ENZYMATIC BLEACHING OF PAPER PULP

Abstract

The use of a GH78 enzyme in the bleaching of pulp for preparing paper materials, such as paper, linerboard, corrugated paperboard, tissue, towels, corrugated containers and boxes.

Description

REFERENCE TO A SEQUENCE LISTING

[0001] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to bleaching of pulp with a GH78 enzyme.

BACKGROUND

[0003] It is well-known to use enzymes in the manufacture of paper materials. Examples of enzymes used for this purpose are proteases, lipases, xylanases, amylases, cellulases, as well as various oxidizing enzymes such as laccases and peroxidases.

[0004] The effects of these enzymes are wide-spread, e.g., control of various deposits such as pitch, strength-improvement, de-inking, drainage improvement, tissue softening, bleaching etc.

SUMMARY OF THE INVENTION

[0005] In a first aspect, the present invention provides a method for increasing the brightness and/or decreasing the kappa number of a paper pulp, comprising contacting the paper pulp with a GH78 enzyme.

[0006] Other aspects and embodiments of the invention are apparent from the description and examples.

[0007] In one aspect the invention relates to a method for increasing the brightness and/or decreasing the kappa number of a paper pulp, comprising contacting the paper pulp with a GH78 enzyme, wherein the GH78 enzyme

a) belongs to glycoside hydrolase family 78 and/or, b) exhibits esterase and rhamnosidase activities and/or c) consists or comprises an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1.

[0008] In another aspect the invention relates to a bleaching composition, comprising a paper pulp and a GH78 enzyme, wherein the GH78 enzyme

a) belongs to glycoside hydrolase family 78 and/or b) exhibits esterase and rhamnosidase activities and/or c) consists or comprises an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1.

[0009] An additional aspect of the invention relates to use of a GH78 enzyme for bleaching and/or decreasing the kappa number of a paper pulp, wherein the GH78 enzyme

a) belongs to glycoside hydrolase family 78 and/or b) exhibits esterase and rhamnosidase activities and/or c) consists or comprises an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1.

DETAILED DESCRIPTION

[0010] The present inventors surprisingly found that lignocellulosic materials (e.g. pulp and the resulting paper material) can be bleached efficiently by contacting the lignocellulosic material with a GH78 enzyme.

Paper and Pulp

[0011] The term "paper material" refers to products, which can be made out of pulp, such as paper, linerboard, corrugated paperboard, tissue, towels, packaging materials, corrugated containers or boxes.

[0012] The term "pulp" or "paper pulp" means any pulp which can be used for the production of a paper material. For example, the pulp can be supplied as a virgin pulp, or can be derived from a recycled source. The pulp may be a wood pulp, a non-wood pulp or a pulp made from waste paper. A wood pulp may be made from softwood such as pine, redwood, fir, spruce, cedar and hemlock or from hardwood such as maple, alder, birch, hickory, beech, aspen, acacia and eucalyptus. A non-wood pulp may be made, e.g., from flax, hemp, bagasse, bamboo, cotton or kenaf. A waste paper pulp may be made by re-pulping waste paper such as newspaper, mixed office waste, computer print-out, white ledger, magazines, milk cartons, paper cups etc.

[0013] In a particular embodiment, the pulp to be treated comprises both hardwood pulp and softwood pulp.

[0014] The wood pulp to be treated may be mechanical pulp (such as ground wood pulp, GP), chemical pulp (such as kraft pulp or sulfite pulp), semichemical pulp (SCP), thermomechanical pulp (TMP), chemithermomechanical pulp (CTMP), or bleached chemithermomechanical pulp (BCTMP).

[0015] Mechanical pulp is manufactured by the grinding and refining methods, wherein the raw material is subjected to periodical pressure impulses. TMP is thermomechanical pulp, GW is groundwood pulp, PGW is pressurized groundwood pulp, RMP is refiner mechanical pulp, PRMP is pressurized refiner mechanical pulp and CTMP is chemithermomechanical pulp.

[0016] Chemical pulp is manufactured by alkaline cooking whereby most of the lignin and hemicellulose components are removed. In kraft pulping or sulphate cooking sodium sulphide or sodium hydroxide are used as principal cooking chemicals.

[0017] The kraft pulp to be treated may be a bleached kraft pulp, which may consist of softwood bleached kraft (SWBK, also called NBKP (Nadel Holz Bleached Kraft Pulp)), hardwood bleached kraft (HWBK, also called LBKP (Laub Holz Bleached Kraft Pulp)) or a mixture of these.

[0018] The pulp to be used in the process of the invention is a suspension of mechanical or chemical pulp or a combination thereof. For example, the pulp to be used in the process of the invention may comprise 0%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% of chemical pulp. In a particular embodiment, a chemical pulp forms part of the pulp being used for manufacturing the paper material. In the present context, the expression "forms part of" means that in the pulp to be used in the process of the invention, the percentage of chemical pulp lies within the range of 1-99%. In particular embodiments, the percentage of chemical pulp lies within the range of 2-98%, 3-97%, 4-96%, 5-95%, 6-94%, 7-93%, 8-92%, 9-91%, 10-90%, 15-85%, 20-80%, 25-75%, 30-70%, 40-60%, or 45-55%.

[0019] In a particular embodiment of the use and the process of the invention, the chemical pulp is a kraft pulp, a sulfite pulp, a semichemical pulp (SCP), a thermomechanical pulp (TMP), a chemithermomechanical pulp (CTMP), a bleached chemithermomechanical pulp (BCTMP). In particular embodiments the kraft pulp is bleached kraft pulp, for example softwood bleached kraft (SWBK, also called NBKP (Nadel Holz Bleached Kraft Pulp)), hardwood bleached kraft (HWBK, also called LBKP (Laub Holz Bleached Kraft Pulp and)) or a mixture thereof.

GH78 Enzyme

[0020] A GH78 enzyme, according to the present invention, is a glycoside hydrolase exhibiting esterase and rhamnosidase activities; preferably exhibiting feruloyl esterase (EC 3.1.1.73) and .alpha.-L-rhamnosidase (EC 3.2.1.40) activities. The GH78 enzyme belongs to the glycoside hydrolase family 78, as defined by the CAZy classification (Henrissat B, Davies G J (1997), Structural and sequence-based classification of glycoside hydrolases, Current Opinion in Structural Biology, 7:637-644).

[0021] In an embodiment, the GH78 enzyme is derived from a Xylaria sp., preferably Xylaria polymorpha. As used herein, "derived from", as in, e.g., "derived from Xylaria polymorpha" means a wild-type alpha-amylase enzyme and variants thereof. Such enzymes can also be prepared synthetically, as is well-known in the art.

[0022] In an embodiment, the amino acid sequence of the GH78 enzyme has at least 60% identity, preferably at least 65% identity, more preferably at least 70% identity, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95%, 96%, 97%, 98%, 99%, and most preferably 100% identity to the amino acid sequence of SEQ ID NO: 1, or to the amino acid sequence of a Xylaria sp. (preferably Xylaria polymorpha) GH78 enzyme.

[0023] In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature GH78 polypeptide of SEQ ID NO: 1 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or up to 5, e.g., 1, 2, 3, 4, or 5.

[0024] In an embodiment, the amino acid sequence of the GH78 enzyme has one or several substitutions, deletions or insertions compared to SEQ ID NO: 1. In particular, the amino acid sequence of the GH78 enzyme is identical to SEQ ID NO: 1.

[0025] The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

[0026] Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

[0027] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for GH78 enzyme activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

[0028] Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0029] The relatedness between two amino acid sequences is described by the parameter "sequence identity". For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues.times.100)/(Length of Alignment--Total Number of Gaps in Alignment).

[0030] The concentration of the GH78 enzyme is typically in the range of 0.01-100 ppm enzyme protein, preferably 0.05-50 ppm enzyme protein, more preferably 0.1-50 ppm enzyme protein, more preferably 0.1-30 ppm enzyme protein, more preferably 0.5-20 ppm enzyme protein, and most preferably 0.5-10 ppm enzyme protein.

[0031] In an embodiment, the concentration of the GH78 enzyme is typically in the range of 1-40 ppm enzyme protein, preferably 1-20 ppm enzyme protein, more preferably 1-10 ppm enzyme protein.

Feruloyl Esterase (EC 3.1.1.73) Activity

[0032] Feruloyl esterase activity is determined by hydrolytic demethylation of 1 mM methyl ferulate to ferulic acid in 100 mM MOPS buffer at pH 6.0. The reaction is initiated by the incubation of reaction mixtures at 37.degree. C. for a suitable time depending on enzyme sample (10-30 min) and then terminated by an equal volume of acetic acid/acetonitrile (11.3% v/v) as stop solution (see also Faulds & Williamson (1994), Microbiology, vol. 140, pp. 779-787).

[0033] After centrifugation, the released ferulic acid is analyzed by HPLC using a reversed phase C18-column (Synergi Fusion-RP 80A, 4.6 mm.times.125 mm, Phenomenex.RTM., Aschaffenburg, Germany). The elution by the isocratic mobile phase, which consists of 60% phosphoric acid (15 mM H.sub.3PO.sub.4, pH 2.6) and 40% acetonitrile (v/v), is monitored at 323 nm over 7 min.

.alpha.-L-Rhamnosidase (EC 3.2.1.40) Activity

[0034] .alpha.-L-rhamnosidase activity is measured using p-nitrophenyl .alpha.-L-rhamnopyranoside (p-NPRP) as substrate (see also Gallego et al. (2001), J. Food Sci., vol. 66, pp. 204-209).

[0035] The assay is performed in a 48-well microtiter plate containing 160 .mu.l of the substrate (2.5 mM p-NPRP in 50 mM sodium acetate buffer, pH 5.0) and 40 .mu.l of enzyme sample. The reaction is performed for 5-20 min at 40.degree. C. and stopped by adding 100 .mu.l of 1 M Na.sub.2CO.sub.3. The enzyme activity is spectrophotometrically measured (.lamda.=405 nm; .epsilon..sub.405=2.5 mM.sup.-1 cm.sup.-1) through the liberation of p-nitrophenol using a microplate reader.

Bleaching

[0036] Bleaching is defined as a process aimed at removal of colour in pulps derived from residual lignin or other colored impurities. Native wood is only slightly colored, whereas residual lignin of a chemical pulp after cooking is highly colored. Traditional concepts for bleaching of pulp include chlorine and oxygen based oxidants which selectively remove chromophore structures present in the pulp. The progress in bleaching is followed by measuring the brightness, which is defined as the reflectance of visible blue light from a pad of pulp sheets using a defined spectral band of light having an effective wavelength of 457 nm. Official ISO standard methods are ISO 2469 or ISO 2470. Bleaching to full brightness (>88% ISO) requires multi-stage application of bleaching chemicals. The first stages in a bleaching sequence are often conceived as delignification, where the majority of residual lignin is removed. The latter stages are often referred to brightening stages, in which the chromophores in the pulps are eliminated to attain a high brightness level.

[0037] According to the invention, the brightness of the pulp is determined according to TAPPI test method T 452 om-98.

Kappa Number

[0038] The Kappa number is an indication of the residual lignin content or bleachability of pulp by a standardized analysis method. The Kappa number is determined by ISO 302, which is applicable to all kinds of chemical and semi-chemical pulps and gives a Kappa number in the range of 1-100.

[0039] According to the invention, the kappa number of the pulp is determined according to TAPPI test method T 236 om-06.

Process Conditions

[0040] The process of the invention is particularly applicable to the bleaching of pulp in a process for making paper material.

[0041] In the case of paper and pulp processing, the process according to the invention can be carried out at any pulp production stage. The enzyme can be added to any holding tank, e.g. to a pulp storing container (storage chest), storage tower, mixing chest or metering chest. The enzyme treatment can be performed before the bleaching of pulp, in connection with the pulp bleaching process or after the bleaching. When carried out in connection with pulp bleaching the enzyme preparation may be added together with bleaching chemicals such as chlorine or chlorine dioxide. Applying oxygen gas, hydrogen peroxide or ozone or combinations thereof may also carry out the bleaching of pulp. The enzyme preparation may also be added together with these substances. Preferably the enzyme preparation is added prior to such bleaching steps. The enzyme can also be added to the circulated process water (white water) originating from bleaching and process water (brown water) originating from the mechanical or chemimechanical pulping process. In a particular embodiment of a kraft pulping process, the enzyme is added during the brown-stock washing.

[0042] In the present context, the term "process water" comprises inter alia 1) water added as a raw material to the paper manufacturing process; 2) intermediate water products resulting from any step of the process for manufacturing the paper material; as well as 3) waste water as an output or by-product of the process. In a particular embodiment, the process water is, has been, is being, or is intended for being circulated (re-circulated), i.e., re-used in another step of the process. The term "water" in turn means any aqueous medium, solution, suspension, e.g. ordinary tap water, and tap water in admixture with various additives and adjuvants commonly used in paper manufacturing processes. In a particular embodiment the process water has a low content of solid (dry) matter, e.g. below 20%, 18%, 16%, 14%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 20% or below 1% dry matter.

[0043] The process of the invention may be carried out at conventional conditions in the paper and pulp processing. The process conditions will be a function of the enzyme(s) applied, the reaction time and the conditions given.

[0044] The enzyme of the invention should be added in an effective amount. By the term "effective amount" is meant the amount sufficient to achieve the desired and expected effect, such as oxidizing pitch components, obtaining a desired bleaching and/or de-inking etc.

[0045] In a particular embodiment, the dosage of the GH78 enzyme and additional enzymes, if any, is from about 0.1 mg enzyme protein to about 100,000 mg enzyme protein (of each enzyme) per ton of paper pulp.

[0046] In further particular embodiments, the amount of the GH78 enzyme and additional enzymes, if any, is in the range of 0.00001-20; or 0.0001-20 mg of enzyme (calculated as pure enzyme protein) per gram (dry weight) of pulp material, such as 0.0001-10 mg/g, 0.0001-1 mg/g, 0.001-1 mg/g, 0.001-0.1, or 0.01-0.1 mg of enzyme per gram of pulp material. Again, these amounts refer to the amount of each enzyme.

[0047] The enzymatic treatment can be done at conventional consistency, e.g. 0.5-10% dry substance. In particular embodiments, the consistency is within the range of 0.5-45%; 0.5-40%; 0.5-35%; 0.5-30%; 0.5-25%; 0.5-20%; 0.5-15%; 0.5-10%; 0.5-8%; 0.5-6%; or 0.5-5% dry substance.

[0048] The enzymatic treatment may be carried out at a temperature of from about 10.degree. C. to about 100.degree. C. Further examples of temperature ranges (all "from about" and "to about") are the following: 20-120.degree. C., 30-120.degree. C., 35-120.degree. C., 37-120.degree. C., 40-120.degree. C., 50-120.degree. C., 60-120.degree. C., 70-120.degree. C., 10-100.degree. C., 10-90.degree. C., 10-80.degree. C., 10-70.degree. C., 10-60.degree. C., and 30-60.degree. C., as well as any combination of the upper and lower values here indicated. A typical temperature is from about 20 to 90.degree. C., or 20 to 95.degree. C., preferably from about 40 to 70.degree. C., or 40 to 75.degree. C. Usually, the enzymatic treatment is carried out at atmospheric pressure. But when the temperature exceeds 100.degree. C., the treatment is carried out at a pressure of 1-2 bar (up to 1 bar above atmospheric pressure).

[0049] The enzymatic treatment is carried out at a pH of from about 2 to about 7, preferably at a pH from about 2.5 to about 6, more preferably at a pH from about 3 to about 5.5, and most preferably at a pH from about 3.5 to about 5.

[0050] A suitable duration of the enzymatic treatment may be in the range from a few seconds to several hours, e.g. from about 30 seconds to about 48 hours, or from about 1 minute to about 24 hours, or from about 1 minute to about 18 hours, or from about 1 minute to about 12 hours, or from about 1 minute to 5 hours, or from about 1 minute to about 2 hours, or from about 1 minute to about 1 hour, or from about 1 minute to about 30 minutes. A typical reaction time is from about 10 minutes to 3 hours, 10 minutes to 10 hours, preferably 15 minutes to 1 hour, or 15 minutes to 2 hours.

[0051] Molecular oxygen from the atmosphere will usually be present in sufficient quantity, if required. Therefore, the reaction may conveniently be carried out in an open reactor, i.e. at atmospheric pressure.

[0052] Various additives over and above the GH78 enzyme and additional enzymes, if any, can be used in the process or use of the invention. Surfactants and/or dispersants are often present in, and/or added to a pulp. Thus the process and use of the present invention may be carried out in the presence of an anionic, non-ionic, cationic and/or zwitterionic surfactant and/or dispersant conventionally used in a pulp. Examples of anionic surfactants are carboxylates, sulphates, sulphonates or phosphates of alkyl, substituted alkyl or aryl. Examples of non-ionic surfactants are polyoxyethylene compounds, such as alcohol ethoxylates, propoxylates or mixed ethoxy-/propoxylates, poly-glycerols and other polyols, as well as certain block-copolymers. Examples of cationic surfactants are water-soluble cationic polymers, such as quartenary ammonium sulphates and certain amines, e.g. epichlorohydrin/dimethylamine polymers (EPI-DMA) and cross-linked solutions thereof, polydiallyl dimethyl ammonium chloride (DADMAC), DADMAC/Acrylamide co-polymers, and ionene polymers, such as those disclosed in U.S. Pat. Nos. 5,681,862; and 5,575,993. Examples of zwitterionic or amphoteric surfactants are betains, glycinates, amino propionates, imino propionates and various imidazolin-derivatives. Also the polymers disclosed in U.S. Pat. No. 5,256,252 may be used.

[0053] Also according to the invention, surfactants such as the above, including any combination thereof may be used in a paper making process together with a GH78 enzyme, as defined herein, and included in a composition together with such enzyme. The amount of each surfactant in such composition may amount to from about 1 to about 1000 ppm of the composition. In particular embodiments the amount of each surfactant is from about 10 to about 1000 ppm, or from about 10 to about 500 ppm, or from about 50 to about 500 ppm.

[0054] In another particular embodiment, each of the above ranges refers to the total amount of surfactants.

[0055] In further particular embodiments of the above method, and of the process of the invention, the GH78 enzyme is used in an amount of 0.005-50 ppm (mg/L), or 0.01-40, 0.02-30, 0.03-25, 0.04-20, 0.05-15, 0.05-10, 0.05-5, 0.05-1, 0.05-0.8, 0.05-0.6, or 0.1-0.5 ppm. The amount of enzyme refers to mg of a well-defined enzyme preparation.

[0056] In the process of the invention, the GH78 enzyme may be applied alone or together with an additional enzyme. The term "an additional enzyme" means at least one additional enzyme, e.g. one, two, three, four, five, six, seven, eight, nine, ten or even more additional enzymes.

[0057] The term "applied together with" (or "used together with") means that the additional enzyme may be applied in the same, or in another step of the process of the invention. The other process step may be upstream or downstream in the paper manufacturing process, as compared to the step in which the pulp is bleached with a GH78 enzyme.

[0058] In particular embodiments the additional enzyme (see also below) is an enzyme which has protease, lipase, xylanase, cutinase, oxidoreductase, cellulase, endoglucanase, amylase, mannanase, steryl esterase, and/or cholesterol esterase activity. Examples of oxidoreductase enzymes are enzymes with laccase, and/or peroxidase activity. In a preferred embodiment, the additional enzyme is lipase.

[0059] The term "a step" of a process means at least one step, and it could be one, two, three, four, five or even more process steps. In other words the GH78 enzyme may be applied in at least one process step, and the additional enzyme(s) may also be applied in at least one process step, which may be the same or a different process step as compared to the step where the GH78 enzyme is used.

[0060] The term "enzyme preparation" means a product containing at least one GH78 enzyme. The enzyme preparation may also comprise enzymes having other enzyme activities, preferably lipolytic enzymes. In addition to the enzymatic activity such a preparation preferably contains at least one adjuvant. Examples of adjuvants, which are used in enzyme preparations for the paper and pulp industry are buffers, polymers, surfactants and stabilizing agents.

[0061] In an embodiment, the process of the invention also includes an alkaline peroxide bleaching stage (E stage and/or P stage), such as described by Camarero, S. et al., Enzyme and Microbial Technology, 35 (2004), pp. 113-120 (see in particular paragraph 2.4). Preferably, the alkaline peroxide bleaching is carried out after the enzymatic bleaching method of the invention. Typical conditions for an alkaline peroxide bleaching stage are initial pH values in the range of 10-11 and end pH above 8.5; temperatures typical ranges from 70-90.degree. C. and peroxide charges from 0.5-1% for 1.5 hours. Peroxide stabilizer may be added and metal management may be handled in previous stage or simultaneously with peroxide bleaching.

Additional Enzymes

[0062] Any enzyme having protease, lipase, xylanase, cutinase, oxidoreductase, cellulose, endoglucanase, amylase, mannanase, steryl esterase, and/or cholesterol esterase activity can be used as additional enzymes in the use and process of the invention. Below some non-limiting examples are listed of such additional enzymes. The enzymes written in capitals are commercial enzymes available from Novozymes NS, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark. The activity of any of those additional enzymes can be analyzed using any method known in the art for the enzyme in question, including the methods mentioned in the references cited.

[0063] Examples of cutinases are those derived from Humicola insolens (U.S. Pat. No. 5,827,719); from a strain of Fusarium, e.g. F. roseum culmorum, or particularly F. solani pisi (WO 90/09446; WO 94/14964, WO 94/03578). The cutinase may also be derived from a strain of Rhizoctonia, e.g. R. solani, or a strain of Alternaria, e.g. A. brassicicola (WO 94/03578), or variants thereof such as those described in WO 00/34450, or WO 01/92502.

[0064] Examples of proteases are the ALCALASE, ESPERASE, SAVINASE, NEUTRASE and DURAZYM proteases. Other proteases are derived from Nocardiopsis, Aspergillus, Rhizopus, Bacillus alcalophilus, B. cereus, B. natto, B. vulgatus, B. mycoide, and subtilisins from Bacillus, especially proteases from the species Nocardiopsis sp. and Nocardiopsis dassonvillei such as those disclosed in WO 88/03947, and mutants thereof, e.g. those disclosed in WO 91/00345 and EP 415296.

[0065] Examples of amylases are the BAN, AQUAZYM, TERMAMYL, and AQUAZYM Ultra amylases. An example of a lipase is the RESINASE A2X lipase. An example of a xylanase is the PULPZYME HC hemicellulase. Examples of endoglucanases are the NOVOZYM 613, 342, and 476 enzyme products.

[0066] Examples of mannanases are the Trichoderma reesei endo-beta-mannanases described in Stahlbrand et al, J. Biotechnol. 29 (1993), 229-242.

[0067] Examples of steryl esterases, peroxidases, laccases, and cholesterol esterases are disclosed in the references mentioned in the background art section hereof. Further examples of oxidoreductases are the peroxidases and laccases disclosed in EP 730641; WO 01/98469; EP 719337; EP 765394; EP 767836; EP 763115; and EP 788547. In the present context, whenever an oxidoreductase enzyme is mentioned that requires or benefits from the presence of acceptors (e.g. oxygen or hydrogen peroxide), enhancers, mediators and/or activators, such compounds should be considered to be included. Examples of enhancers and mediators are disclosed in EP 705327; WO 98/56899; EP 677102; EP 781328; and EP 707637. If desired a distinction could be made by defining an oxidoreductase enzyme system (e.g. a laccase, or a peroxidase enzyme system) as the combination of the enzyme in question and its acceptor, and optionally also an enhancer and/or mediator for the enzyme in question.

Compositions, Methods and Uses

[0068] In a first aspect, the invention provides a method for increasing the brightness and/or decreasing the kappa number of a paper pulp, comprising contacting the paper pulp with a GH78 enzyme.

[0069] Suitable GH78 enzymes are described above. Preferably, the GH78 enzyme belongs to the glycoside hydrolase family 78, and exhibits esterase (feruloyl esterase) and rhamnosidase (.alpha.-L-rhamnosidase) activities.

[0070] The amino acid sequence of the GH78 enzyme may have at least 60% identity, preferably at least 65% identity, more preferably at least 70% identity, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95%, 96%, 97%, 98%, 99%, and most preferably 100% identity to the amino acid sequence of SEQ ID NO: 1.

[0071] In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 1 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or up to 5, e.g., 1, 2, 3, 4, or 5.

[0072] In an embodiment, the GH78 enzyme is derived from a Xylaria sp., such as Xylaria polymorpha.

[0073] In an embodiment, the pulp is wood pulp.

[0074] In an embodiment, the pulp is a chemical pulp, such as kraft pulp.

[0075] In an embodiment, the method of the invention further comprises a step of chlorine dioxide treatment (D stage) and/or alkaline peroxide treatment (E stage).

[0076] In an embodiment, the method of the invention further comprises contacting the pulp with one or more additional enzyme(s), such as a lipase.

[0077] In an embodiment, the method of the invention further comprises a final step of preparing a paper material from the pulp.

[0078] In a second aspect, the invention provides a bleaching composition, comprising a paper pulp and a GH78 enzyme.

[0079] Suitable GH78 enzymes are described above. Preferably, the GH78 enzyme belongs to glycoside hydrolase family 78 and exhibits esterase (feruloyl esterase) and rhamnosidase (.alpha.-L-rhamnosidase) activities.

[0080] The amino acid sequence of the GH78 enzyme may have at least 60% identity, preferably at least 65% identity, more preferably at least 70% identity, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95%, 96%, 97%, 98%, 99%, and most preferably 100% identity to the amino acid sequence of SEQ ID NO: 1.

[0081] In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 1 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or up to 5, e.g., 1, 2, 3, 4, or 5.

[0082] In an embodiment, the GH78 enzyme is derived from a Xylaria sp., such as Xylaria polymorpha.

[0083] In an embodiment, the pulp is wood pulp.

[0084] In an embodiment, the pulp is a chemical pulp, such as kraft pulp.

[0085] In a further aspect, the invention provides a paper material, which is made from the bleaching composition of the invention, or which is made from a paper pulp subjected to the method of the invention.

[0086] The invention also provides for use of the methods and compositions above for bleaching and/or decreasing the kappa number of paper pulp

[0087] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0088] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0089] One specific embodiment of the present invention is described in the set of items herein below.

Items:

[0090] 1. A method for increasing the brightness and/or decreasing the kappa number of a paper pulp, comprising contacting the paper pulp with a GH78 enzyme. 2. The method of item 1, wherein the GH78 enzyme belongs to glycoside hydrolase family 78 and exhibits esterase and rhamnosidase activities. 3. The method of item 1 or 2, wherein the GH78 enzyme is a glycoside hydrolase exhibiting feruloyl esterase and .alpha.-L-rhamnosidase activities. 4. The method of any of items 1 to 3, wherein the amino acid sequence of the GH78 enzyme has at least 60% identity, preferably at least 65% identity, more preferably at least 70% identity, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95%, 96%, 97%, 98%, 99%, and most preferably 100% identity to the amino acid sequence of SEQ ID NO: 1. 5. The method of any of items 1 to 4, wherein the GH78 enzyme is derived from a Xylaria sp., such as Xylaria polymorpha. 6. The method of any of items 1 to 5, wherein the pulp is wood pulp. 7. The method of any of items 1 to 6, wherein the pulp is a chemical pulp, such as kraft pulp. 8. The method of any of items 1 to 7, which further comprises a step of chlorine dioxide treatment (D stage) and/or alkaline peroxide treatment (E stage). 9. The method of any of items 1 to 8, which further comprises contacting the pulp with one or more additional enzyme(s), such as a lipase. 10. A bleaching composition, comprising a paper pulp and a GH78 enzyme. 11. The composition of item 10, wherein the GH78 enzyme belongs to glycoside hydrolase family 78 and exhibits feruloyl esterase and .alpha.-L-rhamnosidase activities. 12. The composition of item 10 or 11, wherein the amino acid sequence of the GH78 enzyme has at least 60% identity, preferably at least 65% identity, more preferably at least 70% identity, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95%, 96%, 97%, 98%, 99%, and most preferably 100% identity to the amino acid sequence of SEQ ID NO: 1. 13. The composition of any of items 10 to 12, wherein the pulp is wood pulp. 14. The composition of any of items 10 to 13, wherein the pulp is a chemical pulp, such as kraft pulp. 15. Use of a GH78 enzyme for bleaching and/or decreasing the kappa number of a paper pulp.

EXAMPLES

[0091] Chemicals used as buffers and substrates were commercial products of at least reagent grade.

[0092] The amino acid sequence of the Xylaria polymorpha GH78 enzyme is shown as SEQ ID NO: 1.

Example 1

Effect of GH78 Enzyme on Bleaching of Oxygen-Bleached Eucalyptus Kraft Pulp

[0093] The Xylaria polymorpha GH78 enzyme was assessed for its ability to bleach oxygen-bleached eucalyptus kraft pulp. Feruloyl esterase and .alpha.-L-rhamnosidase activities were tested for SEQ ID NO: 1.

[0094] Xylaria polymorpha GH78 enzyme treatment of oxygen-bleached eucalyptus kraft pulp was carried out in a 1000 ml Lab-O-Mat beaker (Werner Mathis AG, Zurich, Switzerland) at 4% consistency, pH 8 and 40.degree. C. for 18 hours. The amount of pulp was 15 g and the enzyme dosage was 50 mg and 1000 mg of Xylaria polymorpha GH78 enzyme per kg dry pulp. The reference pulp (negative control) was kept under the same conditions but without enzyme addition. After the enzyme treatment, the water in the pulp was removed by filtration through a Buchner funnel and bleaching was conducted with a DEp-sequence, where D stands for chlorine dioxide and Ep stands for hydrogen peroxide reinforced alkaline extraction. Pulp samples (8 g dry pulp) were DEp-bleached in BA 6040 standard stomacher bags (Seward). The conditions for the D-stage were: 10% consistency, 11.5 kg chlorine dioxide/ton (dry pulp) at 80.degree. C. initial pH 3.5 (final pH 2.5) for 110 min. After the bleaching, the chlorine dioxide was removed from the pulp samples by using a Buchner funnel. The samples were then washed with tap water thoroughly. Conditions for the Ep-stage were 10% consistency, 5 kg H.sub.2O.sub.2/ton (dry pulp), 10.5 kg NaOH/ton (dry pulp) at 85.degree. C. for 80 min. After the Ep-stage, the samples were filtered by using a Buchner funnel.

[0095] After the bleaching, the samples were diluted to 2 liter with deionized water and the slurry was disintegrated for 15,000 revolutions in a standard Pulp Disintegrator (Type 8-3; Lorentzen & Wettre, Kista, Sweden). Handsheets were prepared according to TAPPI test method, "Forming Handsheets for Reflectance Testing of Pulp (Sheet Machine Procedure)" TAPPI T 272. For the determination of brightness, analyses were conducted according to TAPPI test method T 452 om-98. The kappa number of the pulp was determined according to TAPPI test method T 236 om-06. The results of the bleaching experiments are shown in Table 1.

TABLE-US-00001 TABLE 1 Brightness and kappa number after DEp- (Reference) and GH78DEp- bleaching of oxygen-bleached eucalyptus kraft pulp. Pulp Brightness (%) Kappa number Reference (DEp-bleached pulp) 86.3 3.92 GH78DEp-bleached pulp 87.3 3.75 (50 mg GH78/kg) GH78DEp-bleached pulp 86.9 3.42 (1000 mg GH78/kg)

Sequence CWU 1

1

11866PRTXylaria polymorpha 1Met Ala Val Ser Ile Phe Gln Val Ser Phe Glu His His Arg Thr Ala 1 5 10 15 Leu Gly Ile Gly Glu Ala Ser Pro Arg Ile Ser Trp Gln Phe Glu Gly 20 25 30 Asp Ala Gly Asn Trp Ser Gln Ser Gly Tyr Ser Ile Glu Val Ser Arg 35 40 45 Gln Gly Lys Ala Asp Val Phe Asn Val Thr Ser Ser Asp Ser Val Leu 50 55 60 Val Asp Trp Pro Thr Val Ala Leu Ser Ser Ala Glu Ser Ala Leu Val 65 70 75 80 Arg Val Lys Ala Tyr Gly Ala Asp Ser Val Asp Thr Asp Trp Ser Glu 85 90 95 Ala Phe Pro Val Glu Thr Gly Leu Leu Ala Asp Asp Asp Trp Ser Gly 100 105 110 Ala Gln Val Ile Ala Ala Asn Lys Thr Thr Glu Gln Asn Val Pro His 115 120 125 Gln Pro Ile Leu Leu Arg Lys Asp Phe Ser Leu Asp Gly Asp Val Ser 130 135 140 Ser Ala Arg Leu Tyr Ile Thr Ala Tyr Gly Ile Tyr Glu Ala Phe Ile 145 150 155 160 Asn Gly Asp Arg Val Gly Asp Ala Val Leu Ala Pro Gly Trp Gln Ser 165 170 175 Tyr Asn His Arg Leu Val Tyr Asp Thr Tyr Asp Val Thr Ser Leu Leu 180 185 190 Gln Ser Gly Ser Asn Ala Ile Gly Val His Val Gly Glu Gly Trp Tyr 195 200 205 Ala Gly Arg Ile Gly Phe Ser Thr Ser Arg Asn Ile Trp Gly Asp Thr 210 215 220 Leu Gly Ala Phe Ala Leu Leu Val Val Thr Lys Ala Asp Gly Ser Lys 225 230 235 240 Glu Thr Ile Pro Thr Asp Leu Thr Trp Ser Ser Ser Thr Gly Ala Ile 245 250 255 Ile Thr Ser Glu Ile Tyr Asp Gly Glu Leu Tyr Asn Ser Thr Leu Asp 260 265 270 Gln Pro Gly Trp Ala Thr Val Asp Phe Lys Ala Pro Glu Ser Ala Glu 275 280 285 Trp Ile Gly Val Lys Glu Leu Lys Ser Pro Leu Gly Arg Leu Ser Ala 290 295 300 Pro Asp Gly Pro Pro Ile Gln Arg Val Glu Glu Val Gln Leu Gln Glu 305 310 315 320 Val Ile Thr Thr Pro Ser Gly Ala Thr Val Leu Asp Phe Gly Gln Asn 325 330 335 Leu Val Gly Trp Leu Gln Leu Asn Val Thr Gly Pro Ala Gly Thr Ala 340 345 350 Ile Lys Met Val His Val Glu Val Leu Glu Lys Gly Glu Ile Ala Ile 355 360 365 Gly Pro Leu Arg Ser Ala Lys Gln Thr Asp Thr Val Ile Leu Ser Gly 370 375 380 Ser Ala Gln Thr Trp Glu Pro Thr Phe Thr Tyr His Gly Phe Arg Tyr 385 390 395 400 Val Gln Val Asp Asn Trp Pro Val Glu Gln Thr Pro Leu Asp Gln His 405 410 415 Ala Val Lys Ala Ile Val Val His Ser Asp Met Gln Arg Thr Gly Asp 420 425 430 Leu Glu Thr Ser Asp Ser Leu Leu Asn Lys Leu Ile His Asn Val Leu 435 440 445 Trp Ser Leu Lys Gly Asn Phe Met Ser Ile Pro Thr Asp Cys Pro Gln 450 455 460 Arg Asp Glu Arg Leu Gly Trp Thr Gly Asp Ala His Ala Phe Ala Pro 465 470 475 480 Thr Ala Asn Phe Leu Tyr Asn Pro Ala Gly Phe Trp Arg Gly Trp Met 485 490 495 Lys Asp Val Val Ser Glu Gln Leu Glu Met Asp Asn Ile Val Pro Val 500 505 510 Val Val Pro Tyr Val Pro Ala Val Gly Pro Lys Met Pro Thr Ala Val 515 520 525 Trp Gly Asp Val Val Val Ala Asn Ala Trp Asn Ala Tyr Gln Ala Phe 530 535 540 Gly Asp Val Gly Ala Leu Arg Ala Gln Tyr Val Gly Ala Lys Ala Trp 545 550 555 560 Ile Asp Gly Gly Ile Pro Arg Gly Ala Asp Gly Leu Trp Asp His Thr 565 570 575 Tyr Phe Gln Phe Gly Asp Trp Leu Asp Pro Lys Ala Pro Ala Asp Asn 580 585 590 Pro Gly Ala Ala Thr Thr Asp Ser Gly Leu Val Ala Asp Ala Tyr Leu 595 600 605 Val Tyr Val Thr Gly Leu Val Ser Lys Met Ala Ala Val Leu Gly Leu 610 615 620 Asp Ala Glu Ala Ala Glu Tyr Ala Ala Ser Val Ala Ala Leu Lys Lys 625 630 635 640 Ala Tyr Gln Ala Ala Trp Ile Ser Ala Asp Gly Ile Val Lys Tyr Glu 645 650 655 Thr Gln Thr Gly Leu Ala Leu Ser Leu Tyr Phe Gly Leu Phe Pro Asp 660 665 670 Gly Gln Asp Gly Pro Ala Ala Gln Arg Leu Gln Asn Ile Val Glu Ala 675 680 685 Asn Asp Tyr Arg Val Gly Thr Gly Phe Ala Gly Thr His Leu Leu Gly 690 695 700 Gln Thr Leu Thr Ala Tyr Asn Leu Thr Asp Ala Phe Tyr Ser Met Leu 705 710 715 720 Met Gln Thr Ser Val Pro Ser Trp Leu Tyr Gln Val Val Glu Asn Gly 725 730 735 Thr Thr Thr Trp Glu Arg Trp Asp Ser Leu Leu Pro Asp Gly Ser Leu 740 745 750 Asn Ala Asn Met Met Thr Ser Phe Asn His Tyr Ala Phe Gly Ser Val 755 760 765 Val Asn Trp Met Val Arg Thr Ile Gly Gly Leu Ala Pro Ala Glu Pro 770 775 780 Gly Trp Lys Thr Ile Ser Val Ala Pro Ile Pro Gly Gly Gly Leu Thr 785 790 795 800 Ser Ala Lys Thr Thr Tyr Leu Ser Pro Tyr Gly Arg Val Ser Ala Glu 805 810 815 Trp Thr Val Asn Glu Gly Thr Phe Asn Leu Lys Leu Ile Val Pro Pro 820 825 830 Asn Ser Arg Ala Glu Val Ala Leu Pro Gly Asp Asn Gly Arg Val Met 835 840 845 Asn Val Gly Ser Gly Thr His Thr Phe Lys Leu Ser Gly Val Ala Gly 850 855 860 Lys Glu 865

Claims

1. A method for increasing the brightness and/or decreasing the kappa number of a paper pulp, comprising contacting the paper pulp with a GH78 enzyme, wherein the GH78 enzyme a) belongs to glycoside hydrolase family 78, b) exhibits esterase and rhamnosidase activities and c) consists or comprises an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1.

2. The method of claim 1, wherein the GH78 enzyme is a glycoside hydrolase exhibiting feruloyl esterase and .alpha.-L-rhamnosidase activities.

3. The method of claim 1, wherein the amino acid sequence of the GH78 enzyme has at least 65% identity, more preferably at least 70% identity, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95%, 96%, 97%, 98%, 99%, and most preferably 100% identity to the amino acid sequence of SEQ ID NO: 1.

4. The method of claim 1, wherein the GH78 enzyme is derived from a Xylaria sp., such as Xylaria polymorpha.

5. The method of claim 1, wherein the pulp is wood pulp.

6. The method of claim 1, wherein the pulp is a chemical pulp, such as kraft pulp.

7. The method of claim 1, which further comprises a step of chlorine dioxide treatment (D stage) and/or alkaline peroxide treatment (E stage).

8. The method of claim 1, which further comprises contacting the pulp with one or more additional enzyme(s), such as a lipase.

9. A bleaching composition, comprising a paper pulp and a GH78 enzyme, wherein the GH78 enzyme a) belongs to glycoside hydrolase family 78, b) exhibits esterase and rhamnosidase activities and c) consists or comprises an amino acid sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1.

10. The composition of claim 9, wherein the GH78 enzyme belongs to glycoside hydrolase family 78 and exhibits feruloyl esterase and .alpha.-L-rhamnosidase activities.

11. The composition of claim 9, wherein the amino acid sequence of the GH78 enzyme has at least 65% identity, more preferably at least 70% identity, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95%, 96%, 97%, 98%, 99%, and most preferably 100% identity to the amino acid sequence of SEQ ID NO: 1.

12. The composition of claim 9, wherein the pulp is wood pulp.

13. The composition of claim 9, wherein the pulp is a chemical pulp, such as kraft pulp.

14. (canceled)

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