The Effects of Recombinant Cellulomonas fimi β-1,4-glycanases on Softwood Kraft Pulp Fibre and Paper Properties

Biotechnology in the Pulp and Paper Industry L. Viikari and R. Lantto (Editors) 9 2002 ElsevierScience B.V. All rights reserved.

301

The Effects o f R e c o m b i n a n t Cellulomonas fimi 13-1,4-glycanases on Softwood Kraft Pulp Fibre and Paper Properties Shawn D. Mansfield 1, Neff R. Gilkes 2, R. Antony J. Warren 2 and Douglas G. Kilburn 2 Department of Wood Science and 2 Department of Microbiology and Immunology University of British Columbia, Vancouver, British Columbia, Canada ABSTRACT Recombinant Cellulomonas fimi 13-1,4-endoglucanases (Cel5A and Cel6A) and cellobiohydrolases (Cel6B and CeI48A) were assessed for their capacity to selectively modify the physical and optical properties of handsheets derived from a fully bleached, never-dried softwood pulp. The isolated binding domain (Cel6A CBD) and catalytic domain (Cel6A CD) were also evaluated. Treatment with endoglucanases, particularly CeI6A CD, caused substantial damage to the intrinsic fibre strength of the kraft furnish, and consequently compromised the physical handsheet properties. In contrast, treatment with Cel48A produced beneficial modifications, including improvements in handsheet tensile strength following mechanical refining and various degrees of wet pressing. CeI6B and Cel6A CBD had very limited effects on the fibre characteristics, and therefore did not alter the quality of the handsheets produced. An examination of carbohydrate solubilization and changes in the degree of polymerisation of the polysaccharides indicated that the capacity for Ce148A to beneficially modify pulp and paper characteristics seems to be related to its capacity to selectively degrade cellulose-hemicellulose linkages, and release and modify xylan moieties. Furthermore, this enzyme preparation demonstrated limited carbohydrate dissolution, indicating that the observed modifications were attained with very little yield loss. These results suggest that CeI48A, and enzymes with similar glycanase activity should be considered and further evaluated for their potential to treat pulp fibres to improve paper properties. 1. INTRODUCTION The process of mechanically beating or refining pulp imparts desirable alterations to fibres morphology and enhances the papermaking properties. These changes in fibre morphology (i.e. cutting and fibrillation) and the disruption of bonds within the secondary wall all contribute to the improvements in paper strength and optical parameters. However, this process requires substantial energy input, particularly in mills manufacturing paper containing mechanical pulps. The potential of hydrolytic enzymes to decrease refining energy requirements and enhance fibrillation of pulps has been recognized for many years. A process using a cellulase complex from the white-rot fungus Trametes suaveolens to treat chemical pulp fibres was patented in 1968 [1]. Later, a mixture of xylanases and cellulases was shown to reduce the refining energy requirement for secondary fibre processing [2-4]. However, the use cellulase mixtures never gained acceptance because reductions in refining energy were always accompanied by lowered strength and yield due to the aggressive attack on fibres produced by the synergistic action of endoglucanases and cellobiohydrolases. Subsequently, various researchers evaluated individual monocomponent cellulases in attempts to

302 modify pulp fibres specifically and circumvent the reductions in strength or yield [5-7]. Studies on isolated Trichoderma reesei cellulase monocomponents suggest that endoglucanases are more destructive than the predominantly exo-acting cellobiohydrolases. For example, no major differences in strength resulted from treatment with Tr Cel7A (previously called cellobiohydrolase I), while Tr CeI5A (previously endoglucanase II) caused severe fibre damage that could not be restored by beating [5]. Further research confirmed that endoglucanase treatment lowered both fibre and paper strength, but also showed that the extent of degradation varied with the type of endoglucanases employed [7]. These data provide a rationale to screen other, related enzymes for useful fibre modifying properties. To date, the search for appropriate "fibre modifying" enzymes has focused on the components of fungal cellulase systems, while bacterial cellulases remain relatively unexplored. The extent to which bacterial cellulase systems resemble those of fungi is still unclear. However, some differences between fungal and bacterial systems are evident. For example, cellulolytic fungi such as Trichoderma and Humicola spp. use the concerted action of cellobiohydrolases belonging to glycosyl hydrolase families 6 and 7, together with a range of endoglucanases, to solubilise cellulose. Although many cellulolytic bacteria produce a family 6 cellobiohydrolase, they appear to lack family 7 enzymes. However, several well-characterized bacterial systems contain cellulases from family 48, not represented in fungi, which appear to be functional equivalents of the family 7 enzymes [8]. Given such differences, bacterial cellulase systems may prove to be promising sources of enzymes with novel fibre-modifying properties. This study examines the effects of cellulases from the bacterium Cellulomonas fimi on the properties of kraft pulp. The enzymes studied include CeI48A and CeI6B (cellobiohydrolases belonging to families 48 and 6, respectively) and CeI6A and CeI5A (endoglucanases belonging families 5 and 6). In the case of CeI6A, the effects of its isolated catalytic and cellulose-binding domains were also examined independently. The properties of handsheets prepared from enzymetreated pulp, with and without refining, and after application of various levels of wet pressing pressure, were examined. The observed changes in handsheet properties were related to changes in the degree of polymerisation and amounts of polysaccharide dissolution in an attempt to elucidate mechanisms of pulp fibre modification at the molecular level. 2. MATERIALS AND METHODS

2.1. Pulp Never-dried, fully bleached kraft pulp containing approximately 85% Douglas-fir was obtained from Weyerhaeuser Inc. (Tacoma, Washington). The carbohydrate composition of the pulp was 0.6% arabinose, 0.8% galactose, 82.4% glucose, 7.0% xylose, and 7.2% mannose residues, as determined by HPLC following secondary acid hydrolysis [9]. 2.2. Enzymes C. fimi genes encoding CeI6A (previously called CenA), Cel5A (previously CenD), CeI6B (previously CbhA) and Ce148A (previously CbhB) were expressed in Escherichia coli, and the corresponding enzymes purified as previously described [10-13]. The Cel6A catalytic domain (CeI6A CD) and cellulose-binding domain (CeI6A CBD) were isolated by digestion with C. fimi protease and size-exclusion chromatography [10]. 2.3. Enzymatic treatment of pulps Pulp slurry in 5 mM potassium phosphate buffer was adjusted to pH 7 with sulphuric acid and autoclaved for 5 rain. Enzyme (1.0 mg/g dry weight of fibre for CeI6A, CeI5A, CeI6A CD and

303 CeI6A CBD; 0.5 mg/g for Cel6B and CeI48A) were added in the potassium phosphate to bring the preheated (37~ pulp sample to the target consistency of 3% (w/v). The slurries were incubated for 2 h at 37~ with continuous stirring. Following enzymatic treatment, the filtrates were collected for analysis and the pulps were washed 3 times with water to remove residual protein, and then autoclaved for 15 minutes to inactivate any residual enzyme. Control pulps were prepared under the same conditions, without enzyme addition.

2.4. Papermaking and testing Handsheets were prepared and tested according to standard Tappi Test Methods, with the white water recirculated to ensure that both the primary and secondary fines were included in the resultant handsheet. Handsheets of unrefined and refined furnishes were subject to wet pressing at 40, 90 and 140 psi during sheet making. A PFI laboratory refiner (Tappi Test Method T 248 cm-85) was used to prepare the refined furnish to 2000 revolutions. Pulp freeness was measured at 20~ according to Tappi Test Method T227 om-94. 2.5. Fibre analysis Fibre coarseness was determined by passing more than 20,000 fibres, in exact aliquots of approximately 5 mg of pulp through a Kajaani FS-200 (Kajaani, Finland) fibre analyzer. 2.6. Analysis of soluble carbohydrates Filtrates were analysed by high performance anion-exchange chromatography (HPAEC) after secondary acid hydrolysis on a CarboPac PA-1 column using a Dionex DX-500 system equipped with a pulsed amperometric detector (Dionex, Sunnyvale, CA, USA). 2.7. Determination of degree of polymerisation Molecular mass distributions were determined by gel permeation chromatography of tricarbanyl derivatised samples. Pulp samples were carbanylated [14] and the derivatised material was recovered by evaporation. Samples were redissolved in tetrahydrofuran to approximately 0.2 mg/mL and filtered through a 0.45 ~tm pore Teflon membrane. Samples were analysed on a Waters 625 liquid chromatography system (Millipore Corp., Milford, MA) equipped with four TSK-gel columns (Varian, Sunnyvale, CA). The columns (G1000 HXL, G3000 HXL, G4000 HXL and G6000 HXL) were connected in series and had nominal molecular weight cut-offs of 1x 103, 6x 104, 4x105, and 4x107, respectively. Samples were eluted with tetrahydrofuran at a flow rate of 1 mL/min and tricarbanylates quantified by absorption at 254 nm using a Waters 486 UV spectrophotometer. The column series was calibrated using polystyrene standards [15]. The degree of polymerisation (DP) was calculated by dividing the molecular weight of the tricarbanylated derivative by the molecular weight of tricarbanylated anhydroglucose (DP -- Mw/519). 3. RESULTS AND DISCUSSION The primary function of refining is to modify the structure of the fibre in order to enhance their papermaking potential. Subsequently, wet pressing removes water and consolidates the wet web prior to drying. Wet pressing results in several physical and topographical changes in the fibre network, which affect the properties of paper and the recycling potential of the fibre. Refining and wet pressing both significantly influence sheet formation and wet web strength and hence machine performance. The investigations described below are concerned with the effects of individual bacterial cellulases (or their isolated functional domains) on handsheets produced from unrefined and refined fibres using varying degrees of wet pressing during sheet formation.

304 3.1. Effect of enzymatic treatments on properties of unrefined handsheets

The most significant effect of wet pressing is sheet densification [16], and it is clear that higher pressing pressure resulted in increased sheet density in all samples (Fig 1A). Pretreatment with endoglucanase monocomponents (CeI5A, CeI6A or CeI6A CD) enhanced densification at both low and medium pressing pressures, but had no significant effect at higher pressure. Similar effects following endoglucanase pretreatment have been reported previously [5-7]. Pretreatment with cellobiohydrolases (CeI6B and Ce148A) had little or no effect on sheet density (Fig.lA), in agreement with previous studies using fungal cellobiohydrolases [5, 7]. CeI6A CBD was similarly ineffective. Air resistance increased with sheet density in all samples, in agreement with previous data showing that wet pressing results in substantial intra- and interfibre pore closure [17]. It was apparent that at a given sheet density, the air resistance of the resultant handsheets was enhanced slightly by selective enzyme treatment, namely CeI5A, CeI6A CD and CeI48A (Fig. 1B). The degree of interfibre bonding within a handsheet can be inferred from measurement of light scattering: a decrease in the scattering coefficient indicates greater fibre-fibre contact within the sheet, as light is scattered at air-fibre interfaces [18]. As expected, scattering coefficients were reduced in all samples at high sheet densities due to enhanced bonding of densely packed fibres (Fig. 1C). However, treatment with the endoglucanases (CeI5A, CeI6A, CeI6A CD) and the cellobiohydrolase CeI48A resulted in significantly larger reductions at higher sheet densities, relative to control handsheets. The observed changes in densities and scattering coefficients suggested that some enzyme treatments enhanced intrinsic fibre flexibility, making the fibres more collapsible during papermaking. To investigate these effects further, various handsheet strength parameters were determined and plotted against apparent sheet density. In general, all enzyme-treated samples showed improved tensile strength at a given density relative to the control at the lowest pressing pressure (Fig. 2A). However, at the mid-range pressing pressure (90 psi), only the Cel6A, Cel6A CD, CeI5A and CeI48A preparations showed improvements, while at the high pressing pressure (120 psi) only the CeI6A CD demonstrated higher values than the corresponding control. The greatest improvements observed (CeI6A, CeI6A CD and CeI5A), corresponded to those enzymes shown to produce the largest increases in densification. In contrast, tear strength (Fig. 2B), a parameter directly influenced by intrinsic fibre strength and enhanced inter-fibre bonding, was adversely affected by endoglucanase treatment, while treatment with cellobiohydrolase CeI48A or the Cel6A CBD gave marginal improvements. Wet zero-span breaking strength determinations (Fig. 2C) showed that the observed tear strength reductions in endoglucanse-treated samples were the direct result of significant losses in fibre strength. No losses in fibre strength were seen in other samples. 3.2. Effect of enzymatic treatment on properties of refined handsheets

Having established that treatment with C. fimi cellulases caused significant modifications to handsheet properties; the response to refining was investigated. Samples were subjected to 2000 revolutions in a laboratory PFI refiner, and then wet pressed at different levels prior to drying. As with unrefined fibre, treatment with endoglucanases (CeI6A, CeI6A CD and CeI5A) produced significant increases in sheet density relative to the control, while the effects of CeI6A CBD and the cellobiohydrolases CeI6B were minimal (Fig. 3A). Generally, the degree to which an enzyme affected density was correlated with its ability to increase air resistance (Fig. 3B) and decrease the scattering coefficient (Fig. 3C), strongly supporting the theory of enzyme-induced fibre collapsibility and flexibility previously proposed [19].

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Following refining, the extent of wet pressing did not significantly alter the strength properties of either the enzyme-treated control fibres (Fig. 4). However, it was clear that the tensile strength of the sheets was significantly compromised by the application of endoglucanase preparations with the greatest damage resulting from treatment with Cel6A CD, followed by Cel6A and CeI5A (Fig. 4A). In contrast, treatment with the cellobiohydrolase Cel48A resulted in improved tensile strength, while CeI6B treatment was ineffective. As with unrefined fibre, the tear strength (Fig. 4B) and intrinsic fibre strength (Fig. 4C) of the refined furnish was substantially degraded by endoglucanase treatments, while the cellobiohydrolase-treated and Cel6A CBD-treated samples retained similar properties to those of the control.

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3.3. Fibre modifications The influence of fibre morphology on the physical and optical properties of paper has been thoroughly investigated. In general, finer fibres are more flexible and collapsible and produce denser sheets during papermaking. It has previously been shown that reductions in the coarseness of Douglas-fir kraft pulp by treatment with fungal cellulase enhanced subsequent densification [9, 20]. Similarly, in this investigation, C. fimi cellulases also caused small reductions in the coarseness of the unrefined furnish (Table 1). PFI refining alone slightly reduced control pulp coarseness, but the combination of refining and endoglucanase treatment resulted in significant (up to 20% with Cel6A CD) reduction in fibre coarseness (Table 1) and fibre length distribution (data not shown). In contrast, no discernable changes were detected with cellobiohydrolase treatments. These data

307 indicate that the endoglucanase component(s) of complete cellulase complexes may be responsible for the reductions in fibre coarseness previously observed with cellulase complexes.

Table 1. Fibre coarseness and freeness of kraft pulp treated with C. fimi cellulases Enzyme Control Cel6A Cel5A Cel6A CBD CeI6A CD CeI6B CeI48A

Unrefined coarseness .(mg/m) . 0.310 _+0.02 0.294 + 0.05 0.282 + 0.01 0.286 + 0.06 0.286 + 0.07 0.295 + 0.04 0.300 + 0.09

Refined coarseness (mg/m) 0.293 + 0.06 0.242 _+0.04 0.253 +_0.01 0.298 + 0.05 0.229 + 0.07 0.289 + 0.04 0.303 + 0.07 ,,,

Unrefined freeness Refined freeness (mL) (mL) 738 457 734 135 747 176 717 464 755 128 744 450 752 498

3.4. Pulp Freeness Pulp freeness or drainability, as measured by Canadian Standard Freeness (CSF) is an empirical measure of the ease with which water drains from a pulp suspension. This parameter is directly influenced by both the extent of refining and the amount of recycled fibre found within pulp suspensions, as the presence of fines and/or highly fibrillated fibers decreases pulp freeness. Comparative studies have been initiated to determine what effects individual cellulase monocomponents had on freeness [6]. These results indicated that endoglucanases were more effective than cellobiohydrolases. Furthermore, cellobiohydrolases have been shown to act synergistically with endoglucanases to increase freeness, and their combined actions resulted in increased sugar liberated and yield loss [21 ]. The employment of C. fimi cellulase components did not reveal any discernible differences in the effects that endoglucanases and cellobiohydrolases have on the freeness of the unrefined furnish. Pulp freeness following refining indicated that only the endoglucanases (CeI5A and Cel6A) and the catalytic domain (Cel6A CD) could significantly alter this process parameter. Pretreatment with Cel6B and CeI48A, as well as the purified cellulose binding domain (Cel6A CBD) had practically no effect on the development of pulp freeness (Table 1), corroborating with previous studies [21, 22] examining the effects of Trichoderma reesei monocomponent cellulases on pulp properties. 3.5. Carbohydrate solubilisation and modification Analysis of the carbohydrates solubilised by C. fimi cellulases showed that all enzymes, with the exception of CeI48A, were highly selective for glucan (Table 2). Trace quantities of arabinose and xylose liberated by Cel6A and Cel5A indicates either low level cross-reactivity for arabinoxylan or the solubilisation of cellulose molecules associated with soluble hemicellulose, while the absence of mannose is consistent lack of Cel6A and CeI5A cross-reactivity on glucomannan [23]. Release of glucose by Cel5A was about twice that for Cel6A (Table 2), but Cel5A was less effective than CeI6A in reducing fibre strength (Figs. 2 and 4) and degree of polymerisation (Fig. 5A). Similar results obtained by Kleman-Leyer et al., [24] using cotton fibres led to the suggestion that CeI6A preferentially attacks kinks in fibres, resulting in short fibre fragments of low DP, while CeI5A degrades all regions of the fibre surface equally so that only small shifts in DP are observed, even after extensive degradation.

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Table 2. Carbohydrates solubilised by enzymatic treatment of kraft pulp with C. fimi cellulases Enzyme

Carbohydrates Solubilised (% dry weight of pulp)*

Arabinose Galactose Glucose CeI6A 0.02 0 0.68 CeI5A 0 0 1.12 CeI6A CBD 0 0 0.05 CeI6A CD 0 0 0.38 CeI6B 0 0 0.29 Ce148A 0.08 0 0.18 * Values represent treatment minus control

Xyl0se 0.08 0.03 0 0.01 0 1.39

Mannose 0 0 0 0 0 0.04

Total Solubilisation 0.68 1.15 0.05 0.39 0.29 1.69

Regarding the handsheet properties, CeI6B had a very limited effect on any of the pulp/paper properties, while CeI48A improved a number of handsheet parameters, including tensile strength. The ability to improve sheet strength was also maintained after refining, while all the other monocomponent preparations resulted in deleterious effects to both fibre and sheet properties

309 following the mechanical processing of the fibres. The beneficial modifications observed with CeI48A treatments may be related to its ability to modify the xylan component, or the xylancellulose linkages, which could effectively enhance the fibre flexibility and consequently improve the resultant handsheet properties. 4. CONCLUSIONS The effects of purified recombinant Cellulomonasfimi p-l,4-glucanases on the physical and optical properties of handsheets derived from a fully bleached, never-dried softwood were determined. It was apparent that endoglucanases cause substantial damage to the intrinsic fibre strength of the kraft furnish, and consequently the physical handsheet properties. The catalytic domain, Cel6A CD, proved to be even more detrimental to the fibre than its intact protein analog, while solubilising noticeably less polysaccharide. In contrast, the cellulose binding domain, CeI6A CBD, had a very limited effect on the fibre characteristics, and therefore did not compromise the quality of the handsheets produced. Both of the cellobiohydrolases (CeI6B and CeI48A) demonstrated limited carbohydrate dissolution, little or no change in cellulose DP, and had different effects on pulp properties. Cel48A demonstrated beneficial paper properties, including improvements in handsheet tensile strength following mechanical refining and wet pressing. This latter result seems to be related to the release and modification of the xylan moieties. These results suggest that Ce148A should be further considered for treating pulp fibres to improve paper properties, and future investigations should be directed at elucidating the mechanism(s) of CeI48A induced fibre modification. 5. ACKNOWLEDGEMENTS The authors would like to thank Dr. Scott Stephens of Weyerhaeuser Inc., for his valuable input into this manuscript. We would also like to acknowledge Weyerhaeuser Technical Center (Tacoma WA) for its help with papermaking and testing. REFERENCES

1.

W.D. Yerkes, Process for the digestion of cellulosic materials by enzymatic action of Trametes

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310 13. H. Shen, N.R. Gilkes, D.G. Kilburn, R.C. Miller and R.A.J. Warren, Biochem. J. 311 (1995) 67. 14. L.R. Schroeder and F.C. Haigh, Tappi 62 (1979) 103. 15. L. Valtasaari and K. Saarela, Paperi ja Puu - Papper och Tr~i 57 (1975) 5. 16. T.W. Bither and J.F. Waterhouse, Tappi J. 75 (1992) 201. 17. T.C. Maloney, T.-Q. Li, U. Weise and H. Paulapuro, Proc. 50th Appita Ann. Gen. Conf. Vol. 2 (1996) 647. 18. A.M. Scallan and J. Borch, In The fundamental properties of paper related to its uses. Trans. Symp. Cambridge (F. Bolam Ed.) Vol. 1. (1973) 152. 19. S.D. Mansfield and A.R. Dickson, Appita J. 54 (2001) 239. 20. M.S. Lumme, S.D. Mansfield and J.N. Saddler, Wood Fibre Sci. 31 (1998) 385. 21. T. Oksanen, J. Pere, J. Buchert and L. Viikari, Cellulose 4 (1997) 329. 22. Y. Kamaya, J. Ferment. Bioengin. 82 (1996) 549. 23. P. Tomme, E. Kwan, N.R. Gilkes, D.G. Kilbum and R.A.J. Warren, J. Bacteriol. 178 (1996) 4216. 24. K. Kleman-Leyer, N.R. Gilkes, R.C. Miller and T.K. Kirk, Biochem. J. 302 (1994) 463. 25. H. St~lbrand, S.D. Mansfield, J.N. Saddler, D.G. Kilbum, R.A.J. Warren and N.R. Gilkes, Appl. Environ. Microbiol. 64 (1998) 2374.