The Influence of Lignin on the Enzymatic Hydrolysis of Pretreated

Chapter 6

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The Influence of Lignin on the Enzymatic Hydrolysis of Pretreated Biomass Substrates Seiji Nakagame, Richard P. Chandra, and Jack N. Saddler* Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, British Columbia, Canada, V6T1Z4 *Corresponding author: [email protected]

The bioconversion of lignocellulosic biomass to ethanol has been proposed as a potential process to provide a more sustainable transportation fuel substitute for fossil fuels such as petrol and gasoline. However, the recalcitrance of lignocellulosic substrates remains a major challenge for the pretreatment and enzymatic hydrolysis steps of the overall biomass-to-ethanol process. Lignin, one of the major components of lignocellulosic biomass presents both chemical and physical barriers to the enzymatic hydrolysis of pretreated substrates. The two primary mechanisms include, lignins role in limiting access of the enzymes to the cellulose by its physical/mechanical role and location within the substrate and through the non-productive binding of cellulases to the lignin. Although hydrophobic, electrostatic, and hydrogen bonding interactions have been implicated in these cellulase-lignin interactions, the exact nature of this interaction remains to be more fully elucidated. To try to limit the non-productive binding of cellulases to lignin we and other groups have tried to determine the main factors that influence cellulase-lignin interaction. In the work described in this review we show how targeted changes to the chemical and physical properties of lignin, though varying both the pretreatment and biomass feedstock, can decrease the non-productive binding of cellulases to lignin.

© 2011 American Chemical Society In Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass; Zhu, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


Introduction The development of biorefineries that are capable of producing fuels and commodity chemicals from lignocellulosic biomass is viewed as a potential alternative to the world’s current reliance on fossil fuels (1). Driving forces, including potentially higher fossil fuel prices and their contribution to CO2 emissions, have catalyzed many groups to develop cost effective processes to produce biofuels such as ethanol from lignocellulosic biomass (2). The “typical” biomass-to-ethanol process usually consists of three main process steps including pretreatment, which involves the physical, mechanical or chemical breakdown of the cell wall structure to improve recovery of the hemicellulose and lignin components in a useful form as well as enhancing access of the cellulose to the cellulase complex. The other two process steps are enzymatic hydrolysis of the cellulose and the fermentation of the cellulose and hemicellulose derived sugars to ethanol. Several technical and economic issues have slowed the commercialization of the various biomass-to-ethanol processes (3). It is apparent that many of the technical challenges stem from the inherent recalcitrance of the biomass itself. Although cellulose, hemicellulose, and lignin are the primary structural components of most lignocellulosic materials, there are considerable differences between grasses, hardwoods and softwoods in terms of their content, composition and structure (4). Cellulose and hemicelluloses, which comprise about 65-75% of most biomass, substrates, can be broken down to their component sugars for fermentation to ethanol (1a,5). Lignin is generally composed of polymers of phenylpropane units, guaiacyl (G, coniferyl alcohol), syringyl (S, sinapyl alcohol), and p-Hydroxypheny units (H, p-coumaryl alcohol). The monolignols have been shown to be biosynthesized from glucose via phenylalanine (4a). The lignin composition varies depending upon the biomass source and type. For example, for the softwood Pinus sylvestris, the lignin’s H and G content are 2% and 98% (6), for the hardwood Populus euramericana, the lignin’s G and S content are 39% and 61%, and for corn stalks, the H, G, and S content of the lignin are 4%, 35%, and 61%, respectively. In addition, the cell walls in corn stover contain up to 4% ferulate and up to 3% p-coumarate (7). The lignin content also varies depending on the source of the lignocellulosic materials. Softwoods generally contain about 26-32% lignin, hardwoods contain 20-25% lignin (4a), while an agricultural residue such as corn stover generally has a lower lignin content of about 16-22%, when compared to wood derived biomass (4a, 5a, 8). The bond types of the various lignins subunits, also vary depending on the source of the lignocellulosic material (4a). Although in some situations, the residual hemicellulose within the pretreated biomass substrate can significantly limit the ease of enzymatic hydrolysis of the cellulose (8, 9), it is primarily the lignin and the various roles it plays in limiting access to the cellulose and in binding with the cellulase enzymes that have proven to be the main impediments to achieving effective enzymatic hydrolysis (10). For example, the removal of lignin has also been shown to facilitate the complete hydrolysis of the cellulose component regardless of the presence or absence of hemicellulose (11). As mentioned earlier, an effective pretreatment is one which both provides the ready fractionation and recovery of the hemicelluloses and 146 In Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass; Zhu, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


lignin components in a useful form while enhancing access of the cellulose to the cellulase complex (2a, 12). An effective pretreatment should also be economical and robust enough such that it can be applied to a range of biomass substrates. Although an ideal pretreatment process would enhance the ready and complete separation and isolation of each component of the lignocellulosic substrate, this is likely to be impractical for both technical and economic reasons. As a result, pretreated substrates will typically contain at least some amount of hemicellulose and a lot more residual lignin associated with the cellulose rich, water or solvent insoluble stream obtained after pretreatment. The amount of each component remaining associated with the cellulosic fraction will vary depending on the type and severity of the pretreatment (8, 13).

Pretreatment and Enzymatic Hydrolysis of Lignocellulosic Biomass Several pretreatments have been advocated over the years, including steam/dilute acid pretreatment (14), organosolv (15), ammonia fiber expansion/ explosion (AFEX) (16), lime (17), dilute acid (18) and SPORL (33) pretreatments. However, each of the pretreatment processes has its own particular drawbacks and advantages (2a, 16, 19). As mentioned earlier, the lignin content of the residual cellulosic substrate will vary, depending on the pretreatment method used (20). In general, solvent based or alkaline pretreatments result in a more targeted removal of the lignin component, whereas, acidic pretreatments primarily solubilise the hemicelluloses leaving a cellulosic fraction with a high lignin content. For example, earlier comparative pretreatment studies showed that only a small amount of the lignin present in corn stover could be removed by dilute acid, whereas substantial amounts of lignin were removed by lime pretreatment (21). In other cases, the composition of the substrate after AFEX pretreatment was essentially the same as that of the original biomass (21), while the lignin content of steam pretreated substrates increased when compared to the initial substrates, mainly because of the solubilization and removal of the hemicellulose (13). Other work has shown that organosolv pretreatment decreased the lignin content in pretreated substrates due to the solubilization of the lignin into the organic solvent (22). In addition to affecting the amount of lignin in the substrate, the choice of pretreatment will also affect the physical and chemical structure of lignin, altering both the accessibility to the cellulose and the interaction of cellulases with the pretreated substrate. There has been little work done on the effects of pretreatments on lignin structure and even fewer studies on the influence that lignin might play in binding to cellulases. However, some general trends can be anticipated. For example, it is likely that acidic pretreatments such as steam and dilute acid treatment would result in a net increase in condensed lignin, although simultaneous lignin depolymerization and condensation reactions are known to occur (23). It has also been suggested that “external lignin” that is often observed as droplets or re-precipitates on the surfaces of dilute acid, hydrothermal and steam pretreated substrates act as a physical barrier to cellulases 147 In Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass; Zhu, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


(24) . Although the exact mechanisms have not yet been fully elucidated, liquid hot water (hydrothermolysis) or flow-through type pretreatment processes have been shown to result in the removal of lignin. This is likely due to the liberation of acids in an autocatalysis type reaction by cleaving the hemiacetal O-acetyl and uronic acid substitutions of the hemicelluloses, resulting in a similar lignin depolymerization reaction pattern as has been observed with steam pretreatment. However, significant lignin removal during liquid hot water pretreatment is usually obtained at the expense of decreased carbohydrate recovery and seems to vary depending on the source of the pretreated biomass (25). In the case of alkaline pretreatments such as lime, NaOH or ammonia recycle percolation, lignin is solubilized by the deprotonation of the phenolic lignin subunits combined with alkaline induced breakage of the α and β-aryl ether bonds (26). Solvent pretreatments such as ethanol organosolv result in the cleavage of α and β- aryl ether bonds which solubilizes the lignin and increases the free phenolic groups present in the solid substrate (27). Although there are not many examples of the effect that the lignin structure in a given pretreated substrate can have on enzymatic hydrolysis, components such as the phenolic groups in lignin are likely to play an important role in lignin cellulase interactions (28). The enzymatic hydrolysis of cellulose is known to be affected by both enzyme- and substrate-related factors (2a, 29). Although considerable progress has been made by enzyme companies such as Novozymes and Genencor in reducing the cost of enzymatic hydrolysis (30), further improvements are still required. It has been reported that an approximate further 3-fold enzyme cost reduction (from 0.32 to 0.10 $/gal Ethanol) is necessary to reach cost targets for the eventual commercialization of bioconversion of pretreated corn stover to ethanol (31). It is recognized that efficient enzymatic hydrolysis of cellulose is determined by several factors such as the specific surface area, pore size, crystallinity, and degree of polymerization of the cellulose (2a, 32). In addition, it is known that the structure and location of hemicellulose and lignin can also affect the hydrolysis efficiency (2a, 33). Although hemicellulose has been shown to play a role in limiting accessibility of enzymes to the substrate and in enzyme end product inhibition, of the three main components of lignocellulose, hemicelluloses are known to be the most easy to remove by pretreatment (34). Instead, pretreatment studies have tended to focus on minimizing the degradation of hemicelluloses, to products such as furfural and hydroxymethyl furfural, so that the sugars can be obtained in a “useable” form. This can be achieved by using less severe steam pretreatment conditions which, through mild acid hydrolysis (sometimes termed autohydrolysis in unanalyzed conditions) solublizes the hemicellulose resulting in about 80-85% recovery of the original hemicellulose sugars. However, these less severe pretreatment conditions mean that the lignin associated with the cellulose in the water insoluble fraction may still prove to be an impediment to achieving effective enzymatic hydrolysis. Lignin has been implicated in decreasing the efficiency of enzymatic hydrolysis either by physically decreasing accessibility to or by the adsorption of cellulases (2a, 10b, 35). Although the removal of most of the lignin component in lignocellulose is technically possible, using current pulping and/or 148 In Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass; Zhu, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


bleaching processes, these processes are currently not economically feasible for bioconversion applications. For example, as the current price of kraft pulp is approximately US $1000 per ton (northern bleached softwood kraft pulp according to the PIX Pulp Benchmark Index), and the theoretical ethanol yields from glucose is 51%, it is clearly not economically feasible to use kraft pulp as a substrate for the ethanol production as the equivalent ethanol would sell for approximately only $350.00 (assuming the bleached pulp has a cellulose content of 90%), without including any costs for making the ethanol from the pulp. The benchmark for producing “cellulosic ethanol” would be to try and make it cost competitive with corn-ethanol which currently sells for approximately $2.26 per gallon. In summary, it is highly likely that no matter which type of biomass or pretreatment/fractionation process used, the resulting cellulosic substrate will always contain some amounts and types of lignin.

Cellulase-Lignin Interactions One of the major obstacles to efficient hydrolysis is the fact that cellulases work in a heterogeneous system where the enzymes are soluble while acting on an insoluble substrate. Thus, additional difficulties are presented to the enzymes when they encounter ancillary components of the substrate such as lignin which may restrict their access to cellulose. As mentioned earlier, the two possible mechanisms by which lignin might decrease the yield of cellulose hydrolysis are either “physical”, i.e. steric hindrance of the cellulose (36) or through the adsorption of cellulases to lignin (either reversibly or irreversibly) rather than cellulose (10c, 28b, 35a). Cellulases have been suggested to adsorb to lignin via hydrophobic (37), ionic bond (10c) and hydrogen bonding interactions (10c, 28). However, the exact mechanisms by which cellulases interact with lignin and result in the reduction in the efficiency of hydrolysis have yet to be fully resolved. Thus, by studying the effects that lignin has on enzymatic hydrolysis this should help us understand both the fundamental, mechanistic enzyme-substrate interactions as well as contributing to the development of efficient pretreatment methods that should improve the action of the cellulase enzymes. To gain a better sense of how cellulases may potentially interact with lignin we have reviewed some of the general theories that have been advocated to explain protein-solid surface interactions as protein adsorption is known to be affected by the properties of both the protein and the solid surface (38). Protein adsorption is a complex process that depends on a number of parameters, such as electrostatic attraction, hydrogen bonding and the hydrophobic/hydrophilic characteristics of both the protein and the surface on which it is adsorbed (39). Although protein adsorption seems to primarily involve hydrophobic interactions (38, 40), electrostatic contributions also play an important role, particularly for more hydrophilic surfaces (38). Of the protein properties that can potentially affect adsorption, molecular weight, hydrophobicity, electric charge, and stability are reported to be key (38). 149 In Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass; Zhu, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.

Like all proteins, the components of the cellulase enzyme system vary in their characteristics such as their molecular weight, hydrophobicity, electric charge and stability, all of which will affect their adsorption to the lignin component of the pretreated substrates.


Properties of Cellulases T. reesei has a long history of safe use for industrial enzyme production and is currently used as an important model and commercial system for assessing lignocellulose degradation (41). The properties of cellulases from T. reesei are summarized in Table I. The molecular weight of the cellulases varies from 23 to 75 kDa and the pI of most cellulases ranges from 3.5 to 7.8. In the case of the T. reesei cellulase complex, CBHI and CBHII are the major cellulase components, comprising 60% and 20% respectively of the total protein content (42). Most cellulases from T. reesei (except EGIII which is < 3% total protein) consist of two domains, a catalytic domain (CD) and cellulose binding domain (CBD), or cellulose biding module (CBM) . The CD works to hydrolyze cellulose into oligosaccharides or glucose, while the CBD or the CBM is thought to increase the adsorption of cellulases onto the surface of cellulose (43). The mechanism of cellulase adsorption on pure cellulose has been widely studied and it is reported that the adsorption involves both hydrophobic and hydrogen bonding interactions (44). Structure-based site directed mutagenesis studies have indicated that conserved aromatic amino acids are essential for the function of fungal CBD’s (45). It has also been shown that the CD is involved in adsorption of the enzyme onto lignin (46). In addition to the CD, the CBD or the CBM is reported to also play a role in increasing the adsorption of the enzymes onto the lignin (35a, 47). The papers cited above describe most of the work that has been carried out on this topic so far and indicates the limited extent of our knowledge with regard to cellulase interactions with solid surfaces. Thus, one of the goals of this paper was to look in more detail at the various charges involved in lignin-cellulose interactions.

Hydrophobic Interactions Hydrophobic interactions have been shown to be the primary driving force that governs protein adsorption (38, 40). Thus, as the hydrophobicity of both the protein and solid surface increases there is a greater tendency for adsorption to occur as, when protein molecules are dissolved in water, they tend to minimize the exposure of their hydrophobic groups to the aqueous environment (38). However, the protein exterior is often partly hydrophobic. Dehydration of hydrophobic portions of the protein and the sorbent surface is driven by entropy gain and, therefore, promotes adsorption to occur rapidly (38). As a rule, the amount of protein adsorbed is larger at hydrophobic surfaces.

150 In Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass; Zhu, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


Table I. The properties of cellulases from T. reesei Enzyme

Number of total amino acids

Molecular weight (kDa)

Isoelectric point (pI)

CBHI (Cel7A)

497

59-68

3.5-4.2

CBHII (Cel6A)

447

50-58

5.1-6.3

EGI (Cel7B)

437

50-55

3.9-6.0

EGII (Cel5A)

397

48

4.2-5.5

EGIII (Cel12A)

218

25

6.8-7.5

EGV (Cel45A)

225

23

-

β-GI

713

75

7.4-7.8

Actual data was adapted from Hui et. al (2001), Medve et. al (1997), and Valjamae et al. (2001) (48).

As the adsorption of protein onto substrates is generally enhanced by increasing the hydrophobicity of the protein (49), it is reasonable to expect that cellulases with different hydrophobicities will adsorb differently onto various lignocellulosic substrates. The hydrophobicity of cellulases can be calculated depending on their amino acid sequence (50). The specific amino acids present within some CBM’s are reported to promote the adsorption of cellulases on cellulose as well as onto lignin . When Park et al. (2002) modified cellulases with copolymers containing polyoxyalkylene and maleic anhydride (51) they found that, as the hydrophilicity of modified cellulases increased, the amounts of free modified enzyme in the supernatant also increased. In addition to the hydrophobicity of cellulases, the hydrophobicity of the “solid surface” i.e. the lignocellulosic substrate itself, will also affect the tendency of the enzymes adsorb to lignin or cellulose. Consequently, the presence of lignin in the lignocellulose tends to increase the substrates hydrophobicity. When Hodgson and Berg (1988) measured the contact angle of various wood fibers they showed that the contact angle of the α-cellulose was 14.0°, while that of thermomechanical pulps was 42.8° and 51.2°, indicating that the hydrophobicity of lignin is higher than that of cellulose (52). When Maximova et al. (53) examined the effect of the wetting properties of cellulose fibers and mica (which is a smooth, non-porous, hydrophilic surface), they found that the adsorption of lignin onto cellulose fibers and mica increased the contact angle, indicating that the lignin has a higher hydrophobicity than does the cellulose. When the hydrophobicity of lignin and cellulose were also measured using a thin film prepared by a spin coater (54), the contact angle between water and softwood kraft lignin, softwood milled wood lignin (MWL), and hardwood MWL was 46, 52.5, 55.5° respectively, while the contact angle of the cellulose alone was about 20 to 30° (55). Thus, pretreated substrates that have a high lignin content will likely be more hydrophobic when compared to cellulose in the absence of lignin. We recently assessed the properties of lignin from steam and organosolv pretreated corn stover, poplar, and lodgepole pine in which we compared lignin yields (56). Our work showed that, regardless of the pretreatment method used (steam or 151 In Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass; Zhu, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


organosolv), the isolation of lignin from the pretreated corn stover substrates consistently resulted in lower lignin yields than those of poplar and lodgepole pine. This suggested that the hydrophobicity of the lignin in corn stover substrates was lower when compared to lignins from poplar and lodgepole pine (56). It is apparent that the presence of lignin increases the non-productive adsorption of cellulases and that the hydrophobicity of lignin is higher than that of cellulose, strongly suggesting that hydrophobic interactions are one of the main factors governing the adsorption of cellulases to lignin. When Palonen et al. (2004) prepared three kinds of lignin, (alkali-lignin, cellulolytic enzyme lignin (CEL) lignin and acid-lignin) from steam pretreated spruce (SPS) and compared the adsorption properties of the intact enzymes and the CD from the CBHI and EGII cellulases to the SPS and isolated lignins (35a), they showed that the amount of CD adsorbed onto the substrates was lower than observed for the intact enzymes. They thought this was likely due to the greater hydrophobicity of the CBM component of the complete enzyme. As the amount of EGII adsorbed onto the lignins was higher than that of CBHI, they suggested that this difference was a result of the more open nature of the active site of EGII, which lead to greater adsorption through hydrophobic interactions. In related work, the different modules of two cellulase component enzymes, Cel7A (CBHI) and Cel7B (EGI), were compared with regard to their interaction with lignin (47). When the full-length enzymes were used, about 50% of the Cel7A and about 65% of the Cel7B were adsorbed onto the isolated lignin from spruce sawdust, while about 15% of the CDs were adsorbed onto the isolated lignin, suggesting that it was primarily the CBMs which contributed to the proteins adsorption onto the lignin. It was also thought that the difference in the hydrophobicity between the CBM of Cel7A and Cel7B was a result of their different adsorption behavior onto the lignin preparations. The CBM of Cel7A has four aromatic residues, all of which are tyrosine. Three of these tyrosines form the flat surface of the CBM giving the enzyme its affinity for crystalline cellulose. The CBM of Cel7B has five aromatic residues, four of which are tyrosine and one is tryptophan. Borjesson et al. (2007) suggested that the presence of a tryptophan in the CBM of Cel7B instead of a tyrosine on the flat surface increased the hydrophobicity of this part of the protein, based on the hydrophobicity scale of amino acid side chains that has been proposed by Roseman (57). The difference in the hydrophobicity of the tyrosine is caused by the phenolic hydroxyl group in its side chain, while tryptophan has an indole ring. In other related work, Berlin et al. (2006) isolated two types of organosolv lignin from Douglas-fir, with one fraction defined as dissolved lignin (DL), as it was precipitated from the organosolv liquor, while the other fractions was termed enzymatic residual lignin (ERL), as it was the insoluble residue remaining after the enzymatic hydrolysis of the organosolv pulp (10c). These workers then tried to correlate the functional groups in the lignin preparations with their inhibitory effects on cellulose hydrolysis. The low amounts of carboxyl and aliphatic hydroxyl groups found in the DL when compared to the ERL lignin indicated the higher hydrophobicity of the DL lignin, which inhibited hydrolysis to a greater degree than did the ERL lignin (Table II). However, this observation still needs to be confirmed as the DL was dissolved in 50% (w/w) ethanol while 152 In Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass; Zhu, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


the ERL was not dissolved in the ethanol solvent. The empirical parameter, ET(30) of ethanol/water (80:20), which is similar to the chemical composition of the organosolv washing solvent, is 53.7 kcal/mol, while the ET(30) of dioxane, which is used for dissolving lignin, is 36.0 kcal/mol (58). This means that the hydrophobicity of dioxane is higher than that of the ethanol/water (80:20) mixture. Although these workers did not measure the hydrophobicity of the isolated lignins, it is possible that the hydrophobicity of the DL was lower than that of the ERL. Other related work has reported that the addition of additives such as Bovine Serum Albumin (BSA), or polyethylene glycol to the reaction mixture can decrease the non-productive binding of cellulases onto lignin (37a, 47, 59). Palonen et al. (2004) reported that adding an excess amount of BSA to the cellulose mixture that included CBHI or EGII, resulted in a decrease in the amount of cellulases that bound to the SPS and the CEL-lignin (35a). Other workers have shown that cellulases adsorbed to both cellulose and lignin, while BSA adsorbed only onto the lignin (60). The reasons for the difference in adsorption properties between the BSA and cellulases are not well understood, although Eriksson et al. (2002) suggested that the BSA has hydrophobic sites, which will bind with fatty acids and will readily adsorb onto hydrophobic surfaces (37a). The addition of surfactants into the reaction mixture has been shown to increase the saccharification of lignocellulosic materials and to decrease the non-productive binding of cellulases to lignin (37a). When steam-pretreated spruce substrates were used to compare the action of several non-ionic, anionic, and cationic surfactants for their potential to increase enzymatic hydrolysis (37a) the addition of non-ionic or anionic surfactant (sodium dodecyl sulfate; SDS) to the reaction mixture reduced the adsorption amounts of Cel7A onto the SPS. In the related work, Borjesson et al. (2007) showed that non-ionic surfactants and polymers containing poly(ethylene oxide) effectively increased the enzymatic hydrolysis of lignocellulosic substrates (59). The adsorption of polyethylene glycol (PEG) onto the SPS was also thought to be through hydrophobic interaction (the dominant action) as well as through hydrogen bond interactions between the PEG and the lignin component in the lignocellulose. It should be noted that the effect of surfactants on hydrolysis was only evident when lignin was present in the substrate (37a, 59). It is most likely that hydrophobic interactions between cellulases and lignin play an important role in the non-productive binding of cellulases, which consequently contributes to the decrease in efficiency of lignocellulosic hydrolysis.

Electrostatic Interactions Compared to the effect of potential hydrophobic interactions between cellulases and lignin, the role of possible electrostatic effects has not been examined in any detail. In general, both the protein molecule and polymer surface carry charged groups and it is known that dissociation or association of the surface groups is one of the major mechanisms causing a surface charge that originates 153 In Sustainable Production of Fuels, Chemicals, and Fibers from Forest Biomass; Zhu, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2011.


from the carboxyl, amino, phosphate and imidazole groups. If both acidic and basic groups are present, the surface may become positively or negatively charged, depending on the experimental conditions that are used. Typically, these materials have an electronic neutral point called the pI or point of zero charge. If both the protein molecule and polymer surface have the same charge, they repulse each other. If they possess opposite charges, they adsorb. The maximum amounts of adsorbed protein onto polymers are sometimes the same as the pI of the protein or the pI of protein-polymer complex (61). Several groups have looked at possible electrostatic interactions between proteins and solid surfaces. For example, Fukuzaki et al. (1996) examined the interaction between BSA and metal oxide surfaces: silicon dioxide (SiO2, silica), titanium dioxide (TiO2, titania), zirconium oxide (ZrO2, zirconia), and aluminium oxide (Al2O3, alumina) (61a). The pI of BSA was found to be pH 5.1, at which maximum adsorption was observed for all of the metal oxides. There was a direct correlation between the amount of adsorbed BSA and surface charge density. All of the adsorbed BSA on the metal oxides showed charge shifts towards the positive site, suggesting the possible involvement of the carboxylic acids groups on the BSA molecules. When Elgersma et al. (1990) examined the adsorption of BSA on positively and negatively charged polystyrene lattices with different surface charges (61b), they found that maximum adsorption was attained at the pI of the protein-covered polystyrene (PS) particles rather than that of the pI of the protein itself. It is possible that the BSA may be preferentially adsorbed onto lignin when compared to Cel7A, because the pIs of these proteins are quite different (Cel7A: 3.5-4.2, BSA: 4.7-5.3), suggesting that Cel7A carried a negative charge at hydrolysis condition (~pH 5.0), while BSA would carry a positive charge or be electrically neutral.

Table II. Comparison of inhibitory effects on cellulases and amounts of reactive groups in isolated lignins from organsolv pretreated Douglas-fir Dissolved Lignin

Enzymatic residual lignin

Inhibitory effects on cellulases

>

CO

>

COOH

The Influence of Lignin on the Enzymatic Hydrolysis of Pretreated

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