Applications of Enzymes to Lignocellulosics - American Chemical

Chapter 8

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Modulation of Wood Fibers and Paper by CelluloseBinding Domains 1

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Oded Shoseyov, Ilan Levy , Ziv Shani , and Shawn D. Mansfield 1

The Institute of Plant Sciences and Genetics in Agriculture, the Faculty of Agricultural, Food and Environmental Quality Sciences. The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 Israel CBD-Technologies Ltd., Tamar Science Park, P.O. Box 199, Rehovot 76100, Israel C R C Chair in Wood and Fibre Quality, Department of Wood Science, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada 2

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Recombinant cellulose-binding domains (CBD) have previously been shown to modulate the elongation of different plant cells in vitro. Using Acetobacter xylinum as a model system, CBD was found to increase the activity of the cellulose synthase, up to fivefold, in a dose-dependent manner. In Populus, the introduction of a cbd gene under the control of the elongation specific cel1 promoter led to significant increases in biomass production in selected clones compared with wild-type plants. An analysis of the ensuing wood characteristics from the transgenic trees demonstrated significant increases in bothfibercell length and the average degree of cellulose polymerization. Additionally, a significant decrease in microfibril angle was observed. These results coincided with increased burst, tear and tensile indexes of paper preparedfromthese wood fibers. The mechanism by which CBD affects cell wall metabolism remains unknown. A physio-mechanical mechanism was postulated whereby CBD 116

© 2003 American Chemical Society Mansfield and Saddler; Applications of Enzymes to Lignocellulosics ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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localizes between adjacent cellulose microfibrils and separates them in a wedge-like action. In vitro experiments with petunia cell suspensions supports this hypothesis, as increasing concentrations of CBD displayed an abnormal shedding of cell wall layers, indicating that CBD has the potential to cause nonhydrolytic cell wall disruption activity in-vivo. Additionally, CBD fusion proteins may also be used to cross-link and introduce functional molecules into and onto lignocellulosicbased fibre networks. Consequently, these CBD fused molecules can be used to improve the physio-mechanical performance of paper sheets, and to alter its surface properties.

Early studies of CBD-cellulose interactions clearly suggest that the presence of CBD increases the effective concentration of enzyme on insoluble cellulosic substrates, and thereby assists the enzyme through the phase transfer from solublefraction(the enzyme) to insolublefraction(the substrate) (1-7). It was later reported that CBDs were present in both hydrolytic and non-hydrolytic proteins. In proteins that possess hydrolytic activity (cellulases, xylanases), the CBD has been shown to be a discrete domain that concentrates the catalytic domains on the surface of the insoluble substrate (5, 8-12). In contrast, the CBDs in proteins that do not exhibit hydrolytic activity compose part of a scaffolding subunit that organizes the catalytic subunits into a cohesive multi-enzyme complex known as a cellulosome. This enzymatic complex was found to function more efficiently in the degradation of cellulosic substrates (/, 13-17). Removal of the CBDfromthe cellulase molecule orfromthe scaffolding in cellulosomes has been shown to dramatically decrease enzymatic activity (18-23). Today, more than 200 putative sequences, in over 40 different species, have been identified. The binding domains are classified into 16 different families based on amino acid sequence, binding specificity and structure (5, 8-11). CBDs can contain anywherefrom30 to 180 amino acids, and exist as a single, double or triple domain in one protein. Their location within the parental protein can be eitherfromthe C- or N-terminus, and occasionally centrally positioned in the polypeptide chain. The affinity and specificity towards different cellulose allomorphs can also vary significantly (for an extended review on CBDs, see 8, 9, 11, 16, 17, 24). The three-dimensional structure of representative members of different CBD families has been resolved by crystallography and NMR (25-31% and indicate that CBDsfromdifferent families are structurally similar and that their cellulose binding capacity can be attributed, at least in part, to several aromatic amino acids that compose their hydrophobic surface. CBDsfromthe same organism have also been shown to differ in their binding specificity (23)

Mansfield and Saddler; Applications of Enzymes to Lignocellulosics ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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and occasionally two CBDs located on the same enzyme, can also exhibit this distinction (32). Biochemical studies have shown that CBD binding to cellulosic substrates is characterized in decrease entropy. For example, in the case of CBD from C fimi, which binds to crystalline cellulose, the decrease in entropy can be attributed to a net loss in conformationalfreedomof the polysaccharide and protein side chains. Water hydration upon binding may be another factor leading to lower entropy (33). On the other hand, binding of CBD ifromCellulomonas fimi to amorphous cellulose is characterized by decreased enthalpy. This phenomenon can be ascribed to heat release, which occurs upon complex formation that transpires through hydrogen and van der Waals bonding between the equatorial hydroxyl of the glucopyranosyl ring and the polar amino acids (32). Although the interaction of CBDs with their corresponding cellulosic substrates is occasionally irreversible, contact with the cellulose surface is dynamic. Usingfluorescencerecovery techniques Jervis et al. (34) demonstrated that CBDcex is mobile on the surface of crystalline cellulose when it appears in isolated form or as a module of a xylanase protein. Furthermore, it was hypothesized that the binding of family Ila CBDs from C. fimi to cellulose occurs either along or across the chain (35). This review will describe the potential applications of CBDs in fiber modification, both in vivo and in vitro.


Cex

N

Wood Fiber Modification Using CBD The gram-negative bacterium Acetobacter xylinum has long been regarded as a model of cellulose biosynthesis primarily because cellulose microfibril synthesis is set apartfromcell wall formation (36). Consequently, cellulose is produced as separate ribbons composed of microfibrils and their interactions with other polysaccharides do not exist as in the plant cell wall. Since polymerization and crystallization of cellulose is a coupled process in A. xylinum cellulose biosynthesis, any interference during the crystallization phase results in accelerated polymerization (37). It has also been shown that some organic substances with affinity for cellulose can also alter cell growth and cellulosemicrofibril assembly in vivo. For example, carboxymethylcellulose (CMC) and fluorescent brightening agents (calcofluor white ST) prevent microfibril crystallization, thereby enhancing polymerization. These molecules bind to the polysaccharide chains immediately following extrusion from the cell surface, thus preventing normal assembly of the microfibrils and cell walls (38). Shpigel et al. (39) have shown that like other organic cellulose-binding substances, family III CBD derived from Clostridium cellulovorans could modulate



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cellulose biosynthesis. It was apparent that CBD increased the rate of cellulose synthesis activity in A. xylinum up to fivefold compared to a control. Electron microscopy of the cellulose synthesized in the presence of CBDs revealed that the newly formed fibrils are spread out into a splayed ribbon instead of the uniform, thin, packed ribbon as was evident in the control fibers. The mechanism by which CBD affects cell wall metabolism remains unknown. A physiomechanical mechanism has been proposed whereby; CBDs adhere to, and slides between adjacent cellulose fibers and separate them in a wedge-like action (40). This hypothesis is supported by in vitro experiments that show that Petunia cell suspensions treated with increasing concentrations of CBD displayed abnormal shedding of cell wall layers, suggesting that CBDs can cause non-hydrolytic cell wall disruption in vivo (40). Several protocols were tested to analyze the effect of CBDs on living plant cells. In these studies it was found that Family III CBDsfromC. cellulovorans could modulate cell elongation. At low concentrations, this CBD enhanced elongation of Prunus persica L. pollen tubes and A. thaliana root seedlings, whereas at high concentrations, CBD inhibited root elongation in a dosedependent manner. It was demonstrated that cellulose-xyloglucan networks, similar to plant cell walls, could be formed when employing the A. xylinum model system in a medium containing xyloglucan (41-44). NMR analysis indicated that 80 to 85% of the xyloglucan adopts a rigid conformation; in all probability aligned with the cellulose chain, whereas, the remainder is more mobile. The xyloglucan, when present during cellulose synthesis in the A. xylinum model system, causes the cellulose to become more amorphous and increases its tensile strength (45). When CBDs were present, it was shown that the CBDs could compete with xyloglucan for the binding of cellulose (39). Thesefindingssupport the hypothesis that, at least part of the effect CBD exerts on the plant cell wall, is via cellulose-xyloglucan interactions. Table I. Fibre properties of wild type and transgenic Populus trees Clone

LWIf (mm)

Coarseness (mg/m)

Microfibril angle Crystallinity (degrees) (CI)

b

b

b

b

~WT 0.829 ± 0.005 0.914 ± 0.01 l CBDl 0.920 ± 0.004 0.941 ± 0.014 CBD2 0.966 ±0.006 0.962 ±0.010 Length weighted fiber length. Number of fibers analyzed: 13500. dumber of fibers analyzed: 100. Two different clones d

d

b

b

30.59 ± 0.5\\T 28.48 ± 0.554 27.38 ±0.397° c

39.1 ±3.12 43.0 ± 3.12 45.3 ±3.12

a

b

d



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Shoseyov et al. (46) have also shown that CBDs can also modulate plant growth in transgenic plants. The introduction of the Family III cbd genefromG cellulovorans under the control of the elongation-specific cell promoter (47-48) into Populus resulted in significant alteration infiberproperties. The transgenic woodfiberswere significantly longer they displayed higher coarseness and smaller microfibril angles (Table I). An analysis offibercrystallinity by FT-IR (Table I) also suggests that there was a trend of greater crystallinity within the modified fibers. Additional changes were detected in the nature of the cellulose polymers themselves. Figure 1 demonstrates that the transgenic trees contain a larger degree of cellulose polymers rangingfrom2000 to 6000 MW, while exhibiting a lower concentration of cellulose molecules ranging from 200 to 900 MW compared with wild type trees. Therefore, CBD expression in the transgenic trees resulted in a significant shift in the cellulose polymers to a higher molecular weight. Although many morphological and ultrastructural changes were detected in the transgenicfibers,an extensive comparison with the wild type (Table II) revealed that the chemical composition had not changed.

Table II. Chemical composition of wild type and transgenic Populus trees Clone

Natural sugars (%) Gal

Ara

Glu

Xyl

Man

Lignin (%) Ash Ext (%) (%) ISL SL

Total

WT 0.8 0.6 43.6 19.8 2.5 17.3 4.1 0.6 10.1 99.4 CBD1 0.8 0.6 42.5 19.8 2.4 17.2 4.0 0.6 10.9 98.8 CBD2 0.8 0.6 43.3 20.4 2.4 17.1 3.9 0.6 10.0 99.1 NOTE: Gal- Galactos; Ara- Arabinose; Glu- Glucose; Xyl- Xylose; Man- Mannose ISL- Acid Insoluble Lignin; SL- Acid Soluble Lignin

Pulp producedfromthe modified and wild type trees were used to make standard laboratory handsheets, and these sheets were tested for standard paper strength properties. It was apparent that no differences in the pulp yield were observed between the transgenic and wild type trees (data is not shown). The results (Figure 2) clearly show that paper madefromfibersoriginatingfromthe transgenic trees, have improved strength properties as measured by tensile, burst, and tear indices when compared with sheets producedfromthe wild type trees.


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60000

100

1000

10000

Molecular Weight Figure 1. Molecular weight distribution of isolated cellulose polymers in wild type and transgenic trees expressing CBD.

Modulating Cellulose Containing Materials with CBD Din et al. (49) reported that CBD AfromC.fimiendoglucanase A is capable of non-hydrolytic disruption of cellulosic fibers, which results in the release of small particles. In addition, it was shown that CBD nA could prevent the flocculation of microcrystalline bacterial cellulose (50). Similar phenomena were observed for other CBDs (40, 51-54), including C B D (Figure 3). However, this observation is not common to all CBDs (6). Thefirstdirect evidence for the involvement of CBDs infibersurface alteration was reported by Lee et al. (55), who demonstrated using atomic force microscopy that the Cel7A (formally CBH I) generated distinct tracks along the longitudinal axis of cellulosicfibers,while treatment with EG II caused the peeling and smoothing of thefibersurfaces. When cellulases that lacked CBDs were subsequently used, no effect on the surface of the cottonfiberwas detected. Additional information to support this observation camefromthe study carried out by Suurnakki et al. (7). In this application the actions of endoglucanases, cellobiohydrolases and the catalytic domainsfromT. reesei on bleached chemical pulp were compared. According to Cen

Ce

aos



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Figure 2. Mechanical properties ofpapers made offibers from both wild type and transgenic plants expressing CBD (A) Burst Index, (B) Tensile Index and (C) Tear Index.



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Figure 2. Continued.



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Figure 3: Non-hydrolytic fiber disruption of cellulose filter paper at different concentrations of cellulose-binding domain (CBD) originatedfrom Clostridium cellulovorans.

this research, the presence of CBD in the intact enzyme had a beneficial effect on pulp properties such as viscosity and strength after PFI refining. The tensile strength of paper is imparted by both the inherent intrinsic strength of thefibres,as well as by the amount and strength of the fiber-to-fiber bonds (56). Intra-fiber interaction has been shown to improve stress transfer between the adjoiningfibersand is considered to be one of the most important factors affecting overall stress development in thefibernetwork under tensile deformation (57, 58). Earlier studies have shown that the low strength of dryformed structures can be improved by adding binder materials or bicomponent fibers (59). Recently, we demonstrated that CBDs could modify paper properties. Two CBDs belonging to family III (from C. cellulovorans) that had been fused together to form a cellulose cross-linking protein (CCP) were applied ontofilterpaper. These treatments significantly improved the tensile strength (Figure 4A and B) (60). A potential explanation to the effect of CBD and CCP is that these molecules may change the interfacial properties of thefibersand thereby alter thefiber-to-fiberinteraction. Additionally, applying CBDs to cellulosic fibers has the potential to improve paper recycling. It has been demonstrated that the application of CBD on secondary fibers, such as old paperboard containers, results in increased tensile and burst indexes as well as improvement in pulp drainage (61).



125 Although CCP application has been shown to improve mechanical properties of paper sheets (Figure 4C and D), further investigation on the effect(s) of CCP demonstrated that CCP did not interfere with the effect of cationic starch on the dry strength of the paper but rather indicated a synergistic effect (unpublished data). Recently, Kitaoka and Tanaka (62) reported on the production of a novel papermaking reagent by covalently binding activated anionic polyacrylamide (A-PAM) to CBD originatingfromTrichoderma viride 1,4-P-glucanase (CBD-A-PAM). In this manner they were able to produce a molecule, which contained more than one CBD that is capable of cellulose fiber cross-linking. It was subsequently shown that the dry and wet tensile strength of the ensuing paper to which the CBD-A-PAM was added internally increased We propose that the increase in stress to failure caused by CBD and CCP is related to the nature of the CBD's binding site, which is a large hydrophobic planar surface with several attachment sites (3, 25, 26, 63, 64). CCP is an efficient cross-linker due to its larger size and to the number of attachment sites it contains that enable it to cross-link cellulosic materials. Applying a single CBD molecule to the paper also improved its mechanical properties, but to a lesser extent than CCP. The CCP construct can also be employed as a sizing agent. The application of CCP to fibre networks using different methods (imbibing, spraying or blade coating) results in a hydrophobic paper surface (Figure 5). It is hypothesized that at relatively high CCP concentrations most of the binding sites on the cellulose are occupied by a single CBD moiety, and therefore, the second CBD moiety (the non-bound moiety) of CCP exposes its hydrophobic amino acids and consequently increases the surface hydrophobicity (40, 60, 65). Another study demonstrated that polysaccharide structure modification could be achieved using isolated CBDs. It has been suggested that the surface of cellulosic polysaccharide (ramie cotton fibers) was roughened after treatment with CBD (CBDcenAfromC.fimi).It was proposed that these treatments could be used in order to alter dyeing characteristics of cellulose fibers (66). CavacoPaulo et al. (67) demonstrated the effect of CBD on the dye affinity to cotton fibers, and showed increased levels of dye affinity following treatments with family II CBDfromC.fimi.

Conclusions

Clearly there is strong indications that cell wall modification, mediated by CBD, could provide an effective mean for fiber improvement in vivo and the consequent improved fiber properties can significantly enhance the quality of ensuing paper products. In addition, the binding of cellulose-binding domains to cellulosic polymers, under a wide range of environmental conditions, without the



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Figure 4. The effect ofprotein treatment on the mechanical properties ofpapers treated with cellulose-binding domain (CBD) or cellulose cross-linking protein (CCP). Stress at failure (A) and energy adsorption (B) of Whatmanfilterpaper No. 1 coated with CBD or CCP, and papers hand sheets prepared with CBD or CCP in the furnish (C and D). Protein concentration relates to the coating solution (in A and B), or to the furnish (in C and D) as percent protein relative to fibers (w/w).



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Figure 5. Water-absorption time of papers treated with cellulose-binding domain (CBD) or cellulose cross-linking protein (CCP) at different concentrations. Whatman filter paper No. 1 was immersed in protein solution and then left to dry at room temperature. Protein concentration relates to the amount of protein in the coating solution.

need for chemical reactions, makes them attractive moieties for the design of a new class of paper modifying agents that are environmentally friendly.

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Applications of Enzymes to Lignocellulosics - American Chemical

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