Bleaching process

Abstract

Disclosed is a process for bleaching a pulp comprising the delignification of the pulp by the following sequence: an enzymatic treatment step comprising contacting the pulp with a GH11 xylanase of the glycoside hydrolase family at a pH of at least 9.5 and a temperature of at least 85 DEG C; an oxidation step, wherein 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 is capable of reducing the viscosity of the xylan-containing mixture by at least 20% at 90 DEG C and at pH 10.5 as compared to the same mixture without any xylanase.

Description

Technical Field

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

[0002] This section provides useful background information and is not an admission that any of the art described herein represents prior art.

[0003] Bleached pulp is used for the production of, for example, paper, board, tissue, dissolving pulp, fluff pulp, market pulp and, more recently, also for the production of new products defined as MFC (microfibrillated cellulose) and NCF (nanocellulose fibers). Pulp is usually processed by kraft pulping, in which free cellulose fibers are obtained by dissolving lignin in an alkaline cooking liquor. Residual lignin and lignin derivatives attach to the carbohydrate part of the pulp, giving the pulp an undesirable brown color, after which the pulp is usually bleached.

[0004] The pulp bleaching process can be divided into two subsequent parts: delignification and brightening. In delignification, the wood components lignin and hemicellulose are at least partially dissolved and removed from the pulp via the use of bleaching chemicals. During delignification, the brightness of the pulp also increases. In the subsequent brightening part, the delignified pulp is treated with bleaching chemicals that react with chromophores, which causes the elimination of the chromophores, thereby producing a bleached or white pulp.

[0005] Typically, delignification has at least two stages, an oxidation step in which a bleaching agent is added and another alkaline extraction step in which an alkaline agent is added. In addition, the oxidation step may be preceded by an oxygen delignification step (O), in which oxygen is mixed into the pulp and in which most of the lignin in the pulp is removed.

[0006] Bleaching agents added in the oxidation step further aid in lignin removal, and several bleaching agents may be employed in the process. Pulp is typically bleached (whitened) with chemicals containing chlorine (e.g., elemental chlorine), which can result in the production of chlorine-containing organic byproducts that have toxic and mutagenic effects in the environment. Therefore, pulp bleaching methods that do not contain elemental chlorine are utilized to avoid the release of chlorogenic compounds. Completely chlorine-free bleaching utilizes bleaching agents such as hydrogen peroxide.

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

[0008] Xylanases are enzymes that digest xylans to produce 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), have been classified in the CAZy database into glycoside hydrolase families GH5, GH8, GH10, GH11, GH30, and GH43. Xylanases from different glycoside hydrolase families differ structurally from each other, with GH11 xylanases having a β-jelly roll structure, GH5, GH10, and GH30 xylanases having a (β / α) 8-barrel structure, GH8 xylanases having a (α / α) 6-barrel structure, and GH43 xylanases having a 5-fold β-propeller structure. Many different xylanases are known, for example, there are 5949 and 2187 different enzymes in the GH10 and GH11 families, respectively. In addition, many xylanase variants found in nature have been engineered. In pulp bleaching processes, xylanases should depolymerize xylans, break the bonds between xylan and lignin, promote lignin reduction, and improve the accessibility of bleaching chemicals to 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, and therefore the xylanases used in such processes must possess excellent stability and activity under biologically challenging conditions of very alkaline pH and high temperature.

[0009] There is an increasing need for such xylanases that can remain active and functional in pulp bleaching processes, particularly with respect to improving pulp brightness, reducing bleaching chemical consumption, and reducing the generation of toxic chlorine-containing compounds. Several xylanases have been reported that have significant differences in pulp bleaching performance, pH and temperature characteristics, enzyme stability, specific activity, and other enzyme properties, but typically xylanases are unstable and do not function under the very high temperature and alkaline conditions of the pulp bleaching process. Therefore, there is a need for xylanases that are both very thermostable and alkali-resistant and that are active and functional in industrial processes such as pulp bleaching. Therefore, it is an object of the present disclosure to provide a bleaching process with a xylanase that is capable of functioning at high temperatures and alkaline pH in the process. Summary of the invention

[0010] The attached claims define the scope of protection. Any examples and / or technical descriptions of devices, systems, products and / or methods in the specification and / or drawings that are not covered by the present claims are not presented as embodiments of the present invention, but are presented as examples that can be used to understand the present invention.

[0011] According to a first aspect, there is provided a bleaching process comprising:

[0012] Provide pulp;

[0013] Delignification of pulp is carried out by carrying out the following sequence:

[0014] a) an enzyme treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase at a pH of at least 9.5 and a temperature of at least 85°C;

[0015] b) an oxidation step (D) wherein a bleaching agent is added;

[0016] c) an alkaline extraction step (E), wherein an alkaline agent is added; and

[0017] d) recovering bleached pulp;

[0018] The GH11 xylanase is capable of causing a reduction in viscosity of the xylan-containing mixture by at least 20% at 90°C, pH 10.5 and at a concentration of 3.0 μg or less of GH11 xylanase protein per gram of the xylan-containing mixture when compared to a xylan-containing mixture without any xylanase, wherein the xylan-containing mixture comprises 0.07 g / ml or less of xylan.

[0019] In one embodiment, a bleaching process is provided, comprising:

[0020] Provide pulp;

[0021] Delignification of pulp is carried out by carrying out the following sequence:

[0022] a) an enzyme treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase at a pH of at least 9.5 and a temperature of at least 85°C;

[0023] b) an oxidation step (D) wherein a bleaching agent is added;

[0024] c) an alkaline extraction step (E), wherein an alkaline agent is added; and

[0025] d) recovering bleached pulp;

[0026] Among them, the GH11 xylanase has the ability to cause at least a 20% reduction in the viscosity of a mixture containing xylan at 90°C and at pH 10.5 compared to the same mixture without any xylanase.

[0027] The inventors surprisingly found, and show in the examples below, that xylanases suitable for use in the present bleaching process can be selected based on the molecular mechanism of their pulp bleaching properties, rather than just xylanase activity or the release of xylan hydrolysis products from the pulp. This is rationalized by the pre-bleaching effect of xylanases, which is primarily a result of xylan depolymerization, rather than solubilization.

[0028] In one embodiment, GH11 xylanases that depolymerize xylan without solubilizing xylan are particularly advantageous in pulp bleaching processes because xylan solubilization may result in an undesirable increase in chemical oxygen demand (COD) and a loss in pulp yield. Therefore, the selection of a suitable GH11 xylanase according to pulp bleaching performance should be based on the molecular mechanism of pulp bleaching performance, and / or alternative xylanase activity or the release of xylan hydrolysate from the pulp. When polymers such as xylan are depolymerized, the viscosity of the xylan-containing mixture in question is reduced. However, because xylan is only a minor component of pulp, the viscosity reduction of the pulp induced by xylanase may be rare and almost unmeasurable. Surprisingly, the xylanase-induced viscosity reduction, which indicates the depolymerization potential of xylanase, can be measured in a xylan-containing mixture containing pure or purified xylan, rather than in pulp. Furthermore, it was surprisingly found that xylanase induced viscosity reduction is linked to xylanase performance in pulp bleaching processes at high temperature and high pH.

[0029] The bleaching process provides an efficient way to produce delignified and bleached pulp at high temperature and high pH in the presence of GH11 xylanases.

[0030] According to a second aspect, there is provided a pulp composition comprising:

[0031] (a) pulp having a pH of at least 9.5; and

[0032] (b) 0.01-200 g of glycoside hydrolase family GH11 xylanase protein per 1000 kg of dry matter pulp;

[0033] The GH11 xylanase protein has:

[0034] Xylanase activity, and

[0035] Such ability: at 90°C, pH 10.5 and 3.0 μg or less of GH11 per gram of the xylan-containing mixture when compared to a xylan-containing mixture without any xylanase

[0036] The invention relates to a method for preparing a xylanase containing a mixture comprising: a xylanase protein at a concentration of at least 100 μg / ml to at least 200 μg / ml of xylan to cause a viscosity reduction of the xylan-containing mixture by at least 20% within 20 minutes, wherein the xylan-containing mixture comprises 0.07 g / ml or less of xylan.

[0037] In one embodiment, a pulp composition is provided, comprising:

[0038] (a) pulp having a pH of at least 9.5; and

[0039] (b) 0.01-200 g of glycoside hydrolase family GH11 xylanase protein per 1000 kg of dry matter pulp;

[0040] The GH11 xylanase protein has:

[0041] Xylanase activity, and

[0042] The ability to cause a reduction in viscosity of a mixture containing xylan by at least 20% at 90°C and at pH 10.5, compared to the same mixture without any xylanase, wherein the mixture containing xylan comprises 0.07 g / ml or less of xylan and 3.0 μg or less of GH11 xylanase protein per gram of said mixture; and wherein the reduction in viscosity occurs within 20 minutes.

[0043] According to another aspect, there is provided a bleaching process comprising:

[0044] Provide pulp;

[0045] Delignification of pulp is carried out by carrying out the following sequence:

[0046] a) an enzyme treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase at a pH of 9.5-11.5 and a temperature of 85-100° C., wherein the xylanase has a residual xylanase activity of at least 25% after 20 minutes of step X;

[0047] b) an oxidation step (D) wherein a bleaching agent is added;

[0048] c) an alkaline extraction step (E), wherein an alkaline agent is added; and

[0049] d) Recovering the delignified pulp.

[0050] According to another aspect, there is provided a pulp composition comprising:

[0051] (a) a pulp having a pH of at least 9.5;

[0052] (b) 0.25-4 wt. % of a bleaching agent, based on the total weight of the dry matter pulp, the bleaching agent being selected from the group consisting of ClO2, O3, H2O2 and peroxides; and

[0053] (c) 0.01-200 g of glycoside hydrolase family GH11 xylanase protein per 1000 kg of dry matter pulp;

[0054] wherein the GH11 xylanase protein has xylanase activity and the ability to cause at least a 20% reduction in the viscosity of a mixture containing xylan at 90°C and at pH 10.5, compared to the same mixture without any xylanase, wherein the mixture containing xylan comprises 0.07 g / ml or less of xylan and 3.0 μg or less of the GH11 xylanase protein per gram of said mixture; and wherein the reduction in viscosity occurs within 20 minutes.

[0055] The present bleaching process comprising GH11 xylanase is conducive to reducing the total amount of bleaching agent used in the bleaching process of pulp. The present bleaching process is conducive to reducing the amount of ClO2, O3, H2O2 or peroxide consumption in the pulp bleaching process. When compared with the bleaching process without GH11 xylanase, the present bleaching process is conducive to achieving higher pulp brightness and / or higher bleached product output when the same amount of bleaching agent is used in the pulp bleaching process. Compared with the bleaching process without GH11 xylanase, the present bleaching process is conducive to achieving higher pulp brightness and / or higher bleached product output when the reduced amount of bleaching agent is used in the pulp bleaching process. The present bleaching process is conducive to reducing the production cost of bleached pulp. The present bleaching process allows for more efficient bleaching of pulp, making more efficient use of wood raw materials. The present bleaching process is conducive to integrating the use of GH11 xylanase with high pH and thermal stability into the bleaching process, thereby reducing or avoiding the need to change the existing process conditions and / or the settings / hardware commonly used in pulp bleaching. The present bleaching process has the advantage of reducing corrosion, wear and erosion of metal pulp bleaching tanks due to the reduced need for acidic agents to lower the pH of the pulp. The present bleaching process has the advantage of reducing the fresh water consumption of the bleaching process due to the reduced need for additional water washing. The present bleaching process has the advantage of reducing the generation of AOX (adsorbable organic halides, also known as chloroorganic compounds), which are discharged with the wastewater bleach effluent. The present bleaching process has the advantage of reducing the amount of energy required to raise the pulp temperature after washing and before oxygen delignification or before the oxidation step due to the reduced need to reduce the temperature of the enzyme treated pulp.

[0056] The foregoing describes various non-binding example aspects and embodiments. The foregoing embodiments are only used to explain selected aspects or steps that may be utilized in different implementations. Some embodiments may be presented with reference only to certain example aspects. It should be understood that the corresponding embodiments may also be applied to other example aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Some example embodiments will be described with reference to the accompanying drawings, in which:

[0058] Figure 1 Flow chart showing process steps of a pulp delignification bleaching process according to an example embodiment.Process steps X, O, Q within dashed boxes represent optional and / or alternative process steps.

[0059] Sequence Listing

[0060] SEQ ID NO: 1AM24, Thermopolyspora flexuosa Xyn11A protein

[0061] The amino acid sequence of a truncated form of (AM35). AM24 contains amino acids D44-L263 of the full-length wild-type AM35.

[0062] SEQ ID NO:2 Amino acid sequence of AM24 variant C31-4 comprising mutations T3C, A23S, S28I, T30C

[0063] SEQ ID NO: 3AM35, amino acid sequence of full-length mature Thermopolyspora porphyria Xyn11A protein DETAILED DESCRIPTION

[0064] The present invention relates to a bleaching process utilizing in the delignification step of the process a glycoside hydrolase family GH11 xylanase having stability, activity and performance under biologically challenging conditions of high temperature and alkaline pH, resulting in improved bleaching of pulp.

[0065] The term "bleaching process" means a chemical treatment process in which the brightness or whiteness of pulp is increased by removing chemical elements that impart color to the pulp, such as lignin and chromophoric double bonds.

[0066] The term "delignification" means the removal and extraction of lignin contained in pulp. The K value can be used as a measure of the lignin content in pulp.

[0067] The term "alkaline extraction step" means a bleaching step in which the pH of the pulp is increased to a level that extracts oxidized lignin from the pulp.

[0068] The term "glycoside hydrolase" means a hydrolase capable of hydrolyzing a glycosidic bond between two or more carbohydrates or a glycosidic bond between a carbohydrate and a non-carbohydrate moiety.

[0069] The term "glycoside hydrolase family 11" or "GH11" refers to GH11 xylanases defined according to the CAZy database http: / / www.cazy.org / GH11.html, GH11 xylanases have a β-jelly roll structure, and thus GH11 xylanases are structurally different from other xylanases.

[0070] The term "xylan" refers to a matrix polysaccharide or heteropolymer consisting of a repeating backbone of β-1,4-linked xylose residues with various side chain groups. Xylan polysaccharides can be classified based on substituted side chain groups as O-acetylglucuronoxylan (AcGX), O-acetylarbinoxylan (AcAX), O-acetylglucuronoxylan (AcGAX) and arabinoglucuronoxylan (AGX). Materials containing xylans include plant-based or plant-derived materials.

[0071] As used herein, the term "xylanase" means a xylanase defined according to that known in the art as an endo-1,4-β-xylanase or a 4-β-D-xylan hydrolase that catalyzes the endohydrolysis of (1→4)-β-D-xylosidic bonds in xylans. Xylanases have the alternative names endo-(1→4)-β-xylan 4-xylan hydrolase, 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 xylan hydrolase, β-xylanase, β-1,4-xylan xylan hydrolase, endo-1,4-β-xylanase, and β-D-xylanase. Xylanases are classified according to enzyme nomenclature as EC 3.2.1.8.

[0072] As used herein, the term "GH11 xylanase protein" refers to pure GH11 xylanase protein. According to some embodiments, the GH11 xylanase protein can be added as one component of the composition in the enzyme treatment step X, but in such embodiments, the term GH11 xylanase protein refers to pure GH11 xylanase protein as one of the components of the composition. In some embodiments, the amount of GH11 xylanase protein refers to the dry weight (grams) of the GH11 xylanase protein, which can be determined according to the test procedure described in the Examples.

[0073] As used herein, the term "pulp" means a fibrous material containing water that is originally obtained from plants. Pulp can be derived from wood, fiber crops, bagasse, straw, waste paper or rags, or any combination thereof. Therefore, in the context of the present application, "pulp" can refer to wood pulp derived from wood materials and / or to non-wood pulp derived from non-wood materials.

[0074] As used herein, the term "wood pulp" means a fibrous material containing water that is derived from wood materials and is produced by digesting hard or soft wood chips with a solution of sodium hydroxide and sodium sulfide at a temperature above about 120°C.

[0075] As used herein, the term "non-wood pulp" means a fibrous material containing water derived from non-wood materials produced by digesting the non-wood materials, such as grass, cereal straw, corn stover, bamboo, kenaf or bagasse, or any combination thereof.

[0076] As used herein, the term "cellulose pulp" means a fibrous material comprising water that comprises cellulose fibers.

[0077] As used herein, the term "xylan-containing pulp" means wood pulp or non-wood pulp containing xylan. The term "xylan-containing pulp" or "pulp containing / comprising xylan" does not refer to, i.e. does not mean the same thing as the term "mixture containing wood polysaccharides".

[0078] As used herein, the term "hardwood pulp" means wood pulp derived from wood fibers of hardwood trees, such as oak, beech, birch, poplar, eucalyptus and / or aspen.

[0079] As used herein, the term "softwood pulp" means wood pulp derived from wood fibers of coniferous trees, such as pine, spruce, fir, and / or hemlock.

[0080] As used herein, the term "kraft pulp" means wood pulp produced by the Kraft process (also known as the kraft process), in which wood chips are treated with sodium sulfide (Na2S) and sodium hydroxide (NaOH).

[0081] As used herein, the term "thermostable enzyme" or "temperature-resistant enzyme" refers to an enzyme having a good ability to endure and resist structural changes resulting in enzyme inactivation caused by high temperatures, such as temperatures of 85°C or higher.

[0082] As used herein, the term "enzyme pH stability" or "enzyme pH tolerance" describes the property of an enzyme to tolerate and resist structural changes resulting in enzyme inactivation, either caused by high pH, ​​such as pH 9.5 or higher, or alternatively caused by low pH.

[0083] The term "process water" means any water used in the bleaching process of pulp.

[0084] The term "process wash water" means any water used for the purpose of washing pulp in a pulp bleaching process.

[0085] As used herein, the term "viscosity" or "relative viscosity" means the viscosity of a specified material, such as a mixture, suspension or solution containing xylan. As used herein, a decrease in viscosity of a mixture containing GH11 xylanase and xylan is an indicator of the xylan depolymerizing activity of the GH11 xylanase. In one embodiment, the lower the viscosity of the mixture containing xylan decreases over time, the higher the xylan depolymerizing activity of the GH11 xylanase. Viscosity is measured using a rolling ball viscometer.

[0086] As used herein, the term "mixture containing xylan" refers to a mixture containing xylan, and the mixture may refer to a solution, a suspension, or a mixture of a liquid and a solid. In one embodiment, the mixture containing xylan also contains a xylanase, such as a GH11 xylanase. The mixture containing xylan is suitable for assessing the xylan degrading activity of the xylanase. Therefore, the mixture containing xylan does not contain any material that would distort or tamper with the assessment of the xylan degrading activity of the xylanase. For example, the mixture containing xylan must be suitable for assessing the influence of the GH11 xylanase on the viscosity of the mixture containing xylan.

[0087] As used herein, the term "adsorbable organic halide (AOX)" refers to a measure of the organic halogen chlorine, bromine, and iodine loading in a sample, such as a water sample.

[0088] As used herein, the term "chemical oxygen demand (COD)" means a measure of the amount of oxygen that can be consumed by a reaction in a solution. COD represents the amount of oxygen required to completely decompose organic matter in a solution into CO2. COD measurements are also used to determine the amount of oxidizable pollutants and thereby determine toxicity to biological life, with high COD being associated with high toxicity of organic matter.

[0089] As used herein, the term "sequence identity" means the percentage of exact matches of amino acid residues between two optimally aligned sequences to the number of positions where the residues are present in both sequences. When one sequence has a residue that has no corresponding residue in the other sequence, the alignment program used allows for gaps in the alignment, and such positions are not counted in the denominator of the identity calculation.

[0090] As used herein, the term "corresponding position" or "corresponding amino acid position" means that at least two amino acid sequences are aligned as a two-sequence alignment or a multiple sequence alignment according to the identified region of similarity or identity, thereby pairing corresponding amino acids. As used herein, the term "corresponding amino acid" means the amino acid in question in a first amino acid sequence, when the first amino acid sequence is aligned with a second amino acid sequence as a two-sequence alignment or a multiple sequence alignment according to the identified region of similarity or identity, the amino acid in question can be paired with the corresponding amino acid in the second sequence. The sequence identity between the first and second amino acid sequences consisting of corresponding amino acids can be 33-100%.

[0091] As used herein, the term "xylanase activity" refers to xylan hydrolyzing activity.

[0092] The term "residual xylanase activity" means an activity of a xylanase that is not the initial activity of the enzyme, but is altered, optionally reduced, by the process conditions to which the enzyme in question is exposed. Thus, residual xylanase activity refers to the amount, e.g., percentage amount, of the initial activity remaining after exposure to specific process conditions.

[0093] The term "functional fragment" means a fragment or portion of a current GH11 xylanase polypeptide that retains the same or substantially the same enzymatic function or effect as the entire GH11 xylanase polypeptide.

[0094] In the context of enzyme or xylanase stability, the term "stability" describes the property of an enzyme to tolerate and / or function under process conditions that are challenging for the activity and function of the enzyme in question, such process conditions being, for example, high temperature, pH or radiation, a certain concentration of inorganic salts or organic solvents, or a specific reaction mixture composition comprising, for example, proteases, stabilizers, builders, surfactants, etc. The term "stability" reflects the stability of a xylanase according to the present disclosure over time, e.g., how much activity is retained when the xylanase is exposed to process conditions that are challenging for the activity and function of the enzyme in question.

[0095] As used herein, Xyn11A means a Thermopolyspora porgyii xylanase from glycoside hydrolase family 11 (GH11), also known as AM35. As used herein, AM35 means a wild-type mature GH11 xylanase, which is the parent polypeptide for the AM24 polypeptide. The amino acids of the mature AM35 protein correspond to the amino acid sequence of SEQ ID NO: 3.

[0096] As used herein, AM24 (also referred to herein as AM24 protein) is a truncated form of xylanase AM35. The AM24 protein comprises a catalytic module (core) and lacks a carbohydrate binding module (CBM) and a portion of the joint region between the core and the CBM. The amino acids of the mature polypeptide of AM24 correspond to the amino acids of the sequence SEQ ID NO:1, comprising amino acids D1-L220. The "core polypeptide" of AM24 comprises amino acids D1-G191 of SEQ ID NO:1, while the "core polypeptide" of AM24 comprises amino acids T3-S180 of SEQ ID NO:1.

[0097] As used herein, C31-4 refers to a variant of AM24 comprising substitutions T3C, A23S, S28I, T30C. The amino acids of the mature polypeptide of C31-4 correspond to the amino acids of the sequence SEQ ID NO:2 comprising amino acids D1-L220. The "core polypeptide" of C31-4 comprises amino acids D1-G191 of SEQ ID NO:2, and the "core polypeptide" of C31-4 comprises amino acids C3-S180 of SEQ ID NO:2.

[0098] As used herein, the term "carbohydrate binding domain" or "CBM" refers to a specific domain or module in a polypeptide that is capable of facilitating the binding of the polypeptide to carbohydrates.

[0099] As used herein, the term "mature polypeptide" means any polypeptide wherein at least one signal sequence or signal peptide or signal peptide and putative propeptide are cleaved. For example, the "mature polypeptide" of AM24 comprises amino acids D1-L220 of SEQ ID NO: 1, while the "mature core polypeptide" of AM24 comprises amino acids D1-G191 of SEQ ID NO: 1.

[0100] As used herein, "peptide" and "polypeptide" are amino acid sequences comprising a plurality of consecutive polymerized amino acid residues. For the purposes of this disclosure, a peptide is a molecule comprising up to 20 amino acid residues, and a polypeptide comprises more than 20 amino acid residues. A peptide or polypeptide may comprise modified amino acid residues, naturally occurring amino acid residues not encoded by codons, 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, a protein, an antibody, a membrane protein, a peptide hormone, a regulatory factor, or any other protein.

[0101] As used herein, the term "variant" means a sequence or sequence fragment (nucleotide or amino acid) that is inserted, substituted or deleted by one or more nucleotides / amino acids, or a sequence or sequence fragment (nucleotide or amino acid) that is chemically modified. In some embodiments, the term variant may also include variant xylanase polypeptides, fusion proteins comprising variant xylanase polypeptides, or recombinant xylanases.

[0102] The terms "xylanase variant" and "variant xylanase" and "variant of xylanase" mean any xylanase molecule obtained by site-directed or random mutagenesis, insertion, substitution, deletion, recombination and / or any other protein engineering method, which produces a xylanase whose amino acid sequence is different from that of a parent xylanase, which is a wild-type xylanase or a xylanase variant itself. According to the present disclosure, the terms "wild-type xylanase", "wild-type enzyme", "wild type" or "wt" describe a xylanase having an amino acid sequence found in nature or a fragment thereof. The gene encoding the variant can be synthesized or the parent gene can be modified using genetic methods, for example by site-directed mutagenesis, a technique that introduces one or more mutations at one or more defined sites in a polynucleotide encoding a parent polypeptide.

[0103] As used herein, the term "disulfide bridge" refers to a bond formed between sulfur atoms of cysteine ​​residues in a polypeptide or protein. The disulfide bridge may be naturally occurring or non-naturally occurring, and may be introduced, for example, by amino acid substitution.

[0104] The term "catalytic domain" or "catalytic module" or "core" or "catalytic core domain" refers to a domain of an enzyme that may or may not be modified or altered, but which retains at least part of its original activity.

[0105] The term "linker" or "spacer" means a polypeptide comprising at least two amino acids, which can be present between domains of a multidomain protein, such as 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, such as a fusion protein comprising two ribozymes. For example, a fusion protein of a catalytic domain and a CBM is provided by fusing a DNA sequence encoding a catalytic domain, a DNA sequence encoding a linker, and a DNA sequence encoding a CBM in sequence into one open reading frame and expressing this construct.

[0106] As used herein, the term "dry matter" or "dry matter pulp" or "OD pulp" means pulp dried using an oven drying process, wherein the dry matter pulp contains no moisture, ie no water. The dry matter content of oven dried pulp can be determined according to ISO 638:2008.

[0107] As used herein, "amino acid substitution" means the replacement of an amino acid residue at a particular replacement position with an amino acid residue different from the original amino acid. The term "amino acid substitution" may refer to both conservative and non-conservative amino acid substitutions, meaning that the amino acid residue is replaced with an amino acid residue having a similar side chain (conservative) or a different side chain (non-conservative) to the original amino acid residue at that position. The following nomenclature is used to describe substitutions: amino acid residue in a protein scaffold; position; substituted amino acid residue. According to this nomenclature, for example, a single residue of alanine at position 23 is replaced with a serine residue as Ala23Ser or A23S.

[0108] As used herein, the term "comprising" includes the broader meanings "including" and "containing", as well as the narrower expressions "consisting of" and "consisting only of. The words "comprising," "including," and "containing" are each used as open-ended expressions and are not intended to be exclusive.

[0109] In one embodiment, the material derived from the wood material or non-wood material is bleached in a bleaching process. In one embodiment, the wood material or non-wood material is digested in a digester before the bleaching process for obtaining pulp. In one embodiment, the pulp is treated with chemicals to dissolve lignin in the pulp.

[0110] 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 cellulosic pulp, i.e., the pulp comprises cellulose. In one embodiment, the pulp is a kraft pulp cooked in alkali. In one embodiment, the bleaching process comprises a plurality of steps and one or more water washes between the steps, or alternatively, each or some stages comprises a water wash as the final step of the stage.

[0111] 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.

[0112] In one embodiment, the bleaching process comprises providing pulp and delignifying the pulp by carrying out in the following order: a) an enzyme treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase at a pH of at least 9.5 and a temperature of at least 85°C; b) an oxidation step (D) wherein a bleaching agent is added; c) an alkaline extraction step (E) wherein an alkaline agent is added; and d) recovering the bleached pulp. In one embodiment, the GH11 xylanase used in the bleaching process is capable of causing a viscosity reduction of at least 20% in a mixture containing xylan at 90°C and at pH 10.5, compared to the same mixture without any xylanase.

[0113] In one embodiment, the GH11 xylanase has the ability to cause a decrease in viscosity of a xylan-containing mixture by at least 20% when compared to a xylan-containing mixture that does not contain the GH11 xylanase. In one embodiment, the GH11 xylanase has the ability to cause a decrease in viscosity of a xylan-containing mixture by at least 20% when compared to a xylan-containing mixture that does not contain the GH11 xylanase at 90°C, pH 10.5, and at a concentration of 3.0 μg or less of GH11 xylanase protein per gram of xylan-containing mixture that contains 0.07 g / ml or less of xylan. This means that a xylan-containing mixture that contains the GH11 xylanase is compared to an aliquot of a xylan-containing mixture that does not have any GH11 xylanase. In one embodiment, the GH11 xylanase is capable of causing a reduction in 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 at a concentration of 3.0 μg or less of GH11 xylanase protein per gram of the xylan-containing mixture containing 0.07 g / ml or less of xylan, when compared to a xylan-containing mixture to which no GH11 xylanase is added.

[0114] In one embodiment, the GH11 xylanase is a glycoside hydrolase family 11 xylanase.

[0115] In one embodiment the GH11 xylanase is capable of causing a reduction in viscosity of a mixture containing xylan 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.

[0116] In one embodiment the GH11 xylanase is capable of causing a reduction in viscosity of a mixture containing xylan by at least 17%, preferably at least 20%, more preferably at least 25%, more preferably at least 30%, more preferably up to 40%, even more preferably at least 50%, most preferably at least 60% at 85-100°C and at pH 9.5-11.5, compared to the same mixture without any xylanase.

[0117] In one embodiment, the GH11 xylanase has the ability to cause a decrease in the viscosity of a mixture containing xylan 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%, most preferably at least 70% at 85-95°C and at pH 8-11, compared to the same mixture without any xylanase. In one embodiment, the GH11 xylanase has the ability to cause a decrease in the viscosity of a mixture containing xylan by at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, even more preferably at least 70%, most preferably at least 75% at 85-90°C and at pH 8-11, compared to the same mixture without any xylanase.

[0118] In one embodiment, the GH11 xylanase has the ability to cause a decrease in the viscosity of a mixture containing xylan 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%, most preferably at least 60% at 90°C and at pH 10-11, compared to the same mixture without any xylanase. In one embodiment, the GH11 xylanase has the ability to cause a decrease in the viscosity of a mixture containing xylan by at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65%, even more preferably at least 70%, most preferably at least 75% at 90°C and at pH 10-11, compared to the same mixture without any xylanase.

[0119] In one embodiment the GH11 xylanase is capable of causing a reduction in viscosity of a mixture containing xylan 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%, most preferably at least 69% at 90°C and at pH 10.5, compared to the same mixture without any xylanase.

[0120] In one embodiment, the GH11 xylanase is capable of causing at least a 25% reduction in viscosity of a mixture containing xylan at 90°C and at pH 10.5 compared to the same mixture without any xylanase.

[0121] In one embodiment, the GH11 xylanase used in step X is capable of causing a decrease in viscosity of a mixture containing xylan by at least 20% at 85-100°C and at pH 9.5-11.5, compared to the same mixture without any xylanase. In one embodiment, the GH11 xylanase used in step X is capable of causing a decrease in viscosity of a mixture containing xylan 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%, most preferably at least 70% at 85-100°C and at pH 9.5-11.5, compared to the same mixture without any xylanase.

[0122] In one embodiment, the ability of a GH11 xylanase to cause a strong reduction in the viscosity of a mixture containing xylan is an indicator of the xylan depolymerization activity of the enzyme. Therefore, a strong reduction in the viscosity of a mixture containing xylan indicates that the GH11 xylanase used has a high xylan depolymerization activity. In one embodiment, the more strongly the viscosity of a mixture containing xylan is reduced in the presence of a GH11 xylanase, the higher the xylan depolymerization activity of the GH11 xylanase. In one embodiment, the xylan depolymerization activity of a GH11 xylanase is an indicator of the thermostability and pH stability of the enzyme. In one embodiment, the xylan depolymerization activity of a GH11 xylanase at 90°C and at pH 10.5 is an indicator of the thermostability and pH stability of the enzyme. The high xylan depolymerizing activity indicates that the GH11 xylanase in question is suitable for pulp bleaching accompanied by high pH and temperature, such as pH 9-12 and temperature of 85-100°C, preferably pH 9.5-11.5 and temperature of 85-100°C.

[0123] In one embodiment, the xylan depolymerizing activity of the GH11 xylanase is tested in a xylan-containing mixture other than wood pulp.

[0124] In one embodiment, the mixture containing xylan comprises xylan and an aqueous solution, an aqueous suspension, water or an aqueous mixture of a liquid and a solid. In one embodiment, the mixture containing xylan comprises: no more than 0.07g of xylan per ml of the mixture, no more than 3.0μg of GH11 xylanase protein per gram of the mixture; and wherein the reduction 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 mixture containing xylan comprises 0.07g / ml or less xylan. In one embodiment, the mixture containing xylan comprises: 0.07g / ml or less xylan and 3.0μg or less GH11 xylanase protein per gram of the mixture. In one embodiment, the mixture containing xylan comprises 0.07g or less of beechwood xylan in an aqueous pH buffer per ml, and the aqueous pH buffer optionally contains glycine and sodium hydroxide or KH2PO4-K2HPO4 buffer.

[0125] In one embodiment, the reduction in viscosity of the mixture containing xylan occurs within 20 minutes, preferably within 10 minutes, more preferably within 5 minutes and most preferably within 2 minutes.

[0126] In one embodiment, the mixture containing xylan comprises ≤0.07 g / ml, ≤0.06 g / ml, ≤0.05 g / ml, ≤0.04 g / ml, ≤0.03 g / ml, ≤0.02 g / ml, ≤0.01 g / ml or less xylan. In one embodiment, the concentration of the GH11 xylanase in the mixture containing xylan is ≤3.0, ≤2.9, ≤2.5, ≤2.0, ≤1.9, ≤1.5, ≤1.0, ≤0.5, ≤0.3 or ≤0.1 μg xylanase protein per gram of the mixture. In one embodiment, the viscosity of the mixture containing xylan is reduced with the GH11 xylanase in 20 minutes, 15 minutes, 10 minutes, 5 minutes, 2 minutes or less.

[0127] In one embodiment, step X comprises contacting the pulp with a GH11 xylanase at a pH of 9.5-11.5 and a temperature of 85-100°C.

[0128] In one embodiment, step X comprises contacting the pulp with a GH11 xylanase at a pH of at least 9.5 and a temperature of at least 85° C. In one embodiment, step X comprises a pH of 9.5-11.5 and a temperature of 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.

[0129] In one embodiment, the GH11 xylanase is added to the pulp at a pH selected from ≥9.5, ≥10, ≥10.5, ≥11 and 11.5. In one embodiment, the GH11 xylanase is added to the pulp at a temperature selected from ≥85°C, ≥90°C, ≥95°C and 100°C.

[0130] In one embodiment, reducing the alkaline pH of the pulp and / or reducing the process temperature before adding the GH11 xylanase is omitted. In one embodiment, before adding the GH11 xylanase, no acid and / or acid washing step is used to reduce the alkaline pH of the pulp, and / or no water is used to reduce the temperature of the pulp because the GH11 xylanase tolerates the high delignification pH and temperature of the bleaching process. In one embodiment, since it is not necessary to reduce the process temperature with excess water before adding the GH11 xylanase, the water consumption of the bleaching process is reduced. In one embodiment, since it is not necessary to reduce the process pH with acid before adding the GH11 xylanase, the acid consumption of the bleaching process and the wear of the bleaching process hardware are reduced.

[0131] In one embodiment, the GH11 xylanase is capable of causing a reduction in viscosity of a mixture containing xylan by at least 20% at 90°C and at pH 10.5, compared to the same mixture without any xylanase, wherein the mixture containing xylan comprises: ≤ 0.07 g / ml xylan and ≤ 3.0 μg GH11 xylanase protein per gram of said mixture; and wherein the reduction in viscosity occurs within 20 minutes.

[0132] In one embodiment, the GH11 xylanase is capable of causing a decrease in the viscosity of the 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%, most preferably at least 65% within 20 minutes at a pH of 10.5 and at a temperature of 90°C when compared to a xylan-containing mixture without any xylanase, wherein the GH11 xylanase protein concentration is ≤3.0 μg / g of the xylan-containing mixture.

[0133] In one embodiment, the GH11 xylanase is capable of causing the viscosity of the mixture containing xylan to decrease by at least 10% within 20 minutes at a pH of 11 and at a temperature of 90°C when compared to a mixture containing xylan without any xylanase, wherein the GH11 xylanase concentration is 1.9 μg xylanase protein per g of the mixture containing xylan. In one embodiment, the GH11 xylanase is capable of causing the viscosity of the mixture containing xylan to decrease by at least 53% within 20 minutes at a pH of 11 and at a temperature of 90°C when compared to a mixture containing xylan without any xylanase, wherein the GH11 xylanase concentration is 1.9 μg xylanase protein per g of the mixture containing xylan. In one embodiment, the GH11 xylanase is capable of causing the viscosity of the mixture containing xylan to decrease by at least 17% within 20 minutes at a pH of 10 and a temperature of 90°C when compared to a mixture containing xylan without any xylanase, wherein the GH11 xylanase concentration is 2.9 μg xylanase protein per g of the mixture containing xylan. In one embodiment, the GH11 xylanase is capable of causing the viscosity of the mixture containing xylan to decrease by at least 58% within 20 minutes at a pH of 10 and a temperature of 90°C when compared to a mixture containing xylan without any xylanase, wherein the GH11 xylanase concentration is 1.9 μg xylanase protein per g of the mixture containing xylan.

[0134] In one embodiment the GH11 xylanase has a residual xylanase activity of at least 25%, preferably at least 50% and more preferably at least 75% after 10 minutes of step X, preferably after 20 minutes of step X, more preferably after 30 minutes of step X. In one embodiment the GH11 xylanase has a residual xylanase activity of at least 25%, preferably at least 50%, more preferably at least 75% after 20 minutes of step X, wherein step X comprises a pH of 9.5-11.5 and a temperature of 85-100°C.

[0135] In one embodiment the GH11 xylanase has at least 25% residual xylanase activity after 30 minutes of step X, preferably after 40 minutes of step X, more preferably after 50 minutes of step X, wherein step X comprises a pH of 9.5-11.5 and a temperature of 85-100°C.

[0136] In one embodiment, the xylanase retains at least 25% of its xylanase activity after 20 minutes at 85°C and at pH 9.5.

[0137] In one embodiment, the xylanase retains at least 25% of its xylanase activity after 20 minutes at 100°C and at pH 11.5.

[0138] In one embodiment, the GH11 xylanase has a residual xylanase activity of at least 75%, preferably at least 80%, more preferably at least 85% after at least 30 minutes at a pH of 10.9 and at a temperature of 85°C. In one embodiment, the GH11 xylanase in hardwood 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 a pH of 10.9 and at a temperature of 85°C.

[0139] In one embodiment, the xylanase has a residual xylanase activity of at least 75%, preferably at least 80%, more preferably at least 85% at a pH of 10.5 and a temperature of 85°C after at least 30 minutes. In one embodiment, the GH11 xylanase in softwood pulp bleaching process water has a residual xylanase activity of at least 75%, preferably at least 80%, more preferably at least 85% at a pH of 10.5 and a temperature of 85°C after at least 30 minutes.

[0140] In one embodiment, the bleaching process comprises: providing pulp; delignifying the pulp by carrying out in the following order: (a) an enzyme treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase at a pH of 9.5-11.5 and a temperature of 85-100°C, wherein the xylanase has a residual xylanase activity of at least 25% after 20 minutes of step X; (b) an oxidation step (D) wherein a bleaching agent is added; (c) an alkaline extraction step (E) wherein an alkaline agent is added; and (d) recovering the delignified pulp.

[0141] In one embodiment, the pulp is provided from the digester to delignification of a bleaching process.

[0142] In one embodiment at least 0.01 g, preferably at least 0.1 g, more preferably at least 0.2 g, most preferably at least 0.5 g GH11 xylanase protein is added in step X per 1000 kg of dry matter pulp.

[0143] In one embodiment, the GH11 xylanase is added to 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 protein per 1000 kg dry matter pulp. In one embodiment, the concentration of the GH11 xylanase depends on the type of pulp used.

[0144] In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a glycoside hydrolase family GH11 xylanase and at least one additional enzyme or a combination thereof, wherein the additional enzyme is selected from the group consisting of: proteases, amylases, cellulases, β-glucosidases, lipases, xylanases, mannanases, cutinases, esterases, α-glucuronidases, phytases, nucleases, pectinases, pectin hydrolases, pectin lyases, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanases, polysaccharide monooxygenases, laccases, peroxidases and oxidases, with or without a mediator.

[0145] In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a GH11 xylanase and a cellulase. In one embodiment, the enzyme treatment step X comprises contacting the pulp with a GH11 xylanase and a mannanase. In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a GH11 xylanase and a peroxidase. In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a GH11 xylanase and a lignin peroxidase (EC 1.11.1.14). In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a GH11 xylanase and a manganese peroxidase (EC1.11.1.13). In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a GH11 xylanase and a multifunctional peroxidase (EC 1.11.1.16). In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a GH11 xylanase and a dye decolorizing peroxidase (EC 1.11.1.19). In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a GH11 xylanase and a heme peroxidase. In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a GH11 xylanase and a copper-based laccase (EC 1.10.3.2).

[0146] In one embodiment, the bleaching process comprises contacting the pulp with a surfactant, a surfactant, an antifoam, a defoamer, an emulsifier, a dispersant and / or a detergent. In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with a GH11 xylanase and a surfactant, a surfactant, an antifoam, a defoamer, an emulsifier, a dispersant and / or a detergent.

[0147] In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with an enzyme composition comprising one or more enzyme activities, which may be isolated and purified from one or more microbial species. In one embodiment, the enzymes of the enzyme composition may be derived from different species, preferably fungal or bacterial species, or they are present in the fermentation product of one microorganism acting as a host cell for enzyme production, for example in the production of GH11 xylanase, when the microorganism simultaneously produces other enzymes in addition to GH11 xylanase.

[0148] In one embodiment, the enzyme treatment step (X) comprises contacting the pulp with an enzyme composition comprising a GH11 xylanase protein.

[0149] In one embodiment, the pulp is an aqueous mixture comprising at least 0.5-35 wt.% pulp fibers, wherein the pulp fibers are wood fibers and / or non-wood fibers. In one embodiment, the pulp fibers contain xylan, and thus the pulp is a xylan-containing pulp.

[0150] In one embodiment, delignification is performed in the presence of a GH11 xylanase, which has high pH and thermostability.

[0151] In one embodiment, the contacting of the pulp with the GH11 xylanase in step X means that the GH11 xylanase is contacted with xylan contained in the pulp.

[0152] In one embodiment, the delignification further comprises an oxygen delignification step (O) prior to the oxidation step D, wherein oxygen is added in step O; and wherein step X precedes step O or follows step O.

[0153] In one embodiment, oxygen is added as O2 gas in the oxygen delignification step, wherein said step is referred to as the O2 step.

[0154] 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 2 minutes-3 hours, 5 minutes-2 hours, 15-60 minutes or 20-30 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.

[0155] 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 be changed during step O.

[0156] In one embodiment, the pH of the pulp during step O is 10-12 and / or the temperature is 75-130°C.

[0157] In one embodiment, the xylanase is added to the pulp before step O as its own enzyme treatment step X. In one embodiment, the xylanase is added to the pulp before step O and the xylanase retains at least 25% of the residual xylanase activity during at least 20 minutes of enzyme treatment step X, said enzyme treatment step X comprising a pH of 9.5-11.5 and a temperature of 85-100° C. Step O releases a high amount of lignin from the pulp. Therefore, in embodiments wherein the bleaching process comprises step X before step O, washing the pulp between step X and step O is not necessary. In one embodiment wherein the GH11 xylanase is added to the pulp before step O, the GH11 xylanase in the pulp retains some of its activity in step O.

[0158] In one embodiment, the xylanase is added to the pulp after step O and before step D as its own enzyme treatment step X. In one embodiment, the xylanase is added to the pulp after step O and the xylanase retains at least 25% of the residual xylanase activity during at least 20 minutes of enzyme treatment step X, said enzyme treatment step X comprising a pH of 9.5-11.5 and a temperature of 85-100°C. In one embodiment, the GH11 xylanase may be added at any stage before step D, as long as step X is performed in the presence of a GH11 xylanase having a residual xylanase activity of at least 25% 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 is active at any pH between 5.6-11.5 and / or at any temperature between 55-100°C.

[0159] In one embodiment, adding the GH11 xylanase to the pulp before or after step O and before step D allows step X to be performed in a pre-existing storage tank or storage tower of the pulp processing facility without the need to build a separate treatment vessel / tank when integrating the enzyme treatment step X into a bleaching process for pulp that did not previously contain an enzyme treatment step X.

[0160] In one embodiment, each step of the delignification of the bleaching process occurs in separate tanks, such as Figure 1 As shown. 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) can be carried out in separate storage tanks S1 and / or S2 before step O and / or after step O, respectively. In one embodiment, carrying out step X in separate storage tanks S1 and / or S2 allows for sufficient retention time for the enzyme treatment of the pulp. However, depending on the process, the presence of storage tanks S1 and / or S2 may not be necessary, because the enzyme treatment step X can be carried out in another process tank before the bleaching process sequence tank D, in which the oxidation step D is carried out.

[0161] In one embodiment, the oxidation step D is carried out in tank D ( Figure 1 ). In one embodiment, wherein the bleaching process does not comprise step O, 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, wherein the bleaching process comprises step O, 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 tanks O and D to remove dissolved lignin. In one embodiment, wherein the bleaching process comprises a process sequence comprising steps O, X, D, and a water washing step of the pulp is performed after step X, at least a portion of the washing water comprising the GH11 xylanase is recycled back to step O. In one embodiment, wherein the bleaching process comprises a process sequence comprising steps O, X, D, and a water washing step of the pulp is performed between steps O and X, washing the pulp between steps X and D is optional. However, in this case, if a water washing step of the pulp is performed after step X, at least a portion of the washing water comprising the GH11 xylanase is recycled back to step O. In one embodiment, if no washing step is performed between steps X and D, the GH11 xylanase in the pulp retains some of its activity in step D.

[0162] In one embodiment, the bleaching agent in the oxidation step D comprises an oxygen-containing oxidizing agent, preferably the bleaching agent is selected from ClO2, O3, H2O2 and peroxides. Examples of non-oxygen-containing oxidizing agents are Cl2 and other halogens. In one embodiment, the bleaching agent is a compound containing elemental 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 referred to as step D. o In one embodiment, the pulp is directed into tank D for step D and the bleaching agent is added into tank D.

[0163] In one embodiment, both GH11 xylanase and bleaching agent participate in the bleaching of the pulp during the bleaching process, and thus the amount of bleaching agent required in the bleaching process is reduced when compared to a bleaching process without any xylanase. In one embodiment, in step D the pulp comprises GH11 xylanase with residual activity, and both GH11 xylanase and bleaching agent participate in the bleaching of the pulp. In one embodiment, in step D the pulp comprises GH11 xylanase with residual activity, and the bleaching agent in step D is preferably H2O2. In another embodiment, in step D the pulp no longer comprises GH11 xylanase, because the enzyme is removed in the washing step prior to step D. Also in such embodiments, the amount of bleaching agent required in the oxidation step is reduced when compared to a bleaching process without any xylanase, because the pulp entering step D has already been bleached to a certain extent in the enzyme treatment step X. In one embodiment, to achieve a specific pulp brightness, at least 5%, preferably at least 7.5%, more preferably at least 10% less bleaching agent is used compared to a corresponding process without a GH11 xylanase. In one embodiment, the reduction in the amount of bleaching agent results in a reduction in the amount of adsorbable organic halides (AOX) in the effluent of the bleaching process and reduces the need for subsequent effluent treatment.

[0164] In one embodiment, the GH11 xylanase used in the bleaching process is highly tolerant to high pH and temperature. In one embodiment, the use of a temperature and pH tolerant xylanase in a bleaching process results in higher pulp brightness when compared to a bleaching process without the xylanase, or when compared to a process comprising a xylanase with less temperature and pH tolerance. On the other hand, the use of a temperature and pH tolerant xylanase in a bleaching process reduces the need for bleaching agents when a specific pulp brightness is desired.

[0165] In one embodiment, the bleaching agent used is H2O2, a metal chelation step (Q) for removing metals is performed before the oxidation step D, and the GH11 xylanase 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 for removing redox-active metal ions from the pulp. In one embodiment, the duration of step Q is the same as the duration of step X.

[0166] In one embodiment, step Q occurs in a separate metal chelating tank Q before step D in the bleaching process sequence ( Figure 1 ). The purpose of step Q is to remove metals that reduce the bleaching activity of H2O2 from the pulp, such metals are, for example, copper, manganese and iron. In one embodiment, the enzyme treatment step X of the pulp is carried out in the same tank Q as the metal chelation step. In one embodiment, the enzyme treatment step X of the pulp is carried out in the same tank Q as the metal chelation step before the metal chelation step, after the metal chelation step or during the metal chelation step. Alternatively, step X is carried out in another tank before step Q and / or after step Q. In one embodiment, step Q occurs after oxygen delignification of the pulp in tank O. In one embodiment, the pulp is directed from the metal chelation in tank Q directly or via other tanks to the oxidation step in tank D. In one embodiment, the pulp is directed from tank Q to a storage tank (not shown), wherein the enzyme treatment step X is carried out, after which the pulp is directed to the oxidation step in tank D. Preferably, the pulp is washed between steps Q and D to remove dissolved metals from the pulp before the oxidation step.

[0167] In one embodiment, the GH11 xylanase may be added to the pulp before step Q, during step Q or after step Q. In one embodiment, wherein the xylanase is added to the pulp before step Q or during step Q and the process comprises a water washing step of the pulp in tank D between step Q and the oxidation step. In this case, at least a portion of the wash water comprising the GH11 xylanase having residual activity may be recycled back to the metal chelation in tank Q, preferably at least a portion of the wash water is recycled back to the pulp washer upstream of tank Q. In one embodiment, wherein the pulp is washed with water between steps Q and D, the GH11 xylanase may be added to the pulp in tank D or to the pulp in a separate storage tank (not shown) after the washing step and before the addition of the bleaching agent H2O2. In such embodiments, the GH11 xylanase in the pulp may retain some of its activity in step D.

[0168] 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 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 oxidized pulp.

[0169] In one embodiment, after step D, the pulp is directed to an alkaline extraction step E in tank E ( Figure 1 ). In one embodiment, the bleaching process further comprises a water washing step of the pulp between the oxidation step in tank D and the alkaline extraction step in tank E. In such embodiments, the wash water comprising the GH11 xylanase is optionally recycled back to the water washing step before step D. In one embodiment, wherein the bleaching agent in step D is H2O2, the xylanase in the wash water recycled back to the water washing step still has residual activity and is able to contribute to the xylan degradation in tank D.

[0170] In one embodiment, an alkaline agent is added to the alkaline extraction step E in tank E. In one embodiment, the alkaline agent of the 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.

[0171] In one embodiment, the reduction in the amount of ClO2 required as a bleaching agent results in a reduction in the amount of NaOH required as an alkaline agent in step E. In one embodiment, the reduction in the amount of NaOH as an alkaline agent results in a reduction in the chemical oxygen demand (COD) measurement of the bleaching process effluent.

[0172] In one embodiment, the activity of the GH11 xylanase in delignification process water is higher than in normal tap water when process conditions including temperature and pH are the same. In one embodiment, the process water of the delignification stage induces enzyme stability.

[0173] 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, the pH of the pulp is alkaline at the beginning of the alkaline extraction step, and is less alkaline at the end of the alkaline extraction step.

[0174] In one embodiment a final brightness of the pulp can be achieved with the present bleaching process which is at least 1 unit, preferably at least 2 units, more preferably at least 2.5 units higher than a corresponding process without the GH11 xylanase.

[0175] In one embodiment, the delignification of the bleaching process is followed by a brightening part of the delignified pulp, which may also include several process steps. The brightening part may include at least one or more brightening steps (Db, P) in which a bleaching agent is added, and / or one or more brightening alkaline extraction steps (Eb) in which an alkaline agent is added. In one embodiment, the main purpose of the brightening part is not to remove the remaining lignin from the pulp, but to decolorize the remaining lignin, thereby obtaining a higher final brightness.

[0176] In one embodiment, the glycoside hydrolase family GH11 xylanase used in the bleaching process according to the present invention can be of various origins. In one embodiment, the xylanase used in the bleaching process according to the present invention is a wild-type xylanase. In another embodiment, the xylanase used in the bleaching process according to the present invention is a variant of a modified xylanase and / or a wild-type xylanase.

[0177] In one embodiment, the GH11 xylanase is derived from a thermophilic bacterium. In one embodiment, the GH11 xylanase is derived from a bacterium, preferably from Actinobacteria, more preferably from Streptosporangiales, more preferably from Streptosporangiaceae, even more preferably from Thermopolyspora species, most preferably from Boqu Thermopolyspora. In one embodiment, the xylanase is derived from the terrabacteria group. In one embodiment, the xylanase is derived from Actinobacteria. In one embodiment, the xylanase is derived from Actinomycetes. In one embodiment, the xylanase is derived from Streptosporangiales. In one embodiment, the xylanase is derived from Streptosporangiaceae. In one embodiment, the xylanase is derived from Thermopolyspora.

[0178] In one embodiment, the GH11 xylanase is derived from Nonomuraea flexuosa. Nonomuria flexuosa is a synonym for Nonomuraea flexuosa, Nonomuria flexuosa, Nocardia flexuosa, Microtetraspora flexuosa, Acetomadura flexuosa and / or Actinomadura flexuosa.

[0179] In one embodiment, the glycoside hydrolase family GH11 xylanase for bleaching process comprises a catalytic core domain and a carbohydrate binding module (CBM) connected together by a joint. In one embodiment, the glycoside hydrolase family GH11 xylanase for bleaching process is a truncated enzyme, which comprises a catalytic core domain and a joint domain, or a part of a catalytic core domain and a joint domain, without the presence of a CBM. In another embodiment, the glycoside hydrolase family GH11 xylanase for bleaching process only comprises a catalytic core domain, without a joint domain and a CBM. In one embodiment, the GH11 xylanase for bleaching process comprises a mature catalytic core domain.

[0180] In one embodiment, the GH11 xylanase is a polypeptide, wherein the polypeptide or a 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 has a molecular mass of 19-40 kDa, preferably 20-30 kDa, more preferably 23-25 ​​kDa, and most preferably 24 kDa, which is calculated based on its unglycosylated amino acid sequence. In one embodiment, the GH11 xylanase has a β-jelly roll structure. In one embodiment, the GH11 xylanase has a β-jelly roll structure, xylanase activity, and an amino acid sequence having amino acids corresponding to amino acids 3-180 of SEQ ID NO: 2.

[0181] In one embodiment, the GH11 xylanase is a polypeptide, wherein the polypeptide or a functional fragment of the polypeptide has a molecular mass of 31 kDa. In one embodiment, the GH11 xylanase is a polypeptide, wherein the polypeptide or a functional fragment of the polypeptide has a molecular mass of 27 kDa. In one embodiment, the GH11 xylanase is a polypeptide, wherein the polypeptide or a functional fragment of the polypeptide has a molecular mass of 24 kDa. The molecular mass of the GH11 xylanase polypeptide or the functional fragment of the polypeptide can be determined by SDS-PAGE. In one embodiment, the molecular mass of the GH11 xylanase polypeptide or the functional fragment of the polypeptide is calculated based on its unglycosylated amino acid sequence. In one embodiment, the GH11 xylanase polypeptide or the functional fragment of the polypeptide has a molecular mass of 24 kDa, and the molecular mass is calculated based on its unglycosylated amino acid sequence. In one embodiment, the GH11 xylanase lacks xylosidase and cellulase activity.

[0182] In one embodiment, the GH11 xylanase is a variant polypeptide. In one embodiment, the GH11 xylanase is a wild-type polypeptide.

[0183] In one embodiment, the GH11 xylanase has at least one or at least two disulfide bridges between two cysteine ​​residues in its amino acid sequence. In one embodiment, at least one disulfide bridge stabilizes the GH11 xylanase, thereby increasing the pH stability and thermostability of the GH11 xylanase.

[0184] In one embodiment, the GH11 xylanase is a polypeptide comprising residues 3C and 30C, the positions of these residues corresponding 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 is a polypeptide comprising at least one disulfide bridge between two cysteine ​​residues in the 1-191 amino acid region of the polypeptide, at least one of the two cysteine ​​residues being at a position other than the positions corresponding to positions 3 and 30 of SEQ ID NO:2. In one embodiment, the GH11 xylanase is a polypeptide comprising at least one disulfide bridge between cysteine ​​residues in the 1-30 amino acid region of the polypeptide, the positions of the amino acid region corresponding to the positions of SEQ ID NO:2.

[0185] In one embodiment, the GH11 xylanase protein has xylanase activity and has an amino acid sequence corresponding to amino acids 3-180 of SEQ ID NO: 2, or a functional fragment thereof.

[0186] In one embodiment, the GH11 xylanase protein has xylanase activity, has an amino acid sequence corresponding to amino acids 3-180 of SEQ ID NO: 2, or a functional fragment thereof, and is capable of causing a viscosity reduction of at least 20% in a xylan-containing mixture comprising 0.07 g / ml or less of xylan within 20 minutes at 90° C., pH 10.5, and at a concentration of 3.0 μg or less of GH11 xylanase protein per gram of the xylan-containing mixture comprising 0.07 g / ml or less of xylan, when compared to a xylan-containing mixture without any GH11 xylanase. In such embodiments, the xylan-containing mixture comprises xylan and an aqueous solution or water.

[0187] In one embodiment, the GH11 xylanase 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 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; the GH11 xylanase has at least one disulfide bridge between two cysteine ​​residues of the polypeptide. In one embodiment, the GH11 xylanase 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, the GH11 xylanase has at least one disulfide bridge between two cysteine ​​residues of the polypeptide. In one embodiment, at least one disulfide bridge stabilizes the GH11 xylanase, thereby improving the pH stability and thermostability of the GH11 xylanase, compared to a GH11 xylanase without a disulfide bridge. In one embodiment, the GH11 xylanase is a variant polypeptide comprising an amino acid sequence having amino acids corresponding to amino acids 1-191 of SEQ ID NO: 1; the GH11 xylanase has at least one disulfide bridge between two cysteine ​​residues of the polypeptide, and one or two amino acid substitutions at one / more positions corresponding to positions 23 and / or 28 of SEQ ID NO: 1.

[0188] In one embodiment, the GH11 xylanase 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.

[0189] In one embodiment, the GH11 xylanase is a polypeptide comprising an amino acid sequence having a sequence identity of 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% to amino acids 1-191 of SEQ ID NO: 1.

[0190] In one embodiment, the GH11 xylanase is a polypeptide comprising an amino acid sequence having an amino acid sequence identity of at least 79%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% to amino acids 3-180 of SEQ ID NO: 2, or a functional fragment thereof. In one embodiment, the GH11 xylanase is a polypeptide comprising an amino acid sequence having an amino acid sequence identity of at least 79%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% to amino acids 1-191 of SEQ ID NO: 2, or a functional fragment thereof.

[0191] In one embodiment, the GH11 xylanase is a polypeptide comprising an amino acid sequence having a sequence identity of 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% to amino acids 3-180 of SEQ ID NO:2.

[0192] In one embodiment, the GH11 xylanase 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 comprises the inner core polypeptide of C31-4. In one embodiment, the GH11 xylanase comprises the inner core polypeptide of C31-4. In one embodiment, the GH11 xylanase comprises the mature polypeptide of C31-4. In one embodiment, the GH11 xylanase is a variant polypeptide having the amino acid sequence of SEQ ID NO:2.

[0193] In one embodiment, the GH11 xylanase is a polypeptide comprising an amino acid sequence having an amino acid sequence identity of at least 79%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% to 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 is a polypeptide comprising an amino acid sequence having an amino acid sequence identity of at least 79%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% to 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.

[0194] In one embodiment, the GH11 xylanase 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.

[0195] In one embodiment, the GH11 xylanase 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, and wherein the amino acid sequence has at least one disulfide bridge between two cysteine ​​residues of the polypeptide and has an amino acid substitution at position 23 or 28 or at positions 23 and 28, the positions corresponding to positions 23 and 28 of SEQ ID NO: 1.

[0196] In one embodiment, the GH11 xylanase 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, the amino acid positions corresponding to the positions of SEQ ID NO: 1. In one embodiment, the GH11 xylanase 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, the amino acid positions corresponding to the positions of SEQ ID NO: 1. In one embodiment, the GH11 xylanase 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, the amino acid positions corresponding to the positions of SEQ ID NO: 1.

[0197] In one embodiment, a pulp composition is provided, comprising: 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 protein per 1000 kg of dry matter pulp; wherein the GH11 xylanase protein has xylanase activity. In one embodiment, the GH11 xylanase that is part of a pulp composition is capable of causing a reduction in viscosity of a mixture containing xylan by at least 20%, preferably at least 25%, more preferably at least 30%, even more preferably at least 40%, most preferably at least 50% at 90°C and pH 10.5, compared to the same mixture without any xylanase, wherein the mixture containing xylan comprises 0.07 g / ml or less xylan and 3.0 μg or less GH11 xylanase protein per gram of said mixture; and wherein the reduction in viscosity occurs within 20 minutes, preferably within 10 minutes, more preferably within 5 minutes and most preferably within 2 minutes.

[0198] In one embodiment, the pulp composition has a pH of 9.5-11.5 and / or a temperature of 85-100° 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.

[0199] In one embodiment, the pulp composition comprises a GH11 xylanase protein.In one embodiment, the pulp composition comprises a GH11 xylanase protein having an amino acid sequence having amino acids corresponding to amino acids 3-180 of SEQ ID NO:2.

[0200] In one embodiment, the GH11 xylanase protein included in the pulp composition is a polypeptide comprising an amino acid sequence having an amino acid sequence identity to amino acids 3-180 of SEQ ID NO: 2 of at least 79%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably 98%, or a functional fragment thereof.

[0201] 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.

[0202] In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and at least one additional enzyme or a combination thereof, wherein the additional enzyme is selected from the group consisting of: proteases, amylases, cellulases, β-glucosidases, lipases, xylanases, mannanases, cutinases, esterases, α-glucuronidases, phytases, nucleases, pectinases, pectin hydrolases, pectin lyases, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanases, polysaccharide monooxygenases, laccases, peroxidases and oxidases, with or without a mediator.

[0203] In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and a cellulase. In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and a mannanase. In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and a laccase. In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and a peroxidase. In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and a lignin peroxidase (EC 1.11.1.14). In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and a manganese peroxidase (EC1.11.1.13). In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and a multifunctional peroxidase (EC 1.11.1.16). In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and a dye decolorizing peroxidase (EC 1.11.1.19). In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and a heme peroxidase. In one embodiment, the pulp composition comprises an enzyme composition comprising a GH11 xylanase and a copper-based laccase (EC 1.10.3.2).

[0204] In one embodiment, the pulp composition further comprises a surfactant, a surfactant, an antifoaming agent, a defoaming agent, an emulsifier, a dispersant, a detergent, or any combination thereof.

[0205] In one embodiment, the pulp composition comprises 0.25-4 wt% of a bleaching agent, based on the total weight of the dry matter pulp, the bleaching agent being selected from the group consisting of ClO2, O3, H2O2 and peroxides.

[0206] Example

[0207] Xylanases and xylanase variants were designed, prepared and tested for xylanase enzyme stability as described in application EP 20217335. All xylanases were expressed in Trichoderma reesei.

[0208] Xylanase activity was measured as described in Bailey, MJ and Poutanen, K. 1989, Appl. Microbiol. Biotechnol. 30:5-10, but instead of 10 minutes at 50°C and pH 5.3, after 5 minutes at 70°C and pH 7, the amount of reducing carbohydrates released from beech xylan was determined spectrophotometrically using dinitrosalicylic acid and compared with a xylose standard solution. One thermoxylanase unit, abbreviated TXU, is defined as the amount of xylanase producing reducing carbohydrates having a reducing power corresponding to obtaining 1 nmol of xylose from beech xylan in one second at pH 7 and 70°C (1 TXU = 1 nkat).

[0209] The protein amount of the purified enzyme samples was measured by absorption at 280 nm. Xylanases AM24 and C31-4 had an unglycosylated molecular mass of approximately 24 kDa calculated from the amino acid composition and an absorption of 2.852 at 280 nm in a 1 cm cuvette and 1 g of pure protein / liter.

[0210] Example 1. Viscosity measurement to select enzymes suitable for pulp bleaching process

[0211] A viscous xylan suspension was prepared by weighing 7 g of beechwood xylan into a 100 ml volumetric flask, adding an aqueous pH buffer, stirring and heating to boil the suspension for about 20 minutes. A buffer containing 7.5 g / l glycine (equivalent to 0.1 M glycine) and sodium hydroxide was used for pH 10.5. A 0.1 M KH2PO4-K2HPO4 buffer was used for pH 7.0.

[0212] After cooling the xylan-containing mixture to 25° C., the volumetric flask was filled to the calibration mark with buffer and stirred overnight. The next day these mixtures were centrifuged and the supernatant was transferred to a beaker and placed in an ultrasonic bath for 15 minutes.

[0213] Then, 9 g of these supernatants were weighed into each 15 ml test tube, which was then sealed with a rubber stopper and placed in a water bath preheated to a temperature 5° C. above the measurement temperature. Then, 1 g of enzyme solution or 1 g of water without enzyme addition was added as a reference to the preheated supernatant, mixed briefly and quickly injected into the viscometer chamber preheated to the measurement temperature. The viscosity was measured over a period of 20 minutes with a falling ball viscometer of the DMA 4100M type from Anton Paar.

[0214] The viscosity of the mixture without added enzyme remained high, whereas the viscosity of the mixture with added specific enzyme decreased rapidly within the first few minutes until reaching a nearly constant viscosity final value within 20 minutes.

[0215] The following table summarizes the final viscosity values ​​reached within 20 minutes at the temperature and pH indicated in the respective table (average of at least duplicate measurements).

[0216] Table 1. High performance differences of enzymes applied under biologically challenging conditions of 90°C and pH 10.5 were measured.

[0217]

[0218]

[0219] The xylanase concentrations of AM24 and C31-4 used for these experiments were approximately 29 μg and 19 μg of xylanase protein per g of applied enzyme solution, respectively. Because 1 g of enzyme solution was mixed with 9 g of xylan supernatant, the mixture injected into the viscometer resulted in a ten-fold dilution of the enzyme concentration; and the xylan concentration was diluted from 7 g / 100 ml to 6.3 g / 100 ml.

[0220] As a control experiment, inactivated C31-4 was prepared by heating the 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 as described above for viscosity measurement. As expected, the viscosity of this mixture did not decrease, indicating that the viscosity decrease was caused by active enzyme.

[0221] Table 2. Almost no performance differences were measured between enzymes AM24 and C31-4 applied at 80°C and pH 7.0.

[0222]

[0223] Example 2. Further viscosity measurements

[0224] The following experiments were performed as described in Example 1. Buffers containing 7.5 g / l glycine (which is equivalent to 0.1 M glycine) and sodium hydroxide were used for pH 10 and 11. 0.1 M Tris buffer containing 12.1 g / l 2-amino-2-(hydroxymethyl)-1,3-propanediol and hydrochloric acid was used for pH 8. 0.1 M NaHCO3-Na2CO3 buffer was used for pH 9. The pH was adjusted to the desired value at about 60°C.

[0225] The following table summarizes the final viscosity values ​​reached within 20 minutes at the temperature and pH indicated in each table.

[0226] Table 3. High performance differences of enzymes applied at 90°C and pH 11 were measured.

[0227]

[0228] Table 4. High performance differences of enzymes applied at 90°C and pH 10 were measured.

[0229]

[0230]

[0231] Table 5. Performance of enzymes applied at 90°C and pH 8 measured.

[0232]

[0233] Table 6. Almost no difference in performance of enzymes applied at 85°C and pH 8 was measured.

[0234]

[0235] Example 3. High temperature and high pH application of xylanase in kraft pulp from eucalyptus

[0236] 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. The 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 a brightness of 38.0%-ISO. A constant ClO2 dosage (0.2X kappa number) was used. The bleaching trials were carried out in bleaching reactors. An anchor-type Kemu reactor (batch size 0.15-1.0 kg, Tmax 90°C, consistency 25%) was used in X and step E, and an air-heated Hellsten reactor (batch size 0.3-0.8 kg, Tmax 85°C, consistency 12%) was used in step D0.

[0237] About 2 g of enzyme protein / ton of pulp was used. Enzyme solutions of AM24 and C31-4 were applied in equal amounts. The enzyme treatment time was 60 minutes.

[0238] After enzyme step X, the enzymes were inactivated by adding hot water and immediately adjusting the pH of the pulp suspension to pH 2.5-3. After step E, brightness and Kappa number were analyzed.

[0239] Table 7. Results of XDE bleaching with eucalyptus pulp. In the reference (REF) sample, no enzyme was added. Enzyme pretreatment was performed at 90°C using a 60 minute reaction time. aCl denotes active chlorine; ΔBr denotes the difference in brightness; and Δκ denotes the difference in κ number.

[0240]

[0241]

[0242] Pretreatment with both xylanases AM24 and C31-4 increased the brightness of eucalyptus pulp at pH 8.3. The pulp pretreated with C31-4 at pH 8.3 had a brightness 2.8 units higher than the reference pulp and 1.0 unit higher than the pulp treated with AM24. The active chlorine (aCl) used per Δκ ​​and per ΔBr at pH 8.3 in the C31-4 treated pulp was 0.2 kg / t lower than in the AM24 treated pulp and 0.07 kg / t lower than in the AM24 treated pulp, and 0.28 kg / t and 0.19 kg / t lower than in the reference pulp, respectively (Table 7).

[0243] When xylanase pretreatment was carried out at pH 10.5 and 90°C, the C31-4 treated pulp had 2.7 units higher brightness than in the reference pulp, and 0.09 kg / t less active chlorine (aCl) used per Δκ ​​and 0.18 kg / t less active chlorine (aCl) used per ΔBr than in the reference pulp (Table 7).

[0244] The measured brightness (%-ISO) increase was significant and showed that the application of xylanase C31-4 in kraft pulp was beneficial and was effective at 90°C and pH 10.5.

[0245] Example 4. High temperature and high pH application of xylanase in kraft pulp from softwood

[0246] The efficiency of xylanase C31-4 in the bleaching of softwood pulp was tested at high temperature and high pH by using a short bleaching sequence XDE (X, enzyme; D, ClO2 (chlorine dioxide); E, extraction with sodium hydroxide) and a constant ClO2 dosage (0.2X kappa number). The bleaching trials were carried out with a full sequence in a bleaching reactor. An anchor-type Kemu reactor (batch size 0.15-1.0 kg, T max 90°C, consistency 25%) was used in step X and step E, and an air-heated Hellsten reactor (batch size 0.3-0.8 kg, T max 85°C, consistency 12%) was used in step D0.

[0247] The pulp used in the test was oxygen delignified SW kraft pulp 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 about 2 g of enzyme protein per ton of pulp. Tables 8-10 describe the conditions used at different stages of bleaching.

[0248] After step X, the pulp was washed three times. The first wash was done with water at reaction temperature (90°C), followed by two washes with cold (4°C) water. After the washing steps, centrifugation (2600 rpm, 10 s) was performed to remove the water and collect about 10-15 g (measured as oven dry) pulp samples for analysis. In addition, dry matter analysis was done from C31-4 treated samples using an infrared dryer in order to better regulate the amount of pulp. Kappa number and brightness were analyzed from the pulp samples.

[0249] The results from the experiments are shown in detail in Table 11.

[0250] Table 8. Conditions in step X. The pH of the pulp was measured at the beginning (target / initial pH) and after enzyme treatment (final pH).

[0251]

[0252] Table 9. Bleaching conditions in step D. The amounts of pulp were measured as oven dry.

[0253]

[0254]

[0255] Table 10. Bleaching conditions in step E.

[0256]

[0257] Table 11. Results from bleaching experiments.

[0258]

[0259] The softwood kraft pulp pretreated with C31-4 at pH 10.5 had a final brightness that was 2.7 units higher than the reference pulp (no enzyme used) corrected for the same 5.4% active chlorine consumption.

[0260] Example 5. Further application of xylanase at high temperature and high pH in kraft pulp from softwood

[0261] Bleaching tests were repeated with AM24 and C31-4 xylanases using conditions and softwood pulp similar to those in Example 4. Prior to characterization and experiments with the pulp, the pulp was washed at pH 11 and 60°C for about 30 minutes. After the washing step, the pulp had a Kappa number of 16.7, a viscosity of 1050 ml / g and a brightness of 32.6%-ISO. The dosage of both enzymes (C31-4 and AM24) was 2 g of enzyme protein per ton of pulp. The conditions used in the xylanase treatment, bleaching and extraction stages are as described in Tables 12-14. In the xylanase treatment stage (X stage), the two enzymes were applied at different pH and temperatures because the AM24 xylanase was assumed to be inactivated under such pulp mill conditions, while based on previously completed experiments, the C31-4 variant was expected to be able to work under these conditions (pH 10.5 / 90°C).

[0262] Table 12. Conditions for step X. The pH of the pulp was measured at the beginning (target / initial pH) and after enzyme treatment (final pH).

[0263]

[0264] Table 13. Bleaching conditions for step D

[0265]

[0266] Table 14. Conditions in step E

[0267]

[0268] The results from the experiments are included in Table 15.

[0269] Table 15. Results after step E

[0270]

[0271] Scandinavian softwood kraft pulp pretreated with C31-4 at pH 10.5 / 90°C had 2.8 units of brightness higher than the reference pulp (no enzyme used) under the same conditions throughout the XDE bleaching process. The results confirm that C31-4 improves brightness under high pH and high temperature conditions. AM24 xylanase showed a 1.2 unit brightness increase under the conditioned conditions of the X stage (pH 7 / 80°C) and the same conditions as used for C31-4 in the D and E stages.

[0272] The foregoing description provides a complete and informative description of the best mode currently considered by the inventors for carrying out the invention by way of non-limiting examples of specific implementations and embodiments. However, it will be clear to those skilled in the art that the invention is not limited to the details of the embodiments presented in the foregoing, but it can be implemented in other embodiments using equivalent means or in different combinations of embodiments without departing from the features of the invention.

[0273] In addition, some features of the exemplary embodiments disclosed above can be used to advantage without the corresponding use of other features. Therefore, any appropriate combination of embodiments and aspects can be formed. Therefore, the foregoing description should be regarded as merely illustrating the principles of the present invention, rather than limiting it. Therefore, the scope of the present invention is limited only by the attached patent claims.

Claims

1. A bleaching process, comprising: Provide pulp; Delignification of pulp is carried out by carrying out the following sequence: a) an enzyme treatment step (X) comprising contacting the pulp with a glycoside hydrolase family GH11 xylanase at a pH of at least 9.5 and a temperature of at least 85°C; b) an oxidation step (D) wherein a bleaching agent is added; c) an alkaline extraction step (E), wherein an alkaline agent is added; and d) recovering bleached pulp; wherein the GH11 xylanase is capable of causing a reduction in viscosity of the xylan-containing mixture by at least 20% at 90°C, pH 10.5 and at a concentration of 3.0 μg or less of GH11 xylanase protein per gram of the xylan-containing mixture when compared to a xylan-containing mixture without any xylanase, wherein the xylan-containing mixture comprises 0.07 g / ml or less of xylan.

2. The process according to claim 1, wherein the GH11 xylanase is capable of causing a decrease in viscosity of a mixture containing xylan 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.

3. The process of claim 1 or 2, wherein the xylan-containing mixture comprises: no more than 0.07 g of xylan per ml of the mixture, and no more than 3.0 μg of GH11 xylanase protein per gram of the mixture; and Wherein the viscosity reduction occurs within 20 minutes, preferably within 10 minutes, more preferably within 5 minutes and most preferably within 2 minutes.

4. A process according to any preceding claim, wherein step X comprises contacting the pulp with a GH11 xylanase at a pH of 9.5-11.5 and a temperature of 85-100°C.

5. A process according to any preceding claim, wherein in step X at least 0.01 g, preferably at least 0.1 g, more preferably at least 0.2 g, most preferably at least 0.5 g GH11 xylanase protein is added per 1000 kg dry matter pulp.

6. A process according to any preceding claim, wherein the delignification further comprises an oxygen delignification step (O) prior to the oxidation step D, wherein oxygen is added in step O; and Wherein step X is before step O or after step O.

7. A process according to any preceding claim, wherein the bleaching agent in oxidation step D comprises an oxygen-containing oxidizing agent, preferably the bleaching agent is selected from ClO2, O3, H2O2 and peroxides.

8. A process according to any preceding claim, wherein the bleaching agent used is H2O2, a metal chelation step (Q) for removing metals is carried out before the oxidation step D, and a GH11 xylanase is added to the pulp before, during or after the chelation step Q.

9. A process according to any preceding claim, wherein the alkaline agent of alkaline extraction step E is selected from NaOH, MgO or a combination thereof.

10. A process according to any preceding claim, wherein the GH11 xylanase is of bacterial origin, preferably from the phylum Actinomycetes, more preferably from the orders Sporangiales, more preferably from the family Neurospora, even more preferably from a species of the genus Thermopolyspora, most preferably from Thermopolyspora boqu 11. A process according to any preceding claim, wherein the GH11 xylanase has a molecular mass calculated from its unglycosylated amino acid sequence of 19-40 kDa, preferably 20-30 kDa, more preferably 23-25 ​​kDa, most preferably 24 kDa.

12. A process according to any preceding claim, wherein the GH11 xylanase 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; GH11 xylanases have at least one disulfide bridge between two cysteine ​​residues in the polypeptide.

13. A process according to any preceding claim, wherein the GH11 xylanase 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 3-180 of SEQ ID NO: 2, or a functional fragment thereof.

14. A process according to any preceding claim, wherein the GH11 xylanase 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 3-180 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.

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

16. The pulp composition of claim 15, wherein the GH11 xylanase 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%, most preferably 98% amino acid sequence identity to amino acids 3-180 of SEQ ID NO: 2, or a functional fragment thereof.

17. The pulp composition according to claim 15 or 16, wherein the pulp is cellulose pulp.

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

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