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.