Thermostable Enzymes as Biocatalysts in the Biofuel Industry

Abstract

Lignocellulose is the most abundant carbohydrate source in nature and represents an ideal renewable energy source. Thermostable enzymes that hydrolyze lignocellulose to its component sugars have significant advantages for improving the conversion rate of biomass over their mesophilic counterparts. We review here the recent literature on the development and use of thermostable enzymes for the depolymerization of lignocellulosic feedstocks for biofuel production. Furthermore, we discuss the protein structure, mechanisms of thermostability, and specific strategies that can be used to improve the thermal stability of lignocellulosic biocatalysts.

Introduction

With the increase in global energy consumption and expected impending shortages of crude oil, there is a considerable and immediate interest in developing alternative energy sources. Plants harness solar energy at the earth's surface to fix atmospheric carbon dioxide and collectively recycle an estimated 10¹¹ tons of carbon annually (Brett and Waldren, 1996). This carbon is utilized in the formation of complex carbohydrates via photosynthesis. Lignocellulose is the most abundant carbohydrate source in plants and has significant potential for conversion into liquid fuels or biofuels. Biofuels provide a means to reduce the dependence on fossil fuels as well as to reduce global emissions of greenhouse gases into the environment. This is because, unlike fossil fuels, biofuels are renewable over more useful time frames. Further, biofuels such as ethanol have higher octane ratings and combust in a cleaner and more efficient manner than gasoline, meaning their atmospheric carbon footprint is inherently low (Demain et al., 2005, Lynd et al., 1991). Consequently, biofuels have the additional potential to reduce CO₂ emissions to the atmosphere.

First-generation biofuels have already resulted in reduced vehicular emissions of greenhouse gases (Hill et al., 2006). However, the production of first-generation biofuels, which are based on the fermentation of corn starch or cane sugar, are neither economically nor ecologically sustainable, as corn and cane require large areas of land for their cultivation and compete with food crops meant for human consumption. Second-generation fuels that utilize lignocellulose, a recalcitrant, but more abundant part of plant material, are therefore more desirable to tackle the looming environmental and social crisis (Tollefson, 2008). The potential energy inherent in plant biomass far exceeds present human usage (Demain et al., 2005). Cellulosic feedstocks already available from agriculture and other sources are estimated to be approximately a billion tons per year in the USA alone (Corr and Hettenhaus, 2009). Many plants that produce large proportions of lignocellulosic material are capable of growth on less desirable land and require less maintenance (Tollefson, 2008). This means crops available for the production of second-generation biofuels may easily be expanded with little impact. Collectively these factors make second-generation biofuels a cost-effective, plentiful, and renewable energy resource. Accordingly, methods for optimizing the deconstruction of plant cell wall polysaccharides into their component sugars for production of biofuels have garnered considerable attention worldwide.

Lignocellulose consists primarily of three major polymers: cellulose, hemicellulose, and lignin. Cellulose accounts for up to 40% of plant biomass and consequently is the most abundant natural polymer on earth. It comprises a linear polymer of glucopyranose molecules linked by β-1-4 glycosidic linkages that have alternating orientations. Cellulose microfibrils form interstrand hydrogen bonds, which along with van der Waals forces result in a highly crystalline structure. This crystalline form limits enzyme accessibility and, therefore, limits the efficiency of enzymatic hydrolysis. Cellulose hydrolysis is further limited by the intimate associations between cellulose, hemicellulose, pectin, and lignin (Brett and Waldren, 1996, Cosgrove, 2005, Popper and Fry, 2008, Vignon et al., 2004, Zykwinska et al., 2007a, Zykwinska et al., 2007b), which further reduces the accessibility of cellulase enzymes to the cellulose fibers. The hemicellulose fraction of lignocellulose represents a significant source of mostly pentose sugars that are potentially important value-added products for fermentation to biofuels. Indeed, there is an increasing focus on engineering pentose utilization, and even xylan and cellulose saccharification, pathways into ethanologenic microorganisms such as yeast, allowing these organisms to ferment multiple monosaccharide products (Pasha et al., 2007, Ryabova et al., 2003, Voronovsky et al., 2009).

Enzymatic release of monosaccharides from cellulose and hemicellulose is mediated by glycoside hydrolases. Glycoside hydrolases (GHs) are a large class of enzymes that exhibit both broad and stringent substrate specificities. GH enzymes selectively catalyze reactions that produce smaller carbohydrate units from polysaccharides (Kobata, 2001). These enzymes are exquisite catalysts that accelerate the rate of hydrolysis of glycosidic linkages by up to 17 orders of magnitude over the uncatalyzed hydrolysis (Wolfenden et al., 1998). They are applied as biocatalysts in the hydrolysis of natural polysaccharides to mono- and oligosaccharides. GHs are classified into different families based on their amino acid sequences and three-dimensional folds (Cantarel et al., 2009). At present, this system comprises 115 families that have been organized into 14 different clans (CAZy; http://www.cazy.org/). GHs, even within the same genome, typically exhibit a diverse array of multimodular configurations. Polypeptides associated with plant cell wall hydrolysis commonly harbor a catalytic GH domain and a carbohydrate-binding module (CBM). CBMs are small domains with affinity for specific carbohydrate linkages and consequently act to target the catalytic portion of the enzyme to its cognate substrate (see Shoseyov et al., 2006 for a pertinent review). Despite the enormous variety and remarkable structural diversity of GH enzymes, as exhibited through analyses of their three-dimensional structures, all GHs, except for those in GH family 4 (Yip and Withers, 2006), hydrolyze glycosidic linkages by either a single displacement (inversion), or a double displacement (retention) of stereochemical configuration at the anomeric carbon (C1) center (Dodd and Cann, 2009), the mechanism being uniform within a GH family (Davies and Henrissat, 1995).

Enzymes that catalyze the depolymerization of cellulose are broadly classified as cellulases. However, complete and efficient hydrolysis of cellulose requires the cooperative action of at least three cellulolytic enzyme activities, namely endoglucanase (1,4-β-D-glucan glucohydrolase [EC 3.2.1.4]), exoglucanase (1,4-β-D-glucan cellobiohydrolase [EC 3.2.1.91]), and β-glucosidase (β-D-glucoside glucohydrolase, [EC 3.2.1.21]). By contrast, complete enzymatic hydrolysis of hemicellulose requires the action of a larger repertoire of enzymes due to a broader diversity in chemical linkages inherent in these heteropolymers. These enzymes include endo-β-1,4-xylanases ([EC 3.2.1.8]), xylan 1,4-β-xylosidases ([EC 3.2.1.37]), α-L-arabinofuranosidases ([EC 3.2.1.55]), α-glucuronidases ([EC 3.2.1.139]), acetylxylan esterases ([EC 3.1.1.72]), feruloyl esterases ([EC 3.1.1.73]), mannan endo-1,4-β-mannanases ([EC 3.2.1.78]), β-1,4-mannosidases ([EC 3.2.1.25]), and arabinan endo-1,5-α-l-arabinosidases ([EC 3.2.1.99]).

Current efforts to improve depolymerization of lignocellulose or search for new biocatalysts (bioprospecting) employ a multifaceted approach. The strategy includes a search for novel enzymes with high specific activities and relatively low levels of end-product inhibition. In order to be useful on an industrial scale, care is being afforded to other characteristics including thermal stability and tolerance of solutions that vary in pH, organic solvents, chemical and oxidative reagents, and detergent composition.

In the optimization of biorefinery-scale lignocellulose deconstruction, thermostable enzymes (enzymes that maintain structural integrity above 55 °C) possess a number of important advantages over their mesophilic counterparts: (1) these enzymes typically have a higher specific activity and higher stability, allowing for extended hydrolysis times and decreasing the amount of enzyme needed for saccharification (Shao and Wiegel, 1995, Viikari et al., 2007); (2) these enzymes are more compatible with nonenzymatic processes designed to decrease the crystallinity of cellulose (Szijarto et al., 2008); (3) the costs associated with process cooling are decreased or eliminated allowing the volatilization of products such as ethanol to be streamlined (Viikari et al., 2007); (4) mass transport costs are decreased due to decreased fluid viscosity; (5) there is an increased flexibility for biorefinery process configurations (Stutzenberger, 1990); (6) microbial contamination risks are significantly reduced; and finally (7) these enzymes can typically be stored at room temperature without inactivation of activity. These advantages are significant because approximately one-half of the projected process costs in biomass conversions are estimated to be associated with enzyme production, and all these benefits attributed to thermostable enzymes will result in an improvement to the overall economy of the process (Haki and Rakshit, 2003).

Various bacteria, archaea, and fungi have received considerable attention as potential sources for thermostable cellulosic enzymes. The breadth of thermophilic microbes with enzymatic characteristics amenable to lignocellulose deconstruction has been reviewed recently (Blumer-Schuette et al., 2008); however, thermostable enzymes are produced both by thermophilic and mesophilic microorganisms. Additionally, the former review largely neglects fungi, which are a valuable source of thermostable enzymes active on lignocellulose. Further, this is a rapidly evolving area that warrants a comprehensive update. The structural and functional characteristics of thermostable enzymes isolated from both mesophilic and thermophilic organisms, including fungi, and their application to improving lignocellulose hydrolysis for the production of second-generation biofuels is the subject of this review. Furthermore, we will evaluate the advantages, and current knowledge regarding the mechanisms, of thermostability. Finally, we will discuss the methods being employed for improving thermostability.