gift of Dr Paul Weimer, United States Department of Agriculture, Agricultural Research Service, United States Dairy Forage Research Center, WI, USA. The cell concentrate from each strain was lysed using a combination of SDS and proteinase K, and the genomic DNA was purified using a phenol/chloroform extraction methodology [18]. Genomic DNA was prepared as described in the previous section. D. turgidum and C. thermocellum genomic libraries were prepared as described in the previous section and screened on plates containing MUC. LX6 was obtained from screening of the D. turgidum library, and LX3 from screening of the C. thermocellum library. DNA inserts of the positive clones were sequenced, the enzymes of interest were subcloned into pET28a and the resulting vectors were used to transform BL21 (DE3) chemically competent cells [19]. Amino acid sequences were deduced from the DNA sequences of the clones using ExPaSy translate tool (http://expasy.org/tools/dna.html) and confirmed by N-terminal sequencing/SDS PAGE. LX4 and LX5 were cloned by polymerase chain reaction (PCR) amplification of the desired genes from C. thermocellum genomic DNA, while LαGl was cloned by PCR amplification of the desired gene from D. turgidum genomic DNA.

Enzyme expression and purification

Purification of fungal enzymes was performed using an FPLC system (GE Healthcare, Buckinghamshire, UK) at room temperature, while the fraction collector was refrigerated. Cellulases (CBH I, CBH II and EG I) and βG were purified from Accellerase (Genencor, NY, USA) and Novo 188 (Novozyme, CA, USA), respectively. Details of the purification methodology are described in our previous work [8].

Enzymatic hydrolysis of pretreated biomass

The hydrolysis experiments were performed in 2.2 mL deep well microplates (Greiner, NC, USA) at 0.2% (w/w) total glucan loading along with 50 mM citrate buffer (pH 4.5) in a total volume of 500 μL per well, based on the microplate hydrolysis protocol described previously [17]. The enzymes mixtures were prepared separately and added simultaneously using a single or eight-channel automated pipetting workstation (epMotion, Hamburg, Germany) and 96-channel automated pipetting workstation (JANUS, Perkin Elmer, MA, USA). The microplates were incubated at 50°C with shaking at 250 rpm for 24 h. The concentrations of glucose and xylose in the hydrolyzates were measured using suitable enzyme based assays described below. All experiments were carried out in triplicates.

Glucose and xylose assays

Glucose and xylose concentrations were measured using enzymatic kits purchased from R-Biopharm (MI, USA) and Megazyme (Bray, Ireland), respectively. D-Glucose is first phosphorylated to D-glucose-6-phosphate using ATP and hexokinase. The reaction of D-glucose-6-phosphate with NADP⁺ is catalyzed by glucose-6-phosphate dehydrogenase to form D-gluconate-6-phosphate and NADPH. The reactions are stoichiometric to the amount of D-glucose and a corresponding increase in NADPH is measured at 340 nm to estimate the glucose concentration. The xylose assay is based on analogous two-step reactions. α-D-xylose is converted to isomeric β-D-xylose by xylose mutarotase. β-D-xylose is then reacted with NAD⁺, in presence of β-xylose dehydrogenase, to form D-xylonic acid and NADH. The corresponding increase of NADH is measured at 340 nm to determine the xylose concentration. The entire protocol (including liquid transfers and mixing), was performed using an automated 96-channel pipetting workstation (Janus, PerkinElmer, CA, USA). Based on the information provided by the manufacturer, β-xylose dehydrogenase has no cross-reactivity on L-arabinose.

Supplementing bacterial hemicellulases along with fungal cellulases

Previous work has shown that Dictyoglomus and Clostridium derived hemicellulases retain significant activity at pH 4.5-5, whereas Trichoderma derived cellulases lose considerable activity at pH greater than five [9]. Hence, all assays were conducted at pH 4.5-5 to maximize performance of both sets of enzymes. Overall strategy used to obtain an optimized cocktail and major findings from this study are highlighted in Figure 1. From step 2 onwards, each experimental step was designed and tested based on conclusions from preceding steps. For step 1, to test performance of the nine different hemicellases on AFEX treated corn stover, all six xylanases (4 mg/g glucan for each xylanase) and other three accessory enzymes (2 mg/g glucan for each of LβX, LαGl and LArb, respectively) were grouped and doped together along with the core cellulases mixture. This avoided the risk of missing any synergism among the endoxylanases (LXs) and also between LXs and accessory enzymes (LβX, LαGl and LArb). Core fungal enzymes consisted of CBH I, CBH II and EG I (4 mg/g glucan loading of each cellulase) plus 2 mg/g glucan βG loading. Previous results have shown the current βG loading is sufficient to prevent cellobiose build-up at the current glucan loading [8]. The protein mass ratio of CBH I, CBH II and EG I was kept at 1:1:1 in this study since previous results have demonstrated that this ratio results in optimum activity [9]. The minimum loading of LβX, LαGl and LArb required was determined after screening for optimal endoxylanase combination.

From Figure 2, core cellulases alone account for 56% glucose yield and minimal (3%) xylose yield within 24 h hydrolysis. Supplementing xylanases along with core cellulases increased glucose yield to as high as 83%. However, only 13% xylose yield was achieved. One possible reason for the improvement in glucan conversion is that xylanase supplementation helps remove xylan [20] sheathing cellulose fibrils and, hence, increases substrate accessibility [21]. LβX supplementation allows cleavage of xylo-oligomers to monomeric xylose and hence mixtures containing LβX have higher monomeric xylose yield. The α arabinofuranosidase removes arabinose side-chains, while α-glucuronidase cleaves the α 1,2-glycosidic bond of the 4-O-methyl-D-glucuronic acid side chain from the xylan backbone [22, 23]. Adding LArb and LαGl along with cellulases and xylanases, further increased xylose yields by 12% and 7%, respectively. Adding both these enzymes, along with core cellulases and xylanases, allowed the xylose yield to reach as high as 74%. Hence, the removal of side chains which impede endoxylanase activity could enhance xylose yields significantly [24–26]. However, the total enzyme loading employed to obtain this conversion was 44 mg/g glucan, which is not viable for industrial processing. Therefore, systematically screening the critical endoxylanases and determining the minimum amounts of accessory enzymes is necessary to decrease the total enzymes loading without sacrificing hydrolysis performance.

Screening endoxylanases

In Figure 2, all 6 xylanases were loaded together to evaluate their performance along with LβX, LαGl and LArb and also to prevent the risk of missing any synergism among the xylanases. However, not all xylanases may be necessary to bring about efficient xylan hydrolysis. In order to determine the most important xylanases or their combinations, a two level (0 and 4 mg/g glucan for low and high level of enzymes loading), six factor (LX 1 to LX 6) full factorial experiment was carried out by fixing the remaining enzyme loading (CBH I, CBH II and EG I at 4 mg/g glucan each; βG, LβX, LαGl and LArb at 2 mg/g glucan each).

Results of the 24 h hydrolysis results for AFEX corn stover are shown in Figure 3 which depicts the glucose versus xylose yields. The xylose yield shows a strong linear relationship with the glucose yield. This reconfirmed previous findings that high glucose yields can be achieved at higher xylose yields for AFEX treated corn stover [9]. In this experiment, xylanase loading increased depending on the number of xylanases involved in the mixture. Experiments performed by adding individual xylanases showed that LX3 gave the highest conversion followed by LX4. For binary LX mixtures, the best mixture contained both LX3 and LX 4. Mixtures containing neither of these two enzymes resulted in 10% or more drop in both glucose and xylose yield. Combinations containing either LX3 or LX4 exhibited moderate conversions. For ternary mixtures, and other higher multiple LX loadings, LX3 and LX4 gave a similar trend which suggests that both LX3 and LX4 are superior compared to other xylanases.

Optimum cellulase to xylanase ratio

Based on previous results, both LX3 and LX4 were chosen as supplementary xylanases for further studies. As xylose and glucose yields are highly correlated, the ratio between cellulases and xylanases loaded was further optimized.

In Figure 4, varying ratios of xylanases (LX3 and LX 4 at equi-mass loading) were supplemented along with fungal core cellulases (CBH I, CBH II and EG I at equi-mass loading) for a fixed total protein loading of 20 mg/g glucan. A fixed amount (2 mg/g glucan) of other enzymes (βG, LβX, LaGl and LArb) were also loaded. Both the glucose and the xylose yields are shown in Figure 4. Without any xylanases, the glucose and xylose yields dropped to 63% and 43%, respectively. As EG I has some xylanase activity [8, 27], even in the absence of any other xylanases, a significant xylose yield was obtained by the endocellulase in the presence of LβX. Supplementing the mixture with 5% xylanase increased both the glucose and xylose yields significantly. At 25% xylanase supplementation, the highest observed glucose and xylose yields were seen. When no cellulases were loaded, the glucose yield drops to less than 10%. Interestingly, xylanase loadings, ranging from 15% to 75% of total enzyme added, gave comparable glucose (>70%) and xylose yields (>60%).

Synergism between endoxylanases (LX3 and LX4)

The optimal endoxylanase loading is one-third of the amount of cellulases added for hydrolysis. It is interesting to compare the synergistic role of each xylanase in the mixture. Based on previous results, cellulases (CBH I, CBH II and EG I) were loaded at 5 mg/g glucan each, xylanases (LX3 and LX4) were loaded at 5 mg/g glucan total and accessory enzymes (βG, LβX, LαGl and LArb) were loaded at 2 mg/g glucan each. Several reports highlight the synergism between exo-glucanases and also between different families of hemicellulases [28–30]. It is interesting to determine if there is similar synergism between the endoxylanases. Figure 5 shows that supplementing only LX4 or LX3 along with core cellulases and accessory enzymes results in xylose yields of 60% and 65%, respectively. However, synergistic combinations of LX3 and LX4 resulted in a greater than 70% xylose yield. In addition, the glucose yield also benefit from including both LX3 and LX4. Table 1 shows that LX3 and LX4 belong to GH family 10 and 11, respectively. These results suggest that both family 10 and 11 glycosyl hydrolases are necessary to achieve complete xylan conversion for AFEX treated corn stover. It is normally believed that family 10 xylanases are more versatile and efficient than family 11 xylanases [24, 28]. Family 10 xylanases cleave β-1,4 linkages from at least one unsubstituted xylopyranosyl residue adjacent to substituted xylopyranolsyl residues towards the reducing end. However, family 11 xylanases only cleave linkages adjacent to at least two unsubstituted xylopyranosyl residue [24]. Our results are also consistent with Banerjee et al., who found that a combination of GH family 10 and 11 fungal xylanases help increase both glucose and xylose yields [25]. Ratios of LX3/(LX3+LX4) between 0.4-0.7 yielded comparable total monosaccharides yields. These results reconfirm previous observations that both LX3 and LX4 are required in order to achieve higher monosaccharides yields.

Minimizing loading of accessory enzymes (LβX, LαGl and LArb)

In all of the above studies, LβX, LαGl and LArb loading was in excess. In order to determine the minimum loading of each enzyme, different amounts of each were loaded while the other two were kept in excess. Glucose yields for all experiments were comparable (data not shown) which suggests that these enzymes do not influence glucan hydrolysis. However, there was a significant change in the xylose yields as shown in Figure 6. The minimum loading for LβX, LαGl and LArb was 0.6, 0.8 and 0.6 mg/g glucan, respectively. Note: LβX plays a very important role on xylose yield. Without LβX, the xylose yield is very low, even when LαGl and LArb are loaded.

Comparison of monosaccharides yield for optimized and crude commercial enzyme preparations

The previous experiments helped define the optimal cellulase and hemicellulase loadings necessary to maximize monomeric glucose and xylose yields from AFEX treated corn stover. It was interesting to compare the hydrolytic activity of optimized cellulase-hemicellulase cocktail with purified core cellulases and crude commercially available enzymes on pretreated biomass with respect to the maximum monosaccharide yield achieved. In Figure 7, four different enzyme mixtures were compared at varying total protein loadings for glucose (Figure 7A), xylose (Figure 7B) and total monosaccharide (Figure 7C) yield. The comparisons were made against: a commercial enzyme preparation (Accellerase 1000, Genencor, NY, USA); a core cellulase cocktail (CBH I, CBH II and EG I at equi-mass loading, along with 2 mg/g glucan βG); a core cellulase plus xylanase cocktail (3:1 mass ratio between cellulases to xylanases, 2 mg/g glucan βG and 0.6 mg/g glucan LβX); and an optimized cellulase-hemicellulase cocktail (core cellulase-xylanase cocktail, along with LαGl and LArb loaded at 0.8 and 0.6 mg/g glucan, respectively).

From Figure 7, at a fixed total protein loading (30 mg/g of glucan), it is clear that the commercial enzyme (Accellerase 1000) has limited hydrolytic activity on both glucan (70%) and xylan (20%) fractions for AFEX treated corn stover due to the lack of suitable hemicellulase activity. The core cellulase mixture (CBH I, CBH II and EG I) had slightly better glucan conversions but lower xylan yields (<5%) compared to the commercial preparations. Interestingly, the inclusion of xylanases and xylosidases enhanced both glucose and xylose yields significantly. When small amounts of accessory hemicellulases (LαGl and LArb) were included in the core cellulase-xylanase mixtures, glucose yields were unchanged while xylose yields increased to >70%. For the total monosaccharide yield (Figure 7C), Accellerase 1000 at 30 mg/g glucan loading has a similar yield (50%) compared to the optimized cocktail at around 7 mg/g glucan.

In order to achieve monosaccharides yields greater than 80%, which is necessary for an economical industrial process, the optimized cocktail concentration needed to be around 22 mg/g glucan while the non-optimal cellulase-xylanase mixtures required more than 33 mg/g glucan. These results demonstrate how trace amounts of important accessory hemicellulases can further enhance the overall polysaccharide hydrolysis yields for AFEX pretreated corn stover. About 1.2 mg/g glucan total loading of LArb and LαGl can decrease the overall enzyme loading by over 33%. Furthermore, if the cocktail does not contain specific activities, such as β-xylosidase, α-arabinofuranosidase and α-glucuronidase, no matter how much of the other enzymes are loaded, it is impossible to achieve high xylose hydrolysis yields.