Bleaching Process

Abstract

1. A process for bleaching a pulp comprising delignifying the pulp by carrying out the following sequence: an enzyme treatment step comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase enzyme at a pH of at least 9.5 and a temperature of at least 85°C, an oxidation step in which a bleaching agent is added, an alkaline extraction step in which an alkaline agent is added, and recovering the bleached pulp, wherein the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture at 90°C and pH 10.5 by at least 20% when compared to the same mixture without any xylanase enzyme.

Description

[Technical field]

[0001] The present disclosure relates generally to bleaching of pulp. The present disclosure particularly, but not exclusively, relates to an improved pulp delignification process in the presence of a GH11 xylanase enzyme at high pH and high temperature. [Background technology]

[0002] This section provides useful background information without qualifying any of the techniques described herein as representative of the state of the art.

[0003] Bleached pulp is used, for example, to produce paper, paperboard, tissue, dissolving pulp, fluff pulp, market pulp, and in recent years new products defined as MFC (Micro Fibrillated Cellulose) and NCF (Nano Cellulose Fibers) are also used. Pulp is generally processed by Kraft pulping, where free cellulose fibers are obtained by dissolving lignin in an alkaline treatment solution. Residual lignin and lignin derivatives bound to the carbohydrate moieties of the pulp give the pulp an undesirable brown color, which is then traditionally bleached.

[0004] The pulp bleaching process can be divided into two subsequent sections: delignification and whitening. In delignification, the wood components lignin and hemicellulose are at least partially dissolved and removed from the pulp by the use of bleaching chemicals. During delignification, the brightness of the pulp is also increased. In the subsequent whitening section, the delignified pulp is treated with bleaching chemicals that react with the chromophores to cause their removal, thereby producing bleached or white pulp.

[0005] Typically, delignification has at least two stages: an oxidation step, where bleaching agents are added, and an alkaline extraction step, where alkaline agents are added. Furthermore, the oxidation step can be followed by an oxygen delignification step (O), where oxygen is mixed with the pulp to remove most of the lignin in the pulp.

[0006] Bleaching agents added in the oxidation step further contribute to lignin removal, and multiple bleaching agents can be utilized in the process. Pulp is traditionally bleached (whitened) with chemicals that contain chlorine, such as elemental chlorine. This allows for the production of chlorinated organic by-products that have toxic and mutagenic effects in the environment. Therefore, to avoid the release of chlorogenic acid compounds, elemental chlorine-free methods for pulp bleaching are utilized. Completely chlorine-free bleaching utilizes bleaching agents such as hydrogen peroxide.

[0007] Pulp contains a complex mixture of polysaccharides, mainly cellulose, lignin and hemicellulose. Xylan is an important component of hemicellulose. Its chemical composition includes a polysaccharide backbone of β-1,4-linked xylose residues and often side chains that can be, for example, acetyl, arabinosyl and / or glucuronosyl groups. As a component of pulp, xylan inhibits substances that consume bleaching chemicals, such as lignin and hexenuronic acid. Such substances can be chemically bound to and / or physically trapped in the xylan. Hexenuronic acid is formed in the pulp material digester.

[0008] Xylanases are enzymes that digest xylan, resulting in a mixture of smaller xylan fragments. Xylanases that hydrolyze β-1,4-linked xylose residues, particularly endo-1,4-β-xylanases (EC 3.2.1.8), are classified in the CAZy database into the glycoside hydrolase families GH5, GH8, GH10, GH11, GH30 and GH43. Xylanases from the various glycoside hydrolase families are structurally distinct from each other, with GH11 xylanase enzymes having a β-jellyroll structure, GH5, GH10 and GH30 xylanase enzymes having a (β / α)8 barrel structure, GH8 xylanase enzymes having a (α / α)6 barrel structure and GH43 xylanase enzymes having a five-fold β-propeller structure. Many different xylanases are known, for example in the GH10 and GH11 families there are 5949 and 2187 different enzymes, respectively. Furthermore, many variants of xylanases found in nature have been modified. In the pulp bleaching process, xylanases depolymerize xylan, cleave the bonds between xylan and lignin, promote lignin reduction, and improve the access of bleaching chemicals to the lignin, thereby contributing to delignification, bleaching efficiency, and final brightness of the pulp. In pulp bleaching, the pulp entering the delignification stage is hot and alkaline, whereby the xylanases used in such processes retain exceptionally good stability and activity in the biologically challenging conditions of extremely alkaline pH and high temperature.

[0009] There is an increasing demand for such xylanase enzymes that can retain activity and functionality in pulp bleaching processes, especially with respect to increasing pulp brightness, reducing consumption of bleaching chemicals, and reducing the generation of toxic chlorine-containing compounds. Although several xylanases with significant differences in pulp bleaching performance, pH and temperature profile, enzyme stability, specific activity, and other enzyme properties have been reported, in general, xylanases are not stable and do not function under the extremely high temperature and alkaline conditions of pulp bleaching processes. Thus, there is a need for xylanase enzymes that are highly thermostable, alkaline tolerant, and active to function in industrial processes such as pulp bleaching. Therefore, it is an object of the present disclosure to provide such processes using xylanase enzymes that are capable of functioning in bleaching processes at high temperatures and alkaline pH. Summary of the Invention

[0010] The appended claims define the scope of protection. Any examples and / or technical descriptions of the apparatus, system, product and / or process in the specification and / or drawings that are not covered by the claims are presented herein as examples useful for understanding the invention, and not as embodiments of the present invention.

[0011] According to a first aspect, Providing a pulp; The following sequence: a) an enzymatic treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase enzyme at a pH of at least 9.5 and a temperature of at least 85° C.; b) an oxidation step (D), in which a bleaching agent is added; c) an alkaline extraction step (E) in which an alkaline agent is added; and d) Recovering the bleached pulp. The pulp is delignified by carrying out the steps of: A bleaching process comprising: A process is provided wherein the GH11 xylanase enzyme has the ability to reduce the viscosity of the xylan-containing mixture by at least 20% when compared to the xylan-containing mixture not comprising any xylanase enzyme at 90°C, pH 10.5 and at a protein concentration of the GH11 xylanase enzyme of 3.0 μg or less per gram of xylan-containing mixture, and wherein the xylan-containing mixture contains 0.07 g / mL or less xylan.

[0012] In one embodiment, Providing a pulp; The following sequence: a) an enzymatic treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase enzyme at a pH of at least 9.5 and a temperature of at least 85° C.; b) an oxidation step (D), in which a bleaching agent is added; c) an alkaline extraction step (E) in which an alkaline agent is added; and d) Recovering the bleached pulp. The pulp is delignified by carrying out the steps of: A bleaching process comprising: A process is provided, wherein the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture by at least 20% at 90° C. and pH 10.5, as compared to the same mixture without any xylanase enzyme.

[0013] The inventors have surprisingly found, and the examples below demonstrate, that xylanase enzymes suitable for the bleaching process of the present invention can be selected based on the molecular mechanism underlying their pulp bleaching performance, rather than simply on the xylanase enzymatic activity or release of xylan hydrolysis products from the pulp. This is rationalized by the pre-bleaching action of xylanases, which is primarily the result of xylan depolymerization, rather than solubilization.

[0014] In one embodiment, GH11 xylanase enzymes that depolymerize xylan without solubilizing it are particularly advantageous in pulp bleaching processes, since xylan solubilization can lead to undesirable increases in chemical oxygen demand (COD) and losses in pulp yield. Therefore, the selection of suitable GH11 xylanase enzymes for pulp bleaching performance must be based on the molecular mechanisms underlying pulp bleaching performance in addition to and / or instead of xylanase enzyme activity or release of xylan hydrolysis products from the pulp. When a polymer such as xylan is depolymerized, the viscosity of the xylan-containing mixture decreases. However, since xylan is only a minor component of pulp, the xylanase-induced viscosity reduction in pulp can be small and difficult to measure. Surprisingly, it has been found that the xylanase-induced viscosity reduction, which indicates the depolymerization ability of the xylanase enzyme, can be measured in xylan-containing mixtures containing pure or purified xylan, rather than in pulp. Moreover, it has been surprisingly found that viscosity reduction by xylanase is related to the performance of the xylanase in the pulp bleaching process at high temperature and high pH.

[0015] The bleaching process provides an effective method for producing delignified bleached pulp in the presence of GH11 xylanase enzyme at high temperature and high pH.

[0016] According to a second aspect, (a) a pulp having a pH of at least 9.5; and (b) 0.01 to 200 g of glycoside hydrolase family GH11 xylanase enzyme protein per 1000 kg of dry pulp. A pulp composition comprising: The GH11 xylanase enzyme protein is Xylanase activity, and the ability to cause at least a 20% decrease in viscosity of said xylan-containing mixture within 20 minutes at 90° C., pH 10.5, and a concentration of said GH11 xylanase enzyme protein or less per gram of xylan-containing mixture, when compared to said xylan-containing mixture without any xylanase enzyme, wherein said xylan-containing mixture contains no more than 0.07 g / mL xylan. A pulp composition is provided having the following structure:

[0017] In one embodiment, (a) a pulp having a pH of at least 9.5; and (b) 0.01 to 200 g of glycoside hydrolase family GH11 xylanase enzyme protein per 1000 kg of dry pulp. A pulp composition comprising: The GH11 xylanase enzyme protein is Xylanase activity, and The ability to reduce the viscosity of a xylan-containing mixture by at least 20% at 90° C. and pH 10.5 compared to the same mixture without any xylanase enzyme, said xylan-containing mixture containing no more than 0.07 g / mL xylan and no more than 3.0 μg GH11 xylanase enzyme protein per gram of the mixture, wherein the viscosity reduction occurs within 20 minutes. A pulp composition is provided having the following structure:

[0018] According to a further aspect, Providing a pulp; The following sequence: a) an enzymatic treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase enzyme at a pH of 9.5-11.5 and a temperature of 85-100° C., the xylanase enzyme having a residual xylanase activity of at least 25% after 20 minutes of step X; b) an oxidation step (D), in which a bleaching agent is added; c) an alkaline extraction step (E) in which an alkaline agent is added; and d) recovering the delignified pulp. The pulp is delignified by carrying out the above steps. A bleaching process is provided, comprising:

[0019] According to a further aspect, (a) a pulp having a pH of at least 9.5; (b) a bleaching agent selected from ClO2, O3, H2O2, and peroxide, the bleaching agent being 0.25 to 4% by weight of the total weight of the dry pulp; and (c) 0.01 to 200 g of glycoside hydrolase family GH11 xylanase enzyme protein per 1000 kg of dry pulp. A pulp composition comprising: A pulp composition is provided in which the GH11 xylanase enzyme protein has xylanase activity and the ability to reduce the viscosity of a xylan-containing mixture by at least 20% at 90°C and pH 10.5 compared to the same mixture without any xylanase enzyme, the xylan-containing mixture containing no more than 0.07 g / mL xylan and no more than 3.0 μg GH11 xylanase enzyme protein per gram of the mixture, and the viscosity reduction occurs within 20 minutes.

[0020] The bleaching process of the present invention comprising the GH11 xylanase enzyme is advantageous for reducing the total amount of bleaching agent used in the pulp bleaching process. The bleaching process of the present invention is advantageous for reducing the amount of ClO2, O3, H2O2 or peroxide consumption in the pulp bleaching process. The bleaching process of the present invention is advantageous for achieving higher pulp brightness and / or higher bleached product yield with the same amount of bleaching agent used in the pulp bleaching process when compared to a bleaching process not comprising the GH11 xylanase enzyme. The bleaching process of the present invention is advantageous for achieving higher pulp brightness and / or higher bleached product yield with a reduced amount of bleaching agent used in the pulp bleaching process when compared to a bleaching process not comprising the GH11 xylanase enzyme. The bleaching process of the present invention is advantageous for reducing the production cost of bleached pulp. The bleaching process of the present invention allows for more efficient bleaching of the pulp, resulting in more efficient utilization of the wood raw material. The bleaching process of the present invention is advantageous in that it integrates the use of a high pH and thermostable GH11 xylanase enzyme into the bleaching process, thereby reducing or avoiding the need to change existing processing conditions and / or settings / hardware commonly used in pulp bleaching. The bleaching process of the present invention is advantageous in reducing corrosion, wear and erosion of metal pulp bleaching tanks, since there is less need to lower the pulp pH using oxidizing agents. The bleaching process of the present invention is advantageous in reducing fresh water consumption of the bleaching process, since there is less need for additional water washing. The bleaching process of the present invention is advantageous in reducing the production of AOX (adsorbable organic halides, also known as chloro-organic compounds) discharged with the wastewater bleach effluent. The bleaching process of the present invention is advantageous in reducing the amount of energy required to raise the temperature of the pulp after washing and before the oxygen delignification or oxidation step, since there is less need to lower the pulp temperature for enzyme treatment.

[0021] Various non-binding exemplary aspects and embodiments are presented above. The above embodiments are merely used to illustrate selected aspects or steps that may be utilized in different implementations. Some embodiments may be presented only with reference to certain exemplary aspects. It should be understood that the corresponding embodiments may be applied to other exemplary aspects as well. [Brief description of the drawings]

[0022] Some example embodiments will now be described with reference to the accompanying drawings.

[0023] [Figure 1] FIG. 1 illustrates a flow chart of process steps for a pulp delignification and bleaching process, according to an exemplary embodiment. Process steps X, O, Q within dashed boxes indicate optional and / or alternative process steps. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention relates to a bleaching process that utilizes a glycoside hydrolase family GH11 xylanase enzyme in the delignification step of the process, which has stability, activity and performance at biologically challenging conditions of high temperature and alkaline pH, resulting in improved bleaching of pulp.

[0025] The term "bleaching process" refers to a chemical treatment process in which the brightness of the pulp is increased by removing chemical elements such as lignin and double bonds of chromophores that impart color to the pulp.

[0026] The term "delignification" refers to the removal and extraction of lignin contained within the pulp. Kappa number can be used as a measure of the lignin content in the pulp.

[0027] The term "alkaline extraction process" refers to a bleaching process in which the pH of the pulp is raised to a level at which oxidized lignin is extracted from the pulp.

[0028] The term "glycoside hydrolase" refers to a hydrolase that is capable of hydrolyzing a glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety.

[0029] The term "glycoside hydrolase family 11" or "GH11" refers to GH11 xylanases as defined by the CAZy database (http: / / www.cazy.org / GH11.html), which have a β-jellyroll structure, making GH11 xylanase enzymes structurally distinct from other xylanases.

[0030] The term "xylan" refers to a matrix polysaccharide or heteropolymer composed of a backbone of repeating β-1,4-linked xylose residues with various side groups. Xylan polysaccharides can be classified into classes based on the substituted side groups, such as O-acetylglucuronoxylans (AcGX), O-acetylarabinoxylans (AcAX), O-acetylglucuronoarabinoxylans (AcGAX) and arabinoglucuronoxylans (AGX). Xylan-containing materials include plant-based or plant-derived materials.

[0031] As used herein, the term "xylanase" refers to a xylanase enzyme defined according to what is known in the art as endo-1,4-β-xylanase or 4-β-D-xylan xylanohydrolase, which is known to catalyze the endohydrolysis of (1→4)-β-D-xylosidic bonds in xylan. Xylanases have alternative names such as endo-(1→4)-β-xylan 4-xylanohydrolase, endo-1,4-xylanase, xylanase, β-1,4-xylanase, endo-1,4-xylanase, endo-β-1,4-xylanase, endo-1,4-β-D-xylanase, 1,4-β-xylan xylanohydrolase, β-xylanase, β-1,4-xylan xylanohydrolase, endo-1,4-β-xylanase and β-D-xylanase. Xylanases are classified as EC 3.2.1.8 according to enzyme nomenclature.

[0032] As used herein, the term "GH11 xylanase enzyme protein" refers to pure GH11 xylanase enzyme protein. According to some embodiments, GH11 xylanase enzyme protein can be added in enzymatic treatment step X as one of the components of the composition, but in such embodiments, the term GH11 xylanase enzyme protein also refers to pure GH11 xylanase enzyme protein as one of the components of the composition. In some embodiments, the amount of GH11 xylanase enzyme protein refers to the dry weight (grams) of GH11 xylanase enzyme protein, and the dry weight can be determined according to the test procedures described in the examples.

[0033] As used herein, the term "pulp" refers to a moist fibrous material originally obtained from a plant. Pulp can originate from trees, fiber crops, bagasse, straw, waste paper or cloth fragments, or any combination thereof. Thus, in the context of this application, "pulp" can refer to wood pulp, which is of wood origin, and / or non-wood pulp, which is of non-wood origin.

[0034] As used herein, the term "wood pulp" means a moist fibrous material of wood origin and produced by digesting hardwood or softwood chips with a solution of sodium hydroxide and sodium sulfide at temperatures above about 120°C.

[0035] As used herein, the term "non-wood pulp" means a moist fibrous material of non-wood origin and produced by digesting said non-woody materials such as grass, cereal straw, corn stalks, bamboo, kenaf or bagasse, or any combination thereof.

[0036] As used herein, the term "cellulose pulp" means a moist fibrous material that includes cellulose fibers.

[0037] As used herein, the term "xylan-containing pulp" refers to wood or non-wood pulp that contains xylan. The terms "xylan-containing pulp" or "pulp that contains / comprises xylan" do not refer to or are not identical to the term "xylan-containing mixture."

[0038] As used herein, the term "hardwood pulp" means wood pulp derived from the wood fibers of hardwood trees such as oak, beech, birch, aspen, eucalyptus, and / or poplar.

[0039] As used herein, the term "softwood pulp" is wood pulp derived from the wood fibers of softwood trees such as pine, spruce, fir and / or Canadian hemlock.

[0040] As used herein, the term "kraft pulp" means wood pulp produced by the kraft process, also known as the sulfate process, in which wood chips are treated with sodium sulfide (NaS) and sodium hydroxide (NaOH).

[0041] As used herein, the term "thermostable enzyme" or "temperature tolerant enzyme" refers to an enzyme that has a good ability to withstand and resist structural changes that lead to inactivation of the enzyme caused by high temperatures, such as temperatures of 85° C. or higher.

[0042] As used herein, the term "enzyme pH stability" or "enzyme pH resistance" describes an enzyme's ability to tolerate and resist structural changes that lead to inactivation of the enzyme caused by high pH, ​​such as pH 9.5 or above, or alternatively, caused by low pH.

[0043] The term "process water" means any water used in the pulp bleaching process.

[0044] The term "process wash water" means any water that is used for the purpose of washing pulp in a pulp bleaching process.

[0045] As used herein, the term "viscosity" or "relative viscosity" refers to the viscosity of a given material, such as a xylan-containing mixture, suspension, or solution. As used herein, the decrease in viscosity of a mixture containing a GH11 xylanase enzyme and xylan is an indication of the xylan depolymerization activity of the GH11 xylanase enzyme. In one embodiment, the more the viscosity of a xylan-containing mixture decreases over time, the higher the xylan depolymerization activity of the GH11 xylanase enzyme. Viscosity is measured with a falling ball viscometer.

[0046] As used herein, the term "xylan-containing mixture" refers to a mixture containing xylan, which may refer to a solution, a suspension, or a mixture of liquids and solids. In one embodiment, the xylan-containing mixture also contains a xylanase enzyme, such as a GH11 xylanase enzyme. The xylan-containing mixture is suitable for evaluating the xylanolytic activity of the xylanase enzyme. Therefore, the xylan-containing mixture does not contain any substance that would distort or falsify the evaluation of the xylanolytic activity of the xylanase enzyme. For example, the xylan-containing mixture should be suitable for evaluating the effect that the GH11 xylanase enzyme has on the viscosity of the xylan-containing mixture.

[0047] As used herein, the term "adsorbable organic halides (AOX)" means a measure of the chlorine, bromine, and iodine content of organic halogens in a sample, such as a water sample.

[0048] As used herein, the term "chemical oxygen demand (COD)" refers to a measure of the amount of oxygen that can be consumed in a reaction in a measurement solution. COD refers to the amount of oxygen required to completely decompose an organic substance in a solution to CO2. COD measurements are also used to determine the amount of pollutants that can be oxidized and therefore their toxicity to biological organisms. A high COD correlates with a high toxicity of the organic substance.

[0049] As used herein, the term "sequence identity" refers to the percentage of exact matches of amino acid residues between two optimally aligned sequences with respect to the number of positions with that residue present in both sequences. When one sequence has a residue with no corresponding residue in the other sequence, the alignment program used will allow gaps in the alignment and that position will not be counted in the denominator of the identity calculation.

[0050] As used herein, the term "corresponding position" or "corresponding amino acid position" refers to aligning at least two amino acid sequences according to regions of identified similarity or identity, either as a pairwise alignment or as a multiple sequence alignment, thereby pairing corresponding amino acids. As used herein, the term "corresponding amino acid" refers to an amino acid in a first amino acid sequence, which may be paired with a corresponding amino acid in a second sequence when the first amino acid sequence is aligned according to regions of identified similarity or identity, either as a pairwise alignment or as a multiple sequence alignment. The sequence identity between the first amino acid sequence and the second amino acid sequence consisting of corresponding amino acids may be 33-100%.

[0051] As used herein, the term "xylanase activity" refers to xylan hydrolysis activity.

[0052] The term "residual xylanase activity" refers to a xylanase enzyme activity that is not the initial activity of the xylanase enzyme, but that is altered, and optionally decreased, by the process conditions to which the enzyme is exposed. Thus, residual xylanase activity refers to the amount, e.g., a percentage amount, of the initial activity remaining after exposure to particular process conditions.

[0053] The term "functional fragment" refers to a fragment or portion of a current GH11 xylanase enzyme polypeptide that retains the same, or substantially the same, enzymatic function or action as the entire GH11 xylanase enzyme polypeptide.

[0054] The term "stability", in the context of enzyme or xylanase stability, describes an enzyme's ability to withstand and / or function under process conditions that are challenging for the activity and functionality of the enzyme, such as high temperature, pH or radiation, certain concentrations of inorganic salts or organic solvents, or a particular reaction mixture composition, including, for example, proteases, stabilizers, builders, surfactants, etc. The term "stability" reflects the stability of xylanases according to the present disclosure as a function of time, e.g., how much activity is retained when the xylanase is exposed to process conditions that are challenging for the activity and functionality of the enzyme.

[0055] As used herein, Xyn11A refers to Thermopolyspora flexuosa xylanase of glycoside hydrolase family 11 (GH11), also designated as AM35. As used herein, AM35 refers to wild-type mature GH11 xylanase, the parent polypeptide used for the AM24 polypeptide. The amino acids of the mature AM35 protein correspond to the amino acid sequence of SEQ ID NO:3.

[0056] As used herein, AM24 (also referred to herein as AM24 protein) is a truncated form of xylanase AM35. The AM24 protein contains the catalytic module (core) and lacks the carbohydrate binding module (CBM) and part of the linker region between the core and the CBM. The amino acids of the mature polypeptide of AM24 correspond to the amino acids of the sequence of SEQ ID NO:1, including amino acids D1-L220. The "core polypeptide" of AM24 includes amino acids D1-G191 of SEQ ID NO:1, while the "inner core polypeptide" of AM24 includes amino acids T3-S180 of SEQ ID NO:1.

[0057] As used herein, C31-4 refers to a variant of AM24 that includes the substitutions T3C, A23S, S28I, T30C. The amino acids of the mature polypeptide of C31-4 correspond to the amino acids of the sequence of SEQ ID NO:2, which includes amino acids D1 through L220. The "core polypeptide" of C31-4 includes amino acids D1 through G191 of SEQ ID NO:2, while the "inner core polypeptide" of C31-4 includes amino acids C3 through S180 of SEQ ID NO:2.

[0058] As used herein, the term "carbohydrate binding domain" or "CBM" refers to a specific domain or module in a polypeptide that is capable of promoting binding of the polypeptide to carbohydrates.

[0059] As used herein, the term "mature polypeptide" refers to any polypeptide from which at least one signal sequence or signal peptide or signal peptide and putative propeptide has been cleaved. For example, the "mature polypeptide" of AM24 includes amino acids D1 to L220 of SEQ ID NO:1, while the "mature core polypeptide" of AM24 includes amino acids D1 to G191 of SEQ ID NO:1.

[0060] As used herein, "peptide" and "polypeptide" are amino acid sequences that contain multiple contiguous polymerized amino acid residues. For purposes of this disclosure, a peptide is a molecule that contains up to 20 amino acid residues, and a polypeptide contains more than 20 amino acid residues. A peptide or polypeptide may contain modified amino acid residues, naturally occurring amino acid residues that are not encoded by a codon, and non-naturally occurring amino acid residues. As used herein, "protein" may refer to a peptide or polypeptide of any size. A protein may be an enzyme, protein, antibody, membrane protein, peptide hormone, regulator, or any other protein.

[0061] As used herein, the term "variant" refers to a sequence or a fragment of a sequence (nucleotides or amino acids) in which one or more nucleotides / amino acids have been inserted, substituted or deleted, or which has been chemically modified. The term variant also includes, in some embodiments, a variant xylanase polypeptide, a fusion protein comprising a variant xylanase polypeptide, or a recombinant xylanase enzyme.

[0062] The terms "xylanase variant" and "variant xylanase" and "variant of xylanase" refer to any xylanase molecule obtained by site-directed or random mutagenesis, insertion, substitution, deletion, recombination and / or any other protein modification method that results in a xylanase that differs in amino acid sequence from a parent xylanase that is a wild-type xylanase or a xylanase variant itself. The terms "wild-type xylanase", "wild-type enzyme", "wild-type" or "wt" according to the present disclosure describe a xylanase enzyme that has an amino acid sequence found in nature or a fragment thereof. Genetically encoded variants can be synthesized or modified using genetic methods, such as, for example, site-directed mutagenesis, a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding a parent polypeptide.

[0063] As used herein, the term "disulfide bridge" refers to bonds formed between the sulfur atoms of cysteine ​​residues of a polypeptide or protein. Disulfide bridges can be naturally occurring or non-naturally occurring and can be introduced, for example, by one or more amino acid substitutions.

[0064] The terms "catalytic domain" or "catalytic module" or "core" or "catalytic core domain" refer to a domain of an enzyme that may or may not be modified or altered, but that retains at least part of its original activity.

[0065] The term "linker" or "spacer" refers to a polypeptide comprising at least two amino acids that may be present between domains of a multidomain protein, e.g., an enzyme comprising a catalytic domain and a binding domain such as a carbohydrate binding module (CBM), or any other enzyme hybrid, or between two proteins or polypeptides produced as a fusion polypeptide, e.g., a fusion protein comprising two core enzymes. For example, a fusion protein of a catalytic domain with a CBM is provided by fusing the DNA sequence encoding the catalytic domain, the DNA sequence encoding the linker, and the DNA sequence encoding the CBM consecutively into one open reading frame and expressing this construct.

[0066] As used herein, the terms "dry matter" or "dry pulp" or "OD pulp" refer to pulp that has been dried by oven drying techniques, where dry pulp is free of moisture, i.e., water. The dry matter content of oven-dried pulp may be determined according to ISO 638:2008.

[0067] As used herein, "amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue that is different from the original amino acid at that particular substitution position. The term "amino acid substitution" refers to both conservative and non-conservative amino acid substitutions, meaning that an amino acid residue is replaced with an amino acid residue having a similar side chain (conservative) or a different side chain (non-conservative) from the original amino acid residue at that position. Substitutions are described using the following nomenclature: amino acid residue in the protein scaffold, position, substituted amino acid residue. According to this nomenclature, for example, the substitution of a single residue of alanine for the serine residue at position 23 is designated as Ala23Ser or A23S.

[0068] As used herein, the term "comprising" includes the broader terms "including" and "containing," as well as the narrower terms "consisting of" and "consisting only of." "Comprise," "include," and "containing" are each used as open-ended expressions where no exclusivity is intended.

[0069] In one embodiment, the wood or non-wood derived material is bleached in a bleaching process. In one embodiment, prior to the bleaching process, the wood or non-wood is cooked in a digester to obtain a pulp. In one embodiment, the pulp is treated with a chemical to dissolve the lignin in the pulp.

[0070] In one embodiment, the pulp is a hardwood pulp or a softwood pulp, or a mixture thereof. In one embodiment, the pulp is a non-wood pulp. In one embodiment, the pulp is a xylan-containing pulp. In one embodiment, the pulp is a cellulose pulp, that is, the pulp contains cellulose. In one embodiment, the pulp is a kraft pulp that has been cooked in an alkaline solution. In one embodiment, the bleaching process includes multiple steps, with one or more water washes between steps, or alternatively, each or some of the steps includes a water wash as the final step of the step.

[0071] In one embodiment, the pulp provided in the bleaching process is a xylan-containing pulp. In one embodiment, the pulp provided in the bleaching process is a cellulose pulp. In one embodiment, the pulp provided in the bleaching process is a cellulose xylan-containing pulp.

[0072] In one embodiment, the bleaching process comprises providing a pulp and delignifying the pulp by carrying out the following sequence: a) an enzymatic treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase enzyme at a pH of at least 9.5 and a temperature of at least 85° C., b) an oxidation step (D) in which a bleaching agent is added, c) an alkaline extraction step (E) in which an alkaline agent is added, and d) recovering the bleached pulp. In one embodiment, the GH11 xylanase enzyme used in the bleaching process has the ability to reduce the viscosity of a xylan-containing mixture by at least 20% at 90° C. and pH 10.5 compared to an identical xylan-containing mixture without any xylanase enzyme.

[0073] In one embodiment, the GH11 xylanase enzyme is capable of reducing the viscosity of a xylan-containing mixture by at least 20% when compared to said xylan-containing mixture without the GH11 xylanase enzyme. In one embodiment, the GH11 xylanase enzyme is capable of reducing the viscosity of a xylan-containing mixture containing 0.07 g / mL or less of xylan at 90° C., pH 10.5 and a concentration of 3.0 μg or less GH11 xylanase enzyme protein per gram of xylan-containing mixture by at least 20% when compared to a xylan-containing mixture without the GH11 xylanase enzyme. This means that the xylan-containing mixture containing the GH11 xylanase enzyme is compared to an aliquot of the xylan-containing mixture without any GH11 xylanase enzyme. In one embodiment, the GH11 xylanase enzyme is capable of reducing the viscosity of a xylan-containing mixture containing 0.07 g / mL or less of xylan by at least 20% at 90° C., pH 10.5 and a concentration of 3.0 μg or less GH11 xylanase enzyme protein per gram of xylan-containing mixture, when compared to a xylan-containing mixture to which the GH11 xylanase enzyme has not been added.

[0074] In one embodiment, the GH11 xylanase enzyme is a glycoside hydrolase family 11 xylanase enzyme.

[0075] In one embodiment, the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture by at least 25%, preferably at least 30%, more preferably at least 40% and most preferably at least 50% compared to the same mixture without any xylanase enzyme.

[0076] In one embodiment, the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture by at least 17%, preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably at least 40%, even more preferably at least 50%, and most preferably at least 60% at 85-100°C and pH 9.5-11.5 compared to the same mixture without any xylanase enzyme.

[0077] In one embodiment, the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture by at least 10%, preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and most preferably at least 70% at 85-90° C. and pH 8-11 compared to the same mixture without any xylanase enzyme. In one embodiment, the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture by at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, even more preferably at least 70%, and most preferably at least 75% at 85-90° C. and pH 8-11 compared to the same mixture without any xylanase enzyme.

[0078] In one embodiment, the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture by at least 10%, preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably at least 40%, even more preferably at least 50%, and most preferably at least 60% at 90° C. and pH 10-11 compared to the same mixture without any xylanase enzyme. In one embodiment, the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture by at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, even more preferably at least 70%, and most preferably at least 75% at 90° C. and pH 10-11 compared to the same mixture without any xylanase enzyme.

[0079] In one embodiment, the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture at 90°C and pH 10.5 by at least 20%, preferably at least 25%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and most preferably at least 69%, when compared to the same mixture without any xylanase enzyme.

[0080] In one embodiment, the GH11 xylanase enzyme is capable of reducing the viscosity of a xylan-containing mixture by at least 25% at 90° C. and pH 10.5, compared to the same mixture without any xylanase enzyme.

[0081] In one embodiment, the GH11 xylanase enzyme used in step X is capable of reducing the viscosity of a xylan-containing mixture by at least 20% at 85-100° C. and pH 9.5-11.5 compared to the same mixture without any xylanase enzyme. In one embodiment, the GH11 xylanase enzyme used in step X is capable of reducing the viscosity of a xylan-containing mixture by at least 10%, preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and most preferably at least 70% at 85-100° C. and pH 9.5-11.5 compared to the same mixture without any xylanase enzyme.

[0082] In one embodiment, the ability of the GH11 xylanase enzyme to cause a strong viscosity reduction of the xylan-containing mixture is an indication of the xylan depolymerization activity of the enzyme. Thus, a strong viscosity reduction of the xylan-containing mixture indicates that the GH11 xylanase enzyme used has a high xylan depolymerization activity. In one embodiment, the stronger the viscosity reduction of the xylan-containing mixture in the presence of the GH11 xylanase enzyme, the higher the xylan depolymerization activity of the GH11 xylanase enzyme. In one embodiment, the xylan depolymerization activity of the GH11 xylanase is an indication of the thermostability and pH stability of the enzyme. In one embodiment, the xylan depolymerization activity of the GH11 xylanase at 90°C and pH 10.5 is an indication of the thermostability and pH stability of the enzyme. A high xylan depolymerization activity indicates that the GH11 xylanase is suitable for pulp bleaching at high pH and high temperature, such as pH 9-12 and temperature 85-100°C, preferably pH 9.5-11.5 and temperature 85-100°C.

[0083] In one embodiment, the xylan depolymerizing activity of the GH11 xylanase enzyme is tested on a xylan-containing mixture that is not wood pulp.

[0084] In one embodiment, the xylan-containing mixture comprises xylan and an aqueous solution, aqueous suspension, water or aqueous mixture of liquids and solids. In one embodiment, the xylan-containing mixture comprises 0.07 g or less xylan per mL of the mixture and 3.0 μg or less GH11 xylanase enzyme protein per gram of the mixture, and the decrease in viscosity occurs within 20 minutes, preferably within 10 minutes, more preferably within 5 minutes, and most preferably within 2 minutes. In one embodiment, the xylan-containing mixture comprises 0.07 g / mL or less xylan. In one embodiment, the xylan-containing mixture comprises 0.07 g / mL or less xylan and 3.0 μg or less GH11 xylanase enzyme protein per gram of the mixture. In one embodiment, the xylan-containing mixture comprises 0.07 g or less beechwood xylan per mL of an aqueous pH buffer, the aqueous pH buffer optionally containing glycine and sodium hydroxide, or a KH2PO4-K2HPO4 buffer.

[0085] In one embodiment, the reduction in viscosity of the xylan-containing mixture occurs within 20 minutes, preferably within 10 minutes, more preferably within 5 minutes, and most preferably within 2 minutes.

[0086] In one embodiment, the xylan-containing mixture comprises no more than 0.07 g / mL, no more than 0.06 g / mL, no more than 0.05 g / mL, no more than 0.04 g / mL, no more than 0.03 g / mL, no more than 0.02 g / mL, no more than 0.01 g / mL of xylan. In one embodiment, the concentration of GH11 xylanase enzyme in the xylan-containing mixture is no more than 3.0 μg, no more than 2.9 μg, no more than 2.5 μg, no more than 2.0 μg, no more than 1.9 μg, no more than 1.5 μg, no more than 1.0 μg, no more than 0.5 μg, no more than 0.3 μg, or no more than 0.1 μg of xylanase enzyme protein per gram of the mixture. In one embodiment, the viscosity of the xylan-containing mixture is reduced using GH11 xylanase enzyme within 20 minutes, within 15 minutes, within 10 minutes, within 5 minutes, within 2 minutes or less.

[0087] In one embodiment, step X comprises contacting the pulp with a GH11 xylanase enzyme at a pH of 9.5-11.5 and a temperature of 85-100°C.

[0088] In one embodiment, step X comprises contacting the pulp with a GH11 xylanase enzyme at a pH of at least 9.5 and at a temperature of at least 85° C. In one embodiment, step X is 9.5-11.5 and the temperature is 85-100° C. In one embodiment, the pH of the pulp during step X is 9.5-11.5. In one embodiment, the temperature of the pulp during step X is 85-100° C.

[0089] In one embodiment, the GH11 xylanase enzyme is added to the pulp at a pH selected from 9.5 or greater, 10 or greater, 10.5 or greater, 11 or greater, and 11.5. In one embodiment, the GH11 xylanase enzyme is added to the pulp at a temperature selected from 85°C or greater, 90°C or greater, 95°C or greater, and 100°C.

[0090] In one embodiment, the reduction of the alkaline pH of the pulp and / or the reduction of the treatment temperature before the addition of the GH11 xylanase enzyme is omitted. In one embodiment, the alkaline pH of the pulp is not reduced with acid and / or one or more acid washing steps and / or the temperature of the pulp is not reduced with water before the addition of the GH11 xylanase enzyme. This is because the GH11 xylanase enzyme withstands the high delignification pH and temperature of the bleaching process. In one embodiment, the water consumption of the bleaching process is reduced because it is not necessary to reduce the treatment temperature with extra water before the addition of the GH11 xylanase enzyme. In one embodiment, the acid consumption of the bleaching process and the wear on the bleaching process hardware are reduced because it is not necessary to reduce the treatment pH with acid before the addition of the GH11 xylanase enzyme.

[0091] In one embodiment, the GH11 xylanase enzyme has the ability to reduce the concentration of a xylan-containing composition by at least 20% at 90°C and pH 10.5 when compared to the same mixture without any xylanase enzyme, the xylan-containing mixture containing less than 0.07 g / mL xylan and less than 3.0 μg GH11 xylanase enzyme protein per gram of the mixture, and the viscosity reduction occurs within 20 minutes.

[0092] In one embodiment, the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture by at least 20%, preferably at least 25%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and most preferably at least 65% within 20 minutes at pH 10.5 and at a temperature of 90°C, compared to the xylan-containing mixture not containing any xylanase enzyme, and the concentration of GH11 xylanase enzyme protein is 3.0 μg or less per gram of xylan-containing mixture.

[0093] In one embodiment, the GH11 xylanase enzyme is capable of reducing the viscosity of a xylan-containing mixture by at least 10% within 20 minutes at pH 11 and at a temperature of 90° C. when compared to the xylan-containing mixture not comprising any xylanase enzyme, and the concentration of the GH11 xylanase enzyme is 1.9 μg xylanase protein per gram of xylan-containing mixture. In one embodiment, the GH11 xylanase enzyme is capable of reducing the viscosity of a xylan-containing mixture by at least 53% within 20 minutes at pH 11 and at a temperature of 90° C. when compared to the xylan-containing mixture not comprising any xylanase enzyme, and the concentration of the GH11 xylanase enzyme is 1.9 μg xylanase protein per gram of xylan-containing mixture. In one embodiment, the GH11 xylanase enzyme is capable of reducing the viscosity of a xylan-containing mixture by at least 17% within 20 minutes at pH 10 and at a temperature of 90° C. when compared to a xylan-containing mixture not comprising any xylanase enzyme, and the concentration of the GH11 xylanase enzyme is 2.9 μg xylanase protein per gram of xylan-containing mixture. In one embodiment, the GH11 xylanase enzyme is capable of reducing the viscosity of a xylan-containing mixture by at least 58% within 20 minutes at pH 10 and at a temperature of 90° C. when compared to a xylan-containing mixture not comprising any xylanase enzyme, and the concentration of the GH11 xylanase enzyme is 1.9 μg xylanase protein per gram of xylan-containing mixture.

[0094] In one embodiment, the GH11 xylanase enzyme has at least 25%, preferably at least 50%, more preferably at least 75% residual xylanase activity 10 minutes, preferably 20 minutes, more preferably 30 minutes after step X. In one embodiment, the GH11 xylanase enzyme has at least 25%, preferably at least 50%, more preferably at least 75% residual xylanase activity 20 minutes after step X, step X comprising a pH of 9.5-11.5 and a temperature of 85-100°C.

[0095] In one embodiment, the GH11 xylanase enzyme has at least 25% residual xylanase activity 30 minutes, preferably 40 minutes, more preferably 50 minutes after step X, wherein step X comprises a pH of 9.5-11.5 and a temperature of 85-100°C.

[0096] In one embodiment, the xylanase enzyme retains at least 25% of its xylanase activity after 20 minutes at 85° C. and pH 9.5.

[0097] In one embodiment, the xylanase enzyme retains at least 25% of its xylanase activity after 20 minutes at 100° C. and pH 11.5.

[0098] In one embodiment, the GH11 xylanase enzyme has a residual xylanase activity of at least 75%, preferably at least 80%, more preferably at least 85%, after at least 30 minutes at pH 10.9 and a temperature of 85° C. In one embodiment, the GH11 xylanase enzyme in the hardwood pulp bleached water has a residual xylanase activity of at least 75%, preferably at least 80%, more preferably at least 85%, after at least 30 minutes at pH 10.9 and a temperature of 85° C.

[0099] In one embodiment, the xylanase enzyme has a residual xylanase activity of at least 75%, preferably at least 80%, more preferably at least 85%, after at least 30 minutes at pH 10.5 and a temperature of 85° C. In one embodiment, the GH11 xylanase enzyme in the softwood pulp bleaching process water has a residual xylanase activity of at least 75%, preferably at least 80%, more preferably at least 85%, after at least 30 minutes at pH 10.5 and a temperature of 85° C.

[0100] In one embodiment, the bleaching process comprises providing a pulp and performing delignification of the pulp by performing the following sequence: a) an enzyme treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase enzyme at a pH of at least 9.5-11.5 and a temperature of at least 85-100° C., wherein the xylanase enzyme has a residual xylanase activity of at least 25% 20 minutes after step X; b) an oxidation step (D) in which a bleaching agent is added; c) an alkaline extraction step (E) in which an alkaline agent is added; and d) recovering the delignified pulp.

[0101] In one embodiment, the pulp is provided from the digester to a delignification process.

[0102] In one embodiment, in step X, at least 0.01 g, preferably at least 0.1 g, more preferably at least 0.2 g, and most preferably at least 0.5 g GH11 xylanase enzyme protein is added per 1000 kg dry pulp.

[0103] In one embodiment, the GH11 xylanase enzyme protein is added in step X at a concentration of 0.01-200 g, or 0.1-20 g, or 0.2-10 g, or 0.5-5 g GH11 xylanase enzyme protein per 1000 kg dry pulp. In one embodiment, the concentration of GH11 xylanase enzyme varies depending on the type of pulp used.

[0104] In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with a glycoside hydrolase family GH11 xylanase enzyme and at least one further enzyme selected from the group consisting of proteases, amylases, cellulases, β-glucosidases, lipases, xylanases, mannanases, cutinases, esterases, α-glucuronidases, phytases, nucleases, pectinases, pectinolytic enzymes, pectate lyases, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanases, polysaccharide monooxygenases, laccases, peroxidases and oxidases with or without a mediator or combinations thereof.

[0105] In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with a GH11 xylanase enzyme and a cellulase enzyme. In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with a GH11 xylanase enzyme and a mannanase enzyme. In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with a GH11 xylanase enzyme and a peroxidase enzyme. In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with a GH11 xylanase enzyme and a lignin peroxidase enzyme (EC 1.11.1.14). In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with a GH11 xylanase enzyme and a manganese peroxidase enzyme (EC 1.11.1.13). In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with a GH11 xylanase enzyme and a universal peroxidase enzyme (EC 1.11.1.16). In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with a GH11 xylanase enzyme and a dye decolorizing peroxidase enzyme (EC 1.11.1.19). In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with a GH11 xylanase enzyme and a heme peroxidase enzyme. In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with a GH11 xylanase enzyme and a copper-based laccase enzyme (EC 1.10.3.2).

[0106] In one embodiment, the bleaching process comprises contacting the pulp with a surfactant, surface-active agent, anti-foaming agent, defoamer, emulsifier, dispersant and / or detergent. In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a GH11 xylanase enzyme and a surfactant, surface-active agent, anti-foaming agent, defoamer, emulsifier, dispersant and / or detergent.

[0107] In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with an enzyme composition comprising one or more enzymatic activities that may be isolated and purified from one or more species of microorganism. In one embodiment, the enzymes of the enzyme composition may originate from various species, preferably fungal or bacterial species. Alternatively, these enzymes are present in the fermentation product of one microorganism acting as a host cell for enzyme production, such as during the production of the GH11 xylanase enzyme, when the microorganism simultaneously produces other enzymes in addition to the GH11 xylanase enzyme.

[0108] In one embodiment, the enzymatic treatment step (X) comprises contacting the pulp with an enzyme composition comprising a GH11 xylanase enzyme protein.

[0109] In one embodiment, the pulp is an aqueous mixture comprising at least 0.5-35% by weight of pulp fibers, the pulp fibers being wood and / or non-wood fibers, In one embodiment, the pulp fibers contain xylan, such that the pulp is a xylan-containing pulp.

[0110] In one embodiment, delignification is carried out in the presence of a GH11 xylanase enzyme, which has high pH and thermostability.

[0111] In one embodiment, contacting the pulp with a GH11 xylanase enzyme in step X means that the GH11 xylanase enzyme is contacted with the xylan contained in the pulp.

[0112] In one embodiment, the delignification further comprises an oxygen delignification step (O) prior to the oxidation step D, oxygen being added in step O, and step X being before or after step O.

[0113] In one embodiment, oxygen is added as O2 gas in the oxygen delignification process, and the process is referred to as an O2 process.

[0114] In one embodiment, the duration of the enzyme treatment step X is at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 2.5 hours or at least 3 hours. In one embodiment, the duration of the enzyme treatment step X is between 2 minutes and 3 hours, between 5 minutes and 2 hours, between 15 and 60 minutes or between 20 and 30 minutes. In one embodiment, the temperature of step X is at least 80°C, at least 85°C, at least 90°C, at least 95°C or at least 100°C. In one embodiment, the pH of the pulp during step X is at least 8.5, at least 9.0, at least 9.5, at least 10, at least 10.5, at least 11, at least 11.5 or at least 12.

[0115] In one embodiment, the duration of step O is at least 0.5 hours, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 2.5 hours, or at least 3 hours. In one embodiment, the temperature of step O is at least 75° C., at least 80° C., at least 85° C., at least 90° C., at least 95° C., at least 105° C., at least 110° C., at least 115° C., at least 120° C., at least 125° C., or at least 130° C. In one embodiment, the pH of the pulp during step O is at least 9.5, at least 10, at least 10.5, at least 11, at least 11.5, or at least 12. In one embodiment, the pH and temperature of the pulp may vary during step O.

[0116] In one embodiment, the pH of the pulp in step O is from 10 to 12 and / or the temperature is from 75 to 130°C.

[0117] In one embodiment, the xylanase enzyme is added to the pulp before step O as its own enzymatic treatment step X. In one embodiment, the xylanase enzyme is added to the pulp before step O, and the xylanase enzyme retains at least 25% residual xylanase activity during at least 20 minutes of the enzymatic treatment step X, which includes a pH of 9.5-11.5 and a temperature of 85-100°C. Step O releases a large amount of lignin from the pulp. Thus, in embodiments where the bleaching process includes a step X before step O, it is not necessary to wash the pulp between steps X and O. In one embodiment, if the GH11 xylanase enzyme is added to the pulp before step O, the GH11 xylanase enzyme in the pulp retains some of its activity during step O.

[0118] In one embodiment, the xylanase enzyme is added to the pulp after step O and before step D as its own enzymatic treatment step X. In one embodiment, the xylanase enzyme is added to the pulp after step O, wherein the xylanase enzyme retains at least 25% residual xylanase activity for at least 20 minutes of the enzymatic treatment step X comprising a pH of 9.5-11.5 and a temperature of 85-100°C. In one embodiment, the GH11 xylanase enzyme may be added at any stage before step D, so long as step X is carried out in the presence of a GH11 xylanase enzyme having at least 25% residual xylanase activity after at least 20 minutes at a pH of 9.5-11.5 and a temperature of 85-100°C. In one embodiment, the GH11 xylanase enzyme is active at any pH between 5.6-11.5 and / or at any temperature between 55-100°C.

[0119] In one embodiment, the addition of GH11 xylanase enzyme to the pulp before or after step O allows for enzymatic treatment step X to be carried out in existing storage tanks or towers at the pulp processing facility, without the need to construct a separate processing container / tank when incorporating enzymatic treatment step X into a pulp bleaching process that does not already contain enzymatic treatment step X.

[0120] In one embodiment, each delignification step of the bleaching process is carried out in a separate tank, as shown in FIG. 1. In one embodiment, the optional oxygen delignification step (O) is carried out in a separate tank O. In one embodiment, the enzyme treatment step (X) may be carried out in separate holding tanks S1 and / or S2 before and / or after step O, respectively. In one embodiment, carrying out step X in separate holding tanks S1 and / or S2 provides an adequate retention time for the enzyme treatment of the pulp. However, in some processes, the presence of holding tanks S1 and / or S2 may not be required, as the enzyme treatment step X can be carried out in a separate treatment tank preceding the bleaching process sequence tank D where the oxidation step D is carried out.

[0121] In one embodiment, the oxidation step D is carried out in tank D (FIG. 1). In one embodiment, the bleaching process does not include step O, and the pulp is directed to the oxidation step in tank D directly from the digester (not shown) or via other tanks, such as storage tanks S1 and / or S2. In one embodiment, the bleaching process includes step O, and the oxygen-treated pulp is directed to tank D directly from tank O or via other tanks, such as storage tank S2. Preferably, the pulp is washed between tank O and tank D to remove dissolved lignin. In one embodiment, the bleaching process includes a process sequence including steps O, X, and D, and a water washing step of the pulp is carried out after step X, and at least a portion of the wash water containing the GH11 xylanase enzyme is recycled back to step O. In one embodiment, the bleaching process includes a process sequence including steps O, X, and D, and a water washing step of the pulp is carried out after steps O and X, and washing the pulp between steps X and D is optional. However, if a water washing step of the pulp is performed after step X, then again at least a portion of the wash water containing the GH11 xylanase enzyme is recycled back to step O. In one embodiment, the GH11 xylanase enzyme in the pulp retains some of its activity in step D if a washing step is not performed between steps X and D.

[0122] In one embodiment, the bleaching agent in the oxidation step D comprises an oxygen-containing oxidizing agent, the bleaching agent being preferably selected from the group consisting of ClO2, O3, H2O2 and peroxides. Examples for non-oxygen-containing oxidizing agents are Cl2 and other halogens. In one embodiment, the bleaching agent is a chemical compound containing the element oxygen. In one embodiment, the bleaching agent is ClO2. In one embodiment, the bleaching agent is H2O2. In one embodiment, the bleaching agent is an organic peroxide. In one embodiment, the bleaching agent is an inorganic peroxide. In one embodiment, the bleaching agent is O3. In one embodiment, the bleaching agent is ClO2 and the oxidation step is step D. o In one embodiment, the pulp is directed to tank D for step D and bleach is added in tank D.

[0123] In one embodiment, both GH11 xylanase enzyme and bleaching agent are involved in bleaching the pulp during the bleaching process, whereby the amount of bleaching agent required in the bleaching process is reduced when compared to a bleaching process without xylanase enzyme. In one embodiment, the pulp contains GH11 xylanase enzyme with residual activity in step D, and both GH11 xylanase enzyme and bleaching agent are involved in bleaching the pulp simultaneously. In one embodiment, the pulp contains GH11 xylanase enzyme with residual activity in step D, and the bleaching agent in step D is preferably H2O2. In another embodiment, the pulp no longer contains GH11 xylanase enzyme in step D, since the enzyme is removed in a washing step prior to step D. In such an embodiment, the amount of bleaching agent required in the oxidation step is also reduced when compared to a bleaching process without xylanase enzyme, since the pulp entering step D has already been blended to some extent in the enzymatic treatment step X. In one embodiment, to achieve a particular pulp brightness, at least 5%, preferably 7.5%, more preferably at least 10% less bleaching agent is used than would be used in a corresponding process without the GH11 xylanase enzyme. In one embodiment, the reduction in the amount of bleaching agent leads to a reduction in the amount of adsorbable organic halides (AOX) in the effluent of the bleaching process and a reduction in the need for subsequent wastewater treatment.

[0124] In one embodiment, the GH11 xylanase enzyme used in the bleaching process is highly tolerant to high pH and high temperature. In one embodiment, the use of a temperature- and pH-tolerant xylanase enzyme in the bleaching process produces higher pulp brightness when compared to a bleaching process without a xylanase enzyme or when compared to a process that includes less temperature- and pH-tolerant xylanase enzyme. Meanwhile, the use of a temperature- and pH-tolerant xylanase enzyme in the bleaching process reduces the need for bleaching agents when a particular pulp brightness is desired.

[0125] In one embodiment, the bleaching agent used is H2O2, a metal chelation step (Q) to remove metals is carried out before the oxidation step D, and GH11 xylanase enzyme is added to the pulp before, during or after the chelation step Q. In one embodiment, step Q comprises washing the pulp with a chelating agent such as EDTA or DTPA to remove redox active metal ions from the pulp. In one embodiment, the duration of step Q is the same as the duration of step X.

[0126] In one embodiment, step Q is carried out in a separate metal chelation tank Q before step D in the bleaching process sequence (FIG. 1). The purpose of step Q is to remove metals from the pulp that reduce the bleaching activity of H2O2, such metals being, for example, copper, manganese and iron. In one embodiment, the enzymatic treatment step X of the pulp is carried out in the same tank Q as the metal chelation step. In one embodiment, the enzymatic treatment step X of the pulp is carried out in the same tank Q as the metal chelation step before, after or during the metal chelation step. Alternatively, step X is carried out in a separate tank before and / or after step Q. In one embodiment, step Q is carried out after oxygen delignification is carried out on the pulp in tank O. In one embodiment, the pulp is directed from the metal chelation to an oxidation step in tank D directly in tank Q or via another tank. In one embodiment, the pulp is directed from tank Q to a storage tank (not shown) and the enzymatic treatment step X is carried out after the pulp is directed to an oxidation step in tank D. Preferably the pulp is washed between steps Q and D to remove dissolved metals from the pulp prior to the oxidation step.

[0127] In one embodiment, the GH11 xylanase enzyme may be added to the pulp before, during or after step Q. In one embodiment, the xylanase enzyme is added to the pulp before or during step Q and the process includes a water washing step of the pulp between step Q and the oxidation step in tank D. In this case, at least a portion of the wash water containing the residually active GH11 xylanase enzyme is recycled back to the metal chelation in tank Q, and preferably at least a portion of the wash water is recycled from tank Q back to the upstream pulp washing equipment. In one embodiment, the pulp is washed with water between steps Q and D, and the GH11 xylanase enzyme may be initially added to the pulp after the washing step in tank D before the addition of the bleaching agent H2O2 or in a separate holding tank (not shown). In such an embodiment, the GH11 xylanase enzyme in the pulp may retain some of its activity in step D.

[0128] In one embodiment, the duration of the delignification oxidation step in Tank D is at least 0.5 hours, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 2.5 hours or at least 3 hours. In one embodiment, the temperature of the delignification oxidation step in Tank D is at least 50° C., at least 60° C., at least 70° C., at least 80° C., at least 90° C. or at least 100° C. In one embodiment, the pH of the pulp during the delignification oxidation step is 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less or 1 or less. In one embodiment, the bleaching agent added in step D reduces the pH of the pulp after oxidation.

[0129] In one embodiment, after step D the pulp is directed to an alkaline extraction step in Tank E (FIG. 1). In one embodiment, the bleaching process also includes a water washing step of the pulp between the oxidation step in Tank D and the alkaline extraction step in Tank E. In such an embodiment, the wash water, optionally containing GH11 xylanase enzyme, is recycled back to the water washing step before step D. In one embodiment, the bleaching agent in step D is H2O2, and the xylanase enzyme in the wash water recycled back to the water washing step still has residual activity and can contribute to xylan degradation in Tank D.

[0130] In one embodiment, an alkaline agent is added to the alkaline extraction step in Tank E. In one embodiment, the alkaline agent in alkaline extraction step E is selected from NaOH, MgO, or a combination thereof. In one embodiment, the alkaline agent is a mixture of NaOH and H2O2, or a mixture of NaOH and O2, or a mixture of NaOH+O2+H2O2.

[0131] In one embodiment, the reduction in the amount of ClO2 required as a bleaching agent reduces the amount of NaOH as an alkaline agent required in step E. In one embodiment, the reduction in the amount of NaOH as an alkaline agent reduces the measured chemical oxygen demand (COD) of the effluent of the bleaching process.

[0132] In one embodiment, the GH11 xylanase enzyme has higher activity in delignification process water than in normal tap water under the same treatment conditions, including temperature and pH. In one embodiment, the process water in the delignification stage induces enzyme stability.

[0133] In one embodiment, the duration of the delignification alkaline extraction step E is at least 0.5 hours, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 2.5 hours or at least 3 hours. In one embodiment, the temperature of step E is at least 70° C., at least 80° C., at least 90° C. or at least 100° C. In one embodiment, the pH of the pulp during step E is at least 8, at least 9, at least 9.5, at least 10, at least 10.5 or at least 11. In one embodiment, the alkaline agent increases the pH of the pulp, such that the pH of the pulp is alkaline at the start of the alkaline extraction step and less alkaline at the end of the alkaline extraction step.

[0134] In one embodiment, a final brightness of the pulp that is at least 1 unit higher, preferably at least 2 units, and more preferably at least 2.5 units higher can be achieved by the modern bleaching process than a corresponding process without the GH11 xylanase enzyme.

[0135] In one embodiment, the delignification of the bleaching process is followed by a whitening section of the delignified pulp, which may also include several treatment steps. The whitening section may include at least one or more whitening steps (Db, P) where bleaching agents are added and / or one or more whitening alkaline extraction steps (Eb) where alkaline agents are added. In one embodiment, the main purpose of the whitening section is not to remove residual lignin from the pulp, but to decolorize the residual lignin and thereby obtain a high final brightness.

[0136] In one embodiment, the glycoside hydrolase family GH11 xylanase enzyme used in the bleaching process according to the invention can be of various origins. In one embodiment, the xylanase enzyme used in the bleaching process according to the invention is a wild-type xylanase enzyme. In an alternative embodiment, the xylanase enzyme used in the bleaching process according to the invention is a modified xylanase enzyme and / or a variant of the wild-type xylanase enzyme.

[0137] In one embodiment, the GH11 xylanase enzyme originates from a thermophilic bacterium. In one embodiment, the GH11 xylanase enzyme originates from a bacterium, preferably an actinomycete, more preferably from the order Streptosporangiales, more preferably from the family Streptosporangiaceae, even more preferably from Thermopoltspora species, and most preferably from Thermopolyspora flexuosa. In one embodiment, the xylanase enzyme originates from a Terrabacterium. In one embodiment, the xylanase enzyme originates from the phylum Actinomycetes. In one embodiment, the xylanase enzyme originates from the class Actinomycetia. In one embodiment, the xylanase enzyme originates from the order Streptosporangiales. In one embodiment, the xylanase enzyme originates from the family Streptosporangiaceae. In one embodiment, the xylanase enzyme originates from the genus Thermopoltspora.

[0138] In one embodiment, the GH11 xylanase enzyme originates from Thermopolyspora flexuosa, which means Nonomuraea flexuosa, Nonomuria flexuosa, Nocardia flexuosa, Microtetraspora flexuosa, Acetomadura flexuosa, and / or Actinomadura flexuosa.

[0139] In one embodiment, the glycoside hydrolase family GH11 xylanase enzyme used in the bleaching process comprises a catalytic core domain and a carbohydrate binding module (CBM) linked together by a linker. In one embodiment, the glycoside hydrolase family GH11 xylanase enzyme used in the bleaching process is a truncated enzyme comprising the catalytic core domain and the linker domain, or a portion of the catalytic core domain and the linker domain, while the CBM is absent. In yet another embodiment, the glycoside hydrolase family GH11 xylanase enzyme used in the bleaching process comprises only the catalytic core domain, while the linker domain and the CBM are absent. In one embodiment, the GH11 xylanase enzyme used in the bleaching process comprises a mature catalytic core domain.

[0140] In one embodiment, the GH11 xylanase enzyme is a polypeptide, and the polypeptide, or functional fragment of the polypeptide, has a molecular mass of 15-100 kDa, preferably 18-70 kDa, more preferably 19-40 kDa, even more preferably 20-33 kDa. In one embodiment, the GH11 xylanase enzyme has a molecular mass of 19-40 kDa, preferably 20-30 kDa, more preferably 23-25 ​​kDa, and most preferably 24 kDa, calculated from its amino acid sequence in the absence of glycosylation. In one embodiment, the GH11 xylanase enzyme has a β-jellyroll structure. In one embodiment, the GH11 xylanase enzyme protein has a β-jellyroll structure, xylanase activity, and an amino acid sequence having amino acids corresponding to amino acids 3-180 of SEQ ID NO:2.

[0141] In one embodiment, the GH11 xylanase enzyme is a polypeptide and the polypeptide or functional fragment of the polypeptide has a molecular mass of 31 kDa. In one embodiment, the GH11 xylanase enzyme is a polypeptide and the polypeptide or functional fragment of the polypeptide has a molecular mass of 27 kDa. In one embodiment, the GH11 xylanase enzyme is a polypeptide and the polypeptide or functional fragment of the polypeptide has a molecular mass of 24 kDa. The molecular mass of the GH11 xylanase enzyme polypeptide or functional fragment of the polypeptide may be determined by SDS-PAGE. In one embodiment, the molecular mass of the GH11 xylanase enzyme polypeptide or functional fragment of the polypeptide is calculated from the non-glycosylated amino acid sequence. In one embodiment, the GH11 xylanase enzyme polypeptide or functional fragment of the polypeptide has a molecular mass of 24 kDa and is calculated from the non-glycosylated amino acid sequence. In one embodiment, the GH11 xylanase enzyme lacks xylosidase and cellulase activity.

[0142] In one embodiment, the GH11 xylanase enzyme is a variant polypeptide.In one embodiment, the GH11 xylanase enzyme is a wild-type polypeptide.

[0143] In one embodiment, the GH11 xylanase enzyme has at least one, or at least two disulfide bridges between two cysteine ​​residues in its amino acid sequence, hi one embodiment, the at least one disulfide bridge stabilizes the GH11 xylanase enzyme, thereby increasing the pH and thermostability of the GH11 xylanase enzyme.

[0144] In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising residues 3C and 30C, the positions of which correspond to positions 3 and 30 of SEQ ID NO:2, and a disulfide bridge is formed between residues 3C and 30C. In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising at least one disulfide bridge between two cysteine ​​residues in a 1-191 amino acid region of the polypeptide, and the position of at least one of the at least two cysteine ​​residues is different from the positions corresponding to positions 3 and 30 of SEQ ID NO:2. In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising at least one disulfide bridge between cysteine ​​residues in a 1-30 amino acid region of the polypeptide, the positions of which correspond to the positions of SEQ ID NO:2.

[0145] In one embodiment, the GH11 xylanase enzyme protein has xylanase activity and an amino acid sequence having amino acids corresponding to amino acids 3-180 of SEQ ID NO:2, or a functional fragment thereof.

[0146] In one embodiment, the GH11 xylanase enzyme protein has xylanase activity, an amino acid sequence having amino acids corresponding to amino acids 3 to 180 of SEQ ID NO:2, or a functional fragment thereof, and the ability to reduce the viscosity of a xylan-containing mixture containing 0.07 g / ml or less of xylan by at least 20% within 20 minutes at 90° C., pH 10.5, and at a concentration of 3.0 μg or less GH11 xylanase enzyme protein per gram of said xylan-containing mixture, when compared to a xylan-containing mixture not containing any GH11 xylanase enzyme. In one embodiment, the GH11 xylanase enzyme protein has xylanase activity, an amino acid sequence having amino acids corresponding to amino acids 3-180 of SEQ ID NO:2, or a functional fragment thereof, and the ability to reduce the viscosity of a xylan-containing mixture containing 0.07 g / mL or less of xylan by at least 20% within 20 minutes when compared to a xylan-containing mixture not containing any GH11 xylanase enzyme at 90° C., pH 10.5, and a concentration of 3.0 μg or less GH11 xylanase enzyme protein per gram of the xylan-containing mixture. In one such embodiment, the xylan-containing mixture comprises xylan and an aqueous solution or water.

[0147] In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having amino acids corresponding to amino acids 3-180 of SEQ ID NO:2, or a functional fragment thereof. In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having amino acids corresponding to amino acids 3-180 of SEQ ID NO:2, or a functional fragment thereof, wherein the GH11 xylanase enzyme has at least one disulfide bridge between two cysteine ​​residues of the polypeptide. In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having amino acids corresponding to amino acids 1-191 of SEQ ID NO:2, or a functional fragment thereof, wherein the GH11 xylanase enzyme has at least one disulfide bridge between two cysteine ​​residues of the polypeptide. In one embodiment, the at least one disulfide bridge stabilizes the GH11 xylanase enzyme, thereby increasing the pH stability and thermostability of the GH11 xylanase enzyme compared to a GH11 xylanase enzyme not having a disulfide bridge. In one embodiment, the GH11 xylanase enzyme is a variant polypeptide comprising an amino acid sequence having amino acids corresponding to amino acids 1-191 of SEQ ID NO:1, wherein the GH11 xylanase enzyme has at least one disulfide bridge between two cysteine ​​residues of the polypeptide, and one or two amino acid substitutions are present at positions corresponding to positions 23 and / or 28 of SEQ ID NO:1.

[0148] In one embodiment, the GH11 xylanase enzyme is a variant polypeptide comprising an amino acid sequence having at least 79% but less than 100% amino acid sequence identity to amino acids 1-191 of SEQ ID NO:1.

[0149] In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 1-191 of SEQ ID NO:1.

[0150] In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having at least 79%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% amino acid sequence identity to amino acids 3 to 180 of SEQ ID NO: 2, or a functional fragment thereof. In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having at least 79%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% amino acid sequence identity to amino acids 1 to 191 of SEQ ID NO: 2, or a functional fragment thereof.

[0151] In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to amino acids 3-180 of SEQ ID NO:2.

[0152] In one embodiment, the GH11 xylanase enzyme is a variant polypeptide comprising an amino acid sequence having at least 79% but less than 100% amino acid sequence identity to SEQ ID NO:2. In one embodiment, the GH11 xylanase enzyme comprises a C31-4 inner core polypeptide. In one embodiment, the GH11 xylanase enzyme comprises a C31-4 core polypeptide. In one embodiment, the GH11 xylanase enzyme comprises a C31-4 mature polypeptide. In one embodiment, the GH11 xylanase enzyme is a variant polypeptide having the amino acid sequence SEQ ID NO:2.

[0153] In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having at least 79%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% amino acid sequence identity with amino acids 3-180 of SEQ ID NO:2, or a functional fragment thereof, wherein the amino acid sequence has at least one disulfide bridge between two cysteine ​​residues of the polypeptide. In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having at least 79%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% amino acid sequence identity with amino acids 1-191 of SEQ ID NO:2, wherein the amino acid sequence has at least one disulfide bridge between two cysteine ​​residues of the polypeptide.

[0154] In one embodiment, the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having at least 79% amino acid sequence identity to amino acids 3-180 of SEQ ID NO:2, wherein the amino acid sequence has at least one disulfide bridge between two cysteine ​​residues of the polypeptide.

[0155] In one embodiment, the GH11 xylanase enzyme is a variant polypeptide comprising an amino acid sequence having at least 79% but less than 100% amino acid sequence identity to amino acids 3-180 of SEQ ID NO:1, wherein the amino acid sequence has at least one disulfide bridge between two cysteine ​​residues of the polypeptide and amino acid substitutions at positions 23 or 28, or at positions 23 and 28, which positions correspond to positions 23 and 28 of SEQ ID NO:1.

[0156] In one embodiment, the GH11 xylanase enzyme is a variant polypeptide comprising an amino acid sequence having at least 79% but less than 100% amino acid sequence identity to SEQ ID NO:1 and amino acid substitutions at positions 3, 23 and 30, which correspond to the positions in SEQ ID NO:1. In one embodiment, the GH11 xylanase enzyme is a variant polypeptide comprising an amino acid sequence having at least 79% but less than 100% amino acid sequence identity to SEQ ID NO:1 and amino acid substitutions at positions 3, 28 and 30, which correspond to the positions in SEQ ID NO:1. In one embodiment, the GH11 xylanase enzyme is a variant polypeptide comprising an amino acid sequence having at least 79% but less than 100% amino acid sequence identity to SEQ ID NO:1 and amino acid substitutions at positions 3 and 30, which correspond to the positions in SEQ ID NO:1.

[0157] In one embodiment, there is provided a pulp composition comprising a pulp having a pH of at least 9.5 and 0.01-200 g, preferably 0.1-20 g, more preferably 0.2-10 g of glycoside hydrolase family GH11 xylanase enzyme protein per 1000 kg dry pulp, the GH11 xylanase enzyme protein having xylanase activity. In one embodiment, the GH11 xylanase enzyme that is part of the pulp composition has the ability to reduce the viscosity of a xylan-containing mixture by at least 20%, preferably at least 25%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50% at 90° C. and pH 10.5 when compared to the same mixture without any xylanase enzyme, the xylan-containing mixture comprising no more than 0.07 g / mL xylan and no more than 3.0 μg GH11 xylanase enzyme protein per gram of the mixture, and the viscosity reduction occurs within 20 minutes, preferably within 10 minutes, more preferably within 5 minutes, and most preferably within 2 minutes.

[0158] In one embodiment, the pulp composition has a pH of 9.5 to 11.5 and / or a temperature of 85 to 100° C. In one embodiment, the pulp composition has a pH of at least 9.5, at least 10, at least 10.5, at least 11, or at least 11.5. In one embodiment, the pulp composition has a temperature of at least 85° C., at least 90° C., at least 95° C., or at least 100° C.

[0159] In one embodiment, the pulp composition comprises a GH11 xylanase enzyme protein. In one embodiment, the GH11 xylanase enzyme protein included in the pulp composition has an amino acid sequence having amino acids corresponding to amino acids 3-180 of SEQ ID NO:2.

[0160] In one embodiment, the GH11 xylanase enzyme protein contained in the pulp composition is a polypeptide comprising an amino acid sequence having at least 79%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably 98% amino acid sequence identity to amino acids 3 to 180 of SEQ ID NO:2, or a functional fragment thereof.

[0161] In one embodiment, the pulp contained in the pulp composition is a cellulose pulp and / or a xylan-containing pulp. In one embodiment, the pulp contained in the pulp composition is a cellulose pulp. In one embodiment, the pulp contained in the pulp composition is a xylan-containing pulp.

[0162] In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and at least one additional enzyme selected from the group consisting of protease, amylase, cellulase, β-glucosidase, lipase, xylanase, mannanase, cutinase, esterase, α-glucuronidase, phytase, nuclease, pectinase, pectolytic enzyme, pectate lyase, carbohydrase, arabinase, galactanase, xanthanase, xyloglucanase, polysaccharide monooxygenase, laccase, peroxidase and oxidase with or without a mediator, or combinations thereof.

[0163] In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and a cellulase enzyme. In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and a mannanase enzyme. In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and a laccase enzyme. In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and a peroxidase enzyme. In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and a lignin peroxidase enzyme (EC 1.11.1.14). In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and a manganese peroxidase enzyme (EC 1.11.1.13). In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and a universal peroxidase enzyme (EC 1.11.1.16). In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and a dye decolorizing peroxidase enzyme (EC 1.11.1.19). In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and a haem peroxidase enzyme. In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase enzyme and a copper-based laccase enzyme (EC 1.10.3.2).

[0164] In one embodiment, the pulp composition further comprises a surfactant, a surface active agent, an anti-foaming agent, a defoamer, an emulsifier, a dispersant, a detergent, or any combination thereof.

[0165] In one embodiment, the pulp composition comprises 0.25-4% by weight of the total weight of the dry pulp of a bleaching agent selected from ClO2, O3, H2O2, and peroxide. EXAMPLES

[0166] Xylanases and xylanase variants were designed, produced and tested for xylanase enzyme stability as described in EP 20217335. All xylanases were expressed in the filamentous fungus Trichoderma reesei.

[0167] Xylanase enzyme activity was measured as described in Bailey, MJ and Poutanen, K. 1989 in Appl. Microbiol. Biotechnol. 30:5-10, except that after 5 min at 70°C and pH 7 instead of 10 min at 50°C and pH 5.3, the amount of reducing carbohydrates released from beech xylan was determined spectrophotometrically using dinitrosalicylic acid and compared with a xylose standard solution. One unit of thermoxylanase, abbreviated as TXU, is defined as the amount of xylanase enzyme that produces reducing carbohydrates with a reducing power equivalent to 1 nmol of xylose from beech xylan in 1 second at pH 7 and 70°C (1TXU=1 nkat).

[0168] The protein content of the purified enzyme samples was measured by absorbance at 280 nm. Based on the amino acid composition, xylanases AM24 and C31-4 have a molecular mass without glycosylation of approximately 24 kDa and an absorbance of 2.852 at 280 nm for 1 g of pure protein per liter in a 1 cm cuvette.

[0169] Example 1 Viscosity measurements to select suitable enzymes for pulp bleaching processes A viscous xylan suspension was prepared by weighing 7 g of beechwood xylan into a 100 mL volumetric flask, adding aqueous pH buffer, stirring and heating for approximately 20 min until the suspension boiled. For pH 10.5, a buffer containing 7.5 g / L glycine (equivalent to 0.1 M glycine) and sodium hydroxide was used. For pH 7.0, a 0.1 M KH2PO4-K2HPO4 buffer was used.

[0170] After cooling the xylan-containing mixtures to 25°C, the volumetric flasks were filled with buffer up to the calibration mark and stirred overnight. The next day, these mixtures were centrifuged and the supernatants were transferred to a beaker and placed in an ultrasonic bath for 15 min.

[0171] Then, 9 g of the supernatant was weighed into each 15 mL test tube, which was sealed with a rubber stopper and placed in a water bath preheated to 5° C. above the measurement temperature. Then, 1 g of the enzyme solution or 1 g of water as a reference without added enzyme was added to the preheated supernatant, mixed briefly, and rapidly injected into the viscometer chamber, which had been preheated to the measurement temperature. Viscosity was measured using an Anton Paar, Model DMA 4100M, falling ball viscometer, over a period of 20 minutes.

[0172] The viscosity of the mixtures without added enzymes remained high, whereas the viscosity of the mixtures with added specific enzymes decreased rapidly within the first few minutes until reaching an almost constant viscosity end value within 20 min.

[0173] The following table summarizes the end viscosity values ​​(average of at least duplicate measurements) reached within 20 minutes at the temperatures and pHs shown for each in the table.

[0174] [Table 1]

[0175] The xylanase enzyme concentrations of AM24 and C31-4 used in these experiments were approximately 29 μg and 19 μg of xylanase enzyme protein per gram of enzyme solution added, respectively. 1 g of enzyme solution was mixed with 9 g of xylan supernatant, resulting in a 10-fold diluted enzyme concentration with the mixture injected into the viscometer. The xylan concentration was diluted from 7 g per 100 mL to 6.3 g per 100 mL.

[0176] As a control experiment, inactivated C31-4 was prepared by heating enzyme solution C31-4 to boiling for 2 hours and then removing the precipitated protein. 1 g of this solution was mixed with 9 g of supernatant for viscosity measurements as described above. As expected, the viscosity of this mixture did not decrease, indicating that the viscosity decrease was caused by the active enzyme.

[0177] [Table 2]

[0178] Example 2 Further Viscosity Measurements The following experiments were carried out as described in Example 1. For pH 10 and pH 11, a buffer containing 7.5 g / L glycine (equivalent to 0.1 M glycine) and sodium hydroxide was used. For pH 8, a 0.1 M Tris buffer containing 12.1 g / L 2-amino-2-(hydroxymethyl)propane-1,3-diol and hydrochloric acid was used. For pH 9, a 0.1 M NaHCO3-Na2CO3 buffer was used. The pH was adjusted to the desired value at approximately 60°C.

[0179] The following table summarizes the end viscosity values ​​reached within 20 minutes at the temperatures and pHs shown for each of the tables.

[0180] [Table 3]

[0181] [Table 4]

[0182] [Table 5]

[0183] [Table 6]

[0184] Example 3 High temperature and high pH application of xylanase in eucalyptus-derived kraft pulp A short bleaching sequence XDE (step X, enzyme treatment; step D, chlorine dioxide (ClO2) treatment; step E, alkaline extraction with sodium hydroxide) was used to compare the bleaching efficiency of pretreatment with AM24 and C31-4 xylanases at an elevated temperature of 90 °C. Pretreatment was carried out at two pH values, pH 8.3 and pH 10.5. The pulp used was oxygen delignified eucalyptus kraft pulp from an Asian mill with a kappa number of 15.5 and brightness of 38.0%-ISO. A constant ClO2 dosage (0.2 x kappa number) was used. Bleaching tests were carried out in bleaching reactors. An anchored Kemu reactor (batch size 0.15-1.0 kg, Tmax 90 °C, consistency 25%) was used for X and steps E, and an air-heated Hellsten reactor (batch size 0.3-0.8 kg, Tmax 85 °C, consistency 12%) was used for step D0.

[0185] Approximately 2 g of enzyme protein was used per ton of pulp. The enzyme solutions of AM24 and C31-4 were applied in equal amounts. The enzyme treatment time was 60 min.

[0186] After the enzymatic step X, the enzyme was inactivated by adding hot water and the pH of the pulp suspension was immediately adjusted to a pH value of 2.5 to 3. After step E, the brightness and kappa number were analyzed.

[0187] [Table 7]

[0188] Pretreatment with both xylanases AM24 and C31-4 increased the brightness of eucalyptus pulp at pH 8.3. Pulp pretreated with C31-4 at pH 8.3 had 2.8 units higher brightness than the reference pulp and 1.0 unit higher brightness than pulp treated with AM24. The active chlorine (aCl) used at pH 8.3 was 0.2 kg / t per ΔKappa and 0.07 kg / t per ΔBr lower for C31-4 treated pulp than for AM24 treated pulp, and 0.28 kg / t and 0.19 kg / t lower, respectively, than the reference pulp (Table 7).

[0189] When xylanase pretreatment was performed at pH 10.5 and 90°C, the C31-4 treated pulp had 2.7 units higher brightness, 0.09 kg / t lower active chlorine (aCl) usage per ΔKappa and 0.18 kg / t lower active chlorine usage per ΔBr than the reference pulp (Table 7).

[0190] The increase in brightness (%-ISO) measured was significant, revealing that the application of xylanase enzyme C31-4 in kraft pulp was beneficial and was effective at 90°C and pH 10.5.

[0191] Example 4 High temperature and high pH application of xylanase in softwood-derived kraft pulp The efficiency of xylanase enzyme C31-4 in bleaching softwood pulp at high temperature and high pH was tested using a short bleaching sequence XDE (X, enzyme; D, ClO2 (chlorine dioxide); E, extraction with sodium hydroxide) at constant ClO2 dosage (0.2 x kappa number). Bleaching tests were carried out in bleaching reactors for the entire sequence. An anchored Kemu reactor (batch size 0.15-1.0 kg, Tmax 90 °C, consistency 25%) was used for steps X and E, and an air-heated Hellsten reactor (batch size 0.3-0.8 kg, Tmax 85 °C, consistency 12%) was used for step D0.

[0192] The pulp used in the tests was oxygen delignified SW Kraft from a Scandinavian mill with the following properties: Kappa number 25.6, viscosity 1140 mL / g and brightness 26.8%-ISO. The dosage of the C31-4 enzyme preparation was approximately 2 g enzyme protein per tonne of pulp. The conditions used at each stage of bleaching are set forth in Tables 8-10.

[0193] After step X, the pulp was washed three times. The first wash was done with water at reaction temperature (90°C) and the next two washes were done with cold water (4°C). After the washing steps, the water was removed by centrifugation (2600 rpm, 10 sec) and pulp samples of about 10-15 g (measured as oven dried) were collected for analysis. In addition, dry matter analysis was done from the C31-4 treated samples using an IR dryer for better control of pulp quantity. The pulp samples were analyzed for Kappa number and brightness.

[0194] The test results are detailed in Table 11.

[0195] [Table 8]

[0196] [Table 9]

[0197] [Table 10]

[0198] [Table 11]

[0199] Softwood kraft pulp pretreated with C31-4 at pH 10.5 had 2.7 units higher corrected final brightness when compared to the reference pulp (no enzyme used) at the same 5.4% active chlorine consumption.

[0200] Example 5. High temperature and high pH application of additional xylanases in softwood-derived kraft pulp The bleaching test using softwood pulp and similar conditions as in Example 4 was repeated with AM24 and C31-4 xylanases. The pulp was washed at pH 11 and 60°C for about 30 minutes before its characterization and experimentation. After the washing step, the pulp had a kappa number of 16.7, a viscosity of 1050mL / g and a brightness of 32.6%-ISO. The dosage of both enzymes (C31-4 and AM24) was 2g enzyme protein per ton of pulp. The conditions used in the xylanase treatment, bleaching and extraction stages are described in Tables 12 to 14. In the xylanase treatment stage (X stage), the two enzymes were applied at various pH and temperatures since AM24 xylanase is inactive in such paper mill conditions, whereas the C31-4 variant is believed to be capable of functioning in these conditions (pH 10.5 / 90°C) based on previous experiments.

[0201] [Table 12]

[0202] [Table 13]

[0203] [Table 14]

[0204] The experimental results are listed in Table 15.

[0205] [Table 15]

[0206] Scandinavian softwood kraft pulp pretreated with C31-4 at pH 10.5 / 90°C had 2.8 units higher brightness when compared to the reference pulp (no enzyme) at identical conditions throughout the XDE bleaching sequence. The results demonstrated that C31-4 improved brightness at high pH and temperature conditions. AM24 xylanase showed a brightness improvement of 1.2 units at the conditioning conditions in the X stage (pH 7 / 80°C) and at the conditions identical to those used for C31-4 in stages D and E.

[0207] The above description provides a complete and informative description of the best mode currently contemplated by the inventors for carrying out the invention, as non-limiting examples of specific implementations and embodiments, however, it will be apparent to those skilled in the art that the invention is not limited to the details of the embodiments presented above, and that it may be carried out in other embodiments using equivalent means, or in different combinations of the embodiments, without departing from the characteristics of the invention.

[0208] Moreover, some of the features of the exemplary embodiments disclosed above may be used to advantage without the corresponding use of other features. As a result, any suitable combination of embodiments and aspects may be formed. As such, the foregoing description should be considered as merely illustrative of the principles of the invention, and not in limitation of these principles. The scope of the invention is therefore limited only by the appended claims.

[0209] [Sequence table] SEQ ID NO:1 AM24, the amino acid sequence of a truncated form of the Thermopolyspora flexuosa Xyn11A protein (AM35). AM24 contains amino acids D44-L263 of the full-length wild-type AM35. SEQ ID NO: 2: Amino acid sequence of AM24 variant C31-4 containing the mutations T3C, A23S, S28I, T30C SEQ ID NO:3 AM35, the amino acid sequence of the full-length mature Thermopolyspora flexuosa Xyn11A protein

Claims

1. Providing a pulp; The sequence below: a) an enzymatic treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase enzyme at a pH of at least 9.5 and a temperature of at least 85° C.; b) an oxidation step (D), in which a bleaching agent is added; c) an alkaline extraction step (E) in which an alkaline agent is added; and d) Recovering the bleached pulp. The pulp is delignified by carrying out the steps of: A bleaching process comprising:

1. A process wherein the GH11 xylanase enzyme has the ability to reduce the viscosity of the xylan-containing mixture by at least 20% when compared to the xylan-containing mixture not comprising any xylanase enzyme at 90° C., pH 10.5 and a protein concentration of the GH11 xylanase enzyme of 3.0 μg or less per gram of xylan-containing mixture, and wherein the xylan-containing mixture contains 0.07 g / mL or less xylan.

2. 2. The process of claim 1, wherein the GH11 xylanase enzyme has the ability to reduce the viscosity of a xylan-containing mixture by at least 25%, preferably at least 30%, more preferably at least 40% and most preferably at least 50% compared to the same mixture without any xylanase enzyme.

3. the xylan-containing mixture comprising no more than 0.07 g xylan per mL of the mixture and no more than 3.0 μg of the GH11 xylanase enzyme protein per gram of the mixture; and 3. The process of claim 1 or 2, wherein the decrease in viscosity occurs within 20 minutes, preferably within 10 minutes, more preferably within 5 minutes, and most preferably within 2 minutes.

4. 4. The process of any one of claims 1 to 3, wherein step X comprises contacting the pulp with the GH11 xylanase enzyme at a pH of from 9.5 to 11.5 and at a temperature of from 85 to 100°C.

5. 5. The process according to any one of claims 1 to 4, wherein in step X, at least 0.01 g, preferably at least 0.1 g, more preferably at least 0.2 g, and most preferably at least 0.5 g of GH11 xylanase enzyme protein is added per 1000 kg of dry pulp.

6. The delignification further comprises an oxygen delignification step (O) prior to the oxidation step D, wherein oxygen is added in step O; and The process according to any one of claims 1 to 5, wherein said step X is before or after said step O.

7. The bleaching agent in the oxidation step D comprises an oxygen-containing oxidizing agent, preferably the bleaching agent is ClO 2 , O 3 , H 2 O 2 The process of any one of claims 1 to 6, wherein the aryl group is selected from the group consisting of: and peroxides.

8. The bleach used is H 2 O 2 and a metal chelation step (Q) for removing metals is carried out before the oxidation step D, and the GH11 xylanase enzyme is added to the pulp before, during or after the chelation step Q.

9. 9. The process according to any one of claims 1 to 8, wherein the alkaline agent in the alkaline extraction step E is selected from NaOH, MgO or a combination thereof.

10. 10. The process according to any one of claims 1 to 9, wherein the GH11 xylanase enzyme is of bacterial origin, preferably Actinomycete, more preferably Streptosporangiales, more preferably Streptosporangiaceae, even more preferably Thermopoltspora species, most preferably Thermopolyspora flexuosa.

11. 11. The process according to any one of claims 1 to 10, wherein the GH11 xylanase enzyme has a molecular mass of 19-40 kDa, preferably 20-30 kDa, more preferably 23-25 ​​kDa, most preferably 24 kDa, calculated from its amino acid sequence in the unglycosylated state.

12. the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having amino acids corresponding to amino acids 3 to 180 of SEQ ID NO:2, or a functional fragment thereof; The process according to any one of claims 1 to 11, wherein the GH11 xylanase enzyme has at least one disulfide bridge between two cysteine ​​residues of the polypeptide.

13. 13. The process according to any one of claims 1 to 12, wherein the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having at least 79%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% amino acid sequence identity to amino acids 3 to 180 of SEQ ID NO:2, or a functional fragment thereof.

14. the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having at least 79%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% amino acid sequence identity to amino acids 3-180 of SEQ ID NO:2, or a functional fragment thereof; The process according to any one of claims 1 to 13, wherein the amino acid sequence has at least one disulfide bridge between two cysteine ​​residues of the polypeptide.

15. (a) a pulp having a pH of at least 9.5; and (b) 0.01 to 200 g of glycoside hydrolase family GH11 xylanase enzyme protein per 1000 kg of dry pulp. A pulp composition comprising: The GH11 xylanase enzyme protein is Xylanase activity, and the ability to reduce the viscosity of said xylan-containing mixture by at least 20% within 20 minutes at 90° C., pH 10.5 and a concentration of said GH11 xylanase enzyme protein of 3.0 μg or less per gram of xylan-containing mixture, when compared to said xylan-containing mixture without any xylanase enzyme, wherein said xylan-containing mixture contains 0.07 g / mL or less xylan. The pulp composition has the following structure:

16. 16. The pulp composition of claim 15, wherein the GH11 xylanase enzyme is a polypeptide comprising an amino acid sequence having at least 79%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably 98% amino acid sequence identity to amino acids 3 to 180 of SEQ ID NO:2, or a functional fragment thereof.

17. 17. The pulp composition of claim 15 or 16, wherein the pulp is a cellulose pulp.

18. The process according to any one of claims 1 to 14, wherein the pulp is a cellulose pulp.

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