EP0615564A1

EP0615564A1 - Method and enzymatic preparation for treatment of cellulose pulps

Info

Publication number: EP0615564A1
Application number: EP92924742A
Authority: EP
Inventors: Johanna Buchert; Matti Siika-Aho; Marjaana Rättö; Liisa Viikari; Michael Bailey
Original assignee: Valtion Teknillinen Tutkimuskeskus
Current assignee: Valtion Teknillinen Tutkimuskeskus
Priority date: 1991-12-05
Filing date: 1992-12-07
Publication date: 1994-09-21
Also published as: FI915755A0; JPH07501587A; FI89613B; FI89613C; WO1993011296A1; NO942071L; AU3088292A; NO942071D0; BR9206860A; CA2125166A1

Abstract

Procédé de traitement enzymatique de matières lignocellulosiques, notamment les pâtes de cellulose. Selon le procédé, on met les matières en contact avec une préparation enzymatique présentant essentiellement une activité de glucuronidase, afin d'éliminer les ions métalliques de la pâte, d'en modifier la charge superficielle, et d'améliorer l'aptitude de ces matières au traitement enzymatique.Process for the enzymatic treatment of lignocellulosic materials, in particular cellulose pulps. According to the method, the materials are brought into contact with an enzyme preparation essentially exhibiting glucuronidase activity, in order to eliminate the metal ions from the dough, to modify the surface charge, and to improve the ability of these materials to enzymatic treatment.

Description

METHOD ANDENZYMATIC PREPARATIONFORTREATMENT OFCELLULOS PULPS

The present invention concerns a method in accordance with the preamble of claim 1 for treatment of lignocellulosic material, in particular cellulose pulps.

The invention also concerns an enzymatic preparation which is useful for pulp treatment according to the preamble of claim 20.

In traditional chlorine bleaching the residual lignin is solubilized by using chlorine or chlorine dioxide. Presently cellulose pulps are frequently also bleached by oxygen gas, hydrogen peroxide, ozone or by combined sequences including these substances as well as the above-mentioned traditional bleaching chemicals. Enzymatic treatments, carried out by hemicellulases or lignin degrading enzymes, have been combined with the traditional and new bleaching sequences leading to increased bleachability of the fibres. The amounts of enzymes needed to achieve the improved bleachability are low, and the enzymatic treatment can easily be incorporated into the pulp production processes.

According to the prior art, enzymatic treatments have been performed directly on fibres from the pulping processes. According to our results we have found out that the properties of the cellulosic fibres profoundly affect the possibilities of enzymes to act efficiently on the pulps. Thus, e.g. xylanases act unoptimally when the surface charge, i.e. the zeta potential is very low. We have also observed that kraft pulps, treated at a low pH-value, at which the carboxylic groups of the hemicelluloses present in the pulp are in an acid form and do not thus contain metal counter ions, are very poor substrates for enzymatic (hemicellulase) treatments.

In the prior art methods, these factors have not at all been considered, nor has any attempt been made to modify the state of the fibres to make them more suitable as substrates for enzymatic treatment.

It is known that the carboxylic groups present in cellulose pulps affect mainly the surface charge of the pulp (Sjδstrom 1989). The number of these groups depends essentially on the pulping method used. Thus, the carboxylic groups in kraft as well as in mechanical pulps consist mainly of methylglucuronic acid groups in xylan (Sjδstrom 1989). However, in sulphite pulps, in addition to the methyl glucuronic acid groups of xylan, the carboxylic groups comprise sulphonic acids present in lignin, formed during the sulphite pulping process.

The strength and degree of dissociation of the carboxylic groups depends on the counter-ion of the carboxylic group (Scallan and Grignon 1979, Scallan 1983, Lindstrδm and Carlsson 1982). Usually the counter-ion is hydrogen or a metal ion. A great part, in fact most of the metals present in the pulps are bound to these carboxylic groups. This is the case both with chemical and mechanical pulps.

The metal-ions of cellulose pulps may be either useful or harmful to pulp processing. As mentioned earlier, a kraft pulp which is deficient in metal counter-ions of the carboxylic groups is poorly suited to enzymatic treatments. On the other hand, the metal-ions present in the pulps may in many respects be undesirable for the processing of cellulose pulps. Thus, many metal-ions, especially iron and manganese, are detrimental in the peroxide treatment and cause instability of the peroxide. These substances must be removed in order to achieve an optimal bleaching result. The removal of metal-ions is also essential in the production of certain special pulps.

According to known methods, the metal content of pulps can be decreased either by lowering the pH of the pulp to a value, at which the metal-ions are dissociated, or by treating the pulps with known complexing agents, such as DTPA or EDTA (Basta et al. 1991). However, considerable drawbacks are associated with these methods. Thus, after metal removal under acidic conditions, a pulp is formed which is very resistant to the enzymatic treatment prior to bleaching. Some of the unnatural complexing agents may, on the other hand, cause problems e.g. in the waste water purification systems.

It is an aim of the present invention to eliminate the drawbacks of the existing techniques and to develop a completely new concept for the treatment of cellulosic pulps. The invention is based on the idea that by enzymatically removing the carboxylic groups of hemicelluloses from the cellulose pulps, both the surface charge and the metal-ion centent of the pulp can be changed. According to the method of the present invention, the carboxylic groups are therefore removed by treating the cellulose pulp with an enzyme preparation, having an essential glucuronidase enzyme activity.

In particular, the method according to the present invention is characterized by what is stated in the characterizing part of claim 1.

The enzyme preparation according to the present invention is characterized by what is stated in the characterizing part of claim 20.

Within the scope of the present application, the term "enzyme preparation" denotes any product which contains at least one enzyme. Thus, such an enzyme preparation may be a culture liquor containing one or more enzymes, an isolated enzyme or a mixture of one or more enzymes.

The term "essential glucuronidase activity" means that the glucuronidase activity of the enzyme preparation is comparatively high when compared with the other enzyme activities in the preparation, in order for it to remove an essential part of glucuronic acid groups from the substrate.

"Glucuronic acid groups" used in this application is an abbreviation of 4-O-methyl-D- glucuronic acid groups.

It is known that some enzyme preparations, which have been used in pretreatment steps for improving pulp bleachability, contain some glucuronidase activity which may promote the hydrolysis of hemicelluloses. The aim of these known methods has been to improve the hydrolysis of xylan containing methylglucuronic acid groups (Kantelinen et al. 1988, Poutanen et al. 1987). However, specific enzymatic removal of the carboxylic groups has not been sought, but rather a more efficient total hydrolysis of hemicelluloses. The specific removal of carboxylic groups, according to the method of the present invention, would not even have been possible with the enzyme preparations previously used, due to their relativel low glucuronidase activity.

It is also known in the art that enzymatic hydrolysis of hardwood xylans can be enhanced by glucuronidase treatment; Ishihara et al. describe the synergistic effect of xylanase, β- xylosidase, and α-glucuronidase on the hydrolysis of 4-O-methylglucuronoxylan (Ishihara et al. 1990). From the expermiental results presented in the prior art it appears that the relative hydrolysis rate of xylan is increased from the 63 % obtained with xylanase to 74 % with xylan together with glucuronidase, 100 % denoting the combined effect of xylanase, xylosidase and glucuronidase. However, these results relate to hydrolysis of isolated xylans only. Thr prior art publication is completely silent about the application of glucuronidase to industrial pulps and to the modification of the metal content and surface charge of such pulps.

According to the method of the present invention, the paper technical properties of pulps can be modified by enzymatically removing the carboxylic groups of the pulps.

According to one preferred embodiment, the glucuronidase treatment is conducted before the bleaching stage of cellulose pulp. In this way, the consumption of bleaching chemicals can be decreased. Because the glucuronidase treatment decreases the metal-ion content of pulps, hydrogen peroxide can be advantageously used as the bleaching chemical.

According to another prefered embodiment, the amounts of carboxylic groups in the pulp are modified by using glucuronidase in such a way that the action of enzymes on fibre materials can be optimized essentially without any hydrolyzation of the hemicelluloses in the fibres.

The higher the relative glucuronidase activity of the enzyme preparation is, the easier it is to reach this goal. Thus, the bleachability of TCF-puIps (totally chlorine free pulps) can be increased.

The glucuronidase treatment can be conducted separately, simultaneously with another enzymatic treatment, or before such a treatment. When the glucuronidase treatment is carried out together with a hemicellulase (e.g. xylanase, mannanase) treatment, it is preferred to use an enzyme preparation to which glucuronidase has been added, or which has been produced by a strain genetically improved to produce high glucuronidase activity, in order to obtain a preparation with an essentially high glucuronidase activity. According to the invention, the glucuronidase treatment can also be combined with other cellulase and/or ligninase treatments.

In addition to chemical pulps, the method of the invention is also suitable for the treatment o any lignocellulosic pulps, i.e. mechanical or chemimechanical pulps. Thus, in our studies, we have unexpectedly found out that the brightness reversion of pulps can be decreased by a glucuronidase treatment.

According to one preferred embodiment of the invention, the enzyme preparation used comprises the cultivation liquid or medium of a glucuronidase-producing microorganism. Preferably, such a cultivation medium is concentrated before use. According to another preferred embodiment, the enzyme preparation comprises a purified enzyme, isolated from a cultivation liquid.

According to still a further preferred embodiment, the glucuronidase enzyme is isolated from the cultivation liquid of the fungus Trichoderma reesei using anionic ion exchangers and purified by hydrophobic interaction chromatography. It has unexpectedly turned out that the enzyme can be easily isolated by means of these methods. The purified glucuronidase, which is new, has a molecular weight of about 95 kDa according to the SDS-PAGE method. The glucuronidase can also be isolated by simple methods from cultivation liquids of Schizophyllum commune or Aspergillus niger strains. These enzymes, which also are new, are described in more detail in working examples 3 and 4.

The enzyme preparation useful for treatment of lignocellulosic materials comprises an essential glucuronidase enzyme activity and contains only minor amounts, if any, of hemicellulases.

The invention is not, however, limited to the indicated origins of the enzyme nor to the isolation method, and the enzyme can also be obtained by other methods. Thus, it is possible to produce the glucuronidase enzyme by strains of the genus Trichoderma or other microorganisms, which have been mutated or genetically constructed to produce the desired enzyme, or by other production host strains, to which the gene encoding this enzyme has been transfered.

The glucuronidase preparation can be derived from a microorganism strain selected from the group essentially consisting of microorganisms of the genera Trichoderma (e.g. T. reesei), Aspergillus (e.g. A. niger, A. awamori, A. terreus, A. oryzae), Schizophyllum (e.g. S. commune), Aureobasidium (e.g. A. pullulans), Phanerochaete (e.g. P. chrysosporium), Fusarium (e.g. F. oxysporum), Agaricus (e.g. A. bisporus), Penicillium (e.g. P. janthinellum, P. digitatwn), Streptomyces (e.g. S. olivochromogenes, S. βavogriseus), and Bacillus (e.g. B. subtilis, B. circulans). It can also be derived from a microorganism strain selected from the group comprising Thermoascus auranticus, Curvularia inequalis, Tyromyces palustris, Cryptonectria parasitica, Myceliophthora thermophila, and Thermobacter auranticus.

All these microorganisms can be used to produce glucuridases, which hydrolyze the glucuronic side groups of xylan.

According to a preferred embodiment, the enzyme preparations are prepared by cultivating on a cultivation medium comprising xylans any of the above-mentioned glucuronidase- producing microorganisms.

The method according to the invention provides remarkable advantages. Thus, by treating pulp with a glucuronidase as described herein prior to or simultaneously with the treatment of. the pulp with another enzyme (e.g. xylanase), the degree of hydrolysis can be increased and the xylanase treatment can be performed at a low pH-value. Also in this case, however, the effect of the treatment is based specifically on the removal of charged groups and not on the. total hydrolysis of hemicelluloses. The effect of the present method is based on the enzymatic removal of glucuronic acid groups in order to change the surface charge into an advantageous form with respect to further treatments either chemical or enzymatical. By changing the described factors (such as surface charge) the action of, e.g., enzymes to affect the most advantageous parts of the fibre substrate can be regulated. These factors, which previously have not at all been considered, can be manipulated in order to achieve most efficient extraction of residual lignin from the fibres. Thus, the regulation of these factors with the method of invention also affects directly the type and amount of chemicals to be used for the industrial scale extraction of lignin from the fibres and can be further used to improve the low-chlorine or chlorine-free bleaching methods, thus reducing environmental pollution.

By treating the pulp with a glucuronidase enzyme the metal binding glucuronic acid groups can be hydrolyzed, which will enhance the removal of the metal-ions of the pulp.

Unexpectedly it has now been found that the present enzyme treatment will make the chemical removal of metal-ions superfluous. This decreases the already low environmental impact of the peroxide or ozone bleaching.

The enzymatic removal of glucuronic acid groups can be used to improve the production of certain pulps, such as metal-free pulps.

It is also typical for the method of invention that the paper technical properties of the chemical and mechanical pulps can be modified by using the glucuronidase treatment.

The method of invention is described in the following by some non-limiting examples.

As mentioned above, the number of carboxylic groups and of counter-ions bound to them affect the electric charge of the pulp. These factors can be described by different chemical and physical parameters and the surface charge of fibres (pulps) can be measured with the zeta-potential (Melzer 1972). The metal content of pulps can be measured by analyzing the metals in pulp with an atom absorption spectrophotometer. The carboxylic acid content of pulps can be measured e.g. by the method of Sjόstrόm (KCL method 192:68). The action of enzymes in the fibres can be described by the liberation of sugars and by the extractability of lignin fragments after the enzymatic treatment. Another method is to measure the increase o brightness, which can be obtained after the enzymatic treatment which changes the surface charge, i.e. converts the zeta-potential and the carboxylic acid content into a desirable form. Example 1. Production of α-glucuronidase by different fungal strains.

A total of 18 different fungal strains were tested for their production of α-glucuronidase. Medium A contained, in g l^"1: wheat bran 10, Solka floe cellulose 10, distiller's spent grain

10, KH₂PO₄ 5 and (NH₄)₂SO₄ 5. This medium composition was considered suitable in general for the production of enzymes degrading lignocellulosic materials. Medium B contained, in g l^"1: beech xylan (Lenzing AG, Austria) 10, birch glucuronoxylan (Roth 7500) 5, oligo- saccharides from steaming of birchwood 5, distiller's spent grain 10 and mineral salts as in medium A. This medium was considered potentially advantageous for the production of α- glucuronidase because of the presence of glucuronic acid-substituted xylo-oligosaccharides and xylans in the substrate.

The screening cultivations were performed in 250 ml shake flasks containing 50 ml medium. Cultivation was carried out for 4 days, but samples were also taken from the cultivations after 2 days. The thermophilic fungus Myceliophthora thermophila was cultivated at 37 °C and all the other strains at 30 °C. α-glucuronidase was assayed in the culture supernatants by incubating 100 μ\ with 900 μ\ 1 % glucuronoxylan (Roth 7500) at pH 4.8 (0.05 M Na- acetate buffer) for 24 hours at 40 °C and measuring the organic acids produced as glucuronic acid using the method of Kandke et al (1989). The results were expressed as relative production units on the basis of the spectrophotometric absorbance of the reaction mixtures at 660 πm. The results of the screening test for production of α-glucuronidase are presented in Table 1.

Table 1. Production of or-glucuronidase by different fungal strains in shake flasks

STRAIN

Aspergillus oryzae VTT-D-85248

A. awamori VTT-D-75028 A. niger VTT-D-85240 A. niger VTT-D-79106 A. niger VTT-D-85246 A. terreus VTT-D-82209

Aureobasidium pullulans VTT-D-89397 Curvularia inequalis VTT-D-79121 Penicillium digitatum VTT-D-87328 Cryptonectria parasitica VTT-D-82190 Fusarium oxysporum VTT-D-80134

Phanerochaete chrysosporium N-TT-D-85242 Penicillium janthinellum VTT-D-78090 Trichoderma reesei VTT-D-86271 Myceliophthora thermophila VTT-D-76003 (37 °C)

MEDIUM A Wheat bran 10 g

Solka floe cellulose 10 Spent grain 10

MEDIUM B: Beech xylan (Lenzing) 10 Birch glucuronoxylan (Roth) 5

Stake xylan (substituted oligosaccharides) 5 Spent grain 10

Example 2. Purification and characterization of an or-glucuronidase from Trichoderma reesei RUT C-30

Trichoderma. reesei RUT C 30 (VTT-D-86271, ATCC 56765) was cultivated in a bioreactor (Chemap LF 20, working volume 16 1) on a medium containing 30 g l^"1 xylan (Lenzing AG, Lenz, Austria) and 15 g l^"1 corn steep solids (Sigma C-8160) as the main carbon and nitroge sources, supplemented with 5 gl^"1 KH₂PO₄ and 5 gl^*1 (NH₄)₂SO₄. Cultivation conditions were temperature 29 °C, pH controlled between 6.0 and 6.5, aeration about 9 1 min^"1 and cultivation time 3 days. The mycelium was separated by centrifugation.

The clarified supernatant was first fractionated by cation exchange chromatography. The sample was equilibrated at pH 4.5 by acetic acid and at a conductivity corresponding to 50 mM sodium acetate buffer, pH 4.5, containing 0.14 mol I^"1 NaCl. The diluted supernatant was applied to a column (113 x 200 mm) of DEAE Sepharose FF (Pharmacia), pre- equilibrated with this buffer. Elution was performed first with the equilibrating buffer to remove unadsorbed proteins and thereafter with a linear addition of sodium chloride from 150 to 400 mM. Fractions (each 450 ml) which contained the or-glucuronidase eluting during the NaCl gradient were combined for the next purification step by hydrophobic interaction chromatography. The other adsorbed proteins were eluted by increasing the NaCl concentration to 1.0 M and the column was washed with 10 mM sodium hydroxide.

The enzyme preparation obtained in the first chromatographic step was adjusted to pH 5.5 by sodium hydroxide and to a conductivity of 122 mScm^"1 by adding (NH₄)₂SO₄. The sample was applied to a column (113 x 110 mm) of Phenyl Sepharose FF (Pharmacia), previously equilibrated with 25 mM sodium acetate buffer, pH 5.5, containing 0.8 mol l^"1 (NH₄)₂SO₄.

Elution was performed first with the equilibrating buffer and thereafter with a linear gradient of ammonium sulphate from 0.8 M to 0.6 M. Fractions which contained the or-glucuronidase eluted by 0.8 M ammonium sulphate and the decreasing gradient were combined. The other adsorbed proteins were eluted by decreasing the (NH₄)₂SO₄ concentration to 0 M and the column was washed with 6 M urea.

The preparation obtained by hydrophobic chromatography was concentrated in smaller batches (150 - 250 ml) to 20 ml by ultrafiltration (Amicon PM-10 membranes) and applied to a gel filtration column of Sephacryl S-100 HR (Pharmacia, 50 x 745 mm). The purified α- glucuronidase protein was eluted with 50 mM sodium acetate buffer, pH 5.0.

α-Glucuronidase activity was assayed on the basis of the method of Khandke et al. (1989). The assay was performed by incubating 40 μl of methylated triuronic acid, 2'-O-(4-O- methyl-αr-(l,2)-glucuronic acid-jS-D-xylobiose (mGluAX₂ 2.5 mg ml^"1, 5.3 mM) with 10 μl o appropriate enzyme dilution in 0.05 mM sodium acetate, pH 4.8 for 10 or 60 min. The reaction was stopped by adding 200 μl of the copper sulphate reagent of Milner and Avigad (1967) whereafter the mixture was heated in a boiling water bath for 10 min. After cooling,

80 μl of arsenomolybdate reagent B of Nelson (1944) was added after which the absorbance was measured at 690 nm using glucuronic acid as a standard.

The characteristics of the purified or-glucuronidase were determined using standard methods of protein chemistry. These characteristics are described in Table 2. Xylanase activity was assayed as described by Bailey et al. (1992) using 1.0 % (w/v) birchwood 4-O-glucurono- xylan (Roth 7500) as substrate in 50 mM sodium citrate buffer, pH 5.3. 0-Xylosidase was assayed as described by Poutanen and Puls (1988). All enzymatic activities were expressed in SI units (katals).

A sample of culture filtrate of T. reesei VTT-D-86271 was investigated for its content of or- glucuronidase proteins by gel filtration chromatography on a 1.5 litre column of Sephacryl S 100 HR (column height 74.5 cm). In addition to the glucuronidase described with a molecular weight of 95 kDa, two minor glucuronidase activities with apparent molecular weights below 50 kDa could be observed.

Table 2. Characteristics of an or-glucuronidase from Trichoderma reesei RUT C-30

Property Value

Molecular mass in denaturing 95 kDa conditions (SDS-PAGE)

Isoelectric point by chromatofocusing pH 5.0 - 6.2 pH-optimum pH 4.5 - 6.5 pH-stability pH 4.5 - 6.5 specific activity 800 nkat mg protein methyl-glucuronic acid liberated from: ^b

- mGluAX 1.9 mmol/1

- mGluAX₂ 3.3 mmol/1

- mGluAX₄ 1.1 mmol/1

- glucuronoxylan A (Roth 7500) 3.3 mg/1

- glucuronoxylan B (Sigma M-5144) 6.3 mg/1 ^a 50 μg ml^"1 of enzyme protein in 50 mM sodium phosphate-citrate buffers, pH from 3.0 to 7.5; incubation at 40 °C for 24 h. ^b substrate concentration in the hydrolysis study: 4 mM for xylo-oligomers or 8 mg ml^"1 for xylans; enzyme dosing: 6 mg protein mmol^"1 xylo-oligomers or 3 mg protein g'¹ glucuronoxylan; incubation for 24 h at 40 °C at pH 4.8.

Example 3. Purification of an or-glucuronidase from Aspergillus niger

Aspergillus niger VTT-D-77050 (ATCC 12846) was cultivated in a bioreactor (Chemap CF

3000, working volume 10 1) on a medium containing 10 g I^"1 wheat bran, 10 g l^"1 Solka floe cellulase and 2 g l^"1 corn steep solids, supplemented with 2 gl^"1 KH₂PO₄ and 2 g l"¹ (NH₄)₂SO₄. Cultivation conditions were: temperature 30 °C, pH controlled between 4.0 and 6.0, aeration about 4 1 min^"1 and cultivation time 3 days. The mycelium was separated by centrifugation. A portion of the supernatant was equilibrated at pH 4.5 by acetic acid and diluted with an equal volume of distilled water. Diatomaceous earth (Celite Standard Super Cel, Manville) was equilibrated with 50 mM sodium acetate buffer, pH 4.5, and the or-glucuronidase protei was adsorbed from the diluted supernatant to this carrier. The diatomaceous earth was washed once with 50 mM sodium acetate buffer after which the or-glucuronidase was eluted with 0.1 M sodium phosphate buffer containing sodium chloride up to a concentration of 0.5 M. The solution containing the eluted proteins was equilibrated with 10 mM sodium phosphate buffer, pH 6.5 using gel filtration and applied to a column of DEAE Sepharose F (Pharmacia), pre-equilibrated with the same buffer. Elution was performed first with the equilibrating buffer to remove unadsorbed proteins and thereafter with a linear addition of sodium chloride from 150 to 0.2 M. Fractions which contained the or-glucuronidase eluting during the NaCl gradient were combined for the next purification step by cation exchange chromatography. The other adsorbed proteins were eluted by increasing the NaCl concentration to 1.0 M.

The enzyme preparation obtained in the first chromatographic step was equilibrated with 10 mM sodium acetate buffer, pH 4.4 using gel filtration and applied to a column of CM Sepharose FF (Pharmacia), pre-equilibrated with the same buffer. Elution was performed first with the equilibrating buffer to remove unadsorbed proteins and thereafter with a linear addition of sodium chloride from 0 to 0.2 M. Fractions which contained the or-glucuronidase eluting at the end of the NaCl gradient were combined and analysed by standard methods for or-glucuronidase, β-xylanase and β-xylosidase activities as described in example 2. The other adsorbed proteins were eluted by increasing the NaCl concentration to 1.0 M.

The preparation obtained by cation exchange chromatography contained or-glucuronidase activity but did not show any detectable -xylanase activity toward glucuronoxylan (Roth 7500) or jδ-xylosidase activity toward p-nitrophenyl-jS-D-xylopyranosidase. In SDS-PAGE it showed a major band corresponding to a molecular weight of about 120 kDa. Example 4. Purification of an or-glucuronidase from Schizophyllum commune

Schizophyllum commune VTT-E-88362 (ATCC 38548) was cultivated in a bioreactor (Chemap CF 2000, working volume 16 1) on a medium containing 10 g l^"1 wheat bran, 10 g l^"1 Solka floe cellulase and 10 gl^"1 distiller's spent grain, supplemented with 5 gl^"1 KH₂PO₄ and 5 gl^"1 (NH₄)₂SO₄. Cultivation conditions were: temperature 30 °C, pH controlled between 4.0 and 6.0, aeration about 4 1 min^"1 and cultivation time 8 days. The mycelium was separated by centrifiigation and the supernatant concentrated 15 times by ultrafiltration (PM- 10 membranes, Romicon).

The solids were removed from the supernatant by centrifugation after which it was fractionated by anion exchange chromatography. The sample at pH 5.6 was applied to a column of DEAE Sepharose FF (Pharmacia), previously equilibrated with 50 mM sodium acetate buffer, pH 5.6, containing 0.05 moll^"1 sodium chloride. Elution was performed first with the equilibrating buffer to remove unadsorbed proteins and thereafter with a linear addition of sodium chloride from 200 to 450 mM. Fractions which contained the αr- glucuronidase eluting during the NaCl gradient were combined for the next purification step by hydrophobic interaction chromatography. The other adsorbed proteins were eluted by increasing the NaCl concentration to 1.0 M.

The sodium chloride concentration of the enzyme preparation obtained in the first chromatographic step was adjusted 1.5 M. The sample was applied to a column of Phenyl Sepharose FF (Pharmacia), previously equilibrated with 10 mM sodium acetate buffer, pH 5.5, containing 1.5 moll^"1 NaCl. Elution was performed first with the equilibrating buffer and thereafter with a linear gradient of sodium chloride from 1.5 M to 0 M. Fractions which contained the or-glucuronidase eluted during the gradient of decreasing salt concentration were combined and concentrated by ultrafiltration. The other adsorbed proteins were eluted by 10 mM sodium acetate buffer and the column was washed with 6 M urea.

The concentrated fractions from hydrophobic interaction chromatography were assayed for or- glucuronidase, β-ϊ ,4-xylanase and jS-xylosidase with standard methods as described in example 2. These activities in the or-glucuronidase preparation were 133 nkat/ml for or- glucuronidase, 22 nkat nkat/ml for xylanase and below 1 nkat/ml for β-xylosidase.

Example 5. Removal of carboxylic groups from kraft pulp by glucuronidase

Birch pulp (kappa number 15.5) was treated with T. reesei glucuronidase at pH 5 in 50 mM acetate buffer for 4 h. The enzyme dose was 100 nkat/g. The reference pulp was incubated in the buffer for 4 h. After the treatment the pulps were washed with distilled water and the amount of carboxylic groups present in the pulp was determined by conductometric titration as described by Katz et al (1984). The results are presented in Table 3.

Table 3. Removal of carboxylic groups by glucuronidase

As the results show, even at low enzyme dosage, the glucuronidase treatment was able to remove about 7 % of the carboxylic acid groups from the pulp. Increasing the amount of enzyme leads to further removal of glucuronic acid groups from the pulp.

Example 6. Peroxide bleaching of kraft pulp pretreated with glucuronidase.

Birch kraft pulp with kappa number of 14.8 was treated with T. reesei glucuronidase for 4 h at 5 % consistency, pH 5. The pH of the pulp was adjusted with 5 N H₂SO₄. Reference pulp was incubated at the same pH without enzyme addition. The glucuronidase dose was 500 nkat/g. After the treatments the pulps were washed with distilled water and bleached with peroxide. The dosages of chemicals were: 3 % H₂O₂, 1.5 % NaOH, 0.5 % MgSO₄, 0.2 % DTPA. The chemicals were dosed as % per o.d. pulp. The bleaching was carried out for 1 h at 10 % consistency. The bleaching temperature was 80 °C. After the bleaching the pulps were acidified and made into handsheets for the measurement of kappa number (SCAN Cl:1977), Brightness (ISO 2470) and viscosity (SCAN-C-15:1988). The results are given in Table 4.

Table 4. Bleaching of pulp pretreated with glucuronidase

The data of Table 4 show that glucuronidase treatment improved the bleachability of kraft pulp remarkably.

Example 7. Xylanase treatment of a metal-free kraft pulp pretreated with glucuromdase

Birch kraft pulp (kappa 14.8) was converted to hydrogen (metal-free) form at 2 % consistency in 0.1 M HC1 at room temperature. The pulp was washed after the acidification. After washing the pH of the pulp was adjusted with 5 N NaOH to pH 5. Enzymatic treatments were carried out at 5 % consistency for 4 h at 45 °C. The enzyme dosages were: T. reesei xylanase pl 9 200 nkat/g; T. reesei glucuronidase 500 nkat/g. After or between the enzymatic treatments the pulps were washed and bleached as described in Example 6. Table 5. Xylanase treatment of pulps pretreated with glucuronidase

As mentioned earlier, xylanases do not act effciently on metal-free pulp. However, by pretreating the pulp with glucuronidase prior to the xylanase treatment, it is possible to improve the action of xylanase treatment by removing the carboxylic groups from pulp.

Example 8. The effect of glucuronidase treatment on the paper technical properties of mechanical and chemical pulps.

The paper technical properties of cellulose pulps can be modified by treating the bleached or unbleached pulp with glucuronidase thus resulting in total or partial removal of carboxylic acid (methylglucuronic acid) groups from the surface of the fibres.

Example 9. The effect of Agaricus bisporus glucuronidase treatment on the brightness reversion of mechanical pulp

CTMP ja PGW pulps were treated with Agaricus bisporus glucuronidase in 0.2 M sodium acetate buffer (pH 5) at 40 °C for 20 hours. Hand sheets (95 cm²) made from the pulps were irradiated at 27 °C and 47 % relative humidity for 3 hours in a Xenotest 150 S apparatus (Heraeus Hanau) equipped with a xenon lamp, total output 1.3 kW. Before and after the irradiation the reflectances (R, and R₂) of the samples were measured with an Elrepho reflectance photometer with a 457 nm brightness filter. Post-color values were calculated as described by Janson and Forsskahl (1989). The results are indicated in tables 6 (PGW) and 7. It appears that glucuronidase treatment reduced the yellowing (PC₂ values) of both pulps.

Table 6. The effect of glucuromdase on yellowing of PGW

Table 7. The effect of glucuronidase on yellowing of CTMP.

Example 10. The effect of Trichoderma reesei glucuronidase treatment on the brightness reversion of mechanical pulp

PGW pulp suspended in water was treated with Trichoderma reesei glucuronidase and xylanase at 40 °C, pH 4.5 for 20 hours. Hand sheets were irradiated and post-color values calculated as in Example 9. The glucuronidase treatment reduced the yellowing (PC₂ value) of the pulp.

Table 8. The effect of Trichoderma reesei glucuronidase on yellowing of PGW.

Example 11. Removal of metal ions from pulp by or-glucuronidase treatment.

Pine kraft (kappa number 19) was treated with T. reesei or-glucuronidase (dosage 100 nkat/g) for 24 h at 45 °C. After the treatment the pulp was washed and the amounts of metal-ions were determined by AAS as described by Buchert et al (in press). The results are shown in Table 9. Table 9. Removal of metal ions by glucuronidase treatemnt of pulp

Even at low enzyme dosage, glucuronidase treatment resulted in a sigmficant decrease in the metal content of the pulp.

Example 12. Improvement of the hydrolysis of pulp xylan by or-glucuronidase.

Birch kraft pulp was treated with T. reesei xylanase (Tenkanen et al 1992) and glucuromdase as described in example 5. The xylanase dosage was 300 nkat/g. The degree of the hydrolysis of pulp xylan was followed by measuring the reducing sugars. The results are presented in Table 10.

Table 10. Hydrolysis of pulp pretreated with glucuromdase

The glucuronidase addition increased the overall hydrolysis of pulp xylan remarkably. References:

Bailey, M.J., Biely, P. and Poutanen, K. (1992) Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257-270.

Buchert J, Tenkanen M, Pitkanen M and Viikari L. The role of surface charge and swelling on the action of xylanases on birch kraft pulp. Tappi J. In press.

Kantelinen A, Rattό M, Sundquist J, Ranua M, Viikari L and Linko M (1988) 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 Herstellung von Papier. Das Papier 26(7): 305-332.

Poutanen K, Rattό 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.

Lindstrόm 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: nro 2: 40-47.

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

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

Sjόstrόm E, Massan karboksyylipitoisuus. KCL method 192:68. Poutanen K, Tenkanen M, Korte H and Puls J (1991) Accessory enzymes involved in the hydrolysis of xylans. ACS Symp. Ser 460, Enzymes in Biomass Conversion, ss. 426-436.

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

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, ss. 237-244.

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

Khandke KM, Vithayathil PJ and Murthy SK (1989) Purification and characterization of an or-glucuronidase from a thermophillic fungus, Thermoascus aurantiacus. Archiv Biochem

Biophys 274:511-517.

Milner, Y. and Avigad, G. A copper reagent for the determination of hexuronic acids and certain ketohexoses. Carbohydr. Res. 1967, 4, 359-361.

Nelson, N. A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 1944, 153, 375-380.

Katz S, Beatson RP and Scallan AM (1984) Svensk Papperstidn. 87(6):R48.

Poutanen, K. and Puls, J. Appl. Microbiol. Biotechnol. 1988, 28: 425-432.

Tenkanen, M., Puls, J. and Poutanen, K. Enzyme Microb. Technol. 1992, 14: 566-574.

Claims

1. A method for treatment of lignocellulosic materials, in particular cellulosic pulps, c h a r a c t e r i z e d in that the pulp is contacted with an enzyme preparation having an essential glucuronidase activity in order at least partially to remove the glucuronic acid groups of the pulp.

2. The method according to claim 1, wherein the pulp is contacted with a glucuronidase preparation containing only minor amounts, if any, of hemicellulases.

3. The method according to claim 1 or 2, wherein the glucuronidase preparation essentially comprises the cultivation medium of a glucuronidase-producing microorganism strain.

4. The method according to claim 1 or 2, wherein the glucuronidase preparation essentially comprises an enzyme isolated from the cultivation medium of a glucuronidase-producing microorganism strain.

5. The method according to any one of claims 1 to 4, wherein the pulp is contacted with a glucuronidase preparation derived from a microorganism strain selected from the group essentially consisting of microorganisms of the genera Trichoderma (e.g. T. reesei),

Aspergillus (e.g. A. niger, A. awamori, A. terreus, A. oryzae), Schizophyllum (e.g. S. commune), Aureobasidium (e.g. A. pullulans), Phanerochaete (e.g. P. chrysosporium), Fusarium (e.g. F. oxysporum), Agaricus (e.g. A. bisporus), Penicillium (e.g. P. janthinellum, P. digitatum), Streptomyces (e.g. S. olivochromogenes, S. flavogriseus), and Bacillus (e.g. B. subtilis, B. circulans).

6. The method according to any one of claims 1 to 4, wherein the pulp is contacted with a glucuronidase preparation derived from a microorganism strain selected from the group comprising Thermoascus auranticus, Curvulaήa inequalis, Tyromyces palustris, Cryptonectria parasitica, Myceliophthora thermophila, and Thermobacter auranticus.

7. The method according to claim 4 or 5 wherein the glucuronidase preparation is derived from a genetically modified strain containing the gene coding for or-glucuronidase.

8. The method according to claim 1 , wherein pulp from a pulping process is treated with an enzyme preparation having an essential glucuronic activity in order to decrease the amount o metals in the pulp.

9. The method according to claim 1, wherein the pulp is treated with an enzyme preparation having an essential glucuronidase activity and xylanase activity.

10. The method according to any one of claims 1, 8 and 9, wherein the pulp is treated with the enzyme preparation in order to enhance chlorine-free bleaching using oxygen, peroxide or ozone of the pulp.

11. The method according to claim 1, wherein the pulp is treated with the enzyme preparation in order to reduce the colour reversion of the pulp.

12. The method according to claim 1 , wherein the pulp is treated with the enzyme preparation in order to improve the paper technical properties of the pulp.

13. The method according to any of the previous claims, wherein the pulp is first treated with an enzyme preparation having an essential glucuronidase activity and subsequently contacted with an enzyme preparation having a hemicellulase, cellulase and/or ligninase activity.

14. The method according to any of the previous claims, wherein the pulp is treated simultaneously with an enzyme preparation having glucuronidase activity and an enzyme preparation having hemicellulase, cellulase and/or ligninase activity.

15. The method according to claims 1, 14 or 15, wherein the pulp is treated with the enzyme preparation in order to enhance the action of other enzymes, such as hemicellulase, cellulase or ligninase, on the pulp.

16. The method according to any of the foregoing claims, wherein an enzyme is used which removes, in particular, glucuronic acid groups of polymeric xylane chains.

17. The method according to claim 5, wherein an enzyme preparation is used, which has been produced by the microorganism strain Trichoderma reesei, said enzyme having been separated from the cultivation medium by anionic ion exchange and hydrophobic interaction chromatography .

18. The method according to claim 17, wherein the cellulose pulp is treated with an - glucuronidase enzyme whose molecular weight determined by the SDS-PAGE method is about 95 kDa.

19. The method according to claim 5, wherein an enzyme preparation is used, which has been produced by the microorganism strain Aspergillus niger and having an or-glucuroni- dase enzyme whose molecular weight determined by the SDS-PAGE method is about 120 kDa.

20. An enzyme preparation useful for treatment of lignocellulosic materials, in particular cellulose pulps, c h a r a c t e r i z e d in that it has an essential glucuronidase enzyme activity and contains only minor amounts, if any, of hemicellulases.

21. The enzyme preparation according to claim 20, wherein the glucuronidase enzyme is derived from a glucuronidase-producing microorganism strain selected from the group essentially consisting of Trichoderma reesei, Schizophyllum commune, Aspergillus niger, Thermoascus auranticus, Agaricus bisporus, Aspergillus awamori, Aspergillus niger,

Curvularia inequalis, Schizophyllum commune, Tyromyces palustris, Streptomyces flavogriseus, S. olivo-chromogenes, and other Streptomyces strains, Cryptonectria parasitica, Myceliophthora thermophila, Penicillium janthinellum, Penicillium digitatum, Aspergillus terreus, Thermobacter auranticus, Aureobasidium pullulans, Aspergillus oryzae, Phanerochaete chrysosporium, Bacillus circulans and B. subtilis.

22. The enzyme preparation according to claim 21, wherein the glucuronidase preparation is derived from a genetically modified strain containing the gene coding for or-glucuronidase.

23. The enzyme preparation according to claims 20 or 21, wherein the glucuronidase preparation essentially comprises the cultivation medium of the glucuronidase-producing microorganism strain, said cultivation medium optionally having been concentrated.

24. The enzyme preparation according to claims 20 or 21, wherein the glucuronidase preparation essentially comprises an enzyme isolated from the cultivation medium of the glucuronidase-producing microorganism strain.

25. The enzyme preparation according to claims 20 or 21, wherein the glucuronidase enzyme is derived from Trichoderma reesei and its molecular weight determined by the SDS-PAGE method is about 95 kDa.

26. The enzyme preparation according to claims 20 or 21, wherein the glucuronidase enzyme is derived from Aspergillus niger and its molecular weight determined by the SDS-PAGE method is about 120 kDa.