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 treatment of dissolving pulp with one or more enzymes. The enzymatic treatment results in reduced viscosity and/or improved viscosity control in the dissolving pulp production process and/or increased reactivity of the final dissolving pulp.

BACKGROUND OF THE INVENTION

Commercial dissolving pulp or dissolving-grade pulp is a chemical bleached pulp with a high cellulose content enough to be suitable for the production of regenerated cellulose and cellulose derivatives. Commercial dissolving pulp has special properties, such as a high level of brightness and uniform molecular-weight distribution. Commercial dissolving pulp is manufactured for uses that require a high chemical cellulose purity, and particularly low hemicellulose content, since the chemically similar hemicellulose can interfere with subsequent processes. Dissolving pulp is so named because it is not made into paper, but dissolved either in a solvent or by derivatization into a homogeneous solution, which makes it completely chemically accessible and removes any remaining fibrous structure. Once dissolved, it can be spun into textile fibers such as viscose or Lyocell, or chemically reacted to produce derivatized celluloses, such as cellulose triacetate, a plastic-like material formed into fibers or films, or cellulose ethers such as methyl cellulose, used as a thickener.

Conventional viscose manufacturing which uses dissolving pulps as raw material requires improvement with respect to its environmental impact as well as its production costs. There is a need in the art to provide a method for treating dissolving pulp with reduced costs and less environmental impact.

The present invention provides a lytic polysaccharide monooxygenase-based solution that reduces the viscosity and/or improves the viscosity control in the production of dissolving pulp, e.g., kraft and sulfite dissolving pulp. The enzyme solution described in this invention allows a more selective depolymerization of cellulose and thus a better control of pulp viscosity as compared to the conventional methods in use that are unselective with many side reactions, such as oxygen, hydrogen peroxide, ozone, sodium hypochlorite and acid hydrolysis. Furthermore, the reactivity of the dissolving pulp in the present invention is improved, thereby reducing the amount of chemicals used in the viscose production process and/or improving the processability in terms of viscose dope filterability in the viscose making process. Savings in the amount of chemicals utilized in the production of regenerated cellulose such as carbon disulfide (CS ₂ ) in the viscose making process will reduce costs and environmental impact. Similarly, the reactivity increase of the dissolving pulp is expected to benefit the production process of cellulose derivatives, such as subsequent esterification and etherification processes.

SUMMARY OF THE INVENTION

The invention provides a method for treating dissolving pulp, comprising a step of subjecting the dissolving pulp to a lytic polysaccharide monooxygenase.

The method of the present invention generates dissolving pulp with reduced viscosity and/or improved viscosity control in the dissolving pulp production process, and/or increased reactivity for viscose making, and/or increased content of oxidized groups, compared to dissolving pulp obtained by the same process where the lytic polysaccharide monooxygenase (LPMO) treatment is omitted. The said dissolving pulp is kraft dissolving pulp and/or sulfite dissolving pulp.

The present invention further provides a dissolving pulp made by the method of the present invention.

The present invention further provides a textile fiber or a derivatized cellulose made of the dissolving pulp of the present invention.

The present invention further provides use of a lytic polysaccharide monooxygenase for treatment of dissolving pulp.

DEFINITIONS

Dissolving pulp: Dissolving pulp is a high-grade cellulose pulp, with low contents of hemicellulose, lignin and resin. This pulp has special properties, such as high level of brightness and uniform molecular weight distribution. It is used to make products that include rayon and acetate textile fibers, cellophane, photographic film and various chemical additives. To a large extent, use of dissolving wood pulp depends on its purity (cellulose content), which depends mainly on the production process. To obtain products of high quality, these so-called“special” pulps must fulfill certain requirements, such as high cellulose content, low hemicellulose content, a uniform molecular weight distribution, and high cellulose reactivity. Most of the commercial dissolving pulps accomplish these demands to a certain extent. Nevertheless, achieving high cellulose accessibility as well as solvent and reagent reactivity is not an easy task due to the compact and complex structure presented by the cellulose. About 77% of all dissolving pulp is used in the manufacture of cellulosic fibers (rayon and acetate).

Two basic processes are used to produce dissolving pulp: (a) the sulfite process; and b) the sulfate process (kraft).

To manufacture dissolving-grade pulps, removing hemicelluloses from the wood fiber is crucial, because hemicelluloses can affect the filterablility of viscose, the xanthation of cellulose and the strength of the end product during the production of viscose. Hemiceluloses are removed during the cooking of wood and the subsequent bleaching. In sulfite pulping, the acidic conditions used are responsible for removing most of the hemicellulose while in sulfate/kraft process usually a prehydrolysis step is required to remove hemicelluloses. Another method to remove hemicelluloses is by treatment of pulps with enzymes that react only with the hemicellulose portion of the pulp.

Kraft dissolving pulp:“Kraft dissolving pulp” is synonymous with“sulphate dissolving pulp”. A preferred example is a prehydrolysis kraft dissolving pulp. Kraft dissolving pulp is produced by digesting wood chips at temperatures above about 120°C with a solution of sodium hydroxide and sodium sulfide. Some kraft pulping is also done in which the sodium sulfide is augmented by oxygen or anthraquinone. As compared with soda pulping, kraft pulping is particularly useful for pulping of softwoods, which contain a higher percentage of lignin than hardwoods. The term “kraft dissolving pulp” is synonymous with“kraft dissolving cellulose” and“kraft dissolving-grade pulp” and refers to pulp that has a high cellulose content. The cellulose content of the kraft dissolving pulp is preferably at least 90% (weight/weight) such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% (w/w). Kraft dissolving pulp is manufactured for uses that require a high chemical purity, and particularly low hemicellulose content. The hemicellulose content of the dissolving pulp is preferably less than 10% (weight/weight) such as less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% (w/w). Kraft dissolving pulp can e.g. be used for generation of regenerated cellulose or for generation of cellulose derivatives.“Kraft dissolving-grade pulp" can also be defined as pulp that has been purified sufficiently for use in the production of viscose, rayon, cellulose ethers, or cellulose esters with organic or inorganic acids. Sulfite dissolving pulp: The sulfite process produces wood pulp which is almost pure cellulose fibers by using various salts of sulfurous acid to extract the lignin from wood chips in large pressure vessels called digesters. The salts used in the pulping process are either sulfites (S0 ₃ ^2- ), or bisulfites (HS0 ₃ ), depending on the pH. The counter ion can be sodium (Na ⁺ ), calcium (Ca ²⁺ ), potassium (K ⁺ ), magnesium (Mg ²⁺ ) or ammonium (NH ⁴⁺ ).

Sulfite pulping is carried out between pH 1.5 and 5, depending on the counterion to sulfite (bisulfite) and the ratio of base to sulfurous acid. The pulp is in contact with the pulping chemicals for 4 to 14 hours and at temperatures ranging from 130 to 160 °C (266 to 320 °F), again depending on the chemicals used.

Most of the intermediates involved in delignification in sulfite pulping are resonance-stabilized carbocations formed either by protonation of carbon-carbon double bonds or acidic cleavage of ether bonds which connect many of the constituents of lignin. It is the latter reaction which is responsible for most lignin degradation in the sulfite process. The sulfite process is not expected to degrade lignin to the same extent that the kraft process does and the lignosulfonates from the sulfite process are useful byproducts.

The spent cooking liquor from sulfite pulping is usually called brown liquor, but the terms red liquor, thick liquor and sulfite liquor are also used (compared to black liquor in the kraft process). Pulp washers, using countercurrent flow, remove the spent cooking chemicals and degraded lignin and hemicellulose.

"Bleaching" is the removal of color from pulp, primarily the removal of traces of lignin which remains bound to the fiber after the primary pulping operation. Bleaching usually involves treatment with oxidizing agents such as chlorine (C-stage), chlorine dioxide (D-stage), oxygen (O-stage), hydrogen peroxide (P-stage), ozone (Z-stage) and peracetic acid (CH ₃ C0 ₃ H; Paa- stage) or a reducing agent such as sodium dithionite (Y-stage). There are chlorine (Cl ₂ ; C- stage) free processes such as the elemental chlorine free (EOF) bleaching where chlorine dioxide (CI0 ₂ ; D-stage) is mainly used and typically followed by an alkaline extraction stage. Totally chlorine free (TCF) bleaching is another process where mainly oxygen-based chemicals are used. The pulp bleaching process thus typically comprise a sequence of bleaching steps with washing in between them to remove the degradation products arising from the bleaching reactions.

Cold Caustic Extraction (CCE) : A cold alkali extraction, also called Cold Caustic Extraction (CCE), is a method used to to remove short-chain noncellulosic carbohydrates (cellulose purification) that is based on physical effects such as swelling and solubilization. Usually, a CCE stage takes place at temperatures below 45°C and using very high NaOH dosage that, in the liquid phase, can reach values up to 100 g/L. Depending on the pulp consistency in use, this will determine the amount of NaOH per dry weight of pulp. Typical conditions for a CCE-stage can be 5-10% w/w NaOH in the liquid phase for at least 10 min.

Hot Caustic Extraction (HCE): the term“Hot Caustic Extraction” (HCE) is synonymous with“hot alkali extraction”. HCE is a method to remove short chain hemicellulose and amorphous cellulose in pulps. A hot caustic extraction (HCE)-stage is a purification process that is based on chemical reactions, in particular alkaline peeling of hemicelluloses, which is carried out at higher temperatures and lower NaOH concentration compared to CCE.

ISO Brightness: ISO Brightness is defined in ISO 2470-1 (method for measuring ISO brightness of pulps, papers and boards), and it is the intrinsic radiance [reflectance] factor measured with a reflectometer having the characteristics described in ISO 2469.

Pulp viscosity: is measured by dissolving the pulp in a suitable cellulose solvent such as in cupri-ethylenediamine (CED) and measuring the solution viscosity. This measurement gives an indication of the average degree of polymerization of the cellulose. This property can be referred as intrinsic viscosity in ml_/g and measured according to ISO 5351 or as TAPPI viscosity in cP and measured according to TAPPI T 230.

Unbleached or partially bleached or alkaline extracted kraft dissolving pulp: is produced by a kraft based cooking process such as pre-hydrolysis kraft (PHK) cooking but not fully bleached and purified until becoming a commercial kraft dissolving pulp and thus it is not a finished product. Typically it has an ISO brightness below 90% (such as below 85%, such as below 80%, such as below 75%, such as below 70%, such as below 65%, such as below 60%, such as below 55%, such as below 50%, such as below 45%, such as below 40%, such as below 35%, and such as below 30%).

Unbleached or partially bleached or alkaline extracted sulfite dissolving pulp: is produced by a sulfite based cooking process but not fully bleached and purified until becoming a commercial sulfite dissolving pulp and thus it is not a finished product. Typically it has an ISO brightness below 90% (such as below 85%, such as below 80%, such as below 75%, such as below 70%, such as below 65%, such as below 60%, such as below 55%, such as below 50%, such as below 45%, such as below 40%, such as below 35%, and such as below 30%).

Bleached kraft dissolving pulp and bleached sulfite dissolving pulp: is produced by a kraft dissolving pulp or a sulfite based cooking process but fully bleached and purified until becoming a commercial dissolving pulp. Typically it has an ISO brightness above 90% (such as above 91%, such as above 92%, such as above 93%, such as above 94%, such as above 95%, such as above 96%, such as above 97%, such as above 98%, such as above 99%, such as 100%).

Sequence identity: The relatedness between two amino acid sequences or between two nucleo tide sequences is described by the parameter“sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequenc es 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 (EM BOSS: 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) sub stitution matrix. The output of Needle labeled“longest identity” (obtained using the -nobrief op tion) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method for treating dissolving pulp, comprising a step of subjecting the dissolving pulp to a lytic polysaccharide monooxygenase (LPMO).

In a preferred embodiment, the method of the present invention further comprises a step of subjecting the dissolving pulp to a cellulase.

In a preferred method of the present invention, the step of subjecting the dissolving pulp to a lytic polysaccharide monooxygenase and the step of subjecting the dissolving pulp to a cellulase are carried out simultaneously or sequentially in any order. In a further preferred embodiment of the present invention, a lytic polysaccharide monooxygenase is added to the dissolving pulp together with the cellulase. In a further preferred embodiment of the present invention, a lytic polysaccharide monooxygenase is added to the dissolving pulp before the addition of a cellulase. In a further preferred embodiment of the present invention, a lytic polysaccharide monooxygenase is added to the dissolving pulp after the addition of a cellulase.

In a preferred embodiment, the present method of the present invention comprises a step of bleaching the dissolving pulp. In a preferred method of the present invention, the step of subjecting the dissolving pulp to a lytic polysaccharide monooxygenase and the step of bleaching the dissolving pulp are carried out simultaneously or sequentially in any order. In a preferred method of the present invention, the step of bleaching the dissolving pulp is performed using a chemical selected from the group consisting of CI0 ₂ , 0 ₂ , 0 ₃ , H ₂ 0 ₂ , CH CO H and NaOCI.

In a preferred embodiment, the method of the present invention further comprises the step of Alkaline Extraction. In the method of the present invention, Alkaline Extraction is an E, HCE or CCE stage. Special alkaline purification treatments such as HCE or CCE treatments can yield higher cellulose levels in sulfite and kraft processes. In the case of sulfite pulps, HCE is typically employed to further purify the pulp after the sulfite cooking.

In a preferred embodiment, the method of the present invention further comprises a step of subjecting the dissolving pulp to a lytic polysaccharide monooxygenase and an electron donor thereof, preferably ascorbic acid, gallic acid, pyrogallol or cysteine. The electron donor can exist in the dissolving pulp to be treated. In one embodiment, no or a little amount of electron donor is added to the dissolving pulp. In another embodiment, an effective amount of electron donor is added to the dissolving pulp.

In a preferred method of the present invention, the dissolving pulp is an unbleached, partially bleached, bleached or alkaline extracted dissolving pulp. In a preferred method of the present invention, the dissolving pulp is kraft pulp or sulfite pulp.

In one embodiment the lytic polysaccharide monooxygenase added in the method of the present invention has a sequence identity of at least 60% [such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least

90%, such as at least 95%, such as at least 99%] to the mature polypeptide of SEQ ID NO: 1 , the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO:3. In one embodiment the cellulase added in the method of the present invention has a sequence identity of at least 60% (such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least

99%) to SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 7.

In a preferred embodiment, the lytic polysaccharide monooxygenase added in the method of the present invention comprises or consists of SEQ ID NO: 1 or the mature polypeptide thereof, or SEQ ID NO: 2 or the mature polypeptide thereof, or SEQ ID NO: 3 or the mature polypeptide thereof; or the cellulase comprises or consists of SEQ ID NO: 4, SEQ ID NO: 5 or the mature polypeptide thereof, SEQ ID NO: 6 or the mature polypeptide thereof, or SEQ ID NO: 7 or the mature polypeptide thereof. In another preferred embodiment, the lytic polysaccharide monooxygenase added in the method of the present invention comprises amino acids 19 to 226 of SEQ ID NO: 1 , or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof. In another preferred embodiment, the lytic polysaccharide monooxygenase added in the method of the present invention comprises amino acids amino acids 20 to 254 of SEQ ID NO: 2, or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof. In another preferred embodiment, the lytic polysaccharide monooxygenase added in the method of the present invention comprises amino acids 22 to 249 of SEQ ID NO: 3, or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof. In another preferred embodiment, the cellulase added in the method of the present invention comprises SEQ ID NO: 4 or the full length thereof, or a homologous sequence thereof, an allelic variant thereof, or a functional fragment thereof. In another preferred embodiment, the cellulase added in the method of the present invention comprises amino acids 22-305 of SEQ ID NO: 5 or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof. In another preferred embodiment, the cellulase added in the method of the present invention comprises amino acids 22 to 293 of SEQ ID NO: 6 or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof. In another preferred embodiment, the cellulase added in the method of the present invention comprises amino acids 19 to 409 of SEQ ID NO: 7 or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof.

The concentration of the lytic polysaccharide monooxygenase added in the method of the present invention is preferably from 0.05 mg/kg oven dry pulp to 100000 mg/kg oven dry pulp such as a concentration selected from the group consisting of from 0.05 mg/kg oven dry pulp to 250 mg/kg oven dry pulp, from 0.25 mg/kg oven dry pulp to 1000 mg/kg oven dry pulp, from 0.25 mg/kg oven dry pulp to 2000 mg/kg oven dry pulp, from 1.0 mg/kg oven dry pulp to 5000 mg/kg oven dry pulp, from 5.0 mg/kg oven dry pulp to 10000 mg/kg oven dry pulp, from 10.0 mg/kg oven dry pulp to 15000 mg/kg oven dry pulp, from 15.0 mg/kg oven dry pulp to 20000 mg/kg oven dry pulp, from 20.0 mg/kg oven dry pulp to 30000 mg/kg oven dry pulp, from 30.0 mg/kg oven dry pulp to 40000 mg/kg oven dry pulp, from 40.0 mg/kg oven dry pulp to 60000 mg/kg oven dry pulp, from 60.0 mg/kg oven dry pulp to 80000 mg/kg oven dry pulp, and from 80.0 mg/kg oven dry pulp to 100000 mg/kg oven dry pulp, or any combination of these intervals.

The concentration of the cellulase added in the present invention is from 0.05 mg/kg oven dry pulp to 100 mg/kg oven dry pulp such as a concentration selected from the group consisting of from 0.05 mg/kg oven dry pulp to 80.0 mg/kg oven dry pulp, from 0.25 mg/kg oven dry pulp to

60.0 mg/kg oven dry pulp, from 0.25 mg/kg oven dry pulp to 40.0 mg/kg oven dry pulp, from 0.25 mg/kg oven dry pulp to 20.0 mg/kg oven dry pulp, from 0.25 mg/kg oven dry pulp to 10.0 mg/kg oven dry pulp, from 0.25 mg/kg oven dry pulp to 5.0 mg/kg oven dry pulp, from 0.50 mg/kg oven dry pulp to 85.0 mg/kg oven dry pulp, from 0.50 mg/kg oven dry pulp to 65.0 mg/kg oven dry pulp, from 0.50 mg/kg oven dry pulp to 45.0 mg/kg oven dry pulp, from 0.50 mg/kg oven dry pulp to 25.0 mg/kg oven dry pulp, from 0.50 mg/kg oven dry pulp to 15.0 mg/kg oven dry pulp and from 0.50 mg/kg oven dry pulp to 5.0 mg/kg oven dry pulp, or any combination of these intervals.

The method according to the invention results in an improved viscosity control, thereby allowing the reduction in the production of dissolving pulp outside final viscosity specification/target, typically more than 50% (such as more than 60% or more than 70%) reduction in the production of off-grade dissolving pulp with respect to viscosity. In one embodiment the method according to the invention results in increased reactivity of the kraft and/or sulfite dissolving pulp, particularly the kraft dissolving pulp having an increased reactivity of at least 10% (such as at least 20% or at least 30%).

In a preferred embodiment, the method results in reduced viscosity and/or improved viscosity control in the dissolving pulp production process; and/or the method results in increased reactivity of the dissolving pulp, preferably increased Fock’s reactivity related to the viscose making process and therefore allowing savings in CS ₂ and thus reducing costs and environmental impact; and/or the method results in increased content of oxidized groups of the dissolving pulp. This increase in oxidized groups can increase the reactivity of the dissolving pulp not only in terms of fiber swelling and chemical accessibility but also considering that more anchor points (carbonyl and/or carboxyl groups) in the cellulose will be available for subsequent derivatization processes in the production of cellulose derivatives.

In a preferred embodiment, the method of the present invention further comprises subjecting the dissolving pulp to a xylanase and/or a mannanase and/or a lipase and/or laccase and/or peroxidase.

A dissolving pulp made by the method described above is also part of the invention. A textile fiber or a derivatized cellulose made of the dissolving pulp described above is also part of the invention.

The invention also relates to use of a lytic polysaccharide monooxygenase for treatment of dissolving pulp.

The term“lytic polysaccharide monooxygenase” means an enzyme that oxidizes sp(3) carbons in polysaccharides such as chitin, cellulose, and starch in the presence of an external electron donor and, as currently hypothesized, utilizes copper at the active site to activate molecular oxygen. At present those enzymes belong to Auxiliary Activity families AA9, AA10, AA1 1 , AA13, AA14 and AA15 as defined in the database of carbohydrate active enzymes (http://www.cazy.org/).

In a first aspect, the LPMO comprises the following motifs:

[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(4)-[HNQ] and [FW]-[TF]-K-[AIV],

wherein x is any amino acid, x(4,5) is any four or five contiguous amino acids, and x(4) is any four contiguous amino acids.

The LPMO comprising the above-noted motifs may further comprise:

H-x(1 ,2)-G-P-x(3)-[YW]-[AILMV],

[EQ]-x-Y-x(2)-C-x-[EHQN]-[FILV]-x-[ILV], or

H-x(1 ,2)-G-P-x(3)-[YW]-[AILMV] and [EQ]-x-Y-x(2)-C-x-[EHQN]-[FILV]-x-[ILV],

wherein x is any amino acid, x(1 ,2) is any one or two contiguous amino acids, x(3) is any three contiguous amino acids, and x(2) is any two contiguous amino acids.

In a preferred aspect, the LPMO further comprises H-x(1 ,2)-G-P-x(3)-[YW]-[AILMV] In another preferred aspect, the LPMO further comprises [EQ]-x-Y-x(2)-C-x-[EHQN]-[FILV]-x-[ILV] In another preferred aspect, the LPMO further comprises H-x(1 ,2)-G-P-x(3)-[YW]-[AILMV] and [EQ]-x-Y-x(2)-C-x-[EHQN]-[FILV]-x-[ILV]

In a second aspect, the LPMO comprises the following motif:

[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A-[HNQ],

wherein x is any amino acid, x(4,5) is any 4 or 5 contiguous amino acids, and x(3) is any 3 contiguous amino acids. In the above motif, the accepted IUPAC single letter amino acid abbreviation is employed.

In one aspect, the LPMO comprises an amino acid sequence that has a sequence identity to the mature polypeptide of SEQ ID NO: 1 ( Thielavia terrestris), SEQ ID NO: 2 ( Lentinus similis), SEQ ID NO: 3 ( Thermoascus aurantiacus), SEQ ID NO: 8 ( Thielavia terrestris), SEQ ID NO: 9 ( Thielavia terrestris), SEQ ID NO: 10 ( Thielavia terrestris), SEQ ID NO: 1 1 ( Thielavia terrestris), SEQ ID NO: 12 ( Thielavia terrestris), SEQ ID NO: 13 ( Thielavia terrestris), SEQ ID NO: 14 ( Trichoderma reesei), SEQ ID NO: 15 ( Myceliophthora thermophila), SEQ ID NO: 16 ( Myceli - ophthora thermophila), SEQ ID NO: 17 ( Myceliophthora thermophila), SEQ ID NO: 18 ( Myceli ophthora thermophila), SEQ ID NO: 19 ( Myceliophthora thermophila), SEQ ID NO: 20 ( Thermo - ascus aurantiacus ), SEQ ID NO: 21 ( Aspergillus fumigatus), SEQ ID NO: 22 ( Penicillium pi- nophilum), SEQ ID NO: 23 ( Thermoascus sp.), SEQ ID NO: 24 ( Penicillium sp.), SEQ ID NO: 25 ( Thielavia terrestris), SEQ ID NO: 26 ( Thielavia terrestris), SEQ ID NO: 27 ( Thielavia terrestris), SEQ ID NO: 28 ( Thielavia terrestris), SEQ ID NO: 29 ( Thielavia terrestris), SEQ ID NO: 30 ( Thielavia terrestris), SEQ ID NO: 31 ( Thielavia terrestris), SEQ ID NO: 32 ( Thielavia terrestris), SEQ ID NO: 33 ( Thielavia terrestris), SEQ ID NO: 34 ( Thielavia terrestris), SEQ ID NO: 35 ( Thielavia terrestris), SEQ ID NO: 36 ( Thermoascus crustaceus), SEQ ID NO: 37 ( Thermoascus crustaceus), or SEQ ID NO: 38 ( Thermoascus crustaceus) of at least 50%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

In another aspect, the LPMO is an artificial variant comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID

NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22,

SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID

NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33,

SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38; or a homologous sequence thereof, an allelic variant thereof, or a functional fragment thereof.

Preferably, amino acid changes are 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 one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 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 group 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. The most commonly occurring exchanges 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, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a parent 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 cellulolytic enhancing 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 identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to the parent 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. Patent 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).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide. The total number of amino acid substitutions, deletions and/or insertions of the mature LPMO of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID

NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 ,

SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID

NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32,

SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In one aspect, the LPMO is used in the presence of a soluble activating divalent metal cation as described in WO 2008/151043, e.g., copper sulfate.

In one aspect, the LPMO is used in the presence of an electron donor thereof. The electron donor can exist in the dissolving pulp to be treated. In one embodiment, no or a little amount of electron donor can be added to the dissolving pulp. In another embodiment, an effective amount of electron donor can be added to the dissolving pulp.

In the present invention, the electron donor can be a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, or a sulfur-containing compound.

The dioxy compound may include any suitable compound containing two or more oxygen atoms. In some aspects, the dioxy compounds contain a substituted aryl moiety as described herein. The dioxy compounds may comprise one or more (several) hydroxyl and/or hydroxyl derivatives, but also include substituted aryl moieties lacking hydroxyl and hydroxyl derivatives. Non-limiting examples of dioxy compounds include pyrocatechol or catechol; caffeic acid; 3,4- dihydroxybenzoic acid; 4-tert-butyl-5-methoxy-1 ,2-benzenediol; ascorbic acid, pyrogallol; gallic acid; methyl-3, 4, 5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone; 2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid; 4-chloro-1 ,2-benzenediol; 4-nitro-1 ,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1 ,4-diol; (croconic acid; 1 ,3-propanediol; tartaric acid; 2,4-pentanediol; 3-ethyoxy-1 ,2-propanediol; 2,4,4’- trihydroxybenzophenone; cis-2-butene-1 ,4-diol; 3,4-dihydroxy-3-cyclobutene-1 ,2-dione; dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate; 4-hydroxybenzoic acid; and methyl-3, 5-dimethoxy-4-hydroxybenzoate; or a salt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ring system as described herein. The compounds may comprise one or more (several) additional rings, and are not limited to a specific number of rings unless otherwise stated. In one aspect, the bicyclic compound is a flavonoid. In another aspect, the bicyclic compound is an optionally subsituted isoflavonoid. In another aspect, the bicyclic compound is an optionally substituted flavylium ion, such as an optionally substituted anthocyanidin or optionally substituted anthocyanin, or derivative thereof. Non-limiting examples of bicyclic compounds include epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as an optionally substituted aromatic or non-aromatic ring comprising a heteroatom, as described herein. In one aspect, the heterocyclic is a compound comprising an optionally substituted heterocycloalkyl moiety or an optionally substituted heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted 5- membered heterocycloalkyl or an optionally substituted 5-membered heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyl or optionally substituted heteroaryl moiety is an optionally substituted moiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl, dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl, piperidinyl, and oxepinyl. In another aspect, the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted furanyl. Non-limiting examples of heterocyclic compounds include (1 ,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one; 4-hydroxy-5-methyl-3- furanone; 5-hydroxy-2(5H)-furanone; [1 ,2-dihydroxyethyl]furan-2,3,4(5H)-trione; a-hydroxy-y- butyrolactone; ribonic g-lactone; aldohexuronicaldohexuronic acid g-lactone; gluconic acid d- lactone; 4-hydroxycoumarin; dihydrobenzofuran; 5-(hydroxymethyl)furfural; furoin; 2(5H)- furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvate thereof.

The nitrogen-containing compound may be any suitable compound with one or more nitrogen atoms. In one aspect, the nitrogen-containing compound comprises an amine, imine, hydroxylamine, or nitroxide moiety. Non-limiting examples of nitrogen-containing compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol; 1 ,2-benzenediamine; 2,2,6,6-tetramethyl-1 -piperidinyloxy; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8- tetrahydropterine; and maleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinone moiety as described herein. Non-limiting examples of quinone compounds include 1 ,4-benzoquinone; 1 ,4- naphthoquinone; 2-hydroxy-1 ,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1 ,4-benzoquinone or coenzyme Q ₀ ; 2, 3, 5, 6-tetramethyl-1 ,4-benzoquinone or duroquinone; 1 ,4- dihydroxyanthraquinone; 3-hydroxy-1 -methyl-5, 6-indolinedione or adrenochrome; 4-tert-butyl-5- methoxy-1 ,2-benzoquinone; pyrroloquinoline quinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprising one or more sulfur atoms. In one aspect, the sulfur-containing comprises a moiety selected from thionyl, thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limiting examples of sulfur-containing compounds include ethanethiol; 2-propanethiol; 2-propene-1 -thiol; 2-mercaptoethanesulfonic acid; benzenethiol; benzene-1 ,2-dithiol; cysteine; methionine; glutathione; cystine; or a salt or solvate thereof.

Cellulases

Cellulases or cellulolytic enzymes are enzymes involved in hydrolysis of cellulose. In the hydrolysis of native cellulose, it is known that there are three major types of cellulase enzymes involved, namely cellobiohydrolase (1 ,4-p-D-glucan cellobiohydrolase, EC 3.2.1 .91 , e.g., cellobiohydrolase I and cellobiohydrolase II), endo-b-1 ,4-glucanase (endo-1 ,4-p-D-glucan 4- glucanohydrolase, EC 3.2.1 .4) and b-glucosidase (EC 3.2.1 .21 ).

In order to be efficient, the digestion of cellulose and hemicellulose requires several types of enzymes acting cooperatively. At least three categories of enzymes are necessary to convert cellulose into fermentable sugars: endo-glucanases (EC 3.2.1 .4) that cut the cellulose chains at random; cellobiohydrolases (EC 3.2.1 .91 ) which cleave cellobiosyl units from the cellulose chain ends and beta-glucosidases (EC 3.2.1 .21 ) that convert cellobiose and soluble cellodextrins into glucose. Among these three categories of enzymes involved in the biodegradation of cellulose, cellobiohydrolases are the key enzymes for the degradation of native crystalline cellulose. The term“cellobiohydrolase I” is defined herein as a cellulose 1 ,4-beta-cellobiosidase (also referred to as exo-glucanase, exo-cellobiohydrolase or 1 ,4-beta-cellobiohydrolase) activity, as defined in the enzyme class EC 3.2.1 .91 , which catalyzes the hydrolysis of 1 ,4-beta-D-glucosidic linkages in cellulose and cellotetraose, by the release of cellobiose from the non-reducing ends of the chains. The definition of the term“cellobiohydrolase II activity” is identical, except that cellobiohydrolase II attacks from the reducing ends of the chains.

Endoglucanases (EC No. 3.2.1 .4) catalyses endo hydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxy methyl cellulose and hydroxy ethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans and other plant material containing cellulosic parts. The authorized name is endo-1 ,4- beta -D- glucan 4-glucano hydrolase, but the abbreviated term endoglucanase is used in the present specification. In a preferred embodiment of the present invention, the cellulase used in the present invention is an endoglucanase.

The cellulases may comprise a carbohydrate-binding module (CBM) which enhances the binding of the enzyme to a cellulose-containing fiber and increases the efficacy of the catalytic active part of the enzyme. A CBM is defined as contiguous amino acid sequence within a carbohydrate-active enzyme with a discreet fold having carbohydrate-binding activity. For further information of CBMs see the CAZy internet server (Supra) or Tomme et al., (1995) in Enzymatic Degradation of Insoluble Polysaccharides (Saddler, J.N. & Penner, M., eds.), Cellulose-binding domains: classification and properties pp. 142-163, American Chemical Society, Washington.

In a preferred embodiment the cellulases may be a preparation as defined in WO 2008/151079 , which is hereby incorporated by reference. The cellulase preparation may further comprise a beta-glucosidase, such as the fusion protein disclosed in US 60/832,51 1 . In an embodiment the cellulase preparation also comprises a CBH II, preferably Thielavia terrestris cellobiohydrolase II CEL6A. In an embodiment the cellulase preparation also comprises a cellulase enzymes preparation, preferably the one derived from Trichoderma reesei. Cellulases are synthesized by a large number of microorganisms which include fungi, actinomycetes, myxobacteria and true bacteria but also by plants. Especially endoglucanases of a wide variety of specificities have been identified.

The cellulase activity may, in a preferred embodiment, be derived from a fungal source, such as a strain of the genus Trichoderma, preferably a strain of Trichoderma reeser, a strain of the genus Humicola, such as a strain of Humicola insolens ; or a strain of Chrysosporium, preferably a strain of Chrysosporium lucknowense or a strain of the genus Thielavia, preferably Thielavia terrestris.

In one aspect, the cellulase comprises an amino acid sequence that has a sequence identity to SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 7 of at least 50%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In another aspect, the cellulase is an artificial variant comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 7; or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof. The total number of amino acid substitutions, deletions and/or insertions of SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 7 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Fungi and bacteria produce a spectrum of cellulolytic enzymes (cellulases) which, on the basis of sequence similarities (hydrophobic cluster analysis), can be classified into different families of glycosyl hydrolases [Henrissat B & Bairoch A; Biochem. J. 1993 293 781 -788]. At present are known cellulases belonging to the families 5, 6, 7, 8, 9, 10, 12, 26, 44, 45, 48, 60, and 61 of glycosyl hydrolases.

Additional enzymes

Any enzyme having xylanase, mannanase, lipase, laccase, and/or peroxidase activity can be used as additional enzymes in the use and process of the invention. The additional enzymes and a lytic polysaccharide monooxygenase are added simultaneously or sequentially in any order. Below some non-limiting examples are listed of such additional enzymes. The enzymes written in capitals are commercial enzymes available from Novozymes A/S, 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.

An example of a xylanase is the PULPZYME HC hemicellulase.

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

An example of a lipase is the RESINASE A2X lipase. An example of a xylanase is the PULPZYME HC hemicellulase.

Examples of peroxidases, and laccases are disclosed in EP 730641 ; WO 01/98469; EP 719337; EP 765394; EP 767836; EP 7631 15; and EP 788547. In the present context, whenever the peroxidase or laccase 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 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. Temperature for the method of the present invention

The temperature for the method of the present invention is typically from 20°C to 100°C such as a temperature interval selected from the group consisting of from 20°C to 30°C, from 30°C to 40°C, from 40°C to 50°C, from 50°C to 60°C, from 60°C to 70°C, from 70°C to 80°C, from 80°C to 90°C, from 90°C to 100°C, or any combination of these intervals.

Incubation time for the method of the present invention

The incubation time used for the mehod of the present invention is typically from 1 minute to 60 hours such as a time interval selected from the group consisting of from 1 minute to 15 minutes, from 15 minutes to 30 minutes, from 30 minutes to 45 minutes, from 45 minutes to 60 minutes, from 1 hour to 3 hours, from 3 hours to 6 hours, from 6 hours to 10 hours, from 10 hours to 12 hours, from 12 hours to 15 hours, from 15 hours to 20 hours, from 20 hours to 22 hours, from 22 hours to 25 hours, from 25 hours to 30 hours, from 30 hours to 40 hours, from 40 hours to 50 hours, from 50 hours to 60 hours, or any combination of these time intervals.

Pulp used and produced in the method according to the invention: The dissolving pulp used in the present invention can be wood pulp coming e.g. from softwood trees (such as spruce, pine, fir, larch and hemlock) and/or hardwoods (such as eucalyptus, aspen and birch) or other plant sources such as bamboo.

In a preferred embodiment the dissolving pulp is selected from the group consisting of dissolving hardwood pulp and dissolving softwood pulp, or a mixture thereof. The invention relates in one embodiment to dissolving pulp made by the method according to the invention. The invention relates in one embodiment to a kraft dissolving pulp or a sulfite dissolving pulp made by the method according to the invention.

The invention further relates to use of the dissolving pulp according to the invention for production of textile fibers. The dissolving pulp produced may be used in the manufacture of regenerated cellulose such as viscose, rayon, lyocell and modal fibers.

Performing the method of the invention in the presence of one or more surfactants

The method of the present invention can be performed in the presence of one or more surfactants such as one or more anionic surfactants and/or one or more nonionic surfactants and/or one or more cationic surfactants. Surfactants can in one embodiment include poly(alkylene glycol)-based surfactants, ethoxylated dialkylphenols, ethoxylated dialkylphenols, ethoxylated alcohols and/or silicone based surfactants.

Examples of poly(alkylene glycol)-based surfactant are polyethylene glycol) alkyl ester, poly(ethylene glycol) alkyl ether, ethylene oxide/propylene oxide homo- and copolymers, or polyethylene oxide- co-propylene oxide) alkyl esters or ethers. Other examples include ethoxylated derivatives of primary alcohols, such as dodecanol, secondary alcohols, polypropylene oxide], derivatives thereof, tridecylalcohol ethoxylated phosphate ester, and the like.

Specific presently preferred anionic surfactant materials useful in the practice of the invention comprise sodium alpha-sulfo methyl laurate, (which may include some alpha-sulfo ethyl laurate) for example as commercially available under the trade name ALPHA-STEP™-ML40; sodium xylene sulfonate, for example as commercially available under the trade name STEPANATE™- X; triethanolammonium lauryl sulfate, for example as commercially available under the trade name STEPANOL™-WAT; diosodium lauryl sulfosuccinate, for example as commercially available under the trade name STEPAN™-Mild SL3; further blends of various anionic surfactants may also be utilized, for example a 50%-50% or a 25%-75% blend of the aforesaid ALPHA-STEP™ and STEPANATE™ materials, or a 20%-80% blend of the aforesaid ALPHA- STEP™ and STEPANOL™ materials (all of the aforesaid commercially available materials may be obtained from Stepan Company, Northfield, III.).

Specific presently preferred nonionic surfactant materials useful in the practice of the invention comprise cocodiethanolamide, such as commercially available under trade name NINOL™- 1 1 CM; alkyl polyoxyalkylene glycol ethers, such as relatively high molecular weight butyl ethylenoxide-propylenoxide block copolymers commercially available under the trade name TOXIMUL™-8320 from the Stepan Company. Additional alkyl polyoxyalkylene glycol ethers may be selected, for example, as disclosed in U.S. Pat. No. 3,078,315. Blends of the various nonionic surfactants may also be utilized, for example a 50%-50% or a 25%-75% blend of the aforesaid NINOL™ and TOXIMUL™ materials.

Specific presently preferred anionic/nonionic surfactant blends useful in the practice of the invention include various mixtures of the above materials, for example a 50%-50% blends of the aforesaid ALPHA-STEP™ and NINOL™ materials or a 25%-75% blend of the aforesaid STEPANATE™ and TOXIMUL™ materials. Preferably, the various anionic, nonionic and anionic/nonionic surfactant blends utilized in the practice of the invention have a solids or actives content up to about 100% by weight and preferably have an active content ranging from about 10% to about 80%. Of course, other blends or other solids (active) content may also be utilized and these anionic surfactants, nonionic surfactants, and mixtures thereof may also be utilized with known pulping chemicals such as, for example, anthraquinone and derivatives thereof and/or other typical paper chemicals, such as caustics, defoamers and the like.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

EXAMPLES

Materials and Methods

The intrinsic viscosity of the pulp was measured according to ISO 5351 (International Organization for Standardization 5351 ).

The pulp viscosity was measured by mViPr according to WO 201 1/107472 A9.

The amount of aldehyde groups (CHO content) was measured spectrophotometrically according to the procedure described by Obolenskaya et al., “Determination of aldehyde groups in oxidized pulps,” Laboratory Manipulations in Wood and Cellulose Chemistry, Ecologia, Moscow, 21 1 -212, 1991 , which is based on the reaction of 2,3,5-triphenyltetrazolium chloride (TTC) with the aldehyde groups leading to the formation of formazan (red colorant).

The Fock’s reactivity is a measure of how much of a known amount of pulp is reacted with CS ₂ as a small-scale simulation of the viscose making process and it was carried out at 9% NaOH.

Example 1 : LPMO treatment of unbleached hardwood kraft dissolving pulp

Unbleached hardwood kraft dissolving pulp with a kappa number of 6.8 (TAPPI T 236 procedure), ISO brightness of 51% and an intrinsic viscosity of 1025 mL/g produced by a pre hydrolysis kraft pulping process and further treated with a cold-caustic extraction stage from a dissolving pulp production process was used. This pulp was treated with several LPMOs in a small-scale assay using 24 mg of oven-dry fiber at 0.4% consistency, 45°C, pH 5.0 (acetate buffer, 50 mM) for 20 hours. The enzyme treatment (denoted as X-stage) was done at a dosage of 5 mg EP (enzyme protein) / g odp (oven-dry pulp).

The pulp suspension at 0.4% consistency was disintegrated with a magnetic bar in glass test tubes placed in a heating block at 45°C. Ascorbic acid, gallic acid or pyrogallol was added as electron donor to a final concentration of 1 mM in the suspension, followed by the addition of the LPMO enzyme to a final volume of 6 mL. After the 20 hour incubation time upon the addition of the enzyme, the tubes were cooled down in ice and then 6 mL of cupri-ethylenediamine (CED) solution added to dissolve the fibers. The pulp dissolution was done in a rotary agitator at a room temperature of 25°C for 25 min. Control experiments were done in the same way but without the addition of the enzyme.

After the dissolution time, the pulp viscosity was measured by mViPr. The mViPr pipette consists of a modified Gilson Concept C300 pipette equipped with a pressure sensor and Diamond D300 Gilson tips. Samples were kept at a constant temperature within ± 0.1 °C. A volume of 200 mI_ dissolved pulp was aspirated and dispensed in and out of the pipette, respectively, while recording the pressure in the pipette headspace. A pipette speed of 4 was applied. Apirations were followed by a 2s delay, and dispensing was followed by a 5s delay. Each sample measurement consisted of 15 aspiration-dispensing cycles, and pressure results were average of 15 aspiration or dispensing pressures, respectively.

The aspiration pressure results from the mViPr measurements are presented in Table 1. It can be seen that the LPMOs can reduce the average size of the cellulose which is expressed as the reduction in the solution viscosity as measured by the reduction in aspiration pressure. The performance of each LPMO is also dependent on the specific electron donor utilized. In addition, the electron donor itself also degrades cellulose when compared to the original pulp, particularly ascorbic acid. Tt LPMO is quite effective in reducing pulp viscosity with all electron donors.

Table 1. Aspiration pressure of the different unbleached pulps dissolved in CED

Example 2: LPMO treatment of bleached hardwood kraft dissolving pulp

Bleached hardwood kraft never dried pulp of acetate-grade produced by a pre-hydrolysis kraft pulping process having an ISO brightness of 93.7% and an intrinsic viscosity of 684 mL/g was used. This pulp was treated with several LPMOs with three different electron donors using the same conditions and procedures as in Example 1.

The aspiration pressure results from the mViPr measurements are presented in Table 2 for the bleached dissolving pulps. LPMOs can reduce the average size of the cellulose molecules as expressed by the reduction in the solution (dissolved pulp) viscosity, measured by the reduction in aspiration pressure. Higher reduction in bleached pulp viscosity compared to controls (no enzyme) were achieved when the LPMOs were used together with gallic acic and pyrogallol as compared to the ascorbic acid which also reduces signfificantly viscosity itself. Tt LPMO is a more robust LPMO regarding viscosity reduction with all the electron donors used.

Table 2. Aspiration pressure of the different bleached pulps dissolved in CED

Example 3: Effect of LPMO treatment with/without endoglucanase at medium pulp consistency on bleached dissolving pulp viscosity, reactivity and CHO content

Bleached hardwood kraft dissolving pulp produced by a pre-hydrolysis kraft pulping process of viscose-grade was used having an intrinsic viscosity of 512 mL/g. This pulp was treated with several LPMOs using gallic acid (1 mM) as electron donor at 1.5% consistency in Distek vessels (Distek model Symphony 7100) with heating and continuous overhead stirring. Once the pulps were disintegrated and at the temperature set-point of 45°C, gallic acid was added and then the enzyme (LPMO and/or endoglucanase). The enzyme treatment of the pulp took 25.5 hour at pH 5.0 (acetate buffer, 50 mM) using 2 mg EP/g odp of LPMO and 1.2 mg EP/kg odp of endoglucanase. After the enzyme treatment, the pulps were filtered and washed in three consecutive steps with 1 L of tap water. Part of the pulp sample was dried before measuring the CHO content and Fock’s reactivity and part of the sample was kept wet in the fridge to test for intrinsic viscosity.

In Table 3, it can be seen that with a lower dosage of 2 mg EP/g odp a small reduction in viscosity reduction is obtained with LPMOs tested, as compared to Examples 1 and 2 with a higher dosage of LPMOs. However, when combined with an endoglucanase, the viscosity reduction is enhanced with a surprising synergistic effect. The same synergy between LPMO and endoglucanase is seen in terms of Fock’s reactivity and the amount of CHO groups.

Table 3. Intrinsic viscosity, Fock’s reactivity and CHO content of the bleached dissolving pulps

Example 4: Effect of combined LPMO and endoglucanase treatment on unbleached hardwood kraft dissolving pulp

The same unbleached hardwood kraft dissolving pulp of Example 1 was used and treated with several LPMOs using gallic acid (1 mM) as electron donor in combination with several endoglucanases in a small-scale assay using 24 mg of oven-dry fiber at 0.4% consistency, 50°C, pH 6.0 (phosphate buffer, 50 mM) for 20 hours. The LPMO treatments were done at dosages of 2.5 mg EP (enzyme protein) / g odp (high dosage) or 0.5 mg EP / g odp (low dosage). The endoglucanase treatments were done at dosages of 0.5 mg EP / kg odp. The assay and viscosity measurements were performed using the same conditions and procedures as described in Example 1 . The aspiration pressure results from the mViPr measurements are presented in Table 4. It can be seen that the higher the dosage of LPMO, the higher the pulp viscosity reduction. A high dosage of LPMO gives a surprisingly higher viscosity reduction than the endoglucanase treatments. The treatment with a combination of LPMO and an endoglucanase shows better viscosity reduction performance than individual treatment with LPMO or endoglucanase. Using this pulp of high viscosity, it is observed a boosting effect on viscosity reduction by the use of the LPMO combined with the endoglucanase, which allows a significant reduction on the amount of LPMO needed. For instance, at a low Tt LPMO dosage (0.5 mg EP/g odp), the co addition of an endoglucanase 2 and 3 gives even higher viscosity reduction than the high dosage of Tt LPMO alone (2.5 mg EP/g odp).

Table 4. Aspiration pressure of the different unbleached pulps dissolved in CED