Chapter 4
Enzymatic Characterization of Pulps J. Buchert, A. Suurnäkki, M. Tenkanen, and Liisa Viikari
Downloaded by NORTH CAROLINA STATE UNIV on October 8, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch004
VTT Biotechnology and Food Research, P.O. Box 1501, FIN-02044 VTT, Finland
The specificity of enzymes acting on pulp carbohydrates can be exploited in structural and chemical analysis of pulp fibres. Using the selective enzymatic solubilization, pulp components can be qualitatively and quantitatively analyzed, without attacking other components in pulps. Furthermore, the role of pulp components, such as xylan or glucomannan, in the technical properties of pulp can be evaluated after their enzymatic removal from pulp. This paper reviews the use of enzymes as analytical tools in the characterization of chemical pulps.
A clear breakthrough of biotechnical methods in the pulp and paper industry is a reality. In addition to methods already used in full industrial scale, several new methods are being developed. In all these applications a very limited enzymatic hydrolysis results in improved processability of fibres. In addition to using carbohydrate degrading enzymes as process aids, the specific hydrolysis obtained with these enzymes can also be exploited in the analysis of structures and structure-function relationships in fibres. The conditions used in enzymatic solubilization of pulp carbohydrates are very mild, generally the temperature is about 45-50°C and the pH 5 (7). Thus, no destruction of the components is occuring. By using specific hemicellulases in one-stage treatment, up to 30-40% of the corresponding hemicellulose in pulp can be selectively solubilized without affecting other components in pulp (2). The degree of solubilization can be adjusted by controlling enzyme dosage and hydrolysis time. The use of enzymes as analytical tools in fibre characterization requires high purity from the enzyme préparâtes. Hence, availability of commercial enzymes for fibre analysis is limited. Furthermore, when enzymes are used in structure-function characterization of pulps, pilot-scale protein purification is a necessity due to the high amount of pulp to be treated.
0097-6156/96/0655-0038$15.00/0 © 1996 American Chemical Society
In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
4.
BUCHERT ET AL.
Enzymatic Characterization of Pulps
39
Downloaded by NORTH CAROLINA STATE UNIV on October 8, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch004
In this paper the use of enzymes as analytical tools in the characterization of the carbohydrate composition and structure-function relationships in different types of pulps is reviewed. In Figure 1 the principle of enzymatic characterization of fibres is presented.
Technical properties
Carbohydrate analysis
Figure 1. Use of carbohydrate degrading enzymes in fibre characterization.
Enzymes in Characterization of the Carbohydrates of Chemical Pulps The analysis of pulp components by acid hydrolysis has proven to be difficult in some cases due to potential degradation of acid labile components and incomplete hydrolysis of certain linkages. Purified Trichoderma reesei xylanase has been used for selective solubilization of pulp xylan from different types of pulps, whereafter the chemical composition of the solubilized fraction has been determined either by proton N M R (5, 4) or by HPLC after a secondary enzymatic hydrolysis to monomers ( i ) . With this selective enzymatic solubilization method a novel type of uronic acid side group, i.e. hexenuronic acid, has been identified and quantified in kraft pulp xylan (3, 4), Hexenuronic acid (HexA) is formed by β-elimination from 4-O-methylglucuronic acid during kraft pulping and also the intermediate isomerization product, i.e. methylidoronic acid (MeldoA) has been detected in enzymatically solubilized kraft xylan (Teleman, A . et al. Carbohydr. Res,, in press). Hexenuronic acid has not been detected after traditional acid hydrolysis of pulps due to its degradation to furanderivatives at acidic conditions (5). Hexenuronic acid is the major uronic acid component in both softwood and hardwood kraft xylan, whereas sulphite pulps cooked at acidic conditions contained only MeGlcA. Small amounts of HexA could, however, be detected in alkaline sulphite pulp, although also in this case MeGlcA was the major uronic acid (Table 1).
In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
ENZYMES FOR PULP AND PAPER PROCESSING
40
Table 1. Structure of enzymatically solubilized xylan in different unbleached pulps. The extensive xylanase treatment and subsequent NMR analysis was carried out as described previously (4). Sulphite pulps were cooked as described previously (tf). nd= not detected
Downloaded by NORTH CAROLINA STATE UNIV on October 8, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch004
Pulp Spruce sulphite (Acid Mg) Spruce sulphite (ASAQ) Birch kraft Pine kraft
Sidegroups moll 100 mol xylose MeGlcA HexA nd 10.6 1.1 11.3 4.1 2.2 4.7 1.1
Ara nd 9.2 8.2
For the total characterization of pulp components, and especially the uronic acids in pulp xylan, a cellulolytic and hemicellulolytic enzyme mixture for complete hydrolysis of pulp carbohydrates to monosaccharides has been developed (7). Enzymatic total hydrolysis is a suitable for the hydrolysis of chemical pulps in which the accessibility of the carbohydrates is sufficient for the enzymatic attack. Compared with acid hydrolysis the yield of neutral sugars are generally slightly lower after enzymatic hydrolysis (7). However, in addition to the presence of HexA in the enzymatically hydrolyzed sample, also the amount of MeGlcA detected was higher than obtained in acid hydrolyzed sample. Thus, enzyme-aided analysis is especially suitable for the quantification of uronic acids in chemical pulps.
Table 2. Comparison of acid hydrolysis and enzymatic hydrolysis combined to HPLC for the analysis of uronic acids in unbleached birch kraft pulp. n.d.= not detected HexA, % ofd.w.
MeGlcA, % ofd.w.
Total, % ofd.w.
Acid
n.d.
0.30
0.30
Enzymatic
1.41
0.88
2.29
Method of hydrolysis
The relative amounts of different types of carboxyl groups in kraft pulps as well as the distribution of acidic groups in different xylans has been determined by measuring the carboxyl groups in the residual pulps by high precision potentiometric titration (8). Approximately 80% and 90% of the carboxyl groups in unbleached pine and birch brownstock kraft pulps were uronic acids (#). Enzymes in Analysis of the Organization of Pulp Components in Fibres The organization of xylan and lignin on the outermost surface of kraft fibres has been studied by combining enzymatic peeling and ESCA analysis (Electron Spectroscopy for Chemical Analysis) (9). After extensive xylanase treatment (about 30 % of the pulp xylan removed) more lignin could be detected on the surface of pine kraft fibres. Thus,
In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
Downloaded by NORTH CAROLINA STATE UNIV on October 8, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch004
4.
BUCHERTETAL.
Enzymatic Characterization of Pulps
41
within the sampling depth of ESCA (about 10 nm), xylan was apparently partly covering lignin in pine kraft fibres. The selective removal of glucomannan did not uncover lignin in pine kraft fibres. Unlike in pine kraft pulp, the removal of as much as about 40 % of the xylan in birch kraft pulp did not expose lignin on the outermost surface of fibres. This was probably due to the relatively high content of xylan on the surface of birch kraft fibres (10). By measuring the effect of extensive xylan or glucomannan removal on the pore size distribution of unbleached kraft pulps, further information on the location of hemicelluloses has been obtained (Suurnàkki, A . et al. Holzforschung, in press). Even with low hydrolysis levels the pore size distributions were increased indicating the presence of hemicelluloses on the surface of pores. This result is consistent with the previous observation of increased leachability of lignin after xylanase or mannanase treatment (77).
Enzymatic Characterization of the Structure-Function Relationships of Pulps The papermaking properties of kraft pulps are generally considered to be dependent on the composition and properties of individual fibres (72). Also for the studies of the structure-function dependancy selective enzymes solubilizing pulp carbohydrates are indispensable tools. The role of hemicelluloses in the properties of kraft pulps has been investigated by removing different amounts of xylan from pulps by T. reesei xylanase. The effect of xylan on the beatability of kraft pulps was found to depend on the pulp origin. In brownstock birch kraft pulp the enzymatic removal of xylan decreased the beatability of fibres whereas after ECF-bleaching the effect was less pronounced indicating that the chemical structure, i.e the uronic acid profile, might have a role in the beatability (Buchert, J. et al., manuscript in preparation). The brightness stability of birch and pine kraft pulps has also been found to be affected by the uronic acid profile. During extensive removal of xylan by T. reesei xylanase, the uronic acid content was significantly decreased due to solubilization of uronic acid substituted xylan and this resulted in improved brightness stability of different kraft pulps. Enzymatic glucomannan removal had no effect on ageing (Buchert, J. et al. Tappi accepted for publication). Basic phenomena occuring during recycling of chemical pulps, i.e. hornification, has been investigated using selective removal of xylan and glucomannan combined to drying and analysis of the pulp properties. By removing the accessible hemicelluloses from the pulp the hornification was increased, which was observed in decreased swelling, strength and flexibility of the pulp (Oksanen et al., submitted).
Conclusions A deep understanding of the role of different pulp components on the final pulp properties requires the combination of novel and traditional techniques. Enzymes have proved to be powerful tools in fibre analysis. Combined to traditional and modern analysis methods enzymatic treatments provide valuable information of the structure and composition of fibre surfaces as well as the overall pulp matrix (Figure 2). The non-destructive conditions used in enzymatic treatments render the enzyme-aided analysis especially suitable for the analysis of labile components in pulp fibres. The
In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
42
ENZYMES FOR PULP AND PAPER PROCESSING
specificity of enzymes can also be exploited in the characterisation of the relationships between pulp components and papermaking properties in an unique manner. Recent reports on the use of purified enzymes in the characterization of kraft pulps are summarized in Table 3.
Downloaded by NORTH CAROLINA STATE UNIV on October 8, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch004
^^M^^
Chemical structure
Location
Role of pulp components on sheet properties
Figure 2. Information obtained from pulp and fibres by enzyme-aided analysis
Table 3. Recent reports on enzymatic characterization of pulps Combined to
Information obtained
Reference
'HNMR
Structure of solubilized pulp xylan
3,4
High precision potentiometric titration, polyelectrolyte adsoption
Distribution of carboxylic groups in pulp xylan
8
*H N M R , solute exclusion
Effect of xylan and glucomannan on pore size distribution
Suurnakki et al., Holzforschung, in press
Thermal ageing
Role of xylan and glucomannan in brightness stability
Buchert et ai Tappi J., accepted
Drying, fibre characterization
Role of xylan and glucomannan in hornification
Oksanenef aJ., submitted
PFI-refining, sheet properties
Role of hemicelluloses on technical properties of pulps
Buchert et al., in preparation
ESCA
Location of lignin and hemicelluloses in pulp surface
9
HPLC
Total carbohydrate composition of pulps
7
In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
4. BUCHERT ET AL.
Enzymatic Characterization of Pulps
43
Downloaded by NORTH CAROLINA STATE UNIV on October 8, 2012 | http://pubs.acs.org Publication Date: November 21, 1996 | doi: 10.1021/bk-1996-0655.ch004
Literature Cited 1. Buchert, J.; Siika-aho, M.; Bailey, M.; Puls, J.; Valkeajärvi, Α.; Pere, J.; Viikari, L. Biotechnol. Tech. 1993, 7, pp 785-790. 2. Suurnäkki, A. Hemicellulases in the bleaching and characterization of kraft pulps. VTT Publications 267; Technical Research Centre of Finland: Espoo, 1996. 3. Teleman, Α.; Harjunpää, V.; Tenkanen, M.; Buchert, J.; Hausalo, T.; Drakenberg, T.; Vuorinen, T. Carbohydr. Res. 1995, 272, pp 55-71. 4. Buchert, J.; Teleman, Α.; Harjunpää, V.; Tenkanen, M.; Viikari, L.; Vuorinen, L. Tappi J. 1995, 78, pp 125-130. 5. Teleman, Α.; Hausalo, T.; Tenkanen, M.; Vuorinen, T. Carbohydr. Res. 1996, 280, pp 197-208. 6. Buchert, J.; Kantelinen, Α.; Suurnäkki, Α.; Viikari, L.; Janson, J. Holzforschung 1995, 49, pp 439-444. 7. Tenkanen, M.; Hausalo, T.; Siika-aho, M.; Buchert, J.; Viikari, L. Proc. 8th Int. Symp. Wood and Pulping Chemistry, Helsinki, 6-9 June 1995. Vol.III,pp 189-194. 8. Laine, J.; Buchert, J.; Viikari, L.; Stenius, P. Holzforschung, 1996, 50, pp 208-214. 9. Buchert, J.; Carlsson, G.; Viikari, L.; Ström, G. Holzforschung, 1996, 50, pp 69-74. 10. Suurnäkki, Α.; Heijnesson, Α.; Buchert, J.; Viikari, L.; Westermark, U.J.Pulp Paper Sci. 1996, 22, pp J43-J47. 11. Hortling, B.; Korhonen, M.; Buchert, J.; Sundquist, J.; Viikari, L. Holzforschung, 1994, 48, pp 441-446. 12. Clark, J. D. A. Pulp Technology and Treatment for Paper, Miller Freeman Publications Inc.: San Fransisco, CA, 1978, 751 p.
In Enzymes for Pulp and Paper Processing; Jeffries, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.
