SYNERGISTIC INTERACTIONS OF PURIFIED XYLANASES ON BAGASSE PULP HEMICELLULOSE

Authors

Sadhvir Bissoon1,2, Suren Singh1 and Lew Christov2,3

Companies and addresses

1Department of Biological Sciences, Durban Institute of Technology, PO Box 1334, Durban 4000, South Africa;
2Sappi Biotechnology Laboratory, Department of Microbiology and Food Sciences, University of the Free State, PO Box 339, Bloemfontein 9300, South Africa
3Sappi Management Services, PO Box 3252, 1560 Springs, South Africa

emails

singhs@dit.ac.za and ChristoL@sci.uovs.ac.za

Keywords

xylanase, synergistic interaction, fibre bound hemicellulose, biobleaching, brightness, chlorine reduction

ABSTRACT

Three purified xylanases from Trichoderma viride, Aureobasidium pullulans and Thermomyces lanuginosus were evaluated individually as well as in combination on extracted hemicelluloses from bagasse pulp, commercial birchwood xylan, untreated bagasse pulp and alkaline-extracted bagasse pulp. Xylanase of T. lanuginosus appeared to be the least efficient of the xylanases under study, with reducing sugars at a concentration of 0.19; 0.25; and 0.31 and 0.37 mg/g detected after 1, 2, 3, and 24 h, respectively.  Application of the xylanases in combination produced the highest concentration of reducing sugars in comparison to individual xylanase treatments.  At enzymes charges of 5 U/g pulp, reductions of 10 % Cl2 was attained with T. lanuginosus xylanase and 20% with A. pullulans and T. viride xylanase in X-CEH bleaching.  The xylanases when used in combination produced a synergistic effect in bleaching of bagasse pulp thereby inducing a brightness gain of 2.2 points at full Cl2 charge. This exceeded the sum of the individual effects by more than 20%. Alternatively, the Cl2 consumption could be reduced by 30 % while still maintaining a final brightness higher than the control.

1. INTRODUCTION

Bagasse pulp contains approximately 30 to 35% of hemicelluloses, compared to hardwood and softwood pulps which contain approximately 15 to 30% and 7 to 12% hemicelluloses, respectively1.  The enzymatic degradation of xylan requires the use of endo-xylanases, enzymes, which randomly split and solubilize the xylan polymer.  As a result, the penetration of bleaching chemicals into pulp fibres could be improved and therefore lignin removal facilitated.

Hardwood kraft pulp contains more xylan, and smaller xylan macromolecules than softwood kraft pulps2.  There is a tendency for hardwood kraft pulp to show higher benefits with regard to chlorine savings during xylanase aided bleaching3.  The bleach enhancing affects of xylanases on pulp types has been attributed to the hydrolysis of hemicelluloses from pulp.  Restrictions in the pentosan removal from pulps have been assigned to retarded accessibility and chemical modification of residual hemicelluose4.  The extent of xylan hydrolysis in pulps is dependent on the substrate specificity of the particular enzyme used as well as chemical composition and physical state of the xylan substrate5

It has been demonstrated that xylanases can produce bleach-boosting effects on bagasse soda pulp6,7,8.  However, limited literature is available with regards to the correlation between xylan removal and brightness gains from bagasse soda pulp. This study investigates the effects of three purified family 11 xylanases from Trichoderma viride, Aureobasidium pullulans and Thermomyces lanuginosus SSBP on extracted and fibre bound xylan from bagasse pulp and commercially available birchwood xylan in relation to their biobleaching effects.

2. MATERIALS AND METHODS

2.1 Purified xylanases
Purified xylanases from T. viride and A. pullulans (Sigma) and Thermomyces lanuginosus SSBP6 belonging to the Family 11 xylanases were evaluated in this study.  The xylanases were evaluated individually and in mixture on the substrates mentioned below.

2.2 Xylanase assays
Xylanase was routinely assayed by measuring the reducing sugar (RS) released from birchwood xylan as described by Bailey et al9

2.3 Extraction of hemicelluloses from bagasse soda pulp
For chemical removal of hemicelluloses, pulp (100 g) was extracted with 4 000 ml of 20 % degassed KOH under stirring over night10.  The combined alkaline filtrate and washings were then acidified with acetic acid to pH 4.5. Hemicelluloses were precipitated with ethanol and water added to the precipitate, which was finally freeze dried. 

2.4 Enzymatic treatment of isolated hemicelluloses from bagasse pulp and commercial xylan
Commercial birchwood xylan (Roth, Karlsruhe) and the extracted hemicellulose from bagasse pulp were treated with the three purified xylanases individually and in combination.  A 1% substrate solution was used and an enzyme charge of 5 U/g xylan applied for a period of 24h, with samples removed and analysed for RS at 0, 1, 2, 3, and 24 h of incubation.

2.5 Enzymatic treatment of untreated bagasse soda pulp and alkali-extracted pulp
Duplicate samples of extensively washed pulp (5 g dry weight) of 10% consistency were treated with purified xylanases (5 U/g pulp and 5 U/g xylan in pulp) at the respective optimum conditions. After incubation with enzymes, the samples were heated in a boiling water bath for 10 min to denature the enzymes and then filtered through membrane filters (0.45 m). The filtrates were then analysed for RS.

2.6 Reducing sugars
Reducing sugars released after enzyme treatment were quantified according to the method as described by Miller11.

2.7 Bleaching conditions
The chemical bleaching conditions on bagasse soda pulp are described in Table 2.1.

Table 2.1 Conditions for CEH bleaching on bagasse soda pulp

Treatment

Charge (%)

Time (min)

Temperature (oC)

Consistency (%)

C

4.75

45

45

3.2

E

4.5

90

70

12

H

1.5

135

70

12

3. RESULTS

3.1 Effect of purified xylanases on RS release from extracted hemicellulose of bagasse pulp and commercial xylan
There was a proportionate increase in the amount of RS released from the extracted hemicellulose with time (Figure 3.1). A. pullulans xylanase produced the highest RS concentration in comparison to the individual xylanase applications at all incubation periods.  After 3h the RS released by A pullulans and T. viride were virtually the same (0.4 mg/g) compared to a 25 % reduction detected with T. lanuginosus xylanase. The RS released by the three xylanases in mixture was 23 % greater than that produced by A. pullulans after 24h.

Figure 3.1

Figure 3.1 Enzyme-mediated release of RS from extracted hemicellulose of bagasse soda pulp

Xylanase treatment of the commercial birchwood xylan released a higher concentration of RS in comparison to treatment of the extracted hemicelluloses from bagasse pulp. There was a corresponding increase in the release of RS with prolonged incubation (Figure 3.2). After the 24h incubation period, A. pullulans xylanase produced a 25.4 % increase in RS released from birchwood xylan in comparison to the alkaline extracted hemicellulose from bagasse pulp. 

Figure 3.2

Figure 3.2 Enzyme-mediated release of RS from commercial birchwood xylan

3.2 Effect of purified xylanases on RS release from alkali-extracted and untreated bagasse pulp
Bagasse soda pulp treated with the individual xylanases produced less RS in comparison to treatment with the combination of the three enzymes (Table 3.1).  The RS released at the charge of 5 U/g pulp was greater than enzyme charges of 5 U/g xylan in pulp. The RS released from pulp was lower than that detected on the extracted hemicelluloses and commercial xylan. Xylanase treatment of the extracted pulp produced RS at negligible concentrations.

Table 3.1 Effect of purified xylanases on RS release from untreated bagasse pulp

Source of xylanase

RS release (mg/g) from untreated bagasse pulp

5 U/g pulp

5 U/g xylan in pulp

T. lanuginosus

0.22

0.19

T. viride

0.27

0.23

A. pullulans

0.32

0.27

Enzyme combination

0.35

0.31

Time 2h, 10 % consistency

3.3 Evaluation of purified xylanases on bagasse pulp in X-CEH bleaching
At enzyme charges of 5 U/g pulp, reductions of 10 % Cl2 was attained with T. lanuginosus xylanase and 20% with A. pullulans and T. viride xylanase (Figure 3.3).  Any further reductions in Cl2 consumption produced brightness levels below the control level.  At full Cl2 charges A. pullulans xylanase produced the highest brightness gain of 0.9 points in comparison to 0.5 and 0.3 achieved with T. viride and T. lanuginosus xylanase, respectively.

Figure 3.3

Figure 3.3 Influence of purified xylanases (5 U/g pulp) on brightness of bagasse soda pulp in sequence X-CEH at reduced Cl2 charges

The enzyme combination produced a brightness gain of 2.2 points to 87. 5 % at full Cl2 charge (Figure 3.4).  On the other hand the Cl2 consumption was reduced by 30 % while maintaining a final brightness 0.9 points higher than the control. The enzyme combination displayed a greater efficiency in improving the brightness of bagasse pulp, with subsequent higher reductions of Cl2 in CEH bleaching.

Figure 3.4

Figure 3.4 Influence of purified xylanase combination from A. pullulans, T. viride and T. lanuginosus (5 U each xylanase/g pulp) on brightness of bagasse soda pulp in sequence X-CEH at reduced Cl2 charges

4. DISCUSSION

The xylanases when treated in combination, produced the highest levels of reducing sugars detected in the filtrates, indicating effective combined treatment with pronounced influence in comparison to the individual enzyme treatments.  This demonstrates the cooperative effects of the xylanases in hydrolyzing the extracted hemicelluloses from bagasse pulp. The commercial xylan was more susceptible to the purified xylanases in terms of RS release compared to the alkali-extracted hemicelluloses from bagasse pulp. This could be due to the nonselective hemicellulose extraction method performed, implying that other extractives and lignins, and the possible presence of residual alkali (KOH) on the extracted hemicellulose, could be a factor that hindered the enzyme hydrolysis efficiency. 

The lower hydrolysis potential of the xylanases on hemicellulose in bagasse pulp as compared to the extracted hemicelluloses and commercial xylan could be attributed to the chemical modifications of the hemicelluloses during pulping, resulting in limited recognition of the polysaccharide by the enzyme as reported by Allison et al12.  All three enzymes displayed varying abilities of hydrolysis on isolated hemicelluloses and bagasse pulp. It is therefore proposed that the enzymes could have varying degrees of specificity for the hemicelluloses.  For instance, A. pullulans xylanase could be more specific to the target xylan than the two other xylanases. The xylanase combination displayed a synergistic effect in bleaching of bagasse pulp, achieving a greater brightness gain of 2.2 points than the sum of the individual effects of the enzymes (1.8 brightness points).  The study demonstrated clearly that the xylanase mediated release of RS as well as compounds contributing to the low brightness of the pulp correlated positively with the bleach enhancing effect with concomitant savings in Cl2 consumption in CEH bleaching sequence.

5. CONCLUSIONS

Birchwood xylan was more susceptible to enzymatic hydrolysis than extracted hemicelluloses from bagasse pulp.  A. pullulans xylanase was the most efficient enzyme when applied to xylan and bagasse pulp, followed by T. viride and T. lanuginosus xylanase in decreasing order of efficiency. The xylanases displayed a greater bleach boosting effect on the untreated bagasse pulp than on the extracted pulp.  A synergistic effect was evident during biobleaching of bagasse pulp, however it was absent during RS release.

6. ACKNOWLEDGEMENTS

We wish to acknowledge the National Research Foundation (NRF), and Sappi Management services for financial support, Sappi Management Services for granting permission to publish this work, Sappi Biotechnology Laboratory for excellent technical assistance.

7. LITERATURE CITED

1. Beg, Q., Kapoor, K., Mahajan, M.L. and Hoondal, G.S., "Microbial xylanases and their industrial applications", Applied Microbiology and Biotechnology, 56:326-338 (2001).

2. Wong, K.K.Y., Nelson, S.L. and Saddler, J.N., "Xylanase treatment for the peroxide bleaching of oxygen delignified kraft pulps derived from three softwood species", Journal of Biotechnology, 48:137 (1996).

3. Karlsson, O. and Westermark, U., "Evidence for chemical bonds between lignin and cellulose in kraft pulps", Journal of Pulp and Paper Science, 22:397 (1996).

4. Paice, M.G. and Juracek, L, "Removing hemicellulose from pulps by specific enzymatic hydrolysis", Journal of Wood Chemistry and Technology, 4:187-198 (1984).

5. Christov, L.P. and Prior, B.A., "Bleaching response of sulfite pulps to pretreatment with xylanases", Biotechnology Progress, 13:695-698 (1997).

6. Bissoon, S., Christov, L. and Singh. S., "Bleachboosting effects of purified xylanase from Thermomyces lanuginosus SSBP on bagasse pulp", Process Biochemistry, 37:567-572 (2002).

7. Bissoon, S., Singh, S. and Christov, L., "Evaluation of the bleach enhancing effect on xylanases on bagasse pulp", In: Biotechnology in the Pulp and Paper Industry, (eds.) L. Viikari and Lantto, R, 247-254 (2002).

8. Madlala, A.M., Bissoon, S., Singh, S. and Christov, L., "Xylanase-induced reduction of chlorine dioxide consumption during elemental chlorine-free bleaching of different pulp types", Biotechnology Letters, 23:345-351 (2001).

9. Bailey, M.J., Biely, P. and Poutanen, K., "Interlaboratory testing of methods for assay of xylanase activity", Journal of Biotechnology, 23:257–270 (1992).

10. Karlsson, O., Pettersson, B. and Westermark, U., "The use of cellulases and hemicellulases to study lignin -celluloses as well as lignin-hemicellulose bonds in kraft pulps", Journal of Pulp and Paper Science, 27:196-201 (2001).

11. Miller, G.L., "Use of dinitrosalicylic acid reagent for determination of reducing sugar", Analytical Chemistry, 31:238-244 (1959).

12. Allison, R.W, Clark, T.A and Suurnäkki, A., "Effect on modified kraft pulping on enzyme assisted bleaching", Appita Journal, 49:18 (1996).

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