The Synergistic Effects of Endoglucanase and Xylanase in Modifying

Chapter 7

The Synergistic Effects of Endoglucanase and Xylanase in Modifying Douglas Fir Kraft Pulp 1

Catherine D. Edgar, Shawn D. Mansfield, Georg M. Gübitz, and John N. Saddler

Downloaded by PURDUE UNIV on November 29, 2016 | http://pubs.acs.org Publication Date: March 31, 1998 | doi: 10.1021/bk-1998-0687.ch007

Chair of Forest Products Biotechnology, Department of Wood Science, University of British Columbia, 270-2357 Main Mall, Vancouver, British Columbia V6T 1Z4, Canada Kraft pulpfromDouglas-fir (Pseudotsuga menziesii) was treated with an enzyme preparation containing both cellulase and xylanase activities, as well as individual endoglucanase and xylanase components to assess their potential to enhance both pulp characteristics and paper properties. The use of individual components greatly reduced both yield and strength losses when compared to treatments with the crude cellulase preparation. Xylanase treatments enhancedfiberflexibility,resulting in denser paper sheets with a lower light scattering coefficient. Although treatments with the endoglucanase decreased individual fiber integrity, enhanced fiber collapsibility was obtained. When the purified xylanase and endoglucanase preparations were used in combination, the action of the xylanase increased fiber accessibility to the endoglucanase. The combined action of the two enzymes resulted in enhanced bonding, indicated by as much as 10% increase in burst index over the level obtained by xylanase alone. Enzyme applications in the pulp and paper industry have been steadily increasing over the last twenty years. Although most areas are still evolving, several applications are being aggressively pursued, including the reduction of pitch deposits (7), drainage enhancements (2), deinking of secondary fibers (5), enhancing pulp bleaching (4), and modifyingfibercharacteristics (5, 6). Enhancing bleaching with the use of xylanases has been the most successful endeavor to date, with several mills routinely using xylanase prebleaching (7). The continued use of xylanases in alternative bleaching strategies has inspired considerable research to try to both elucidate the enzymatic bleaching mechanism (8-10) and provide a better understanding of fiber morphology (11, 12). This increased knowledge about the action of xylanase enzymes can also be used to enhance enzymatic fiber modification. 1

Corresponding author.

© 1998 American Chemical Society

Eriksson and Cavaco-Paulo; Enzyme Applications in Fiber Processing ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

75


76 Various theories have been proposed to explain the enzyme mechanism involved in fiber modification, with the predominant suggestion being that the fibrils and fiber bundles are attacked on the surface, peeling off subsequent layers and eventually leading to disintegration of thefibers(75, 14). This enzymatic cleavage can lead to increased freeness (2, 6,15-17) and enhancedfiberflexibility, resulting in denser paper sheets (6). However, enzyme treatments can also result in a decrease in yield and fiber strength. One probable reason for the observed strength loss is that commercial cellulase systems, originally intended for use in cellulose hydrolysis, have been used to try to obtain selective fiber modification. One possible solution to this problem would be to use individual cellulase components to obtain beneficial fiber changes while minimizing yield and strength losses in the pulp. Previous work by other researchers found that pure endoglucanases alone did not giveriseto high levels of hydrolysis (5) and indicated that the action of endoglucanases was necessary to achieve improved pulp freeness (7 7). Related work also indicated that xylanases may play an important role in fiber modification (18) and that xylanase enzymes selectively attack xylan, leaving the cellulose relatively intact, preserving the strength of thefibers(19). In the work reported in this paper, the effects of a cellulase/hemicellulase preparation were compared to the action of pure endoglucanase and xylanase enzymes when added to Douglas-fir kraft pulp. In this way, the individual components were assessed for their ability to enhance thefibercharacteristics of the pulp while limiting both yield and strength losses. The effects of the xylanase and endoglucanase preparations were assessed both separately and in combination to distinguish their individual actions and determine any synergistic effects. Materials and Methods Pulp. Chemical pulp (kraft) derived from Douglas-fir (Pseudotsuga menziesii) was obtained from the Croften mill (Fletcher Challenge), British Columbia, Canada. The sugar composition of the pulp was determined by acid hydrolysis and subsequent HPLC analysis, yielding 72.6% glucose, 5.95% xylose, 0.41% arabinose, 0.56% galactose, and 6.66% mannose. The total lignin content was 5.4%. Enzymes. Novozyme SP342, SP476, and SP613, derivedfromHumicola insolens, were obtainedfromNovo Nordisk, Denmark. Ecozyme was obtainedfromThomas Swan and Co., England. Xylanase I was obtainedfromPrimalco, Finland. The enzyme activities of these preparations were measured on carboxymethylcellulose (2% CMC, Sigma), xylan (1% birchwood xylan, Sigma), andfilterpaper (No. 1 Whatman) using methods described previously (20). The assays were carried out at pH 7.0. Total protein in solution was determined using the bicinchoninic acid protein assay (27). Enzyme treatment of pulp. The pulp slurries (3% consistency in 50 mM phosphate buffer, pH 7.0) were treated for 1 hour at 50 °C under continuous agitation, with a range of enzyme loadings based on activity (CMC or Xylanase IU) per gram oven dry fiber.


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All enzyme reactions were stopped by boiling for 10 minutes. Control pulps were similarly treated, without the addition of enzymes. The reaction filtrates were acid hydrolyzed and analyzed for sugars released by anion-exchange chromatography on a CarboPac PA-1 column using a Dionex DX-500 HPLC system (Dionex, Sunnyvale, California, USA), using fucose as an internal standard. Pulp Testing. Pulpfreenesswas measured at 20 °C according to TAPPIΤ 227 om-94 (CSF tester, R. Mitchell Co.). Handsheets were prepared according to TAPPI Τ 205 om88, with the white water recycled to ensure that the fines were included in the resultant handsheet. Light scattering coefficient was measured at 681 nm (Carl Zeiss, Elrepho). For the determination of burst index, tear index, zero-span breaking length, and density tests were conducted according to TAPPI standard methods. Results and Discussion Although several endoglucanase and xylanase preparations were initially evaluated, subsequent analysis of the individual activities and specificities of the various enzymes (Table I) indicated the high purity of the SP613 (endoglucanase) and Ecozyme (xylanase) preparations. Two other preparations (SP476 and Xylanase I) were also evaluated, as they were used to compare the effects of different enzymes with similar activity profiles. The crude cellulase enzyme (CX) and the endoglucanase (EG) were loaded at 15 IU CMC/g of pulp. At this dosage, the crude cellulase preparation (CX) had 800 IU xylanase activity. Therefore, the xylanase enzyme (X) was loaded at 800 IU xylanase/g of pulp, to ensure that the activities of the individual components were comparable to those of the crude cellulase preparation. Similarly, xylanase I was loaded at 800 IU xylanase/g of pulp. Table I. Protein content and activities of various enzyme preparations Commercial Description Protein Endoglucanase Xylanase Name (mg/mL) (IU/mL) (IU/mL)

Filter Paper (IU/mL)

SP342 SP613 Ecozyme SP476 Xylanase I

Cellulase/ xylanase (CX) Endoglucanase (EG) Xylanase (X) Endoglucanase (EG4) Xylanase (XI)

44.1

30

1610

2

24.2

20

2.1

0

5.7

0

6010

0

22

17

9.8

0

27

6.0

2690

0



78 The crude cellulase preparation greatly reduced burst index, while the endoglucanase and xylanase components caused no significant decrease, as compared to the control (Figure 1 A). It is probable that the synergistic action of the endoglucanases and cellobiohydrolases within the crude cellulase preparation (CX) contributed to the observed strength reduction. Other workers have also shown that the combined action of cellobiohydrolases and endoglucanases was more damaging to pulp fibers than was the action of the endoglucanase alone (77, 22). The use of the individual enzymes, especially the endoglucanase, successfully curtailed pulp hydrolysis as only 0.37% and 1.9% yield loss was observed after respective endoglucanase and xylanase treatments (Table II). Both the crude cellulase preparation and the xylanase released similar amounts of xylose (Table II), solubilizing 27% and 29% respectively of the original xylose present in the pulp. However, when equal loadings of endoglucanase were added in either the form of the crude cellulase preparation or the purified endoglucanase, the crude cellulase preparation released 30 mg glucose/g pulp (4%), while the endoglucanase only released 1.4 mg glucose/g pulp (0.2%). This emphasized the key role that the synergistic action of the cellobiohydrolases plays in cellulose hydrolysis when it acts in combination with the endoglucanase. Table II. Sugars released by enzymatic treatments of Douglas-fir kraft pulp Enzyme Amount ofsugar soluWmd (mg/gpulp) Preparation Glucose Xylose Arabinose Galactose Mannose

Yield loss

The Synergistic Effects of Endoglucanase and Xylanase in Modifying

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