Elsevier

Bioresource Technology

Volume 101, Issue 24, December 2010, Pages 9624-9630
Bioresource Technology

Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes

https://doi.org/10.1016/j.biortech.2010.06.137Get rights and content

Abstract

Typically, the enzymatic hydrolysis rate of lignocellulosic biomass is fast initially but then slows down more rapidly than can be explained by just consumption of substrate. Although several factors including enzyme inhibition, enzyme deactivation, a drop in substrate reactivity, or nonproductive binding of enzyme to lignin could be responsible for this loss of effectiveness, we recently reported evidence that xylose, xylan, and xylooligomers dramatically decrease conversion rates and yields, but clarification was still needed fors the magnitude of their effect. Therefore, in this study, xylan and various xylooligomers were added to Avicel hydrolysis at low enzyme loadings and found to have a greater effect than adding equal amounts of xylose derived from these materials or when added separately. Furthermore, xylooligomers were more inhibitory than xylan or xylose in terms of a decreased initial hydrolysis rate and a lower final glucose yield even for a low concentration of 1.67 mg/ml. At a higher concentration of 12.5 mg/ml, xylooligomers lowered initial hydrolysis rates of Avicel by 82% and the final hydrolysis yield by 38%. Mixed DP xylooligomers showed strong inhibition on cellulase enzymes but not on β-glucosidase enzymes. By tracking the profile change of xylooligomers, a large portion of the xylooligomers was found to be hydrolyzed by Spezyme CP enzyme preparations, indicating competitive inhibition by mixed xylooligomers. A comparison among glucose sugars and xylose sugars also showed that xylooligomers were more powerful inhibitors than well-established glucose and cellobiose.

Introduction

Cellulosic biomass is uniquely suited for sustainable production of liquid transportation fuels, and the power of modern biotechnology promises competitive advantages (Lynd et al., 2008). However, the cost of cellulase enzymes coupled with the large amounts required to realize commercially viable yields is by far the dominant economic barrier to large scale implementation. A significant contributor to the high dose demands is that hydrolysis rates slow down as reaction proceeds much faster than can be explained by substrate consumption alone for a typical enzymatic hydrolysis (Ramos et al., 1993, Yang et al., 2006). Many reasons have been offered for the loss in enzyme effectiveness with time and the consequent high doses required for good yields including end-product inhibition, enzyme deactivation with time and temperature, drop in substrate reactivity with conversion due to initial removal of more easily hydrolyzed material, and nonproductive binding of enzyme to lignin (Converse et al., 1988, Eriksson et al., 2002, Holtzapple et al., 1990, Scheiding et al., 1984). Although the exact cause is still uncertain and multiple factors are likely responsible, substrate and end-product inhibition are believed to be very important (Tengborg et al., 2001, Xiao et al., 2004), with glucose and cellobiose identified as the principle cellulase inhibitors that bind to cellulase active sites regardless of the inhibition pattern (Holtzapple et al., 1990). However, other hemicellulose sugars, such as mannose, xylose, and galactose, have been also shown to inhibit cellulase (Xiao et al., 2004), and liquid from Ammonia Recycled Percolation (ARP) pretreatment of corn stover that is rich in xylooligomers, soluble lignin, sugar and lignin degradation products, significantly inhibited cellulase and microbial activities (Kim et al., 2006, Zhu et al., 2006).

Enzymatic hydrolysis of cellulose is a multi-step heterogeneous reaction in which insoluble cellulose is initially broken down at the solid–liquid interface via the synergistic action of endo-glucanases (Xiao et al., 2004) (EC 3.2.1.4) and exo-glucanases/cellobiohydrolases (CBH) (EC 3.2.1.91). This initial reaction is accompanied by further liquid-phase hydrolysis of soluble intermediates, i.e., short celluloligosaccharides and cellobiose, that are catalytically cleaved to produce glucose by the action of β-glucosidase (BG) (EC 3.2.1.21) (Messner et al., 1991). Enzyme accessibility to cellulose is postulated to be impeded by lignin and hemicellulose coating cellulose and restricting access by enzymes. In this vein, several studies have shown that removing a high percentage of hemicellulose can facilitate cellulose conversion by enzymatic hydrolysis (Allen et al., 2001, Grohmann et al., 1989, Ishizawa et al., 2007, Kabel et al., 2007, Palonen et al., 2004, Yang and Wyman, 2004, Zhu et al., 2004). Similarly, addition of purified endoxylanase and hemicellulolytic esterase activities enhanced cellulose conversion by cellobiohydrolase I (Cel7A), and a direct relationship was shown between xylan removal by enzymes and enhancement of cellulose hydrolysis (Selig et al., 2008). Such studies attribute the benefits of hemicellulose removal to improving accessibility of cellulase enzymes to hydrolysis substrates, but additional impacts that hemicelluloses and their hydrolysis products could have on enzyme action have received little attention.

A recent paper determined for the first time that xylobiose and higher DP xylooligomers inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, with xylose, xylobiose, and xylotriose having progressively greater impacts on hydrolysis rates (Kumar and Wyman, 2009). This interpretation was supported through showing that addition of xylanase and β-xylosidase improved enzymatic hydrolysis of xylan and pure cellulose. Addition of beta-xylosidase and xylanase also enhanced performance for enzymatic hydrolysis of solids resulting from sulfur dioxide pretreatment and particularly from ammonia fiber expansion (AFEX) pretreatment that leaves virtually all of the xylose in the pretreated solids. These results strongly suggest that xylooligomers inhibit cellulase action and that both glucose and xylose release could be significantly enhanced by supplementation with β-xylosidase and xylanase (Kumar and Wyman, 2009). However, the benefits will likely vary with the amounts of xylose and other hemicellulose oligomers present in the liquid, and the choice of enzyme and enzyme loadings are also expected to vary with substrate composition.

Although this limited evidence suggested that xylooligomers reduce enzyme activity and therefore effectiveness, the mechanism for inhibition of cellulase and other hydrolysis enzymes by xylooligomers was still uncertain, and more information was needed to clarify the degree to which sugars and oligomers released from hemicellulose affect enzymatic hydrolysis of cellulose. Therefore, in this study, pure cellulose was hydrolyzed in the presence of xylose, xylan, and xylooligomers to determine the degree to which these compounds impact cellulose conversion. In addition, the influence of xylooligomer concentration on cellulose hydrolysis was measured, and the effects of these components on the action of cellulase and β-glucosidase were determined. The results showed for the first time that xylooligomers can play a powerful role in slowing enzymatic hydrolysis, thereby potentially explaining to some extent why hydrolysis rates slow with conversion and why removal of hemicellulose improves cellulose conversion yields.

Section snippets

Materials

Avicel PH-101 cellulose (cellulose content > 97%, Lot & filling code: 1300045 32806P01) and xylose (xylose purity > 99%, Lot & filling code: 1403673 33308088) were purchased from Sigma (St. Louis, MO). Birchwood xylan with a xylan content measured to be ∼85% was also purchased from Sigma (Lot # is 038K0751). A xylobiose standard of over 95% purity was obtained from Megazyme International Ireland Ltd. (Bray, Co. Wicklow, Ireland, Cat No. O-XBI). Genencor International (2600 Kennedy Drive, Beloit,

Effects of xylan derivatives on enzymatic hydrolysis of pure cellulose

Xylooligomers are important intermediates in hemicellulose hydrolysis, and we sought to better quantify how inhibitory they are to cellulose hydrolysis by enzymes. Because pure xylooligomers with DPs greater than five are not commercially available and even those available are extremely expensive, a mixture of xylooligomers was produced by water-only hydrolysis of Birchwood xylan, with the distribution of different DP xylooligomers shown in Fig. 1. Then, xylan, xylose, and xylooligomers derived

Conclusion

In summary, our results indicated that xylose, xylooligomers, and xylan strongly inhibited cellulase. In addition, inhibition increased with concentration of these compounds, and xylooligomers were more inhibitory to cellulase than either xylan or xylose for an equivalent amount of xylose at the concentrations studied. Xylooligomers were also far more inhibitory to cellulase than equal molar amounts of glucose or cellobiose. However, additional research is needed to determine which xylooligomer

Acknowledgements

Most of this work was funded by Mascoma Corporation headquartered in Lebanon, NH, and we are grateful for their support and helpful discussions. In addition, the UCR Bourns College of Engineering through the Center for Environmental Research and Technology (CE-CERT) and the Chemical and Environmental Engineering Department provided funding for this research as well as vital equipment and facilities. We appreciate Ford Motor Company sponsorship of the Chair in Environmental Engineering at the

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    Present address: Center for Bioproducts and Bioenergy, Washington State University, 2710 University Dr., Richland, Washington 99354, United States.

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