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Application of xylanases in the pulp and paper industry
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Bioresource Technology 50 (1994) 65-72 © 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/94[$7.00
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APPLICATION OF X Y L A N A S E S IN THE PULP A N D PAPER INDUSTRY Johanna Buchert, Maija Tenkanen, Anne Kantelinen & Liisa Viikari* VTT BiotechnicaI Laboratory, PO Box 1501, FIN-02044 VTT, Finland
application areas of hemicellulases is in the pulp and paper industry. Furthermore, enzymatic methods are especially suited to reduce environmental impacts of the pulp and paper industry. Organic chlorine compounds formed during the bleaching of chemical pulp have probably attracted most attention in recent years. These compounds arise mainly from the reactions between residual lignin present in wood fibres, causing the brown colour of unbleached pulp, and the chlorine used for bleaching. For the same amount of active chlorine used, chlorine dioxide causes the formation of adsorbable organic halogen (AOX) in an amount of only one fifth of that caused by chlorine gas (Germgfird & Larsson, 1983). Earlier measures taken by the forest industry to solve the chlorine problem have focused on improving effluent treatment methods. Today, the emphasis of research and development work in this area has shifted more towards improving the processes. In the search to produce pulp with non-polluting chemicals, more efficient pulping methods, which reduce the amount of residual lignin passing through to the bleaching process, and alternative bleaching methods, are being developed (Kovasin & Tikka, 1992). Among the new bleaching technologies, enzymes have already shown their potential (Koponen, 1991; Lavielle et aL, 1992). Enzymes can be used for specific modifications of pulp for the development of environmentally safe processes. The original concept of using hemicellulases to improve bleachability of kraft pulps was first published in 1986 (Viikari et aL, 1986; 1987). The brightness of the pulp was substantially increased by the enzymatic treatment, and this improved brightness could be exploited for reduction of chlorine chemical consumption. It was observed that the saving could be gained either in the prebleaching stage, as chlorine gas, or in the final bleaching stage, as chlorine dioxide (Viikari et al., 1986; 1987). For environmental reasons, the possibility of minimizing the consumption of chlorine gas in bleaching was of particular interest. Since the end of the 1980s the pulp and paper industry has been forced to consider any new technique available for the reduction of chlorine consumption, or for the increase of brightness of totally chlorine-free pulps. The enzymatic treatment has been successfully combined with more efficient pulping and non-chlorine bleaching methods.
Abstract
Interest in hemicellulolytic enzymes has increased remarkably during recent years. This is mainly due to the new areas of application of these enzymes within the pulp and paper industry. Among these, the most promising seems to be utilization of hemicellulases, especially xylanases, to increase the bleachability of kraft pulps. This is partly due to the great potential of an environmentally safe method. The main enzymes needed in the enzyme-aided bleaching have been shown to belong to the group of endo-fl-xylanases. Xylanases act mainly on the relocated, reprecipitated xylan on the surface of the pulp fibres. Enzymatic hydrolysis of this specific type of xylan renders the structure of the fibres more permeable, allowing enhanced extraction of residual lignin from the fibres. The hydrolysis of hemicelluloses in the inner fibre layers may also enhance the bleachability. The main goals in the enzyme-aided bleaching of kraft pulps have been the reduction of consumption of chlorine chemicals in the bleaching process, and consequent lowering of the A OX of the effluents. In the production of totally clorine-free pulps, enzymes have also been successfully used for increasing the brightness of pulp. Other suggested enzymatic modifications of fibres are aimed at improved drainage (water removal) in the paper machine, improvement of fibre properties or production of dissolving pulps. The xylanolytic enzymes and their application areas are reviewed. Key words: Hemicellulases, xylanases, pulp and paper, bleaching.
INTRODUCTON
Hemicelluloses are major constituents of plant cell wall polysaccharides. The structure of various types of hemicelluloses depends on the type of plant, and may even vary between different parts of the same plant. Hemicelluloses are usually classified according to the main sugar residues in the backbone, e.g. xylans, glucomannans, galactans and glucans. Both wood and pulp contain high amounts of structurally different hemicelluloses. Thus, one of the most important potential *To whom correspondence should be addressed. 65
66
J. Buchert, M. Tenkanen, A. Kantelinen, L. Viikari
SUBSTRATES
Xylan and glucomannan form the basic backbone polymers of wood hemicelluloses. In hardwoods, the main hemicellulose is O-acetyl-O-methylglucuronoxylan, whereas in softwoods, arabino-4-O-methylglucuronoxylan comprises about one third of the total hemicelluloses. Hardwood xylan contains 4-O-methylglucuronic acid and acetyl side-groups. Methylglucuronic acid is linked to the backbone by a( 1 --, 2) glycosidic bonds. Acetic acid is esterified at the hydroxyl group of carbon 2 and/or 3 (Lindberg et al., 1973). Softwood xylan contains 4-O-methylglucuronic acid and L-arabinofuranoside sidegroups linked to the backbone by a(1--, 2)- and a(1--, 3)-glycosidic bonds, respectively. The average molar ratio of xylose:4-Omethylglucuronic acid: acetic acid in hardwood xylan is 10:1: 7, and that of xylose :4-O-methyl-glucuronic acid:arabinose sugar units in softwood xylans is 8:1.6:1 (Sjrstrrm, 1981). Extensive modification of hemicelluloses takes place during pulp production processes. During the heating period of the conventional kraft cooking, when the alkali concentration is comparatively high, part of the xylan is dissolved in the pulping liquor. As the cooking proceeds the alkali concentration decreases, and degraded, short-chain xylan is precipitated in a more or less crystalline form on the surface of cellulose microfibrils (Yllner & Enstrrm, 1956; Yllner et al., 1957). The configuration of the xylose units allows close contact of the xylan chains with the cellulose and it is likely that part of the xylan, after removal of its substituents, tends to cocrystallize with, or become adsorbed on, the cellulose of the pulp. A considerable part of the xylan is readsorbed or reprecipitated onto the cellulose, although a part remains undissolved at its original location in the fibre (Croon & Enstrrm, 1962; Hartler & Lund, 1962). Approximately half of the xylan present in normal pine kraft pulps is estimated to be relocated xylan (Yllner et al., 1957; Meller, 1965). In birch kraft pulps the amount of reprecipitated xylan has been estimated to be about 1-3% of the pulp (Clayton & Stone, 1967), or about 4-12% of the total xylan in pulp (Axelsson et al., 1962). The reprecipitation of xylan is preceeded by the reprecipitation of dissolved lignin during kraft pulping. These redeposited polymers have also been suggested to be chemically linked to each other (Jansson & Palenius, 1972; Jansson et al., 1975; Iversen & W~innstrrm, 1986). Furthermore, hemicelluloses seem to physically restrict the passage of high molecular mass lignin out of the pulp fibre (Ahlgren et al., 1971; Scallan, 1977). XYLANASES Endo-l,4-fl-D-xylanases (EC 3.2.1.8) catalyze the random hydrolysis of 1,4-fl-D-xylosidic linkages in xylans (IUB, 1982). Xylanases are produced by several bacteria, yeast and fungi, although the latter have been extensively studied (see Wong et al., 1988; Eriksson
et al., 1990; Viikari et al., 1993; Coughlan & Hazlewood, 1993). The published information on the induction (constitutive or induced) as well as regulation (under separate or common regulatory control system with cellulases) of xylanases varies, and induction as well as regulation seems to differ between organisms. Some reports demonstrate that xylanases are inducible and the specific induction can occur independently of cellulase synthesis (Coughlan & Hazlewood, 1993). Fungal xylanases of Aspergillus and Trichoderma spp., and bacterial xylanases of Bacillus spp., Streptomyces spp. and Clostridium spp. have been intensively studied (Wong et al., 1988; Eriksson et al., 1990). Several of the xylanases purified are rather small (molecular mass around 20 kDa), monomeric proteins with basic isoelectric point (pI 8-9-5). They also exhibit a great homology on the molecular level and belong to the family G (or 11 ) of glycosyl hydrolases. The other xylanases with higher molecular mass and lower pI value belong to the other identified endoxylanase family F (or 10) (Henrissat, 1991; Henrissat & Bairoch, 1993). The optimum pH for xylan hydrolysis is around five for most fungal xylanases, and they are normally stable between pH values of two and nine. The pH optima of bacterial xylanases are generally slightly higher than the pH optima of fungal xylanases. Alkalophilic Bacillus spp. (Nakamura et al., 1993) and alkalophilic actinomycetes (Tsujibo et al., 1990) have been reported to produce xylanases with high activity at alkaline pH values. Most of the fungi and bacteria produce xylanases which tolerate temperatures below 40-50°C. Xylanases have also been characterized from thermophilic organisms. The most thermostable xylanase described is that of an extremely thermophilic Thermotoga spp. with a half life of 20 min at 105°C (Bragger et al., 1989). Xylanases with half lives from a few minutes up to 90 min at 80°C have been produced by Thermoascus aurantiacus, Bacillus stearothermophilus, Caldocellum saccharolyticum, Clostridium stercorarium and Thermomonospora spp. (Zamost et al., 1991; Ltithi et aL, 1990). Endoxylanases show the highest activity against polymeric xylan, and the rate of the hydrolysis reaction normally decreases with the decreasing chain length of the oligomeric substrates. They do not hydrolyze xylobiose, and the hydrolysis of xylotriose is in most cases negligible or at least limited. The main products formed from the hydrolysis of xylan are xylobiose, xylotriose, and substituted oligomers of two to four xylosyl residues. The length and the type of the substituted products depends on the mode of action of the individual xylanases. Most of the enzymes studied cleave the xylan backbone, leaving the substituent on the nonreducing end of the xylosyl chain of the oligosaccharide. Some xylanases were reported to leave the substituent on the reducing end and in the middle of the oligosaccharide chain (Dekker, 1985; Coughlan, 1992). Of the end products, at least xylotriose has been reported to inhibit the action of xylanases (Royer &
Application of xylanases in the pulp and paper industry
Nakas, 1991). In addition to hydrolytic activity, transferase activity has been detected in several xylanases (Coughlan, 1992; Viikari et al., 1993). Of the xylanases produced by Trichoderma spp., two main groups of enzymes can be identified. Both types have low MW but the pI is different. Xylanases with high pI have been quite extensively studied, whereas the other type of xylanase, with pI near to pH 5, has been purified and characterized only from T. lignorum and T. reesei. More than one basic xylanase has been isolated from T. harzianum, T. koningii and T. longibrachiatum (Wong & Saddler, 1992). However, only one of these enzymes made a major contribution to the total xylanase activity in the culture filtrate. The pI 9"0 and pI 5"5 xylanases of T. reesei have been shown to be different gene products, and were both classified as belonging to the family G (Tenkanen et al., 1992a; Ttrrtnen et al., 1992). Multiple xylanases have also been purified from culture filtrates of A. niger (Wong et al., 1988), A. oryzae (Bailey et al., 1991), A. kawachii (Ito et al., 1992), A. awamori (Kormelink et al., 1993) and other fungi, as well as bacteria (Wong et al., 1988; Viikari et al., 1993). By production of multiple xylanases the microorganisms may maximize the utilization of xylans with different structures.
APPLICATION OF XYLANASES IN BLEACHING
Comparison of different xylanases Several xylanases have been tested for their properties in bleaching experiments. The action of enzymes on pulp bleachability has been studied by different methods. In the first phase, enzymes are usually characterized by isolated substrates, which are used in the determination of their activities. These substrates, however, vary extensively with respect to their origin and composition. Furthermore, comparison of enzyme activities is complicated by the utilization of different analysis methods (Bailey et al., 1992). The action of enzymes can also be compared on the actual substrate, the pulp. In these tests, the solubilization of pulp carbohydrates has usually been the key parameter (Senior et al., 1988; Kantelinen et al., 1993b). In addition, an increase in the liberation of lignin-derived compounds after the enzymatic treatment (Yang & Eriksson, 1992; Pedersen et al., 1992) or after alkaline extraction (Kantelinen et al., 1993a; Hortling et al., 1994) has been used for evaluation of different enzymes. However, the most reliable method to compare different xylanases is the bleaching of enzyme treated pulps. In practice, the most convenient measure is the brightness increase in peroxide delignification. Most of the reports published on application of xylanases are still based on unpurified enzymes. The culture filtrates used, however, have contained xylanases as the main activity and the enzyme preparations are generally dosed according to xylanase activity. Even with unpurified enzymes, identification of the
67
sugars released in the enzymatic treatments have confirmed that xylanase was the cause of the major activity (Viikari et al., 1987; 1990). The main enzymes needed to enhance the delignification of kraft pulp were later shown to be endo-fl-xylanases (Viikari et al., 1991; Paice et al., 1988; Kantelinen et al., 1988; Jurasek & Paice, 1988). Only small differences in the bleaching efficiency have been observed between xylanases from different organisms, although the degree of hydrolysis has varied. The role of xylanase activity in delignification of kraft pine pulp has been studied with purified xylanases of T. reesei (Tenkanen et al., 1992b; Kantelinen, 1992; Buchert et al., 1992). The purified xylanases of T. reesei were shown to have different pI values, pH optima and substrate specificities. Although the xylanase with pI 9 has been shown to be relatively more specific for non-substituted xylans, both purified xylanases were equally efficient in limited hydrolysis of pulp xylans, as well as in subsequent chemical delignification (Buchert et al., 1992). Interestingly, the pHoptimum of the pI 9 xylanase was shifted in pulp treatments to pH 6-7 from the optimum pH of 5.0, determined on isolated xylan (Buchert et al., 1992). Optimization of the amount of enzyme and the hydrolysis time revealed that reduction in the Kappa number of pine kraft pulp (measured after peroxide delignification) could be achieved using low amounts of enzymes and a relatively short incubation time (Table 1). Minimization of the overall hydrolysis of hemicelluloses is necessary in order to maintain a high pulp yield, and the advantageous properties of hemicelluloses in the pulp (Viikari et al., 1986; 1987). It has been suggested that the enzymatic hydrolysis of pulp xylans can be directed by chemical means, i.e. by adjustment of ionic strength, pH and counter ion profile in the pulp (Buchert et al., 1993).
Mechanism of enzyme-aided bleaching Two types of phenomena are probably involved in the enzymatic pretreatment of kraft pulp resulting in improved delignification. Hydrolysis of the reprecipitated xylan renders the pulp more permeable, thus facilitating the removal of residual lignin (Kantelinen et a l., 1991; 1993 a). After the xylanase pretreatment, the average molecular weight of alkali-extracted lignins is increased. The hydrolysis of xylan located in the inner layers and possibly linked to lignin may also affect the delignification (Paice et al., 1992). Due to the inaccessibility of pulp substrates, xylanases are expected to act mainly on the relocated, reprecipitated xylan on the surface of the pulp fibres (Kantelinen et al., 1991; Kantelinen et al., 1993a). The reprecipitation of xylan occurs when almost all of the xylan side-groups are cleaved off, resulting in a very resistant chemical structure. In agreement with this, the accessory enzymes splitting off the xylan side-groups have been found to have only limited effects in hydrolysis and bleaching tests (Kantelinen et al., 1988; Kantelinen, 1992). The hypothesis is supported by the observation that the extraction of xylan by DMSO,
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J. Buchert, M. Tenkanen, A. Kantelinen, L. Viikari
Table 1. Effect of xylanase (from Trichoderma reesei, pI 9, Tenkanen et al., 1992a) dosage on the bleachability of birch kraft pulp (original Kappa number 19.9) with peroxide as bleaching chemical. Bleaching sequence used EnzQP, where Q denotes chelator and P peroxide. Bleaching conditions as described previously by Suurnikki et al. (1994)
Xylanase dosage (nkat/g)
Reducing sugars (% of d.w.)
Brighmess (ISO)
Kappa number
Viscosity (dma/kg)
0 100 300 600 1000
0 1.04 1.98 2.86 3-30
55.4 58.0 58.0 57.5 57.5
12.6 12.3 10.5 10.8 10.9
1180 1230 1260 1270 1280
which is specific for highly substituted xylan, did not improve the bleachability of pulp (Skjold-Joergensen et aL, 1992). Thus it appears that different types of xylans exist in the pulp. New pulping methods may strongly affect the composition and localization of hemicelluloses in pulp. The extent of reprecipitation of xylan has been suggested to diminish in the extended cooks. In some pulps produced by extended cooking, the effect of enzymes in bleaching has been found to decrease (Pedersen et al., 1992; Tolan, 1992). However, preliminary studies with kraft pulps produced by new modified methods to very low Kappa numbers have indicated that enzymes increase brightness after peroxide delignification (Tolan, 1992). Consistently with the suggested mechanism based on hydrolysis of relocated xylans, enzymes have not been observed to increase the brightness of sulphite pulps (Buchert et al., submitted). Most sulphite pulps are produced under acid conditions in which solubilized hemicelluloses are degraded and no reprecipitation occurs. The optimal mixture of xylanolytic enzymes needed for the complete hydrolysis of xylans can be designed according to the chemical composition of the isolated substrate. However, when hemicelluloses are bound to the fibre matrix, other factors such as localization, and the structural organization of the substrate within the fibre, also affect the efficiency of enzymatic treatments (Kantelinen et aL, 1993 b). The surface charge has been observed to be a key parameter in enzymatic treatments (Buchert et aL, 1993). In addition to the chemical composition, all these parameters are changed to varying degrees during different types of pulping processes. Effect of xylanases on bleachability
Unbleached kraft pulps have a low brightness due to the brown coloured lignin, which is removed in successive bleaching stages. The bleaching procedure is chosen with respect to the pulp type in order to attain the target brightness with retention of the strength properties. The most reactive bleaching chemicals are elemental chlorine (C), ozone (Z) and peroxy acids, which react with all the aromatic structures of lignin. Chlorine dioxide (D) and oxygen (0) react with lignin structures which have a free phenolic hydroxyl group. Sodium
hypochlorite (H) and hydrogen peroxide (P) react with certain functional groups, such as double bonds (Gellerstedt & Lindfors, 1991). Degraded lignin is extracted during intermediate alkaline (E) washing stages. The most effective delignification can be achieved by the action of different types of bleaching chemicals active on different sites in lignin (Sj6str6m, 1981). The conventional bleaching sequences contain a D/C prebleaching stage with various ratios of chlorine dioxide and chlorine gas. Oxygen delignification and chlorine dioxide have replaced elemental chlorine in the prebleaching stage. The bleaching chemicals used in the final bleaching depend on the target brightness and the end-use of the pulp. The benefits obtained by using enzymes are dependent on the chemical bleaching sequence used, as well as on the residual lignin content of the pulp (Table 2). Xylanases have generally been used for the treatment of brown stock pulp or oxygen delignified pulp. Owing to the high temperature and alkalinity of the pulp, the enzymes are generally applied after pH adjustment to about 5-7, and cooling of the pulp to 40-50°C (Viikari et al., 1991; 1993). In some basic studies, enzymes were used in the pretreatment of pulp before peroxide delignification or various chlorine bleaching sequences (Viikari et al., 1986; 1987; 1990; 1991; Clark et al., 1991; Buchert et al., 1992; Perrolaz et al., 1991; Sinner et al., 1991; Skerker et al., 1991). Enzyme pretreatment has been reported to result in a higher final brightness or a lower bleaching chemical consumption. The chlorine needed in prebleaching has been shown to be reduced by 20-30%. The saving in chlorine chemicals for the entire bleaching is generally half of this figure (Viikari et al., 1986; 1987). As a result, the AOX load of the prebleaching effluent has been reduced by 15-20% (Koponen, 1991). In elemental chlorine-free (ECF) bleaching sequences the use of enzymes increases the productivity of the bleaching plant, when the production capacity of CIO2 is a limiting factor. This is often the case when the utilization of chlorine gas has been abandoned. In totally chlorinefree bleaching sequences, the addition of enzymes increases the final brightness value, which is a key parameter in marketing of the chlorine-free pulps (Koponen, 1991; Viikari et al., 1991; Lavielle et al., 1992; Farrell & Skerker, 1992). In addition to this,
Appfication of xylanases in the pulp and paper industry
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Table 2. The aims of using xylanases in bleaching. Examples of bleaching sequences (D = chlorine dioxide, C--elementary chlorine, E=aikaline extraction, O = oxygen, P = hydrogen peroxide, Z = ozone)
Bleaching sequence
Major aim
Conventional (D/C)EDED Elementary chlorine free (ECF) D EDED O D EDED (EOP)D(EP)D D(EOP)DED Totally chlorine free (TCF) OPPP OZEP OPZE
Reduction in the use of chlorine AOX decrease Increase of the bleaching plant capacity AOX decrease
savings in the TCF bleaching chemicals is important with respect both to costs and to the strength properties of the pulp. The strength properties of enzymatically-treated pulps have been reported to be rather similar to those of the reference pulps, both in laboratory scale and in mill trials (Viikari et al., 1986; 1987; 1990; Paice et al., 1988; Perrolaz et al., 1991; Koponen, 1991; Chauvet et al., 1987). The treatment of kraft pulp with cellulasefree xylanases increases pulp viscosity due to partial hydrolysis of low DP xylan in the pulp (Paice et aL, 1988; Clark et al., 1990). In the production of TCF pulps the use of enzymes enables the preservation of acceptable strength properties. When using enzymes, lower amounts of unselective bleaching chemicals, such as peroxide and ozone, are needed. The price of enzymatic treatment today is estimated to be about 2-5 USS per ton of pulp (Viikari et al., 1991 ). The dosages of enzymes reported to be used in enzyme-aided bleaching have varied between 30 and about 8300 nkat/g. However, an economically realistic dosage is about 100 nkat/g. The prices of enzymes are expected to decrease as more efficient production strains and technologies are adopted. Usually, the price of the enzyme is only compared with that of the competing bleaching chemicals. However, other facts, such as decreased AOX-loadings and retention of viscosity or other technical pulp properties may confer additional advantages on enzymes. In such cases it may be difficult to specify a price. In the future, all available technologies will complete with regard to both effectiveness and price. In 1992 more than 10 mills were reported to be using xylanases continuously for improved bleachability of kraft pulps. Almost 100 mill trials have been carried out, about half of them in Europe. The production of TCF pulps has increased dramatically during the past two years. The TCF-technologies applied today are often based on bleaching of oxygen-delignifled pulps with enzymes and hydrogen peroxide.
Increase in brightness Improved strength properties
O T H E R APPLICATIONS OF XYLANASES
Previously, the main aim in the application of hemicellulases was the total hydrolysis of hemicellulosic substrates for production of fermentable sugars. Today, most new applications are related to the pulp and paper industry. Water removal on the paper machine has been shown to improve as a result of limited hydrolysis of the fibres in recycled paper. Mixtures of cellulases and hemicellulases were found to decrease the SR-value, which describes the drainage behaviour of the fibres (Pommier et al., 1989). The effect is apparently due to improved fibrillation or change in the composition of fine particles. Mill trials have been carried out successfully using a commercial T. reesei enzyme preparation (Pommier et al., 1990). The enzymes responsible for these effects have not yet been identified. The chemical pulping processes have been designed to produce fibres with optimal properties for various purposes. High hemicellulose content of the pulp results in high yields and is generally regarded as an advantage with respect to the interfibre bonding properties in papermaking. Consistent with this, reduction in interfibre bonding has been observed after treatment of pulps with xylanases (Noe et al., 1986; Roberts et al., 1990). The desired structural changes in the fibre, which are brought about by beating and refining, are external fibrillation and fibre swelling, which improve the flexibility and bonding ability of the fibres. The role of xylans in the modification of fibre properties was studied using xylanases from Sporotrichum dimorphosporum in the treatment of fully bleached spruce sulphite and birch kraft pulps. Electron microscopic examination revealed external fibrillation and good flexibility of fibres, implying internal modification (Mora et al., 1986). The water retention value, which describes fibre swelling, was considerably increased. The conclusion was that xylan was hydrolyzed in the whole, delignified cell walls. The enzymatically-treated
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J. Buchert, M. Tenkanen, A. Kantelinen, L. Viikari
pulps were comparable to slightly beaten pulps. The beatability was generally enhanced and the energy demand was reduced about threefold (Noe et al., 1986). Attempts to remove hemicellulose for production of dissolving pulps with very low hemicellulose contents have shown that complete enzymatic hydrolysis of hemicellulose within the pulp is difficult to achieve. Even with very high loadings of enzymes, only relatively small amounts of xylans could be removed. The xylan content in delignified mechanical aspen pulp was reduced from approximately 20% to 10%, whereas in bleached hardwood sulphite pulp the xylan content was decreased from 4% to only 3"5% (Paice & Jurasek, 1984). The complete removal of residual hemicellulose seems thus unattainable, probably due to the inaccessible location of the substrate.
CONCLUSIONS The new, major, large-scale application area of xylanases is clearly in the pulp and paper industry, in order to increase the bleachability of kraft pulps. The improved bleachability is based mainly on the action of endo-fl-xylanases, which can be produced efficiently on an industrial scale. Partial hydrolysis of xylan facilitates the extraction of lignin from the pulp in higher amounts and with higher molecular masses. The enzymatic pretreatment method is applicable to any traditional or modern bleaching sequence at existing plants without significant investments. The primary goals of the enzymatic treatment have been to decrease chemical consumption, reduce environmental loading, and/ or to increase the final brightness of pulp. In the TCF bleaching sequences, enzymes can improve the brightness, which may otherwise remain below an acceptable level, without loss of viscosity. Enzyme-aided bleaching is thus both environmentally and economically advantageous. Previously, the main aim in the application of hemicellulases has been the total hydrolysis of hemicellulosic substrates for production of fermentable sugars. The knowledge gathered on the hydrolysis mechanism of hemicelluloses, especially xylans, has greatly promoted the rapid application of these enzymes in new areas. The general information already available on individual hemicellulases will help in the development of future applications.
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Bailey, M. J., Puls, J. & Poutanen, K. (1991). Purification and properties of two xylanases from Aspergillus oryzae. Biotechnol. Appl. Biochem., 13, 380-9. Bailey, M. J., Biely, P. & Poutanen, K. (1992). Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol., 23, 257-70. Bragger, J. M., Daniel, R. M., Coolbear, T. & Morgan, H. W. (1989). Very stable enzymes from extremely thermophilic archaebacteria and eubacteria. Appl. Microbiol. Biotechnol., 31,556-61. Buchert, J., Tenkanen, M., Pitkfnen, M. & Viikari, L. (1993). Role of surface charge and swelling on the action of xylanases on birch kraft pulp. TappiJ., 76, 131-5. Buchert, J., Kantelinen, A., Suurn~ikki, A., Viikari, L. & Janson, J. (199?). Evaluation of the effects of hemicellulases on the bleachability of sulphite pulps (submitted). Chauvet, J.-M., Comtat, J. & Noe, P. (1987). Assistance in bleaching of never-dried pulps by the use of xylanases, consequences on pulp properties. In Proc. 4th Int. Congr. on Wood and Pulping Chemistry, Vol. II, pp. 325-7. Clark, T. A., McDonald, A. G., Senior, D. J. & Mayers, P. R. (1990). Mannanase and xylanase treatment of softwood chemical pulps, effects on pulp properties and bleachability. In Biotechnology in the Pulp and Paper Manufacture, ed. K. T. Kirk & H.-M. Chang. ButterworthHeinemann, Boston, pp. 153-67. Clark, T. A., Steward, D., Bruce, M. E., McDonald, A. G., Singh, A. P. & Senior, D. J. (1991 ). In Proc. 45th APPITA Annual General Conference, Vol. I, pp. 193-200. Clayton, D. W. & Stone, J. E. (1967). The redeposition of hemicelluloses during pulping. Part l:The use of a tritiumlabelled xylan. Pulp Paper Mag. Canada, 64T, 459-68. Coughlan, M. P. (1992). Towards an understanding of the mechanism of action of main chain-hydrolyzing xylanases. In Xylans and Xylanases, ed. J. Visser, G. Beldman, M. A. Kusters-van Someren & A. G. J. Voragen. Elsevier, Amsterdam, pp. 111-39. Coughlan, M. P. & Hazlewood, G. P. (1993). fl-l,4-D-Xylandegrading enzyme systems: biochemistry, molecular biology and applications. Biotechnol. Appl. Biochem., 17, 259-89. Croon, I. & Enstr6m, B. (1962). The hemicelluloses in sulphate pulps from Scots pine. Svensk Papperstid., 65, 595-9. Dekker, R. E H. (1985). Biodegradation of the hemicelluloses. In Biosynthesis and Biodegradation of Wood Components, ed. T. Higuchi. Academic Press, Orlando, pp. 505-33. Eriksson, K.-E., Blanchette, R. A. & Ander, P. (1990). Microbial and Enzymatic Degradation of Wood Components. Springer-Verlag, Berlin. Farrell, R. L. & Skerker, P. S. (1992). Chlorine-free bleaching with Cartazyme T M HS treatment. In Xylans and Xylanases, ed. J. Visser, G. Beldman, M. A. Kusters-van Someren & A. G. J. Voragen. Elsevier, Amsterdam, pp. 315-25. Germg~trd, K. & Larsson, S. (1983). Oxygen bleaching in modem softwood kraft pulp mill. Paper Timber, 65, 287-90. Gellerstedt, G. & Lindfors, E.-L. (1991). On the structure and reactivity of residual lignin in kraft pulp fibers. In Proc. Int. Pulp Bleaching Conf., Vol. I. SPCI, pp. 73-88. Hartler, N. &Lund, A. (1962). Sorption of xylans on cotton. Svensk Papperstid., 65, 951-5. Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J., 280,309-16. Henrissat, B. & Bairoch, A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J., 293, 781-8. Hortling, B., Korhonen, M., Buchert, J., Sundquist, J. & Viikari, L. (1994). Leachability of lignin from kraft pulps after xylanase treatment. Holzforschung, 48, 441-6.
Application of xylanases in the pulp and paper industry Ito, K., Ogasawara, H., Sugimoto, T. & Ishikawa, T. (1992). Purification and properties of acid stable xylanases from Aspergillus kawachii. Biosci. Biotech. Biochem., 56, 1338-40. IUB (International Union of Biochemistry)(1982). Enzyme Nomenclature. Academic Press, London. Iversen, T. & Wannstr6m, S. (1986). Lignin-carbohydrate bonds in a residual lignin isolated from pine kraft pulp. Holzforschung, 40, 19-22. Jansson, J. & Palenius, I. (1972). Aspects of the colour of kraft pulp and kraft lignin. Paper Timber, 54, 343-52. Jansson, J., Palenius, I., Stenkud, B. & Sfigforss, P. E. (1975). Differences in colour and strength of kraft pulps from batch and flow cooking. Paper Timber, 57,387-90. Jurasek, L. & Paice, M. G. (1988). Xylanase A of Schizophyllum commune. Methods Enzym., 160A, 659-62. Kantelinen, A. (1992). Enzymes in bleaching of kraft pulp. VTT Publications 114. Technical Research Centre of Finland, Espoo. 86 pp. Kantelinen, A., Rfitt6, M., Sundquist, J., Ranua, M., Viikari, L. & Linko, M. (1988). Hemicellulases and their potential role in bleaching. TappiJ., 1-9. Kantelinen, A., Sundquist, J., Linko, M. & Viikari, L. (1991). The role of reprecipitated xylan in the enzymatic bleaching of kraft pulp. Proc. 6th Int. Symp. Wood and Pulping Chemistry, Vol. I, pp. 493-500. Kantelinen, A., Hortling, B., Sundquist, J., Linko, M. & Viikari, L. ( 1993 a). Proposed mechanism of the enzymatic bleaching of kraft pulp with xylanases. Holzforschung, 47, 318-24. Kantelinen, A., Rantanen, T., Buchert, J. & Viikari, L. ( 1993 b ). Enzymatic solubilization of fibre-bound and isolated birch xylans. J. Biotechnol., 28, 219-28. Koponen, R. (1991). Enzyme systems prove their potential. Pulp Paper Int., 33( 11 ), 20-25. Kormelink, E J. M., Searle-Van Leeuwen, M. J. F., Wood, T. M. & Voragen, A. G. J. (1993). Purification and characterization of three endo-(1,4)-fl-xylanases and one flxylosidase from Aspergillus awamori. J. Biotechnol., 27, 249-65. Kovasin, K. K. & Tikka, P. O. (1992). Superbatch cooking results in superlow Kappa numbers. Paper presented at the 4th Int. Conf. New Available Techniques and Current Trends, Bologna, Italy, 19-22 May. Lavielle, P., Koljonen, M., Piiroinen, P., Koponen, R., Reid, D. & Fredriksson, R. (1992). Three large-scale uses of xylanases in kraft pulp bleaching. Proc. 4th Int. Conf. New Available Techniques and Current Trends, pp. 203-15. Lindberg, B., Rosell, K.-G. & Svensson, S. (1973). Position of the O-acetyl groups in birch xylan. Svensk Papperstid., 76, 30-2. Liithi, E., Jasmat, N. B. & Bergquist, P. L. (1990). Xylanase from the extremely thermophilic bacterium Caldocellum saccharolyticum: overexpression of the gene in Escherichia coli and characterization of the gene product. Appl. Environ. Microbiol., 56, 2677-83. Meller, A. (1965). The retake of xylan during alkaline pulping. A critical appraisal of the literature. Holzforschung, 19, 118-24. Mora, F., Comtat, J., Barnoud, F., Pla, E & Noe, P. (1986). Action of xylanases on chemical pulp fibers. Part I: Investigations on cell-wall modifications. J. Wood Chem. Technol., 6, 147-65. Nakamura, S., Wakabayashi, K., Nakai, R., Aono, R. & Horikoshi, K. (1993). Appl. Environ. Microbiol., 59, 2311-16. Noe, P., Chevalier, J., Mora, E & Comtat, J. (1986). Action of xylanases on chemical pulp fibers. Part II: Enzymatic beating. J. Wood Chem. TechnoL, 6, 167-84. Paice, M. G. & Jurasek, L. (1984). Removing hemiceUulose from pulps by specific enzymic hydrolysis. J. Wood Chem. TechnoL, 4, 187-98.
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Paice, M., Bernier, M. & Jurasek, L. (1988). Viscosity enhancing bleaching of hardwood kraft pulp with xylanase from a cloned gene. Biotechnol. Bioeng., 32,235-9. Paice, M. G., Gurnagul, N., Page, D. H. & Jurasek, L. (1992). Mechanism of hemiceUulose-directed prebleaching of kraft pulps. Enzyme Microb. Technol., 14,272-6. Pedersen, L. S., Kihlgren, P., Nissen, A. M., Munk, N., Holm, H. C. & Choma, P. P. (1992). Enzymatic bleach boosting of kraft pulp. Laboratory and mill scale experiences. Proc. Tappi, 1992 Pulping Conf., Boston, pp. 31-7. Perrolaz, J. J., Davis, S., Gysin, B., Zimmerman, W., Casimir, J. & Fiechter, A. ( 1991). Elemental chlorine free bleaching with a thermostable xylanase, a new alternative to elemental chlorine for bleaching. Proc. 6th Int. Symp. Wood and Pulping Chemistry, Vol. I, pp. 485-9. Pommier, J.-C., Fuentes, J.-L. & Goma, G. (1989). Using enzymes to improve the process and product quality in the recycled paper industry. Part I. The basic laboratory work. TappiJ., 72, 187-91. Pommier, J.-C., Goma, G., Fuentes, J.-L., Rousset, C. & Jokinen, O. (1990). Using enzymes to improve the process and product quality in the recycled paper industry. Part II. Industrial applications. TappiJ., 73, 197-202. Roberts, J. C., McCarthy, A. J., Flynn, N. J. & Broda, P. (1990). Modifications of paper properties by the pretreatment of pulp with Saccharomonospora viridis xylanase. Enzyme Microb. Technol., 12, 21 O- 13. Royer, J. C. & Nakas, J. P. (1991). Purification and characterization of two xylanases from Trichoderma longibrachiatum. Eur. J. Biochem., 202,521-9. Scallan, A. M. (1977). The accommodation of water within pulp fibres. In Proc. Fibre-Water Interactions in Paper Making. Pergamon Press, Oxford, pp. 9-27. Senior, D. J., Mayers, P. R., Miller, D., Sutcliffe, R., Tan, L. U. L. & Saddler, J. N. (1988). Selective solubilization of xylan in pulp using a purified xylanase from Trichoderma harzianum. Biotechnol. Lett., 10, 907-12. Sinner, M., Ditzelmuller, G., Wizani, W., Steiner, W. & Esterbauer, E. (1991). VAI-Biobleiche. Papier, 45,403-10. Sj6str6m, E. (1981). Wood Chemistry, Fundamentals and Applications. Academic Press, Inc., New York, 223 pp. Skerker, P. S., Farrell, R. L. & Chang, H.-M. (1991). Chlorine-free bleaching with Cartazyme HS treatment. Proc. 1991 Int. Pulp Bleaching Conf., Stockholm, pp. 93-105. Skjold-Joergensen, S., Munk, N. & Pedersen, L. S. (1992). Recent progress within the application of xylanases for boosting the bleachability of kraft pulp. In Biotechnology in the Pulp and Paper Industry, ed. M. Kuwahara & M. Shimada. UNI Publishers, Tokyo, pp, 93-100. Suurn~ikki, A., Kantelinen, A., Hortling, B., Buchert, J. & Viikari, L. (1993). Enzymatic solubilization of hemicelluloses in industrial softwood kraft pulps. Holzforschung (submitted). Tenkanen, M., Puls, J. & Poutanen, K. (1992a). Two major xylanases of Trichoderma reesei. Enzyme Microb. Technol., 14,566-74. Tenkanen, M., Buchert, J., Puls, J., Poutanen, K. & Viikari, L. (1992b). Two main xylanases of Trichoderma reesei and their use in pulp processing. In Xylans and Xylanases, ed. J. Visser, G. Beldman, M. A. Kusters-van Someren & A. G. J. Voragen. Elsevier, Amsterdam, pp. 547-50. Tolan, J. S. (1992). The use of enzymes to enhance pulp bleaching. Proc. Tappi, 1992 Pulping Conf., Boston, pp. 13-17. T6rr6nen, A., Mach, R. L., Messner, R., Gonzalez, R., Kalkkinen, N., Harkki, A. & Kubicek, C. P. (1992). The two major xylanases from Trichoderma reesei: characterization of both enzymes and genes. Bio/Technology, 10, 1461-5. Tsujibo, H., Sakamoto, T., Nishino, N. & Hasegawa, T. (1990). Purification and properties of three types of
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xylanases produced by an alkalophilic actinomycete. J. Appl. Bacteriol., 69, 398-405. Viikari, L., Ranua, M., Kantelinen, A., Linko, M. & Sundquist, J. (1986). Bleaching with enzymes. In Biotechnology in the Pulp and Paper Industry. Proc. 3rd Int. Conf., pp. 69-9. Viikari, L., Ranua, M., Kantelinen, A., Linko, M. & Sundquist, J. ( 1987). Application of enzymes in bleaching. Proc. 4th Int. Symp. Wood and Pulping Chemistry, Vol. 1, pp. 151-4. Viikari, L., Kantelinen, A., Poutanen, K. & Ranua, M. (1990). Characterization of pulps treated with hemicellulolytic enzymes prior to bleaching. In Biotechnology in Pulp and Paper Manufacture, ed. K. T. Kirk & H.-M. Chang. Butterworth-Heinemann, Boston, pp. 145-51. Viikari, L., Sundquist, J. & Kettunen, J. (1991). Xylanase enzymes promote pulp bleaching. Paper Timber, 73, 384-9. Viikari, L., Tenkanen, M., Buchert, J., Ritt6, M., Bailey, M., Siika-aho, M. & Linko, M. (1993). Hemicellulases for industrial applications. In Bioconversion of Forest and
Agricultural Plant Residues, ed. J. Saddler. CAB International, Wallingford, UK, pp. 131-82. Wong, K. K. Y. & Saddler, J. N. (1992). Trichoderma xylanases, their properties and application. CRC Crit. Rev. Biotechnol., 12,413-35. Wong, K. K. Y., Tan, L. U. L. & Saddler, J. N. (1988). Multiplicity of fl-l,4-xylanase in microorganisms: functions and applications. Microbiol. Rev., 52,305-17. Yang, J. L. & Eriksson, K.-E. L. (1992). Use of hemicellulolytic enzymes as one stage in bleaching of kraft pulps. Holzforschung, 46, 481-8. Yllner, S. & Enstr6m, B. (1956). Studies of the adsorption of xylan on cellulose fibres during the sulphate cook. Part 1. Svensk Papperstid., 59, 229-32. Yllner, S., Ostberg, K. & Stockman, L. (1957). A study of the removal of the constituents of pine wood in the sulphate process using a continuous liquor flow method. Svensk Papperstid., 60, 795-802. Zamost, B. L., Nielsen, H. K. & Starnes, R. L. (1991). Thermostable enzymes for industrial applications. J. Ind. Microbiol., 8, 71-82.
' ]L,,.
Bioresource Technology 50 (1994) 65-72 © 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/94[$7.00
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0960-8524(94)00044-1
ELSEVIER
APPLICATION OF X Y L A N A S E S IN THE PULP A N D PAPER INDUSTRY Johanna Buchert, Maija Tenkanen, Anne Kantelinen & Liisa Viikari* VTT BiotechnicaI Laboratory, PO Box 1501, FIN-02044 VTT, Finland
application areas of hemicellulases is in the pulp and paper industry. Furthermore, enzymatic methods are especially suited to reduce environmental impacts of the pulp and paper industry. Organic chlorine compounds formed during the bleaching of chemical pulp have probably attracted most attention in recent years. These compounds arise mainly from the reactions between residual lignin present in wood fibres, causing the brown colour of unbleached pulp, and the chlorine used for bleaching. For the same amount of active chlorine used, chlorine dioxide causes the formation of adsorbable organic halogen (AOX) in an amount of only one fifth of that caused by chlorine gas (Germgfird & Larsson, 1983). Earlier measures taken by the forest industry to solve the chlorine problem have focused on improving effluent treatment methods. Today, the emphasis of research and development work in this area has shifted more towards improving the processes. In the search to produce pulp with non-polluting chemicals, more efficient pulping methods, which reduce the amount of residual lignin passing through to the bleaching process, and alternative bleaching methods, are being developed (Kovasin & Tikka, 1992). Among the new bleaching technologies, enzymes have already shown their potential (Koponen, 1991; Lavielle et aL, 1992). Enzymes can be used for specific modifications of pulp for the development of environmentally safe processes. The original concept of using hemicellulases to improve bleachability of kraft pulps was first published in 1986 (Viikari et aL, 1986; 1987). The brightness of the pulp was substantially increased by the enzymatic treatment, and this improved brightness could be exploited for reduction of chlorine chemical consumption. It was observed that the saving could be gained either in the prebleaching stage, as chlorine gas, or in the final bleaching stage, as chlorine dioxide (Viikari et al., 1986; 1987). For environmental reasons, the possibility of minimizing the consumption of chlorine gas in bleaching was of particular interest. Since the end of the 1980s the pulp and paper industry has been forced to consider any new technique available for the reduction of chlorine consumption, or for the increase of brightness of totally chlorine-free pulps. The enzymatic treatment has been successfully combined with more efficient pulping and non-chlorine bleaching methods.
Abstract
Interest in hemicellulolytic enzymes has increased remarkably during recent years. This is mainly due to the new areas of application of these enzymes within the pulp and paper industry. Among these, the most promising seems to be utilization of hemicellulases, especially xylanases, to increase the bleachability of kraft pulps. This is partly due to the great potential of an environmentally safe method. The main enzymes needed in the enzyme-aided bleaching have been shown to belong to the group of endo-fl-xylanases. Xylanases act mainly on the relocated, reprecipitated xylan on the surface of the pulp fibres. Enzymatic hydrolysis of this specific type of xylan renders the structure of the fibres more permeable, allowing enhanced extraction of residual lignin from the fibres. The hydrolysis of hemicelluloses in the inner fibre layers may also enhance the bleachability. The main goals in the enzyme-aided bleaching of kraft pulps have been the reduction of consumption of chlorine chemicals in the bleaching process, and consequent lowering of the A OX of the effluents. In the production of totally clorine-free pulps, enzymes have also been successfully used for increasing the brightness of pulp. Other suggested enzymatic modifications of fibres are aimed at improved drainage (water removal) in the paper machine, improvement of fibre properties or production of dissolving pulps. The xylanolytic enzymes and their application areas are reviewed. Key words: Hemicellulases, xylanases, pulp and paper, bleaching.
INTRODUCTON
Hemicelluloses are major constituents of plant cell wall polysaccharides. The structure of various types of hemicelluloses depends on the type of plant, and may even vary between different parts of the same plant. Hemicelluloses are usually classified according to the main sugar residues in the backbone, e.g. xylans, glucomannans, galactans and glucans. Both wood and pulp contain high amounts of structurally different hemicelluloses. Thus, one of the most important potential *To whom correspondence should be addressed. 65
66
J. Buchert, M. Tenkanen, A. Kantelinen, L. Viikari
SUBSTRATES
Xylan and glucomannan form the basic backbone polymers of wood hemicelluloses. In hardwoods, the main hemicellulose is O-acetyl-O-methylglucuronoxylan, whereas in softwoods, arabino-4-O-methylglucuronoxylan comprises about one third of the total hemicelluloses. Hardwood xylan contains 4-O-methylglucuronic acid and acetyl side-groups. Methylglucuronic acid is linked to the backbone by a( 1 --, 2) glycosidic bonds. Acetic acid is esterified at the hydroxyl group of carbon 2 and/or 3 (Lindberg et al., 1973). Softwood xylan contains 4-O-methylglucuronic acid and L-arabinofuranoside sidegroups linked to the backbone by a(1--, 2)- and a(1--, 3)-glycosidic bonds, respectively. The average molar ratio of xylose:4-Omethylglucuronic acid: acetic acid in hardwood xylan is 10:1: 7, and that of xylose :4-O-methyl-glucuronic acid:arabinose sugar units in softwood xylans is 8:1.6:1 (Sjrstrrm, 1981). Extensive modification of hemicelluloses takes place during pulp production processes. During the heating period of the conventional kraft cooking, when the alkali concentration is comparatively high, part of the xylan is dissolved in the pulping liquor. As the cooking proceeds the alkali concentration decreases, and degraded, short-chain xylan is precipitated in a more or less crystalline form on the surface of cellulose microfibrils (Yllner & Enstrrm, 1956; Yllner et al., 1957). The configuration of the xylose units allows close contact of the xylan chains with the cellulose and it is likely that part of the xylan, after removal of its substituents, tends to cocrystallize with, or become adsorbed on, the cellulose of the pulp. A considerable part of the xylan is readsorbed or reprecipitated onto the cellulose, although a part remains undissolved at its original location in the fibre (Croon & Enstrrm, 1962; Hartler & Lund, 1962). Approximately half of the xylan present in normal pine kraft pulps is estimated to be relocated xylan (Yllner et al., 1957; Meller, 1965). In birch kraft pulps the amount of reprecipitated xylan has been estimated to be about 1-3% of the pulp (Clayton & Stone, 1967), or about 4-12% of the total xylan in pulp (Axelsson et al., 1962). The reprecipitation of xylan is preceeded by the reprecipitation of dissolved lignin during kraft pulping. These redeposited polymers have also been suggested to be chemically linked to each other (Jansson & Palenius, 1972; Jansson et al., 1975; Iversen & W~innstrrm, 1986). Furthermore, hemicelluloses seem to physically restrict the passage of high molecular mass lignin out of the pulp fibre (Ahlgren et al., 1971; Scallan, 1977). XYLANASES Endo-l,4-fl-D-xylanases (EC 3.2.1.8) catalyze the random hydrolysis of 1,4-fl-D-xylosidic linkages in xylans (IUB, 1982). Xylanases are produced by several bacteria, yeast and fungi, although the latter have been extensively studied (see Wong et al., 1988; Eriksson
et al., 1990; Viikari et al., 1993; Coughlan & Hazlewood, 1993). The published information on the induction (constitutive or induced) as well as regulation (under separate or common regulatory control system with cellulases) of xylanases varies, and induction as well as regulation seems to differ between organisms. Some reports demonstrate that xylanases are inducible and the specific induction can occur independently of cellulase synthesis (Coughlan & Hazlewood, 1993). Fungal xylanases of Aspergillus and Trichoderma spp., and bacterial xylanases of Bacillus spp., Streptomyces spp. and Clostridium spp. have been intensively studied (Wong et al., 1988; Eriksson et al., 1990). Several of the xylanases purified are rather small (molecular mass around 20 kDa), monomeric proteins with basic isoelectric point (pI 8-9-5). They also exhibit a great homology on the molecular level and belong to the family G (or 11 ) of glycosyl hydrolases. The other xylanases with higher molecular mass and lower pI value belong to the other identified endoxylanase family F (or 10) (Henrissat, 1991; Henrissat & Bairoch, 1993). The optimum pH for xylan hydrolysis is around five for most fungal xylanases, and they are normally stable between pH values of two and nine. The pH optima of bacterial xylanases are generally slightly higher than the pH optima of fungal xylanases. Alkalophilic Bacillus spp. (Nakamura et al., 1993) and alkalophilic actinomycetes (Tsujibo et al., 1990) have been reported to produce xylanases with high activity at alkaline pH values. Most of the fungi and bacteria produce xylanases which tolerate temperatures below 40-50°C. Xylanases have also been characterized from thermophilic organisms. The most thermostable xylanase described is that of an extremely thermophilic Thermotoga spp. with a half life of 20 min at 105°C (Bragger et al., 1989). Xylanases with half lives from a few minutes up to 90 min at 80°C have been produced by Thermoascus aurantiacus, Bacillus stearothermophilus, Caldocellum saccharolyticum, Clostridium stercorarium and Thermomonospora spp. (Zamost et al., 1991; Ltithi et aL, 1990). Endoxylanases show the highest activity against polymeric xylan, and the rate of the hydrolysis reaction normally decreases with the decreasing chain length of the oligomeric substrates. They do not hydrolyze xylobiose, and the hydrolysis of xylotriose is in most cases negligible or at least limited. The main products formed from the hydrolysis of xylan are xylobiose, xylotriose, and substituted oligomers of two to four xylosyl residues. The length and the type of the substituted products depends on the mode of action of the individual xylanases. Most of the enzymes studied cleave the xylan backbone, leaving the substituent on the nonreducing end of the xylosyl chain of the oligosaccharide. Some xylanases were reported to leave the substituent on the reducing end and in the middle of the oligosaccharide chain (Dekker, 1985; Coughlan, 1992). Of the end products, at least xylotriose has been reported to inhibit the action of xylanases (Royer &
Application of xylanases in the pulp and paper industry
Nakas, 1991). In addition to hydrolytic activity, transferase activity has been detected in several xylanases (Coughlan, 1992; Viikari et al., 1993). Of the xylanases produced by Trichoderma spp., two main groups of enzymes can be identified. Both types have low MW but the pI is different. Xylanases with high pI have been quite extensively studied, whereas the other type of xylanase, with pI near to pH 5, has been purified and characterized only from T. lignorum and T. reesei. More than one basic xylanase has been isolated from T. harzianum, T. koningii and T. longibrachiatum (Wong & Saddler, 1992). However, only one of these enzymes made a major contribution to the total xylanase activity in the culture filtrate. The pI 9"0 and pI 5"5 xylanases of T. reesei have been shown to be different gene products, and were both classified as belonging to the family G (Tenkanen et al., 1992a; Ttrrtnen et al., 1992). Multiple xylanases have also been purified from culture filtrates of A. niger (Wong et al., 1988), A. oryzae (Bailey et al., 1991), A. kawachii (Ito et al., 1992), A. awamori (Kormelink et al., 1993) and other fungi, as well as bacteria (Wong et al., 1988; Viikari et al., 1993). By production of multiple xylanases the microorganisms may maximize the utilization of xylans with different structures.
APPLICATION OF XYLANASES IN BLEACHING
Comparison of different xylanases Several xylanases have been tested for their properties in bleaching experiments. The action of enzymes on pulp bleachability has been studied by different methods. In the first phase, enzymes are usually characterized by isolated substrates, which are used in the determination of their activities. These substrates, however, vary extensively with respect to their origin and composition. Furthermore, comparison of enzyme activities is complicated by the utilization of different analysis methods (Bailey et al., 1992). The action of enzymes can also be compared on the actual substrate, the pulp. In these tests, the solubilization of pulp carbohydrates has usually been the key parameter (Senior et al., 1988; Kantelinen et al., 1993b). In addition, an increase in the liberation of lignin-derived compounds after the enzymatic treatment (Yang & Eriksson, 1992; Pedersen et al., 1992) or after alkaline extraction (Kantelinen et al., 1993a; Hortling et al., 1994) has been used for evaluation of different enzymes. However, the most reliable method to compare different xylanases is the bleaching of enzyme treated pulps. In practice, the most convenient measure is the brightness increase in peroxide delignification. Most of the reports published on application of xylanases are still based on unpurified enzymes. The culture filtrates used, however, have contained xylanases as the main activity and the enzyme preparations are generally dosed according to xylanase activity. Even with unpurified enzymes, identification of the
67
sugars released in the enzymatic treatments have confirmed that xylanase was the cause of the major activity (Viikari et al., 1987; 1990). The main enzymes needed to enhance the delignification of kraft pulp were later shown to be endo-fl-xylanases (Viikari et al., 1991; Paice et al., 1988; Kantelinen et al., 1988; Jurasek & Paice, 1988). Only small differences in the bleaching efficiency have been observed between xylanases from different organisms, although the degree of hydrolysis has varied. The role of xylanase activity in delignification of kraft pine pulp has been studied with purified xylanases of T. reesei (Tenkanen et al., 1992b; Kantelinen, 1992; Buchert et al., 1992). The purified xylanases of T. reesei were shown to have different pI values, pH optima and substrate specificities. Although the xylanase with pI 9 has been shown to be relatively more specific for non-substituted xylans, both purified xylanases were equally efficient in limited hydrolysis of pulp xylans, as well as in subsequent chemical delignification (Buchert et al., 1992). Interestingly, the pHoptimum of the pI 9 xylanase was shifted in pulp treatments to pH 6-7 from the optimum pH of 5.0, determined on isolated xylan (Buchert et al., 1992). Optimization of the amount of enzyme and the hydrolysis time revealed that reduction in the Kappa number of pine kraft pulp (measured after peroxide delignification) could be achieved using low amounts of enzymes and a relatively short incubation time (Table 1). Minimization of the overall hydrolysis of hemicelluloses is necessary in order to maintain a high pulp yield, and the advantageous properties of hemicelluloses in the pulp (Viikari et al., 1986; 1987). It has been suggested that the enzymatic hydrolysis of pulp xylans can be directed by chemical means, i.e. by adjustment of ionic strength, pH and counter ion profile in the pulp (Buchert et al., 1993).
Mechanism of enzyme-aided bleaching Two types of phenomena are probably involved in the enzymatic pretreatment of kraft pulp resulting in improved delignification. Hydrolysis of the reprecipitated xylan renders the pulp more permeable, thus facilitating the removal of residual lignin (Kantelinen et a l., 1991; 1993 a). After the xylanase pretreatment, the average molecular weight of alkali-extracted lignins is increased. The hydrolysis of xylan located in the inner layers and possibly linked to lignin may also affect the delignification (Paice et al., 1992). Due to the inaccessibility of pulp substrates, xylanases are expected to act mainly on the relocated, reprecipitated xylan on the surface of the pulp fibres (Kantelinen et al., 1991; Kantelinen et al., 1993a). The reprecipitation of xylan occurs when almost all of the xylan side-groups are cleaved off, resulting in a very resistant chemical structure. In agreement with this, the accessory enzymes splitting off the xylan side-groups have been found to have only limited effects in hydrolysis and bleaching tests (Kantelinen et al., 1988; Kantelinen, 1992). The hypothesis is supported by the observation that the extraction of xylan by DMSO,
68
J. Buchert, M. Tenkanen, A. Kantelinen, L. Viikari
Table 1. Effect of xylanase (from Trichoderma reesei, pI 9, Tenkanen et al., 1992a) dosage on the bleachability of birch kraft pulp (original Kappa number 19.9) with peroxide as bleaching chemical. Bleaching sequence used EnzQP, where Q denotes chelator and P peroxide. Bleaching conditions as described previously by Suurnikki et al. (1994)
Xylanase dosage (nkat/g)
Reducing sugars (% of d.w.)
Brighmess (ISO)
Kappa number
Viscosity (dma/kg)
0 100 300 600 1000
0 1.04 1.98 2.86 3-30
55.4 58.0 58.0 57.5 57.5
12.6 12.3 10.5 10.8 10.9
1180 1230 1260 1270 1280
which is specific for highly substituted xylan, did not improve the bleachability of pulp (Skjold-Joergensen et aL, 1992). Thus it appears that different types of xylans exist in the pulp. New pulping methods may strongly affect the composition and localization of hemicelluloses in pulp. The extent of reprecipitation of xylan has been suggested to diminish in the extended cooks. In some pulps produced by extended cooking, the effect of enzymes in bleaching has been found to decrease (Pedersen et al., 1992; Tolan, 1992). However, preliminary studies with kraft pulps produced by new modified methods to very low Kappa numbers have indicated that enzymes increase brightness after peroxide delignification (Tolan, 1992). Consistently with the suggested mechanism based on hydrolysis of relocated xylans, enzymes have not been observed to increase the brightness of sulphite pulps (Buchert et al., submitted). Most sulphite pulps are produced under acid conditions in which solubilized hemicelluloses are degraded and no reprecipitation occurs. The optimal mixture of xylanolytic enzymes needed for the complete hydrolysis of xylans can be designed according to the chemical composition of the isolated substrate. However, when hemicelluloses are bound to the fibre matrix, other factors such as localization, and the structural organization of the substrate within the fibre, also affect the efficiency of enzymatic treatments (Kantelinen et aL, 1993 b). The surface charge has been observed to be a key parameter in enzymatic treatments (Buchert et aL, 1993). In addition to the chemical composition, all these parameters are changed to varying degrees during different types of pulping processes. Effect of xylanases on bleachability
Unbleached kraft pulps have a low brightness due to the brown coloured lignin, which is removed in successive bleaching stages. The bleaching procedure is chosen with respect to the pulp type in order to attain the target brightness with retention of the strength properties. The most reactive bleaching chemicals are elemental chlorine (C), ozone (Z) and peroxy acids, which react with all the aromatic structures of lignin. Chlorine dioxide (D) and oxygen (0) react with lignin structures which have a free phenolic hydroxyl group. Sodium
hypochlorite (H) and hydrogen peroxide (P) react with certain functional groups, such as double bonds (Gellerstedt & Lindfors, 1991). Degraded lignin is extracted during intermediate alkaline (E) washing stages. The most effective delignification can be achieved by the action of different types of bleaching chemicals active on different sites in lignin (Sj6str6m, 1981). The conventional bleaching sequences contain a D/C prebleaching stage with various ratios of chlorine dioxide and chlorine gas. Oxygen delignification and chlorine dioxide have replaced elemental chlorine in the prebleaching stage. The bleaching chemicals used in the final bleaching depend on the target brightness and the end-use of the pulp. The benefits obtained by using enzymes are dependent on the chemical bleaching sequence used, as well as on the residual lignin content of the pulp (Table 2). Xylanases have generally been used for the treatment of brown stock pulp or oxygen delignified pulp. Owing to the high temperature and alkalinity of the pulp, the enzymes are generally applied after pH adjustment to about 5-7, and cooling of the pulp to 40-50°C (Viikari et al., 1991; 1993). In some basic studies, enzymes were used in the pretreatment of pulp before peroxide delignification or various chlorine bleaching sequences (Viikari et al., 1986; 1987; 1990; 1991; Clark et al., 1991; Buchert et al., 1992; Perrolaz et al., 1991; Sinner et al., 1991; Skerker et al., 1991). Enzyme pretreatment has been reported to result in a higher final brightness or a lower bleaching chemical consumption. The chlorine needed in prebleaching has been shown to be reduced by 20-30%. The saving in chlorine chemicals for the entire bleaching is generally half of this figure (Viikari et al., 1986; 1987). As a result, the AOX load of the prebleaching effluent has been reduced by 15-20% (Koponen, 1991). In elemental chlorine-free (ECF) bleaching sequences the use of enzymes increases the productivity of the bleaching plant, when the production capacity of CIO2 is a limiting factor. This is often the case when the utilization of chlorine gas has been abandoned. In totally chlorinefree bleaching sequences, the addition of enzymes increases the final brightness value, which is a key parameter in marketing of the chlorine-free pulps (Koponen, 1991; Viikari et al., 1991; Lavielle et al., 1992; Farrell & Skerker, 1992). In addition to this,
Appfication of xylanases in the pulp and paper industry
69
Table 2. The aims of using xylanases in bleaching. Examples of bleaching sequences (D = chlorine dioxide, C--elementary chlorine, E=aikaline extraction, O = oxygen, P = hydrogen peroxide, Z = ozone)
Bleaching sequence
Major aim
Conventional (D/C)EDED Elementary chlorine free (ECF) D EDED O D EDED (EOP)D(EP)D D(EOP)DED Totally chlorine free (TCF) OPPP OZEP OPZE
Reduction in the use of chlorine AOX decrease Increase of the bleaching plant capacity AOX decrease
savings in the TCF bleaching chemicals is important with respect both to costs and to the strength properties of the pulp. The strength properties of enzymatically-treated pulps have been reported to be rather similar to those of the reference pulps, both in laboratory scale and in mill trials (Viikari et al., 1986; 1987; 1990; Paice et al., 1988; Perrolaz et al., 1991; Koponen, 1991; Chauvet et al., 1987). The treatment of kraft pulp with cellulasefree xylanases increases pulp viscosity due to partial hydrolysis of low DP xylan in the pulp (Paice et aL, 1988; Clark et al., 1990). In the production of TCF pulps the use of enzymes enables the preservation of acceptable strength properties. When using enzymes, lower amounts of unselective bleaching chemicals, such as peroxide and ozone, are needed. The price of enzymatic treatment today is estimated to be about 2-5 USS per ton of pulp (Viikari et al., 1991 ). The dosages of enzymes reported to be used in enzyme-aided bleaching have varied between 30 and about 8300 nkat/g. However, an economically realistic dosage is about 100 nkat/g. The prices of enzymes are expected to decrease as more efficient production strains and technologies are adopted. Usually, the price of the enzyme is only compared with that of the competing bleaching chemicals. However, other facts, such as decreased AOX-loadings and retention of viscosity or other technical pulp properties may confer additional advantages on enzymes. In such cases it may be difficult to specify a price. In the future, all available technologies will complete with regard to both effectiveness and price. In 1992 more than 10 mills were reported to be using xylanases continuously for improved bleachability of kraft pulps. Almost 100 mill trials have been carried out, about half of them in Europe. The production of TCF pulps has increased dramatically during the past two years. The TCF-technologies applied today are often based on bleaching of oxygen-delignifled pulps with enzymes and hydrogen peroxide.
Increase in brightness Improved strength properties
O T H E R APPLICATIONS OF XYLANASES
Previously, the main aim in the application of hemicellulases was the total hydrolysis of hemicellulosic substrates for production of fermentable sugars. Today, most new applications are related to the pulp and paper industry. Water removal on the paper machine has been shown to improve as a result of limited hydrolysis of the fibres in recycled paper. Mixtures of cellulases and hemicellulases were found to decrease the SR-value, which describes the drainage behaviour of the fibres (Pommier et al., 1989). The effect is apparently due to improved fibrillation or change in the composition of fine particles. Mill trials have been carried out successfully using a commercial T. reesei enzyme preparation (Pommier et al., 1990). The enzymes responsible for these effects have not yet been identified. The chemical pulping processes have been designed to produce fibres with optimal properties for various purposes. High hemicellulose content of the pulp results in high yields and is generally regarded as an advantage with respect to the interfibre bonding properties in papermaking. Consistent with this, reduction in interfibre bonding has been observed after treatment of pulps with xylanases (Noe et al., 1986; Roberts et al., 1990). The desired structural changes in the fibre, which are brought about by beating and refining, are external fibrillation and fibre swelling, which improve the flexibility and bonding ability of the fibres. The role of xylans in the modification of fibre properties was studied using xylanases from Sporotrichum dimorphosporum in the treatment of fully bleached spruce sulphite and birch kraft pulps. Electron microscopic examination revealed external fibrillation and good flexibility of fibres, implying internal modification (Mora et al., 1986). The water retention value, which describes fibre swelling, was considerably increased. The conclusion was that xylan was hydrolyzed in the whole, delignified cell walls. The enzymatically-treated
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J. Buchert, M. Tenkanen, A. Kantelinen, L. Viikari
pulps were comparable to slightly beaten pulps. The beatability was generally enhanced and the energy demand was reduced about threefold (Noe et al., 1986). Attempts to remove hemicellulose for production of dissolving pulps with very low hemicellulose contents have shown that complete enzymatic hydrolysis of hemicellulose within the pulp is difficult to achieve. Even with very high loadings of enzymes, only relatively small amounts of xylans could be removed. The xylan content in delignified mechanical aspen pulp was reduced from approximately 20% to 10%, whereas in bleached hardwood sulphite pulp the xylan content was decreased from 4% to only 3"5% (Paice & Jurasek, 1984). The complete removal of residual hemicellulose seems thus unattainable, probably due to the inaccessible location of the substrate.
CONCLUSIONS The new, major, large-scale application area of xylanases is clearly in the pulp and paper industry, in order to increase the bleachability of kraft pulps. The improved bleachability is based mainly on the action of endo-fl-xylanases, which can be produced efficiently on an industrial scale. Partial hydrolysis of xylan facilitates the extraction of lignin from the pulp in higher amounts and with higher molecular masses. The enzymatic pretreatment method is applicable to any traditional or modern bleaching sequence at existing plants without significant investments. The primary goals of the enzymatic treatment have been to decrease chemical consumption, reduce environmental loading, and/ or to increase the final brightness of pulp. In the TCF bleaching sequences, enzymes can improve the brightness, which may otherwise remain below an acceptable level, without loss of viscosity. Enzyme-aided bleaching is thus both environmentally and economically advantageous. Previously, the main aim in the application of hemicellulases has been the total hydrolysis of hemicellulosic substrates for production of fermentable sugars. The knowledge gathered on the hydrolysis mechanism of hemicelluloses, especially xylans, has greatly promoted the rapid application of these enzymes in new areas. The general information already available on individual hemicellulases will help in the development of future applications.
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Bailey, M. J., Puls, J. & Poutanen, K. (1991). Purification and properties of two xylanases from Aspergillus oryzae. Biotechnol. Appl. Biochem., 13, 380-9. Bailey, M. J., Biely, P. & Poutanen, K. (1992). Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol., 23, 257-70. Bragger, J. M., Daniel, R. M., Coolbear, T. & Morgan, H. W. (1989). Very stable enzymes from extremely thermophilic archaebacteria and eubacteria. Appl. Microbiol. Biotechnol., 31,556-61. Buchert, J., Tenkanen, M., Pitkfnen, M. & Viikari, L. (1993). Role of surface charge and swelling on the action of xylanases on birch kraft pulp. TappiJ., 76, 131-5. Buchert, J., Kantelinen, A., Suurn~ikki, A., Viikari, L. & Janson, J. (199?). Evaluation of the effects of hemicellulases on the bleachability of sulphite pulps (submitted). Chauvet, J.-M., Comtat, J. & Noe, P. (1987). Assistance in bleaching of never-dried pulps by the use of xylanases, consequences on pulp properties. In Proc. 4th Int. Congr. on Wood and Pulping Chemistry, Vol. II, pp. 325-7. Clark, T. A., McDonald, A. G., Senior, D. J. & Mayers, P. R. (1990). Mannanase and xylanase treatment of softwood chemical pulps, effects on pulp properties and bleachability. In Biotechnology in the Pulp and Paper Manufacture, ed. K. T. Kirk & H.-M. Chang. ButterworthHeinemann, Boston, pp. 153-67. Clark, T. A., Steward, D., Bruce, M. E., McDonald, A. G., Singh, A. P. & Senior, D. J. (1991 ). In Proc. 45th APPITA Annual General Conference, Vol. I, pp. 193-200. Clayton, D. W. & Stone, J. E. (1967). The redeposition of hemicelluloses during pulping. Part l:The use of a tritiumlabelled xylan. Pulp Paper Mag. Canada, 64T, 459-68. Coughlan, M. P. (1992). Towards an understanding of the mechanism of action of main chain-hydrolyzing xylanases. In Xylans and Xylanases, ed. J. Visser, G. Beldman, M. A. Kusters-van Someren & A. G. J. Voragen. Elsevier, Amsterdam, pp. 111-39. Coughlan, M. P. & Hazlewood, G. P. (1993). fl-l,4-D-Xylandegrading enzyme systems: biochemistry, molecular biology and applications. Biotechnol. Appl. Biochem., 17, 259-89. Croon, I. & Enstr6m, B. (1962). The hemicelluloses in sulphate pulps from Scots pine. Svensk Papperstid., 65, 595-9. Dekker, R. E H. (1985). Biodegradation of the hemicelluloses. In Biosynthesis and Biodegradation of Wood Components, ed. T. Higuchi. Academic Press, Orlando, pp. 505-33. Eriksson, K.-E., Blanchette, R. A. & Ander, P. (1990). Microbial and Enzymatic Degradation of Wood Components. Springer-Verlag, Berlin. Farrell, R. L. & Skerker, P. S. (1992). Chlorine-free bleaching with Cartazyme T M HS treatment. In Xylans and Xylanases, ed. J. Visser, G. Beldman, M. A. Kusters-van Someren & A. G. J. Voragen. Elsevier, Amsterdam, pp. 315-25. Germg~trd, K. & Larsson, S. (1983). Oxygen bleaching in modem softwood kraft pulp mill. Paper Timber, 65, 287-90. Gellerstedt, G. & Lindfors, E.-L. (1991). On the structure and reactivity of residual lignin in kraft pulp fibers. In Proc. Int. Pulp Bleaching Conf., Vol. I. SPCI, pp. 73-88. Hartler, N. &Lund, A. (1962). Sorption of xylans on cotton. Svensk Papperstid., 65, 951-5. Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J., 280,309-16. Henrissat, B. & Bairoch, A. (1993). New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J., 293, 781-8. Hortling, B., Korhonen, M., Buchert, J., Sundquist, J. & Viikari, L. (1994). Leachability of lignin from kraft pulps after xylanase treatment. Holzforschung, 48, 441-6.
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Paice, M., Bernier, M. & Jurasek, L. (1988). Viscosity enhancing bleaching of hardwood kraft pulp with xylanase from a cloned gene. Biotechnol. Bioeng., 32,235-9. Paice, M. G., Gurnagul, N., Page, D. H. & Jurasek, L. (1992). Mechanism of hemiceUulose-directed prebleaching of kraft pulps. Enzyme Microb. Technol., 14,272-6. Pedersen, L. S., Kihlgren, P., Nissen, A. M., Munk, N., Holm, H. C. & Choma, P. P. (1992). Enzymatic bleach boosting of kraft pulp. Laboratory and mill scale experiences. Proc. Tappi, 1992 Pulping Conf., Boston, pp. 31-7. Perrolaz, J. J., Davis, S., Gysin, B., Zimmerman, W., Casimir, J. & Fiechter, A. ( 1991). Elemental chlorine free bleaching with a thermostable xylanase, a new alternative to elemental chlorine for bleaching. Proc. 6th Int. Symp. Wood and Pulping Chemistry, Vol. I, pp. 485-9. Pommier, J.-C., Fuentes, J.-L. & Goma, G. (1989). Using enzymes to improve the process and product quality in the recycled paper industry. Part I. The basic laboratory work. TappiJ., 72, 187-91. Pommier, J.-C., Goma, G., Fuentes, J.-L., Rousset, C. & Jokinen, O. (1990). Using enzymes to improve the process and product quality in the recycled paper industry. Part II. Industrial applications. TappiJ., 73, 197-202. Roberts, J. C., McCarthy, A. J., Flynn, N. J. & Broda, P. (1990). Modifications of paper properties by the pretreatment of pulp with Saccharomonospora viridis xylanase. Enzyme Microb. Technol., 12, 21 O- 13. Royer, J. C. & Nakas, J. P. (1991). Purification and characterization of two xylanases from Trichoderma longibrachiatum. Eur. J. Biochem., 202,521-9. Scallan, A. M. (1977). The accommodation of water within pulp fibres. In Proc. Fibre-Water Interactions in Paper Making. Pergamon Press, Oxford, pp. 9-27. Senior, D. J., Mayers, P. R., Miller, D., Sutcliffe, R., Tan, L. U. L. & Saddler, J. N. (1988). Selective solubilization of xylan in pulp using a purified xylanase from Trichoderma harzianum. Biotechnol. Lett., 10, 907-12. Sinner, M., Ditzelmuller, G., Wizani, W., Steiner, W. & Esterbauer, E. (1991). VAI-Biobleiche. Papier, 45,403-10. Sj6str6m, E. (1981). Wood Chemistry, Fundamentals and Applications. Academic Press, Inc., New York, 223 pp. Skerker, P. S., Farrell, R. L. & Chang, H.-M. (1991). Chlorine-free bleaching with Cartazyme HS treatment. Proc. 1991 Int. Pulp Bleaching Conf., Stockholm, pp. 93-105. Skjold-Joergensen, S., Munk, N. & Pedersen, L. S. (1992). Recent progress within the application of xylanases for boosting the bleachability of kraft pulp. In Biotechnology in the Pulp and Paper Industry, ed. M. Kuwahara & M. Shimada. UNI Publishers, Tokyo, pp, 93-100. Suurn~ikki, A., Kantelinen, A., Hortling, B., Buchert, J. & Viikari, L. (1993). Enzymatic solubilization of hemicelluloses in industrial softwood kraft pulps. Holzforschung (submitted). Tenkanen, M., Puls, J. & Poutanen, K. (1992a). Two major xylanases of Trichoderma reesei. Enzyme Microb. Technol., 14,566-74. Tenkanen, M., Buchert, J., Puls, J., Poutanen, K. & Viikari, L. (1992b). Two main xylanases of Trichoderma reesei and their use in pulp processing. In Xylans and Xylanases, ed. J. Visser, G. Beldman, M. A. Kusters-van Someren & A. G. J. Voragen. Elsevier, Amsterdam, pp. 547-50. Tolan, J. S. (1992). The use of enzymes to enhance pulp bleaching. Proc. Tappi, 1992 Pulping Conf., Boston, pp. 13-17. T6rr6nen, A., Mach, R. L., Messner, R., Gonzalez, R., Kalkkinen, N., Harkki, A. & Kubicek, C. P. (1992). The two major xylanases from Trichoderma reesei: characterization of both enzymes and genes. Bio/Technology, 10, 1461-5. Tsujibo, H., Sakamoto, T., Nishino, N. & Hasegawa, T. (1990). Purification and properties of three types of
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