Elsevier

Bioresource Technology

Volume 99, Issue 11, July 2008, Pages 4997-5005
Bioresource Technology

Synergistic enhancement of cellobiohydrolase performance on pretreated corn stover by addition of xylanase and esterase activities

https://doi.org/10.1016/j.biortech.2007.09.064Get rights and content

Abstract

Significant increases in the depolymerization of corn stover cellulose by cellobiohydrolase I (Cel7A) from Trichoderma reesei were observed using small quantities of non-cellulolytic cell wall-degrading enzymes. Purified endoxylanase (XynA), ferulic acid esterase (FaeA), and acetyl xylan esterase (Axe1) all enhanced Cel7A performance on corn stover subjected to hot water pretreatment. In all cases, the addition of these activities improved the effectiveness of the enzymatic hydrolysis in terms of the quantity of cellulose converted per milligram of total protein. Improvement in cellobiose release by the addition of the non-cellulolytic enzymes ranged from a 13–84% increase over Cel7A alone. The most effective combinations included the addition of both XynA and Axe1, which synergistically enhance xylan conversions resulting in additional synergistic improvements in glucan conversion. Additionally, we note a direct relationship between enzymatic xylan removal in the presence of XynA and the enhancement of cellulose hydrolysis by Cel7A.

Introduction

The displacement of a significant fraction of petroleum usage in the United States over the next two to three decades with biomass-derived ethanol is an important goal for the US Department of Energy. To meet the goals outlined by the President of the United States, the feedstock base must be substantially expanded to include lignocellulosic biomass. To this end, the Advanced Energy Initiative (2006) has identified the need to develop technologies that make cellulosic ethanol cost-competitive.

The recalcitrant nature of lignocellulosic biomass necessitates that the transition from grain to lignocellulose-based ethanol production requires the development of advanced conversion technologies. It is the essential nature of biomass structure and composition that results in the natural recalcitrance of plants to microbial and chemical deconstruction. Unlike starch, cellulose and hemicellulose are structural polysaccharides and are difficult to hydrolyze. Furthermore, thermal conversion of biomass is energy intensive and the desired products are typically low-value. Biological conversion is enhanced by a pretreatment step to disrupt the biomass structure, typically releasing lignin and/or five carbon hemicellulosic sugars and rendering the remaining cellulose accessible to enzymatic hydrolysis. The development of an economic but effective pretreatment remains a significant barrier to the lignocellulosic ethanol industry. To address the problem, diverse approaches are being investigated, including steam explosion, ammonia fiber expansion, dilute and concentrated acid, and alkaline-based processes.

Different pretreatment technologies have varying affects on product yield and required subsequent process steps (Wyman et al., 2005a, Wyman et al., 2005b). In general, severe pretreatments result in high sugar degradation, toxic product formation, environmental liabilities, and substantial process costs. During the pretreatment process, released sugars can be degraded to furan derivatives and phenolics that act as fermentation inhibitors (de Mancilha and Karim, 2003, Liu, 2006, Lopez et al., 2004, Weil et al., 2002). As the severity of pretreatment increases (higher acid concentration and/or temperature) the formation of inhibitory compounds increases (Yourchisin and Van Walsum, 2004). Biological pretreatments offer the advantage of lower energy use and mild process conditions but have slow hydrolysis rates (Keller et al., 2003). Mild pretreatments produce decreased sugar losses, lowered degradation product formation, and reduced environmental impact, however, they typically result in products of lower enzyme digestibility (Ohgren et al., 2007). A mild pretreatment coupled with advanced enzyme formulations can allow for decreased production of inhibitory compounds and higher yield from the hemicellulose fraction. Such an approach will require the use of “accessory” enzymes to degrade any remaining hemicellulose and synergize with cellulases, which are typically responsible for a significant portion of sugar production during the enzymatic conversion of biomass.

The term “cellulase” represents a broad group of enzymes with various specificities acting synergistically to hydrolyze cellulose. Industrial hosts for producing cellulases include the saprophytic fungi Trichoderma reesei, Penicillium funiculosum, Humicola grisea, and others. Common among these preparations is the presence of a reducing-end specific cellobiohydrolase from the glycosyl hydrolase (GH) family 7. Of the GH7 enzymes, cellobiohydrolase I (Cel7A) from T. reesei is the most significant and well-studied single enzyme for cellulose hydrolysis. It is secreted at high concentrations and comprises up to 60% of the protein in commercial cellulase preparations (Keranen and Penttila, 1995) and is arguably the most important single enzyme involved in cellulose deconstruction. By itself; however, Cel7A has limited activity on lignocellulosic material.

Cel7A has long been known to act synergistically with other cellulase and hemicellulase activities (Kipper et al., 2005, Tuohy et al., 1994, Wood et al., 1989) and its enhancement by β-d-glucosidase is one of the most widely reported synergistic effects (Wilson et al., 1994). Another of the earliest reported cellulase synergies is the endo/exo effect, where endocellulases are thought to create new, free cellodextrin chain ends for cellobiohydrolase (exocellulase) enzymes (Boisset et al., 2001, Murashima et al., 2002, Zhang and Lynd, 2006). Synergism between reducing and non-reducing end exocellulases has also been reported (Miyazaki et al., 2004, Wood and Mccrae, 1986), as has endo/endo enhancement (Boisset et al., 2000, Zhou and Ingram, 2000). Still other studies have demonstrated the synergy of cellulase with non-cellulase enzymes, including primarily hemicellulases (Han et al., 2004, Koukiekolo et al., 2005, Morgavi et al., 2000, Murashima et al., 2003). Much of the synergism between cellulases and hemicellulases is believed to arise from the ability of hemicellulases to expose the cellulose microfibril core, by either removing the hemicellulose or the hemicellulosic side-chains (Yu et al., 2003). Modern hypotheses regarding cellulase synergy have been summarized in a review by Henrissat (1994).

To study synergies between individual enzyme activities involved in the hydrolysis of lignocellulosic materials, we have used purified enzymes to better understand the one-on-one interactions in the myriad of proteins involved. Cel7A from T. reesei was used as a reporter enzyme to investigate contributions from specific hemicellulolytic activities to glucan conversion of pretreated biomass. For this study we have purified an endoxylanase, XynA, from a commercial xylanase preparation and two esterases, Axe1 and FaeA, which were produced via heterologous expression in Aspergillus awamori. The effects of these enzymes on the performance of Cel7A were assessed on corn stover that has been subjected to hot water pretreatment.

Section snippets

Pretreated corn stover

Corn stover used for this study was harvested in 2003 at the Kramer Farm in Wray, Colorado. The stover was pretreated in a flow-through hot water pretreatment reactor at Dartmouth College under subcontract with the National Renewable Energy Laboratory. The pretreatment was conducted at 200 °C for 16 min with a 2.5% solids loading. Detailed discussion of the equipment utilized for the pretreatment can be found in the published works of Wyman and co-workers (Liu and Wyman, 2003, Wyman et al., 2005a

Pretreated corn stover composition

The chemical composition of the corn stover, before and after pretreatment, is listed in Table 1. It is evident that the hot water pretreatment effectively removed both xylan and lignin from the original stover, of which xylan removal was the most significant (with the residual xylan comprising only about 4.5% of the residual solids). The xylan fraction remaining in this pretreated material is presumably a more hydrolysis-resistant xylan and may not be as susceptible to enzymatic digestion as

Discussion

In general, Cel7A performance with the addition of XynA, Axe1 and/or FaeA exceeded the performance achievable with the same or greater quantities of Cel7A, as measured by cellobiose release. Although this was not an optimization study, we should point out that in all cases, the addition of small quantities of these non-cellulolytic activities allows for a more efficient (Fig. 5) glucan conversion, on the basis of milligrams of glucan converted per milligram of total protein utilized, than

Conclusions

The addition of purified endoxylanase and/or hemicellulolytic esterase activities has been shown to significantly enhance sugar release by purified Cel7A during the enzymatic saccharification of pretreated corn stover. The purified XynA, Axe1 and/or FaeA components in combination with Cel7A enhanced cellobiose release from hot water pretreated corn stover well beyond the additive effects of the individual components. The combinations most effective in enhancing cellobiose release in the

Acknowledgements

The work was funded by the DOE Office of the Biomass Program (1000 Independence Avenue, SW, Washington, DC 20585).

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