WO2016073610A1

WO2016073610A1 - Xylanase based bleach boosting

Info

Publication number: WO2016073610A1
Application number: PCT/US2015/059045
Authority: WO
Inventors: William Widner; Bjoern Lennart Pierre Alexander Cassland; Kristian Bertel Rømer M. KROGH; Jens Tønne ANDERSEN; Henrik Lund
Original assignee: Novozymes A/S
Priority date: 2014-11-07
Filing date: 2015-11-04
Publication date: 2016-05-12

Abstract

The present invention relates to a thermophilic and alkalophilic xylanase for bleach boosting of paper pulp. The invention further relates to a bleach boosting method comprising treatment of paper pulp with the thermophilic and alkalophilic xylanase. A composition comprising paper pulp and the thermophilic and alkalophilic xylanase is also part of the invention.

Description

XYLANASE BASED BLEACH BOOSTING

Reference to sequence listing

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference. FIELD OF THE INVENTION

The present invention relates to methods of bleaching pulp. More specifically, the present invention relates to methods of bleaching pulp using xylanase.

BACKGROUND OF THE INVENTION

The production of bleached chemical pulp is a major industry. Bleached chemical pulp is the largest component of all types of white paper, including that used in photocopy paper, writing paper, and paper packaging. In addition, bleached chemical pulp is also used to impart strength to less expensive grades of paper, such as newsprint. Bleached chemical pulp has large markets because of its high degree of whiteness and cleanliness, the stability of the whiteness, its high strength, and the ease and uniformity of the printing surface it provides. These attributes are obtained when lignin, which is colored and decreases the interfiber bonding of the cellulose, is almost completely removed from the pulp.

In the process of chemical pulping, the furnish (or feedstock) primarily consists of wood chips which are added to a reaction chamber, known as a digester, and are treated with chemicals to dissolve lignin in the pulp.

There are several chemical pulping processes known in the art. Two of the major chemical pulping processes are kraft pulping, in which the pulp is cooked in alkaline liquor, and sulfite pulping, in which the pulp is cooked in acidic liquor. Both kraft pulping and sulfite pulping may be performed in batch or continuous digestors.

One of the main purposes of the pulping process is to release lignin which binds cellu- lose fibers in the feedstock. Pulping dissolves 85 % to 95 % of the lignin in the feedstock material. Following the pulping stage, the pulp is washed with water to remove dissolved lignin.

While pulping removes most of the lignin in the feedstock material, it is not capable of removing all the lignin without destroying the cellulose fibers of the feedstock. The remaining lignin is removed from the pulp by bleaching.

A pulp bleaching process may consist of many stages. For example, following pulping, a pulp bleaching process may comprise an alkaline oxygen delignification stage (0), an enzymatic treatment stage (X), one or more chlorine dioxide bleaching stages (D), and one or more alkaline extraction stages (E). A pulp bleaching process may also comprise one or more water washes or alternatively, each stage may comprise a water wash as a final step of the stage. Thus, a representative pulp bleaching sequence in which pulp is bleached using three chemical bleaching stages and two alkaline extraction stages may be represented as D-E-D-E-D. Similarly, a pulp bleaching sequence wherein pulp is subjected to an alkaline oxygen delignification stage, an enzymatic treatment stage, three chlorine dioxide bleaching stages and two alkaline extraction stages wherein each stage is followed by a water wash may be represented by O-X- D-E-D-E-D.

It is common for mills to perform an alkali-oxygen delignification stage prior to carrying out chemical bleaching of pulp. This process consists of reacting the pulp with oxygen and alkali at high temperatures (e.g. at approximately 100°C) for a period of e.g. about one hour.

Alkali-oxygen delignification reduces the amount of lignin in the pulp by 35-50%, but this process is harsh on the pulp and is often accompanied by destruction of some of the cellulose fibers in the pulp. Following alkali-oxygen delignification, the pulp is typically washed as described earlier to remove solubilized lignin.

The next bleaching stage after alkali-oxygen delignification is usually chemical bleaching with oxidative chemicals, the most prominent being chlorine dioxide (CI0₂). However, several processes have been described which may facilitate or enhance bleaching of pulp prior to chemical bleaching. For example, an enzymatic treatment stage with xylanase may be used to enhance the bleaching of pulp prior to chemical bleaching.

Xylanases are used in the pulp and paper industry to enhance the bleaching of pulp and to decrease the amount of bleaching chemicals used in bleaching stages. There have been several mechanisms proposed for the bleaching action of xylanase. During the kraft cook, a fraction of the solubilized xylan reprecipitates on the surface of the pulp fibres. It is believed that xylanase hydrolysis of the reprecipitated xylan increases the permeability of the unbleached pulp fibre and facilitates the lignin removal in subsequent bleaching stages. Xylanase bleach boosting may also give rise to removal of hexenuronic acid and lignin-carbohydrate complexes which are formed during kraft cooking and remain bound to xylan within the fibre wall.

Regardless of the mechanism, xylanase treatment allows subsequent bleaching chemicals such as chlorine, chlorine dioxide, hydrogen peroxide, or combinations of these chemicals to bleach pulp more efficiently than in the absence of xylanase. Pretreatment of pulp with xylanase prior to chemical bleaching increases the whiteness and quality of the final paper product and reduces the amount of bleaching chemicals such as chlorine-based chemicals which must be used to bleach the pulp. This in turn decreases the presence of adsorbable organic hal- ides (AOX) in effluents produced by such processes.

Xylanases have been isolated from a variety of organisms including bacteria and fungi.

Generally, fungal xylanases exhibit optimal activity at acidic pHs, in the range of about 3.5 to 5.5, and a temperature of about 50°C. In contrast, bacterial xylanases exhibit typically optimal activity at pH 5 to pH 7 and a temperature optimum between 50°C and 70°C.

Following kraft pulping and alkali oxygen delignification the temperature and the pH of the pulp are high, and each of these operations are followed by a washing stage. The condi- tions of the pulp following pulping and alkali oxygen delignification have prompted efforts to identify and isolate thermophilic and alkalophilic xylanases which may be used for enzymatic treatment with minimal adjustment of the temperature and pH of the pulp. For example, US 5,405,789 (Campbell et al.) discloses construction of thermostable mutants of low molecular mass xylanase from Bacillus circulans. US 5,759,840 (Sung et al.) discloses modification of a family 1 1 xylanase from Trichoderma reesei to improve thermophilicity, alkalophilicity and thermostability as compared to the natural xylanase. US 5,916,795 (Fukunaga et al.) discloses a thermostable xylanase from Bacillus.

The object of the present invention is to develop a thermophilic and alkalophilic xylanase for bleach boosting of paper pulp.

SUMMARY OF THE INVENTION

In a first main aspect of the present invention there is provided a xylanase, derived from a gene contained within Dictyoglomus thermophilum, and contained within the family of enzymes known as Xylanases, and having endo-beta-1 ,4-xylanase activity at elevated tempera- tures, wherein the xylanase consists or comprises an amino acid sequence as defined in SEQ ID NO: 1 , and wherein said xylanase can be used for boosting of bleaching of cellulose containing products such as paper pulp.

In a further main aspect of the present invention there is provided a method for boosting of bleaching of cellulose products such as paper pulp wherein the process utilizes a xylanase according to the present invention such as a xylanase consisting of or comprising SEQ ID NO: 1 and having beta- 1 ,4-xylanase activity at elevated temperatures.

The invention relates to a method of bleaching paper pulp comprising the steps of:

a) reacting said paper pulp with one or more bleaching agent; and

b) treating said paper pulp with a xylanase according to the invention such a xylanase consisting of or comprising SEQ ID NO: 1 or a xylanase having at least 95% (such as at least

96%, 97%, 98% or 99%) identity to SEQ ID NO: 1 .

The invention also relates to a composition comprising paper pulp and a xylanase consisting of or comprising SEQ ID NO: 1 or consisting of or comprising one or more variants of SEQ ID NO: 1 such as a xylanase having at least 95% (such as at least 96%, 97%, 98% or 99%) identity to SEQ ID NO: 1. Preferably the xylanase has activity at a temperature between 60°C and 95°C and within the pH range 5 to 10. More preferably the xylanase has activity at approximately 85°C and approximately pH 8.75 e.g. in the presence of pulp.

In a yet further main aspect the invention comprises the use of the xylanase described above as a means though not necessarily the sole means for an at least partial degradation of the xylans in a mass of paper pulp, whereby in use the pulp becomes at least partially bleached and/or bleaching by chemicals is boosted.

DEFINITIONS

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 includes 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 multistage 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.

Bleach boosting: the term "bleach boosting" means that the bleaching of the paper pulp is improved/enhanced by the xylanase treatment of the paper pulp. The improved bleaching means that bleaching of the paper pulp is increased and/or that the amount of chemicals used for bleaching (to obtain the same bleaching result) can be reduced.

Paper and Pulp: The term "paper material" refers to products, which can be made of pulp, such as paper, linerboard, corrugated paperboard, tissue, towels, packaging materials, corrugated containers or boxes. The term "pulp" means any pulp which can be used for the production of a paper material.

SEQ ID NO 1 : SEQ ID NO 1 is defined in the sequence listing as well as herein below: QTSITLTSNA SGTFDGYYYE LWKDTGNTTM TVYTQGRFSC QWSNINNALF RTGKKYNQNW

QSLGTIRITY SATYNPNGNS YLCIYGWSTN PLVEFYIVES WGNWRPPGAT SLGQVTIDGG

TYDIYRTTRV NQPSIVGTAT FDQYWSVRTS KRTSGTVTVT DHFRAWANRG LNLGTIDQIT LCVEGYQSSG SANITQNTFS QGS DETAILED DESCRIPTION OF THE INVENTION

Following is a description of the preferred embodiment of the present invention. It is given by way of example only, and it should be appreciated that a number of modifications may be made to this example without departing from the scope of the invention. The object of the present invention is to develop a thermophilic and alkalophilic xylanase for bleach boosting of paper pulp. The invention relates to methods for bleaching and methods for bleach boosting of pulp using the xylanase according to the invention.

a) reacting said paper pulp with one or more bleaching agent; and

b) treating said paper pulp with a xylanase according to the invention.

Step a) is performed with one or more conventional bleaching agents/methods. Step b) is described in further details herein below. The paper and pulp according to the invention is also described herein below. In one embodiment step a) is performed before step b). In another embodiment step b) is performed before step a). In a further embodiment step a) and b) are per- formed simultaneously.

Paper and Pulp

The term "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 can be 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 chemithermimechanical pulp.

Chemical pulp can be manufactured by alkaline cooking whereby most of the lignin and hemicellulose components are removed. In Kraft pulping or sulphate cooking sodium, sulphide and 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 and)) 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.

Bleaching methods

This invention relates to xylanases from organisms belonging to the species Dictyoglo- mus thermophilum or variants of these xylanases and their use in bleaching of paper pulp. The organisms have been characterised by Saiki et al. (Saiki, T., Kobayashi, Y., Kawagoe, K., and Beppu, T., 1985; Int J. Syst. Bacterid.), Patel et al. (Patel, B. K., Morgan, H. W., Wiegel, J., and Daniel, R. M., 1987; Arch. Microbiol. 147 21 -24), and Love et al. (Love, C. A., Patel, B. K. C, Ludwig, W. and Stackebrant, E., 1993; FEMS Microbiology Letters 107 317-320). The type culture of Dictyoglomus thermophilum is publicly available from the Deutsche Sammlung von Mirkoorganismen und Zellkulturen GmbH, Mascheroder Weg ib, D-3300 Braunschweig, Ger- many, under the accession number DSM 3960. The present invention relates to bleaching of paper pulp comprising use of a xylanase derived from a gene contained within Dictyoglomus thermophilum, and contained within the family of enzymes known as Xylanases, and having beta-1 ,4-xylanase activity at elevated temperatures, wherein the xylanase has an amino acid sequence as defined in SEQ ID NO: 1 or comprises the amino acid sequence as defined in SEQ ID NO: 1 , or is a variant of SEQ ID NO: 1 and wherein said xylanase can be used for bleaching or boosting of bleaching of cellulose products such as paper pulp.

The present invention further relates to a method/process for bleaching and/or boosting of bleaching of cellulose containing products such as paper pulp, wherein the method/process utilizes a xylanase according to the present invention such as a xylanase consisting of SEQ ID NO: 1 or comprising the amino acid sequence as defined in SEQ ID NO: 1 or variants of SEQ ID NO: 1.

In one preferred embodiment the bleaching method comprises use of the xylanase according to the present invention.

In one embodiment the bleaching method comprises use of a xylanase consisting of

SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of SEQ ID NO: 1 and one or more additional amino acids in the C-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acids in the C-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the bleaching method comprises use of a xylanase consisting of SEQ I D NO: 1 and one or more additional amino acids in the N-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acids in the N-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the bleaching method comprises use of a xylanase consisting of SEQ ID NO: 1 and one or more additional amino acids in the C-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acids in the C-terminal of SEQ ID NO: 1 and one or more additional amino acids in the N-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acids in the N-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the bleaching method comprises use of a variant of SEQ ID NO: 1 having one or more amino acid deletions in the C-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions in the C-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the bleaching method comprises use of a variant of SEQ ID NO: 1 having one or more amino acid deletions in the N-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions in the N-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the bleaching method comprises use of a variant of SEQ ID NO: 1 having one or more amino acid deletions in the C-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions in the C-terminal of SEQ ID NO: 1 and one or more amino acid deletions in the N-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions in the N-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the bleaching method comprises use of a xylanase consisting of from 203 to 21 1 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the bleaching method comprises use of a xylanase consisting of from 203 to 210 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of from 203 to 209 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of from 203 to 208 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of from 203 to 207 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of from 203 to 206 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of from 203 to 205 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of from 203 to 204 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of less than 212 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of less than 21 1 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of less than 210 amino acids and comprising SEQ ID NO: 1. In one embodiment the bleaching method comprises use of a xylanase consisting of less than 219 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of less than 208 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of less than 207 amino acids and comprising SEQ ID NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of less than 206 amino acids and comprising SEQ I D NO: 1.

In one embodiment the bleaching method comprises use of a xylanase consisting of less than 205 amino acids and comprising SEQ ID NO: 1.

In one embodiment the xylanase used in the bleaching method is a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 , such as an amino acid sequence that is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 and wherein the xylanase has xylanase activity. The variant can comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitutions (conservative, non-conservative or a mixture of conservative and non- conservative substitutions) and wherein the xylanase has xylanase activity. Preferably, the amino acid substitutions are conservative.

Examples of conservative substitutions are within the groups of basic amino acids (argi- nine, 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/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, LeuA al, 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 DNase 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 DNase 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 pho- toaffinity 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; W095/17413; or W095/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner ei a/., 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 us- ing the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment). In one embodiment the xylanase used in the bleaching method is a variant of SEQ ID NO: 1 having one or more amino acid deletions (C-terminal and/or N-terminal and/or non-terminal) such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions, and wherein the xylanase has xylanase activity.

In one embodiment the xylanase used in the bleaching method is a variant of SEQ ID NO: 1 having one or more amino acid additions (C-terminal and/or N-terminal and/or nonterminal) such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid additions and wherein the xylanase has xylanase activity.

In one embodiment the xylanase used in the bleaching method is a variant of SEQ ID

NO: 1 having one or more amino acid deletions (C-terminal and/or N-terminal and/or nonterminal) such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions and one or more amino acid additions (C-terminal and/or N-terminal and/or nonterminal) such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid additions and wherein the xylanase has xylanase activity.

In one embodiment the xylanase used in the bleaching method is a variant of SEQ ID NO: 1 having one or more amino acid deletions (C-terminal and/or N-terminal and/or non- terminal) and/or one or more amino acid additions (C-terminal and/or N-terminal and/or nonterminal) and/or one or more amino acid substitutions (conservative, non-conservative or a mixture of conservative and non-conservative substitutions) and wherein the xylanase has xylanase activity.

Preferably the xylanase used in the bleaching method has activity between 45°C and

100°C and within the pH range 3 to 1 1. More preferably the xylanase has activity between 75°C and 90°C and within the pH range 7 to 9. More preferably the xylanase has activity between 80°C and 90°C and within the pH range 8 to 9. Even more preferably the xylanase has activity at approximately 85°C and approximately pH 8.75.

Preferably the xylanase used in the bleaching method has activity between 60°C and

95°C and within the pH range 5 to 10. More preferably the xylanase has activity between 75°C and 90°C and within the pH range 7 to 9. More preferably the xylanase has activity between 80°C and 90°C and within the pH range 8 to 9. Even more preferably the xylanase has activity at approximately 85°C and approximately pH 8.75.

95°C. More preferably the xylanase has activity between 75°C and 90°C. More preferably the xylanase has activity between 80°C and 90°C. Even more preferably the xylanase has activity at approximately 85°C.

Preferably the xylanase used in the bleaching method has activity within the pH range 5 to 10. More preferably the xylanase has activity within the pH range 7 to 9. More preferably the xylanase has activity within the pH range 8 to 9. Even more preferably the xylanase has activity at approximately 85°C and approximately pH 8.75.

In a yet further main aspect the invention comprises the use of the xylanase described above as a means though not necessarily the sole means for an at least partial degradation of the xylans in a mass of pulp, whereby in use the pulp becomes at least partially bleached and/or bleaching by chemicals is boosted.

The xylanase according to the invention can be used in ECF (Elemental Chlorine Free) and/or TCF (Totally Chlorine Free) bleaching sequences.

The process of the invention is particularly applicable to the bleaching of pulp in a pro- cess 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 xylanase 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 xylanase 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 xylanase may also be added together with these substances. Preferably the xylanse is added prior to bleaching. The xylanase can also be added to the circu- lated 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 xylanase is added during the brown-stock washing.

In the present context, the term "process water" comprises 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 xylanase applied, the reac- tion time and the conditions given.

The xylanase 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 obtaining a desired bleaching and/or saving of bleaching chemicals.

In a particular embodiment, the dosage of the xylanase is from about 0.1 mg xylanase to about 100,000 mg xylanase per ton of paper pulp.

In further particular embodiments, the amount of the xylanase is in the range of 0.00001- 20; or 0.0001 -20 mg of xylanase (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 xylanase per gram of pulp material.

The xylanase 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 xylanase treatment may be carried out at a temperature of from about 50°C to about 120°C. Further examples of temperature ranges (all "from about" and "to about") are the following: 50-60°C, 60-70°C, 70-80°C, 80-90°C, 90-100°C, 100-1 10°C, 1 1 -120°C, as well as any combination thereof. A typical temperature is from about 60 to 95°C, or 70 to 95°C, preferably from about 80 to 95°C, or 90 to 95°C. Usually, the enzymatic treatment is carried out at atmospheric pressure. But when the temperature exceeds 100°C, the treatment can be carried out at a pressure of 1-2 bar (up to 1 bar above atmospheric pressure).

The xylanase treatment is carried out at a pH of from about 3 to about 12, preferably at a pH from about 7 to about 12, more preferably at a pH from about 8 to about 1 1 , and most preferably at a pH from about 8 to about 10. The pH for the xylanase treatment can alternatively be selected from the group consisting of the pH intervals consisting of pH 7-8, pH 8-9, pH 9-10, pH 10-1 1 , pH 1 1 -12, pH 12-13, pH 13-14, or any combination of these intervals.

A suitable duration of the xylanase 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 5 hours, 30 minutes to 4 hours, preferably 1 hour to 3 hours, or 2 hours to 3 hours.

Various additives and/or additional enzymes 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, polydial- lyl dimethyl ammonium chloride (DADMAC), DADMAC/Acrylamide co-polymers, and ionene polymers, such as those disclosed in US patents nos. 5,681 ,862; and 5,575,993. Examples of zwitterionic or amphoteric surfactants are betains, glycinates, amino propionates, imino propio- nates and various imidazolin-derivatives. Also the polymers disclosed in US patent 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 xylanase 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 par- ticular 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 xylanase 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.

In the process of the invention, the xylanase 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 xylanase.

In particular embodiments the additional enzyme is an enzyme which has protease, lipase, 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 xylanase of the invention 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 xylanase is used.

Compositions

The present invention further relates to a composition comprising cellulose products such as paper pulp and a xylanase according to the present invention such as a xylanase consisting of SEQ ID NO: 1 or comprising the amino acid sequence as defined in SEQ ID NO: 1 or variants of SEQ ID NO: 1 such as a xylanase having at least 95% (such as at least 96%, 97%, 98% or 99%) identity to SEQ ID NO: 1.

In one preferred embodiment the composition comprises a paper pulp and a recombinant xylanase. In one preferred embodiment the composition comprises a paper pulp and a xylanase comprising or consisting of SEQ ID NO: 1.

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of SEQ ID NO: 1 and one or more additional amino acids in the C-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acids in the C-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of SEQ ID NO: 1 and one or more additional amino acids in the N-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acids in the N-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of SEQ ID NO: 1 and one or more additional amino acids in the C-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acids in the C-terminal of SEQ ID NO: 1 and one or more additional amino acids in the N-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional amino acids in the N-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the composition comprises 1 ) pulp and 2) a variant of SEQ ID NO: 1 having one or more amino acid deletions in the C-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions in the C-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the composition comprises 1 ) pulp and 2) a variant of SEQ ID NO: 1 having one or more amino acid deletions in the N-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions in the N-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the composition comprises 1 ) pulp and 2) a variant of SEQ ID NO: 1 having one or more amino acid deletions in the C-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions in the C-terminal of SEQ ID NO: 1 and one or more amino acid deletions in the N-terminal of SEQ ID NO: 1 , such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions in the N-terminal of SEQ ID NO: 1 and wherein the xylanase has xylanase activity.

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of from 203 to 21 1 amino acids and comprising SEQ ID NO: 1.

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of from 203 to 210 amino acids and comprising SEQ ID NO: 1. In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of from 203 to 209 amino acids and comprising SEQ ID NO: 1.

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of from 203 to 208 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of from 203 to 207 amino acids and comprising SEQ ID NO: 1.

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of from 203 to 206 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of from 203 to 205 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of from 203 to 204 amino acids and comprising SEQ ID NO: 1.

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of less than 212 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of less than 21 1 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of less than 210 amino acids and comprising SEQ ID NO: 1.

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of less than 219 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of less than 208 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of less than 207 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of less than 206 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the composition comprises 1 ) pulp and 2) a xylanase consisting of less than 205 amino acids and comprising SEQ ID NO: 1 .

In one embodiment the xylanase in the composition is a variant of SEQ ID NO: 1 com- prising or consisting of an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 , such as an amino acid sequence that is at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 and wherein the xylanase has xylanase activity. The variant can comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid substitu- tions (conservative, non-conservative or a mixture of conservative and non-conservative substitutions) and wherein the xylanase has xylanase activity.

In one embodiment the xylanase in the composition is a variant of SEQ ID NO: 1 having one or more amino acid deletions (C-terminal and/or N-terminal and/or non-terminal) such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions, and wherein the xylanase has xylanase activity.

In one embodiment the xylanase in the composition is a variant of SEQ ID NO: 1 having one or more amino acid additions (C-terminal and/or N-terminal and/or non-terminal) such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid additions and wherein the xylanase has xylanase activity.

In one embodiment the xylanase in the composition is a variant of SEQ ID NO: 1 having one or more amino acid deletions (C-terminal and/or N-terminal and/or non-terminal) such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid deletions and one or more amino acid additions (C-terminal and/or N-terminal and/or non-terminal) such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid additions and wherein the xylanase has xylanase activity.

In one embodiment the xylanase in the composition is a variant of SEQ ID NO: 1 having one or more amino acid deletions (C-terminal and/or N-terminal and/or non-terminal) and/or one or more amino acid additions (C-terminal and/or N-terminal and/or non-terminal) and/or one or more amino acid substitutions (conservative, non-conservative or a mixture of conservative and non-conservative substitutions) and wherein the xylanase has xylanase activity.

Preferably the xylanase in the composition has activity between 45°C and 100°C and within the pH range 3 to 1 1 . More preferably the xylanase has activity between 75°C and 90°C and within the pH range 7 to 9. More preferably the xylanase has activity between 80°C and 90°C and within the pH range 8 to 9. Even more preferably the xylanase has activity at approximately 85°C and approximately pH 8.75.

Preferably the xylanase in the composition has activity between 60°C and 95°C and within the pH range 5 to 10. More preferably the xylanase has activity between 75°C and 90°C and within the pH range 7 to 9. More preferably the xylanase has activity between 80°C and 90°C and within the pH range 8 to 9. Even more preferably the xylanase has activity at approximately 85°C and approximately pH 8.75.

Preferably the xylanase in the composition has activity between 45°C and 100°C. More preferably the xylanase has activity within between 75°C and 90°C. More preferably the xylanase has activity within between 80°C and 90°C. Even more preferably the xylanase has activ- ity at approximately 85°C. Preferably the xylanase in the composition has activity within the pH range 3 to 1 1. More preferably the xylanase has activity within the pH range 7 to 9. More preferably the xylanase has activity within the pH range 8 to 9. Even more preferably the xylanase has activity at approximately 85°C and approximately pH 8.75.

Preferably the xylanase of the composition has xylanase activity.

The composition comprising pulp and the xylanase according to the invention can have a temperature of from about 45°C to about 120°C. Further examples of temperature ranges (all "from about" and "to about") are the following: 50-60°C, 60-70°C, 70-80°C, 80-90°C, 90-100°C, 100-1 10°C, 1 1-120°C, as well as any combination thereof. A typical temperature of the compo- sition is from about 60 to 95°C, or 70 to 95°C, preferably from about 80 to 95°C, or 90 to 95°C.

The pH of the composition comprising pulp and the xylanase according to the invention typically has a pH of from about 3 to about 12, preferably a pH from about 8 to about 12, more preferably a pH from about 8 to about 1 1 , and most preferably a pH from about 8 to about 10. The pH of the composition can alternatively be selected from the group consisting of the pH in- tervals consisting of pH 7-8, pH 8-9, pH 9-10, pH 10-1 1 , pH 1 1 -12, pH 12-13, pH 13-14, or any combination of these intervals.

The composition comprising pulp and the xylanase according to the invention should comprise the xylanase of the invention in an effective amount. By the term "effective amount" is meant the amount sufficient to achieve the desired and expected effect, such as obtaining a de- sired bleaching and/or saving of bleaching chemicals.

In a particular embodiment, the dosage of the xylanase in the composition is from about 0.1 mg xylanase to about 100,000 mg xylanase per ton of paper pulp.

In further particular embodiments, the amount of the xylanase in the composition is in the range of 0.00001 -20; or 0.0001 -20 mg of xylanase (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 xylanase per gram of pulp material.

The composition comprising pulp and the xylanase according to the invention can have a 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.

In further particular embodiments of the above composition, and of the process of the invention, the xylanase 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.

In the process of the invention, the xylanase may be applied alone or together with an additional enzyme. Accordingly, the composition of the present invention can comprise paper pulp, the xylanase according to the invention and 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. In particular embodiments the additional enzyme is an enzyme which has protease, lipase, 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.

In a yet further main aspect the invention comprises the use of the composition described above as a means though not necessarily the sole means for an at least partial degra- dation of the xylans in a mass of pulp, whereby in use the pulp becomes at least partially bleached and/or bleaching by chemicals is boosted.

PREFERRED EMBODIMENTS

Preferred embodiment of the invention are given herein below.

1. A method of bleaching paper pulp comprising the steps of: a) reacting said paper pulp with one or more bleaching agents; and b) treating said paper pulp with a xylanase having at least 95% (such as at least 96%, 97%, 98% or 99%) identity to SEQ ID NO: 1.

2. The method according to item 1 , wherein the xylanase treatment in step b) is performed at a temperature between 70°C to 100°C. 3. The method according to any of items 1 and 2, wherein the xylanase treatment in step b) is performed at a pH between 8 and 10.

4. The method according to any of items 1 to 3, wherein step a) is performed before step b).

5. The method according to any of items 1 to 3, wherein step b) is performed before step a).

6. The method according to any of items 1 to 3, wherein step a) and step b) are performed simultaneously.

7. The method according to any of items 1 to 6, wherein the xylanase is at least 96% identical to SEQ ID NO: 1. 8. The method according to any of items 1 to 7, wherein the xylanase is at least 97% identical to SEQ ID NO: 1.

9. The method according to any of items 1 to 8, wherein the xylanase is at least 98% identical to SEQ ID NO: 1. 10. The method according to any of items 1 to 9, wherein the xylanase is at least 99% identical to SEQ ID NO: 1.

1 1. The method according to any of items 1 to 10, wherein the xylanase is identical to SEQ ID NO: 1 .

12. The method according to any of items 1 to 1 1 , wherein the xylanase consists of less than 212 amino acids and comprises SEQ ID NO: 1.

13. The method according to any of items 1 to 12, wherein the xylanase consists of less than 21 1 amino acids and comprises SEQ ID NO: 1.

14. The method according to any of items 1 to 13 wherein the xylanase consists of less than 210 amino acids and comprises SEQ ID NO: 1. 15. The method according to any of items 1 to 14, wherein the xylanase consists of less than 209 amino acids and comprises SEQ ID NO: 1.

16. The method according to any of items 1 to 15, wherein the xylanase consists of less than 208 amino acids and comprises SEQ ID NO: 1.

17. The method according to any of items 1 to 16, wherein the xylanase consists of less than 207 amino acids and comprises SEQ ID NO: 1.

18. The method according to any of items 1 to 17, wherein the xylanase consists of less than 206 amino acids and comprises SEQ ID NO: 1.

19. The method according to any of items 1 to 18, wherein the xylanase consists of less than 205 amino acids and comprises SEQ ID NO: 1. 20. A composition comprising paper pulp and a xylanase with a sequence identity to SEQ ID NO: 1 of at least 95% (such as at least 96%, 97%, 98% or 99%). 21. The composition according to item 20, wherein the xylanase consists of or is identical to SEQ ID NO:1 .

22. The composition according to any of items 20-21 , wherein the temperature of the composition is between 70°C to 100°C. 23. The composition according to any of items 20-22, wherein the pH of the composition is between pH 8 and 10.

24. The composition according to any of items 20-23, wherein the xylanase is at least 96% identical to SEQ ID NO: 1 .

25. The composition according to any of items 20-24, wherein the xylanase is at least 97% identical to SEQ ID NO: 1 .

26. The composition according to any of items 20-25, wherein the xylanase is at least 98% identical to SEQ ID NO: 1.

27. The composition according to any of items 20-26, wherein the xylanase is at least 99% identical to SEQ ID NO: 1 . 28. The composition according to any of items 20-27, wherein the xylanase is identical to SEQ ID NO: 1.

29. The composition according to any of items 20-28, wherein the xylanase consists of less than 212 amino acids and comprises SEQ ID NO: 1.

30. The composition according to any of items 20-29, wherein the xylanase consists of less than 21 1 amino acids and comprises SEQ ID NO: 1.

31. The composition according to any of items 20-30, wherein the xylanase consists of less than 210 amino acids and comprises SEQ ID NO: 1.

32. The composition according to any of items 20-31 , wherein the xylanase consists of less than 209 amino acids and comprises SEQ I D NO: 1. 33. The composition according to any of items 20-32, wherein the xylanase consists of less than 208 amino acids and comprises SEQ ID NO: 1. 34. The composition according to any of items 20-33, wherein the xylanase consists of less than 207 amino acids and comprises SEQ ID NO: 1.

35. The composition according to any of items 20-34, wherein the xylanase consists of less than 206 amino acids and comprises SEQ ID NO: 1. 36. The composition according to any of items 20-35, wherein the xylanase consists of less than 205 amino acids and comprises SEQ ID NO: 1.

37. A method for making a white paper material from a paper pulp comprising:

a) preparing the composition of any of items 20-36;

b) reacting the paper pulp with a chlorine and/or oxygen based oxidant before, simultaneous with, or after step a); and

c) preparing a paper material from the paper pulp;

wherein steps a) and b) are carried out under conditions sufficient to obtain a brightness of > 88% ISO (such as more than 90% ISO).

38. Use of the composition of any of items 20-36 for making a paper material.

EXAMPLE 1 : DICTYOGLOMUS THERMOPHILUM XYLANASE TREATMENT AT HARSH CONDITIONS GIVES RISE TO SAVINGS IN BLEACHING CHEMICALS DURING TCF BLEACHING OF OXYGEN-DELIGNIFIED EUCALYPTUS KRAFT PULP

To determine whether the novel Dictyoglomus thermophilum GH 1 1 xylanase (SEQ ID NO 1 ) can contribute to savings in bleaching chemicals, oxygen-delignified eucalyptus kraft pulp was bleached in a QP- and XQP-bleaching sequence with different amounts of hydrogen peroxide. The pre-bleaching treatment with the Dictyoglomus thermophilum xylanase (SEQ ID NO 1 ) was conducted at 80°C and pH 9.5 for 2 h. The reference pulps were treated under the same conditions but with no enzyme addition. The conditions for the QP- and the XQP-bleaching sequence are shown in Table 1.

Table 1. Conditions for QP- and XQP-bleaching of pulps.

After the bleaching, the hydrogen peroxide was removed from the pulp samples on a Buchner funnel. The samples were then washed thoroughly. After the washing, the pulp samples were resuspended in water to a consistency of 0.4 %. The pH of the pulp was adjusted with H₂S0₄ (to pH 2). After 20 min the pulp was drained using a Buchner funnel and washed with deion- ized water.

The kappa number was determined on approximately 0.5 to 1 g pulp samples using a scaled- down version of the Technical Association of the Pulp and Paper Industry (TAPPI) standard method T236.

When xylanase treated and untreated pulps were compared, the D. thermophilum xylanase treated pulps demonstrated a lower hydrogen peroxide requirement to reach the same kappa number as the reference (Table 2). As can be seen in Table 2, the untreated pulp which was bleached with 1.5 % hydrogen peroxide reached approximately 9.6 in kappa number. The pulps treated with D. thermophilum xylanase and 1 .28 and 0.9 % hydrogen peroxide were bleached to kappa number 9.2 and 9.6, respectively. The results in Table 2 suggest that D. thermophilum xylanase treatment saves approximately 40 % hydrogen peroxide.

Table 2. The effect of D. thermophilum xylanase treatment on hydrogen peroxide requirements in a XQP bleaching sequence. The pre-bleaching treatment with Dictyoglomus thermophilum xylanase was conducted at 80°C and pH 9.5 for 2 h.

EXAMPLE 2: TCF BLEACHING OF OXYGEN-DELIGNIFIED EUCALYPTUS KRAFT PULP USING THE DICTYOGLOMUS THERMOPHILUM GH11 XYLANASE AT EXTREME TEM- PERATURE CONDITIONS

The effect of the novel Dictyoglomus thermophilum GH 1 1 xylanase (SEQ ID NO 1 ) was evaluated on oxygen-delignified eucalyptus kraft pulp at 90°C and pH 8 in a XQP-bleaching sequence. The reference pulp was treated in the same way as the xylanase treated pulp and bleached in QP-bleaching process. Xylanase treated and untreated pulps were bleached under the conditions summarized in Table 3.

Table 3. Conditions for QP- and XQP-bleaching of pulps.

After the P-stage, the pulp samples were treated in the same way as described in Example 1 . The kappa number was determined using a scaled-down version of the Technical Association of the Pulp and Paper Industry (TAPPI) standard method T236.

As shown in Table 4, the D. thermophilum xylanase contributed to a significant kappa number reduction at 90°C and pH 8.

Table 4. Kappa number after XQP-bleaching of oxygen-delignified eucalyptus kraft pulp. The xylanase pretreatment was conducted at 90°C and pH 8 for 2 h.

EXAMPLE 3: SIGNIFICANT CHLORINE DIOXIDE SAVINGS ARE OBTAINED DURING ECF- BLEACHING OF HARDWOOD KRAFT PULP BY USING THE NOVEL GH 11 XYLANASE FROM DICTYOGLOMUS THERMOPHILUM The savings in chlorine dioxide that can be obtained by using the novel Dictyoglomus thermophilum GH 1 1 xylanase (SEQ ID NO 1 ) was analysed after XD₀E₁D₁D₂-bleaching of unbleached aspen kraft pulp. The reference pulp was bleached in the same way as the xylanase treated pulp but without xylanase addition. The reference pulp was bleached with 2.1 1 % chlorine dioxide in the D₀-stage whereas the xylanase treated pulps were bleached with four different charg- es of chlorine dioxide in the D₀-stage. The treatment conditions for each bleaching stage are shown in Table 5.

Table 5. Bleaching conditions for XD₀E₁D₁D₂-bleaching and D₀E₁D₁D₂-bleaching of aspen kraft pulp.

pH 8 8

Retention time (h) 2 2

Consistency (%) 10 10

Do-stage

CI0₂ (%) 2.1 1 2.1 1 , 1 .89, 1.68 and 1.48

Temperature (°C) 60 60

Final pH 3.5-4.0 3.5-4.0

Retention time (h) 1 1

Consistency (%) 10 10

E₁ -stage

NaOH (%) 1.06 1.06

Temperature (°C) 70 70

Final pH 1 1.0-1 1.5 1 1.0-1 1.5

Retention time (h) 1 1

Consistency (%) 10 10

D₁ -stage

CI0₂ (%) 1.05 1 .05

Temperature (°C) 75 75

Final pH 3.5-4.0 3.5-4.0

Retention time (h) 3 3

Consistency (%) 10 10

D₂-stage

CI0₂ (%) 0.36 0.36

Temperature (°C) 75 75

Final pH 3.5-4.0 3.5-4.0

Retention time (h) 3 3

Consistency (%) 10 10

After the pulp bleaching, the brightness of the D₀E₁D₁D₂- and XD₀E₁D₁D₂-bleached pulps was determined. The hand sheets were made by using TAPPI standard equipment aacording to TAPPI method T272 sp-97. The brightness of the hand sheets was analyzed according to TAP- PI method T-452 om-98. As can be seen in Table 6, the D. thermophilum xylanase treated pulp had 1 .4 units higher brightness than the reference pulp when the pulps were bleached with equal chlorine dioxide charges in the D₀-stage. In addition, the D. thermophilum xylanase treated pulps that were bleached with 10 and 20 % less chlorine dioxide in the D₀-stage showed sig- nificantly higher brightness values than the reference pulp which implies that savings above 20 % chlorine dioxide can be obtained with the D. thermophilum xylanase at these very harsh conditions. Table 6. Brightness values after XD₀E₁D₁D₂-bleaching and D₀E₁D₁D₂-bleaching of aspen hardwood kraft pulp with different chlorine dioxide charges in D₀-stage. The xylanase pretreatment was conducted at 80°C and pH 8 for 2 h.

EXAMPLE 4: SIGNIFICANT CHLORINE DIOXIDE SAVINGS ARE OBTAINED IN ECF BLEACHING OF SOFTWOOD KRAFT PULP BY USING THE NOVEL GH 11 XYLANASE FROM DICTYOGLOMUS THERMOPHILUM.

In order to evaluate the performance of the novel Dictyoglomus thermophilum GH 1 1 xylanase (SEQ ID NO 1 ) on softwood, the savings in chlorine dioxide was analysed after XDOE-I D-I D^ bleaching of oxygen-delignified loblolly pine kraft pulp. The reference pulp was treated identically without the addition of enzyme. The reference pulp was bleached with 2.1 1 % chlorine dioxide in the D₀-stage whereas the xylanase treated pulps were bleached with four different charges of chlorine dioxide in the D₀-stage. The treatment conditions for each bleaching stage are shown in Table 7.

Table 7. Bleaching conditions for XDoE-i D-i D^bleaching and DoE-i D-i D^bleaching of loblolly pine kraft pulp.

Temperature (°C) 80 80

pH 8 8

Retention time (h) 2 2

Consistency (%) 10 10

Do-stage

CI0₂ (%) 1 .8 1 .8, 1 .62, 1 .44 and 1 .26

Temperature (°C) 60 60

Final pH 3.5-4.0 3.5-4.0

Retention time (h) 1 1

Consistency (%) 10 10

Ei -stage

NaOH (%) 0.9 0.9

Temperature (°C) 70 70

Final pH 1 1 .0-1 1 .5 1 1 .0-1 1 .5

Retention time (h) 1 1

Consistency (%) 10 10

Di -stage

CI0₂ (%) 0.8 0.8

Temperature (°C) 75 75

Final pH 3.5-4.0 3.5-4.0

Retention time (h) 3 3

Consistency (%) 10 10

D₂-stage

CI0₂ (%) 0.36 0.36

Temperature (°C) 75 75

Final pH 3.5-4.0 3.5-4.0

Retention time (h) 3 3

Consistency (%) 10 10

After the bleaching, the brightness of the D₀E₁ D₁ D₂- and XD₀E₁ D₁ D₂-bleached pulps were determined. The hand sheets were made by using TAPPI standard equipment aacording to TAPPI method T272 sp-97. The brightness of the hand sheets was analyzed according to TAPPI method T-452 om-98. As can be seen in Table 8, the D. thermophilum xylanase treated pulp had 1 .2 units higher brightness than the reference pulp when the pulps were bleached with equal chlorine dioxide charges in the D₀-stage. When the D. thermophilum xylanase treated pulps were bleached with 10 and 20 % less chlorine dioxide in the D₀-stage, the pulps showed significantly higher brightness values than the reference pulp. These results show that at least 20 % savings in chlorine dioxide can be obtained with the D. thermophilum xylanase during ECF bleaching of softwood.

Table 8. Brightness values after XD₀E₁D₁D₂-bleaching and D₀E₁D₁D₂-bleaching of oxygen- delignified loblolly pine kraft pulp with different chlorine dioxide charges in the D₀-stage. The xylanase pretreatment was conducted at 80°C and pH 8 for 2h.

Claims

1. A composition comprising paper pulp and a xylanase with a sequence identity to SEQ ID NO: 1 of at least 97%.

2. The composition according to claim 1 , wherein the xylanase is at least 98% identical to SEQ ID NO: 1.

3. The composition according to any of claims 1-2, wherein the xylanase is at least 99% identical to SEQ ID NO: 1.

4. The composition according to any of claims 1 -3, wherein the xylanase is identical to SEQ ID NO: 1 .

5. The composition according to any of claims 1 -4, wherein the xylanase consists of less than 207 amino acids and comprises SEQ I D NO: 1.

6. The composition according to any of claims 1 -5, wherein the xylanase consists of less than 206 amino acids and comprises SEQ ID NO: 1.

7. The composition according to any of claims 1 -6, wherein the xylanase consists of less than 205 amino acids and comprises SEQ I D NO: 1.

8. The composition according to any of claims 1 -7, wherein the temperature of the composition is between 70°C to 100°C.

9. The composition according to any of claims 1-8, wherein the pH of the composition is between pH 8 and 10.

10. A method for making a white paper material from a paper pulp comprising:

a) preparing the composition of any of claims 1-9;

c) preparing a paper material from the paper pulp;

wherein steps a) and b) are carried out under conditions sufficient to obtain a brightness of > 88% ISO.

1. Use of the composition of any of claims 1-9 for making a paper material.