Patent Publication Number: US-2016237618-A1

Title: Enzymatic bleaching of paper pulp

Description:
REFERENCE TO A SEQUENCE LISTING 
     This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. 
     FIELD OF THE INVENTION 
     The present invention relates to bleaching of pulp with a GH78 enzyme. 
     BACKGROUND 
     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. 
     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 
     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. 
     Other aspects and embodiments of the invention are apparent from the description and examples. 
     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.
 
     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.
 
     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 
     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 
     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. 
     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. 
     In a particular embodiment, the pulp to be treated comprises both hardwood pulp and softwood pulp. 
     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). 
     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. 
     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. 
     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. 
     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%. 
     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 
     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 α-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). 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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). 
     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×100)/(Length of Alignment—Total Number of Gaps in Alignment). 
     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. 
     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 
     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° 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 &amp; Williamson (1994),  Microbiology, vol.  140, pp. 779-787). 
     After centrifugation, the released ferulic acid is analyzed by HPLC using a reversed phase C18-column (Synergi Fusion-RP 80A, 4.6 mm×125 mm, Phenomenex®, Aschaffenburg, Germany). The elution by the isocratic mobile phase, which consists of 60% phosphoric acid (15 mM H 3 PO 4 , pH 2.6) and 40% acetonitrile (v/v), is monitored at 323 nm over 7 min. 
     α-L-Rhamnosidase (EC 3.2.1.40) Activity 
     α-L-rhamnosidase activity is measured using p-nitrophenyl α-L-rhamnopyranoside (p-NPRP) as substrate (see also Gallego et al. (2001),  J. Food Sci.,  vol. 66, pp. 204-209). 
     The assay is performed in a 48-well microtiter plate containing 160 μl of the substrate (2.5 mM p-NPRP in 50 mM sodium acetate buffer, pH 5.0) and 40 μl of enzyme sample. The reaction is performed for 5-20 min at 40° C. and stopped by adding 100 μl of 1 M Na 2 CO 3 . The enzyme activity is spectrophotometrically measured (λ=405 nm; ε 405 =2.5 mM −1  cm −1 ) through the liberation of p-nitrophenol using a microplate reader. 
     Bleaching 
     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 (&gt;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. 
     According to the invention, the brightness of the pulp is determined according to TAPPI test method T 452 om-98. 
     Kappa Number 
     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. 
     According to the invention, the kappa number of the pulp is determined according to TAPPI test method T 236 om-06. 
     Process Conditions 
     The process of the invention is particularly applicable to the bleaching of pulp in a process for making paper material. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     The enzymatic treatment may be carried out at a temperature of from about 10° C. to about 100° C. Further examples of temperature ranges (all “from about” and “to about”) are the following: 20-120° C., 30-120° C., 35-120° C., 37-120° C., 40-120° C., 50-120° C., 60-120° C., 70-120° C., 10-100° C., 10-90° C., 10-80° C., 10-70° C., 10-60° C., and 30-60° C., as well as any combination of the upper and lower values here indicated. A typical temperature is from about 20 to 90° C., or 20 to 95° C., preferably from about 40 to 70° C., or 40 to 75° C. Usually, the enzymatic treatment is carried out at atmospheric pressure. But when the temperature exceeds 100° C., the treatment is carried out at a pressure of 1-2 bar (up to 1 bar above atmospheric pressure). 
     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. 
     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. 
     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. 
     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. 
     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. 
     In another particular embodiment, each of the above ranges refers to the total amount of surfactants. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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. 
     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° 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 
     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. 
     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. 
     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. 
     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. 
     Examples of mannanases are the  Trichoderma reesei  endo-beta-mannanases described in Ståhlbrand et al,  J. Biotechnol.  29 (1993), 229-242. 
     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 
     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. 
     Suitable GH78 enzymes are described above. Preferably, the GH78 enzyme belongs to the glycoside hydrolase family 78, and exhibits esterase (feruloyl esterase) and rhamnosidase (α-L-rhamnosidase) activities. 
     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. 
     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. 
     In an embodiment, the GH78 enzyme is derived from a  Xylaria  sp., such as  Xylaria polymorpha.    
     In an embodiment, the pulp is wood pulp. 
     In an embodiment, the pulp is a chemical pulp, such as kraft pulp. 
     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). 
     In an embodiment, the method of the invention further comprises contacting the pulp with one or more additional enzyme(s), such as a lipase. 
     In an embodiment, the method of the invention further comprises a final step of preparing a paper material from the pulp. 
     In a second aspect, the invention provides a bleaching composition, comprising a paper pulp and a GH78 enzyme. 
     Suitable GH78 enzymes are described above. Preferably, the GH78 enzyme belongs to glycoside hydrolase family 78 and exhibits esterase (feruloyl esterase) and rhamnosidase (α-L-rhamnosidase) activities. 
     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. 
     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. 
     In an embodiment, the GH78 enzyme is derived from a  Xylaria  sp., such as  Xylaria polymorpha.    
     In an embodiment, the pulp is wood pulp. 
     In an embodiment, the pulp is a chemical pulp, such as kraft pulp. 
     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. 
     The invention also provides for use of the methods and compositions above for bleaching and/or decreasing the kappa number of paper pulp 
     Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. 
     The present invention is further described by the following examples which should not be construed as limiting the scope of the invention. 
     One specific embodiment of the present invention is described in the set of items herein below. 
     Items: 
     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 α-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 α-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 
     Chemicals used as buffers and substrates were commercial products of at least reagent grade. 
     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 
     The  Xylaria polymorpha  GH78 enzyme was assessed for its ability to bleach oxygen-bleached  eucalyptus  kraft pulp. Feruloyl esterase and α-L-rhamnosidase activities were tested for SEQ ID NO: 1. 
       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° 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 Büchner 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° 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 Büchner funnel. The samples were then washed with tap water thoroughly. Conditions for the Ep-stage were 10% consistency, 5 kg H 2 O 2 /ton (dry pulp), 10.5 kg NaOH/ton (dry pulp) at 85° C. for 80 min. After the Ep-stage, the samples were filtered by using a Büchner funnel. 
     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 &amp; 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 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)