FI89613C - Process for enzymatic treatment of cellulose pulp - Google Patents

Description

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Method for the enzymatic treatment of cellulosic pulps

The present invention relates to a process according to claim 1 for the enzymatic treatment of cellulosic pulps.

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Conventional chlorine bleaching uses chlorine or chlorine dioxide to dissolve lignin. Today, cellulosic pulp is often also bleached with oxygen gas, hydrogen peroxide, or combinations of these and the aforementioned bleaching chemicals. These conventional bleaching methods have combined enzymatic treatments with hemic-10 lulases and ligninases in some prior art solutions to provide improved fiber bleaching. The amounts of enzyme required are small to achieve the bleaching effect and the enzyme treatment can be easily incorporated into the pulping process.

Prior art enzymatic treatments have been performed directly on the fibers from the cooking. However, in our studies, we have been able to state that the fiber properties of cellulose pulp have a significant effect on the operating conditions of enzymes. Thus, for example, xylanases function poorly when the surface charge of the fiber i.e. zetapotentiaa-li is very negative. We have also found that low pH treated sulphate pulp, whose hemicellulose carboxyl groups are in acid form and thus do not contain metal counterions, is poorly suited as a substrate for enzymes (hemicellulases).

However, prior art enzyme treatment solutions have not addressed these issues and the prior art has not sought to change the state of the fibers to make the fibers more suitable for enzymatic treatment.

It is known that the surface charge of a cellulosic pulp, i.e. pulp, is mainly affected by the carboxyl groups it contains (Sjöström 1989). The amount of these depends essentially on the method of cooking the pulp. Thus, the carboxyl groups of the sulphate pulp (as well as the mechanical pulp) consist mainly of the methylglucuronic acid groups of the xylan in the pulp (Sjöström 1989). Instead, the carboxyl groups of the sulphite pulp consist not only of the methylgluconic acids of xylan but also of the sulphonic acids formed in the lignin in the boil.

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The charge intensity and degree of dissociation of carboxyl groups vary depending on the form of the counterion of the carboxyl group (Scallan and Grignon 1979, Scallan 1983, Lindström and Carlson 1982). Usually the counterion is a hydrogen or metal ion, with a large proportion - in fact most - of the metal ions within the mass bound to the carboxylic acid groups. This applies to both chemical and mechanical pulps.

The metal ions in the cellulosic pulp can be an advantage or a disadvantage for the treatment of the pulp. As mentioned above, sulphate pulp lacking metal counterions in the carboxylic acid groups of hemisululoses is poorly suited for enzyme treatment. On the other hand, the metals contained in the pulp 10 are in many respects undesirable for further processing of the pulp. Thus, many metal ions, especially iron and manganese, are detrimental to peroxide stability in peroxide bleaching and must be removed to achieve optimal bleaching results.

According to current methods, the metal content of the fiber is reduced either by lowering the pH of the pulp sufficiently to dissociate the metals, or by treating the pulp with known complexing agents, e.g., DTPA or EDTA (Basta et al. 1991). However, there are significant drawbacks to these solutions. Thus, removal of metal under acidic conditions gives a pulp which is difficult to carry out 20: efficient enzyme treatment during bleaching. The use of complex forms that are foreign to nature may again cause problems, for example in connection with biological wastewater treatment.

The object of the present invention is to obviate the drawbacks associated with the prior art and to provide a completely new method for treating cellulosic pulps.

Our invention is based on the idea that by acting on the carboxylic acid groups of the hemisiluloses contained in the cellulose pulp, not only the surface-30 charge of the cellulose pulp but also its metal content can be changed. According to the invention, the carboxylic acid groups are preferably removed from the pulp by treating the pulp with an enzyme preparation having substantial glucuronidase activity.

More specifically, the method according to the invention is mainly characterized by what is set forth in the characterizing part of claim 1.

For the purposes of this application, "enzyme preparation" means any S product containing at least one enzyme. Thus, the enzyme preparation may be e.g.

a culture medium containing the enzyme or enzymes, an isolated enzyme or a mixture of two or more enzymes.

By "substantial glucuronidase activity" is meant that the glucuronid activity of the enzyme preparation 10 is reasonably high relative to any other enzyme activities of the enzyme preparation to cleave a substantial portion of the glucuronic acid groups from the substrate.

According to a preferred embodiment of the invention, it is an object that by specifically changing the amount of carboxyl groups by means of glucuronidase, the optimal activity of the enzymes in the fiber can be regulated without cleaving the other hemicellulose of the fiber.

The higher the relative activity of glucuronidase in the enzyme preparation used, the easier it will be to achieve this goal.

It is known that some enzyme preparations used as pretreatments for bleaching contain glucuronidase, which may have the effect of promoting the hydrolysis of hemicelluloses. In this case, however, their use has been based specifically on the improvement of the hydrolysis result of xylan containing glucuronic acid groups (Kantelinen et al. 1988, Poutanen et al.

1987). However, the prior art does not aim for a specific carboxyl group; -25 not for removal but for more efficient total hydrolysis of hemicelluloses. The specific removal of carboxyl groups according to the invention could not have been achieved with the enzyme preparations used; the relative glucuronidase activity of these preparations is too low.

: 3C * According to the invention, the glucuronidase treatment can be performed either simultaneously with xylanase or the like.

with or before hemicellulase treatment. When the glucuronidase treatment is performed in combination with another hemicellulase treatment, it is most preferred to use an enzyme preparation to which either glucuronidase activity has been added or the production strain has been genetically modified to produce more glucuronidase to provide a preparation with substantial glucuronidase activity. The glucuronidase treatment according to the invention can also be combined with cellulase and / or ligninase treatments known per se.

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It is particularly preferred to carry out the glucuronidase treatment before bleaching the pulp, possibly combined with a standard xylanase treatment. In this way, the consumption of the bleaching chemical can be further reduced. Since glucuronidase lowers the metal content of the pulp, hydrogen peroxide can be advantageously used as a bleaching chemical.

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The process according to the invention is also suitable for use in the treatment of lignin-containing pulp, i.e. mechanical or chemimechanical pulp. In our experiments, we have surprisingly been able to find that glucuronidase treatment can reduce the post-yellowing of such a mass.

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According to a preferred embodiment, a culture medium of a microorganism producing a glucuronidase enzyme is used as the enzyme preparation. According to another equally advantageous alternative, an enzyme isolated from the culture medium of the microorganism is again used as the enzyme preparation.

According to a particularly preferred embodiment of the present invention, the glucuronidase enzyme is separated from the culture medium of THchoderma reesei using anionic ion exchangers and hydrophobic interaction chromatography. With these separation methods, the enzyme can be isolated surprisingly simply and easily.

The resulting α-glucuronidase enzyme has a molecular weight of about 95 kDa as determined by SDS-PAGE. It is well suited for use in the method of the invention.

However, the invention is not limited to the enzyme separation method shown, but it is also possible to separate the enzyme in question using other known methods.

It is also possible to produce a suitable glucuronid enzyme with strains of Trichodema reesei, genetically or mutatively enhanced to produce the exact enzyme desired, or with other genetically modified production hosts into which the gene encoding this enzyme has been transferred. Glucuronidase can also be isolated from other strains, such as Thermoascus auranticus (Khandke et al. 1989), Agaricus Bispo-5 rus, Aspergillus awamori, Aspergillus niger, Schizophyllum commune, Tyromyces palustris, S. Streptomyces flivogriseus, spp., Thermobacter auranticus (Poutanen et al. 1991), Aureobasidium pullulans, Aspergillus oryzae, Bacillus circulars and Bacillus subtilis.

All of these strains of microorganisms yield glucuronidase, which cleaves in particular the glucuronic acid side groups of the polymeric xylan chain. Treatment with xylanase enzymes alone removes xylan fragments which, depending on the composition of the xylan, may contain glucuronic acid groups. However, this treatment alone is not effective or specific enough to substantially reduce glucuronic acid groups. By combining gluco-15 ronidase treatment with xylanase treatment, branched xylan chains can be cleaved into their moieties. In this case, however, the predominantly xylan-removing glucuronidase acts to promote the complete hydrolysis of the xylan fragments detached from the fiber to xylose. However, the removal of xylan is not an object of the present invention, although it may be included in this treatment.

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The invention provides considerable advantages. Thus, treatment of pulp with glucuronidase in accordance with this invention, either before or after enzymatic treatment (e.g., xylanase treatment), causes the level of hydrolysis to increase and the xylanase treatment can be performed at low pH. Again, however, the potency of the enzyme is based on the removal of specifically charged groups rather than the complete hydrolysis of hemicellulose, as in the prior art. The effect of the method according to the invention is based on the fact that by removing the glucuronic acid groups enzymatically, the surface charge of the fiber can be changed to a form which is advantageous for the enzyme. Em. by modifying the factors, it can be influenced which part of the fiber is most appropriately affected by the enzymes. These factors, which have not been considered at all in previous studies, may influence how the lignin in the fiber can most easily be extracted from the fiber. Thus, the regulation of these factors can also directly affect the type and amount of chemicals 8961 3 6 used to industrially extract lignin from fiber and thus further improve environmentally friendly low-chlorine or chlorine-free bleaching methods.

It has further surprisingly been found in the present invention that the treatment of the pulp with glucuronidase can facilitate the removal of metals by removing the metal-binding glucuronic acid groups, leaving the removal of metals required for peroxide bleaching either completely or partially unnecessary. This further reduces the environmental impact of peroxide bleaching.

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It is also typical of the process according to the invention that the paper technical properties of the mechanical and chemical pulp can be modified by means of the glucuronidase treatment.

The number of carboxyl groups in the pulp and the associated counterions affect the charge of the pulp. These factors can be imaged by a variety of known chemical and physical assay methods. The surface charge of a mass can be measured by zeta potential (Melzer 1972). The metal content of the pulp can be measured by analyzing the metals in the pulp by atomic absorption spectrometry. The carboxyl group content of the pulp can be measured, for example, by the Sjöström method (KCL method 192: 68). The activity of the enzymes in the fiber is described by both the sugars released from the fiber and the lignin fragments extracted therefrom, as well as the bleaching result that can be achieved with the enzymes when the surface charge of the fiber, i. the zeta potential and the carboxyl group content of the fiber have been obtained in the desired form.

The invention will now be considered in light of a few non-limiting examples.

Example 1. Production of glucuronidase in certain microbial strains 30 Aspergillus oryzae VTT-D-85248, Trichoderma reesei Rut C-30 and Aureobasidium pullu 8 9 61 3 7

Ians VTT-D-89397 (NRRL Y-2311-1) strains were grown in medium containing 20 g / l xylan (Roth), 20 g / l corn steepwater and nutrients. Glucuronidase activity was measured from the growth solution. The activities obtained were 250 pkat / ml, 1000 pkat / ml and 100 pkat / ml, respectively. The results show that glucuronidase can be produced by conventional hemicellulase-5 glass production organisms.

Example 2. Isolation and characterization of T. reesei glucuronidase

Isolation of the enzyme was initiated by buffering Trichoderma reesei growth solution by gel-10 filtration to pH 5.7 (Sephadex G-25). At this pH, the enzyme was bound to an anion exchange chromatography column (DEAE-Sepharose FF), and the proteins adhering to the column were eluted with pH 5.7 buffer supplemented with sodium chloride to form a linear concentration gradient from 0 to 0.15 M. fractions.

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Enzyme-containing fractions were further purified by hydrophobic interaction chromatography column (Phenyl Sepharose FF). The enzyme was bound e.m. to the material at a salt concentration of 0.8 M (NH 4) 2 SO 4. The enzyme adhering to the column was eluted with pH 5.6 buffer by adding (NH 4) 2 SO 4 to form a decreasing linear concentration gradient between 0.8 and 0.4 M. The purified enzyme was eluted from the fractions collected during the gradient and concentrated by ultrafiltration ( Amicon, PM-10 membrane).

:; : The α-glucuronidase activity of the enzyme has been determined using the method described by: -25 by Puis et al. (1987). The method measures the amount of methyl glucuronic acid released by the enzyme from 4-O-methyl-glucuronosylxylobiose by chromatography. High molecular weight xylan with glucuronic acid substituents (Roth xylan) has also been used as a substrate in the experiments.

The protein properties of the purified enzyme were determined by standard protein chemistry methods. These properties are described in Table 1.

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Table 1. Properties of Trichoderma reesei α-glucuronidase

Property Value Unit 5

Molecular weight (SDS-PAGE) 95 kDa

Molecular weight 88 kDa in gel filtration Specific activity 10 - as substrate 4-O-methyl approx. 100 nkat / mg prot.

glucuronosylxylobiose pH optimum 3-5 15 Example 3. Removal of carboxyl groups of sulphate pulp with glucuronidase Pine and birch sulphate pulp are treated with glucuronidase at pH 5 at a consistency of 5%. The treatment time is 5 hours and the enzyme dose is 1000 nkat / g. After the glucuronidase treatment, the pulp is washed with distilled water and the carboxyl group content of the pulp is determined. Massas-20 ta also measure surface charge (zeta potential) and metal content. From the results, it can be stated that the glucuronidase treatment is able to reduce the number of carboxyl groups in the pulp, the surface charge of the pulp and the metal content of the pulp.

Example 4. Peroxide bleaching of glucuronidase-treated sulphate pulp Pine sulphate pulp is treated with glucuronidase as in Example 3. The treated and untreated pulp is bleached with a 1-step peroxide sequence (3% hydrogen peroxide, 1.5% NaOH, 0.2% MgSO 4, 10% sake, 10% s C). The residual peroxide, the brightness of the pulp and the number of pieces after bleaching are measured from the pulp. Glucuronidase treatment has a clear effect on peroxide stability, viscosity and brightness. The reference mass is the untreated mass and the complexed mass (0.2% DTPA, 30 min, 5% consistency, 50 ° C).

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Example 5. Xylanase treatment of a glucuronidase-treated pulp Pine sulphate pulp is treated with glucuronidase separately or simultaneously with a xylanase treatment as in Example 3. Xylanase-5 purified from T. reesei (Tenkanen et al., Submitted for publication) is used as the xylanase. The pulp is bleached with the D / CEDED sequence.

Untreated pulp and xylanase-only pulp are used as a reference for the enzyme treatment, and after hydrolysis the reducing sugars released during the treatment are measured. It is found that glucuronidase treatment promotes xylanase activity as measured by reducing sugars. This can also be seen as the brightness of the pulp 10 measured after bleaching. It is noted that when glucuronidase treatment is performed before xylanase treatment, the result is as good as when glucuronidase and xylanase are used concomitantly.

Example 6. Improvement of hydrolysis of acidic pulp by glucuronidase 15 Pine sulphate pulp is washed with sulfuric acid at pH 4 for 1 hour. After the treatment, glucuronidase is added to the pulp and the treatment is continued for 2 hours. The enzyme dose is 500 nkat / g. After the glucuronidase treatment, the carboxyl content is measured from the pulp. The glucuronidase-treated pulp is hydrolysed with T. reesei xylanase (100 nkat / g) for 2 h and the ‘reducing sugars’ are measured. Compared to acid-washed pulp alone, glucuronidase-20 treatment is able to improve xylanase activity in the fiber.

Example 7. Effect of glucuronidase on mechanical and chemical pulp papermaking. . properties; -25 Mechanical pulp and sulphate pulp are treated with glucuronidase as in Example 3.

It is found that the treatment can affect the paper properties, tensile strength and bonding ability of the pulps.

Example 8. Effect of glucuronidase on post-yellowing of mechanical pulp

CTMP and PGW pulp suspensions were treated with Agaricus bisporus glucuronidase 0.2 M

3961 3 ίο in sodium acetate buffer (pH 5) at 40 ° C for 20 hours. After the treatment, 95cm2 sheets were made from the pulp. The sheets were exposed (at 27 ° C and 47% relative humidity) for three hours in a Xenotest ISO S (Herauas Hanau) with a xenon lamp. The reflectance of the pulp sheets was measured before and after exposure at 457 nm (brightness) as a filter. 5 PC values describing post-emergence were calculated as presented by Jansson and Forskäl (1989). Glucuronidase treatment was found to reduce post-accumulation of both pulps.

Table 2. Effect of glucuronidase treatment on post-yellowing of PGW pulp

10 Handling Ro R, R, PCI PC2 PC

buffer 59.7 60.3 56.85 -0.53 3.31 2.78 glucuronidase 10 59.7 60.55 57.15 -0.75 3.21 2.46 nkat / g glucuronidase 100 59.7 60 , 35 57.45 -0.57 2.73 2.16 15 nkat / g

Table 3. Effect of glucuronidase treatment on post-yellowing of CTMP pulp

20 Handling Ro R, R, PCI PC2 PC

buffer 57.7 57.6 54.25 0.1 3.68 3.78 glucuronidase 10 57.7 57.55 54.45 0.15 3.39 3.54 nkat / g glucuronidase 100 57.7 58.35 55.15 -0.65 3.38 2.73 25 nkat / g 8 9 61 ό 11

References:

Kantelinen A, Rättö M, Sunquist J, Ranua M, Viikari L and Linko M (1988) 5 Hemicellulases and their potential role in bleaching, TAPPI PRoc 1988 Int Pulp

Bleaching Conf, April 1988, ss. 1-9.

Melzer I (1972) Zeta-potential und Seine Bedeutung bei der Derienung von Papier. Das Papier 26 (7): 305-332.

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Poutanen K, Rättö M, Puls J and Viikari L (1987) J. Biot. 6: 49-60.

Puls, J., Schmidt, O. & Granzow, C., Enzyme Microb. Technol. 1987, 9: 83-88.

15 Lindström T and Carlsson G (1982) The effect of chemical environment on fiber swelling.

Svensk Papperstidn 85 (3): R14-20.

Scallan AM and Grignon J (1979) The effect of cations on pulp and paper properties. Svensk Papperstidn 1979: No. 2: 40-47.

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Scallan (1983) The effect of acidic groups on the swelling of Pulps: a review. Tappi Journal 66 (11): 73-75.

Sjöström E (1989) The origin of charge on cellulosic fibers. Nordic Pulp and Paper 25 Research Journal 4 (2): 90-93.

Sjöström E, Carboxyl content of pulp, KCL method 192: 68

Buchert J and Viikari L (1991) Method for the enzymatic treatment of lignocellulosic materials in particular: 30 cellulose pulps. Finnish Pat. hak. 914780

Poutanen K, Tenkanen M, Korte H, and Puls J (1991) Accessory enzymes involved in the 8961 3 12 hydrolysis of xylans. ACS Symp. Ser 460, Enzymes in Biomass Conversion, ss. 426-436.

Khandke KM, Vithayathil PI, and Murthy SK (1989) Arch Biochem Biophys 274: 511-517.

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Basta J, Holtinger L, and Hook J (1991) Controlling the profile of metals in the pulp before hydrogen peroxide treatment. Proc. 6th Int. Symp. Wood and Pulping Chemistry, Melbourne 1991, p. 237-244.

10 Jansson J and Forsskähl I (1989) Color changes in lignin rich Pulps on irradiation by light. Nordic Pulp and Paper Research Journal no 3/1989: 197-203.

Claims (13)

13 K961i A method for the mechanical treatment of cellulose pulp, characterized in that the pulp is contacted with an enzyme preparation having substantial glucuronidase activity to at least partially remove the glucuronic acid groups of the pulp. Process according to Claim 1, characterized in that an enzyme preparation produced with the microorganism strain Thermoascus auranticus is used. Process according to Claim 2, characterized in that a growth solution of a glucuronidase-producing microorganism is used as the enzyme preparation. Process according to Claim 2, characterized in that an enzyme isolated from a culture medium of the microorganism is used as the enzyme preparation. A method according to claim 4, wherein an enzyme preparation is used which: has been produced with the microorganism strain Trichoderma reesei, characterized in that. The enzyme with .25 α-glucuronidase activity is separated from the culture medium by an anionic ion exchanger and hydrophobic interaction chromatography. Process according to Claim 1 or 5, characterized in that the cellulose pulp is treated with the enzyme α-glucuronidase, which has a molecular weight of about 95 kDa as determined by the SDS-PAGE method. . Process according to one of the preceding claims, characterized in that the pulp 8961 8 is first treated with an enzyme preparation having substantial glucuronidane activity and then contacted with an enzyme preparation having hemicellulase, cellulase and / or ligninase activity. Process according to one of Claims 1 to 6, characterized in that the pulp is treated simultaneously with an enzyme preparation having substantial glucuronidase activity and an enzyme preparation having hemicellulase, cellulase and / or ligninase activity. Process according to Claim 8, characterized in that the pulp is treated with an enzyme preparation having at least substantial glucuronidase activity and xylanase activity. A method according to claim 1, characterized in that the treatment of the cellulose pulp with said enzyme preparation is performed before the bleaching of the pulp. 10 Agaricus bisporus, Aspergillus awamori, Aspergillus niger, Schizophyllum commune, Tyromyces palustris, Streptomyces flavogriseus, S. olivochromogenes, Streptomyces spp., Thermobacter auranticus, Aureobasidium pullulans, Aspergillus oryza, from a strain bred from organisms or any other productive host to which a glucuronidase-producing gene has been added. A method according to claim 10, characterized in that the cellulose pulp is treated with said enzyme preparation before peroxide bleaching to reduce the metal content of the pulp. 20 A method according to claim 1, characterized in that the lignin-containing cellulosic pulp is treated with said enzyme preparation to reduce the post-yellowing of the pulp. Process according to one of the preceding claims, characterized in that the enzyme used is a glucuronidase which in particular cleaves the glucuronic acid side groups of the polymeric xylan chain. 8961 3