Classifications

• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
  • D21H11/00—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only
  • D21H11/16—Pulp or paper, comprising cellulose or lignocellulose fibres of natural origin only modified by a particular after-treatment
  • D21H11/20—Chemically or biochemically modified fibres
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
  • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes
• • D—TEXTILES; PAPER
  • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
  • D01C—CHEMICAL OR BIOLOGICAL TREATMENT OF NATURAL FILAMENTARY OR FIBROUS MATERIAL TO OBTAIN FILAMENTS OR FIBRES FOR SPINNING; CARBONISING RAGS TO RECOVER ANIMAL FIBRES
  • D01C1/00—Treatment of vegetable material
• • D—TEXTILES; PAPER
  • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
  • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
  • D01F2/00—Monocomponent artificial filaments or the like of cellulose or cellulose derivatives; Manufacture thereof
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C3/00—Pulping cellulose-containing materials
  • D21C3/003—Pulping cellulose-containing materials with organic compounds
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C3/00—Pulping cellulose-containing materials
  • D21C3/02—Pulping cellulose-containing materials with inorganic bases or alkaline reacting compounds, e.g. sulfate processes
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C3/00—Pulping cellulose-containing materials
  • D21C3/02—Pulping cellulose-containing materials with inorganic bases or alkaline reacting compounds, e.g. sulfate processes
  • D21C3/022—Pulping cellulose-containing materials with inorganic bases or alkaline reacting compounds, e.g. sulfate processes in presence of S-containing compounds
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C3/00—Pulping cellulose-containing materials
  • D21C3/22—Other features of pulping processes
  • D21C3/26—Multistage processes
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C9/00—After-treatment of cellulose pulp, e.g. of wood pulp, or cotton linters ; Treatment of dilute or dewatered pulp or process improvement taking place after obtaining the raw cellulosic material and not provided for elsewhere
  • D21C9/10—Bleaching ; Apparatus therefor
  • D21C9/1005—Pretreatment of the pulp, e.g. degassing the pulp

Definitions

• the present invention relates to treatment of dissolving pulp with one or more enzymes.
• the enzymatic treatment results in reduced viscosity and/or improved viscosity control in the dissolving pulp production process and/or increased reactivity of the final dissolving pulp.
• Dissolving pulp or dissolving-grade pulp is a chemical bleached pulp with a high cellulose content enough to be suitable for the production of regenerated cellulose and cellulose derivatives.
• Commercial dissolving pulp has special properties, such as a high level of brightness and uniform molecular-weight distribution.
• Commercial dissolving pulp is manufactured for uses that require a high chemical cellulose purity, and particularly low hemicellulose content, since the chemically similar hemicellulose can interfere with subsequent processes. Dissolving pulp is so named because it is not made into paper, but dissolved either in a solvent or by derivatization into a homogeneous solution, which makes it completely chemically accessible and removes any remaining fibrous structure.
• cellulose such as cellulose triacetate, a plastic-like material formed into fibers or films, or cellulose ethers such as methyl cellulose, used as a thickener.
• the present invention provides a lytic polysaccharide monooxygenase-based solution that reduces the viscosity and/or improves the viscosity control in the production of dissolving pulp, e.g., kraft and sulfite dissolving pulp.
• the enzyme solution described in this invention allows a more selective depolymerization of cellulose and thus a better control of pulp viscosity as compared to the conventional methods in use that are unselective with many side reactions, such as oxygen, hydrogen peroxide, ozone, sodium hypochlorite and acid hydrolysis.
• the reactivity of the dissolving pulp in the present invention is improved, thereby reducing the amount of chemicals used in the viscose production process and/or improving the processability in terms of viscose dope filterability in the viscose making process. Savings in the amount of chemicals utilized in the production of regenerated cellulose such as carbon disulfide (CS 2 ) in the viscose making process will reduce costs and environmental impact. Similarly, the reactivity increase of the dissolving pulp is expected to benefit the production process of cellulose derivatives, such as subsequent esterification and etherification processes.
• the invention provides a method for treating dissolving pulp, comprising a step of subjecting the dissolving pulp to a lytic polysaccharide monooxygenase.
• the method of the present invention generates dissolving pulp with reduced viscosity and/or improved viscosity control in the dissolving pulp production process, and/or increased reactivity for viscose making, and/or increased content of oxidized groups, compared to dissolving pulp obtained by the same process where the lytic polysaccharide monooxygenase (LPMO) treatment is omitted.
• the said dissolving pulp is kraft dissolving pulp and/or sulfite dissolving pulp.
• the present invention further provides a dissolving pulp made by the method of the present invention.
• the present invention further provides a textile fiber or a derivatized cellulose made of the dissolving pulp of the present invention.
• the present invention further provides use of a lytic polysaccharide monooxygenase for treatment of dissolving pulp.
• Dissolving pulp is a high-grade cellulose pulp, with low contents of hemicellulose, lignin and resin. This pulp has special properties, such as high level of brightness and uniform molecular weight distribution. It is used to make products that include rayon and acetate textile fibers, cellophane, photographic film and various chemical additives. To a large extent, use of dissolving wood pulp depends on its purity (cellulose content), which depends mainly on the production process. To obtain products of high quality, these so-called“special” pulps must fulfill certain requirements, such as high cellulose content, low hemicellulose content, a uniform molecular weight distribution, and high cellulose reactivity. Most of the commercial dissolving pulps accomplish these demands to a certain extent.
• removing hemicelluloses from the wood fiber is crucial, because hemicelluloses can affect the filterablility of viscose, the xanthation of cellulose and the strength of the end product during the production of viscose.
• Hemicrucoses are removed during the cooking of wood and the subsequent bleaching.
• the acidic conditions used are responsible for removing most of the hemicellulose while in sulfate/kraft process usually a prehydrolysis step is required to remove hemicelluloses.
• Another method to remove hemicelluloses is by treatment of pulps with enzymes that react only with the hemicellulose portion of the pulp.
• Kraft dissolving pulp is synonymous with“sulphate dissolving pulp”.
• a preferred example is a prehydrolysis kraft dissolving pulp.
• Kraft dissolving pulp is produced by digesting wood chips at temperatures above about 120°C with a solution of sodium hydroxide and sodium sulfide. Some kraft pulping is also done in which the sodium sulfide is augmented by oxygen or anthraquinone. As compared with soda pulping, kraft pulping is particularly useful for pulping of softwoods, which contain a higher percentage of lignin than hardwoods.
• the term “kraft dissolving pulp” is synonymous with“kraft dissolving cellulose” and“kraft dissolving-grade pulp” and refers to pulp that has a high cellulose content.
• the cellulose content of the kraft dissolving pulp is preferably at least 90% (weight/weight) such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% (w/w).
• Kraft dissolving pulp is manufactured for uses that require a high chemical purity, and particularly low hemicellulose content.
• the hemicellulose content of the dissolving pulp is preferably less than 10% (weight/weight) such as less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% (w/w).
• Kraft dissolving pulp can e.g.
• “Kraft dissolving-grade pulp” can also be defined as pulp that has been purified sufficiently for use in the production of viscose, rayon, cellulose ethers, or cellulose esters with organic or inorganic acids.
• Sulfite dissolving pulp The sulfite process produces wood pulp which is almost pure cellulose fibers by using various salts of sulfurous acid to extract the lignin from wood chips in large pressure vessels called digesters.
• the salts used in the pulping process are either sulfites (S0 3 2- ), or bisulfites (HS0 3 ), depending on the pH.
• the counter ion can be sodium (Na + ), calcium (Ca 2+ ), potassium (K + ), magnesium (Mg 2+ ) or ammonium (NH 4+ ).
• Sulfite pulping is carried out between pH 1.5 and 5, depending on the counterion to sulfite (bisulfite) and the ratio of base to sulfurous acid.
• the pulp is in contact with the pulping chemicals for 4 to 14 hours and at temperatures ranging from 130 to 160 °C (266 to 320 °F), again depending on the chemicals used.
• the spent cooking liquor from sulfite pulping is usually called brown liquor, but the terms red liquor, thick liquor and sulfite liquor are also used (compared to black liquor in the kraft process).
• Pulp washers using countercurrent flow, remove the spent cooking chemicals and degraded lignin and hemicellulose.
• Bleaching is the removal of color from pulp, primarily the removal of traces of lignin which remains bound to the fiber after the primary pulping operation.
• Bleaching usually involves treatment with oxidizing agents such as chlorine (C-stage), chlorine dioxide (D-stage), oxygen (O-stage), hydrogen peroxide (P-stage), ozone (Z-stage) and peracetic acid (CH 3 C0 3 H; Paa- stage) or a reducing agent such as sodium dithionite (Y-stage).
• oxidizing agents such as chlorine (C-stage), chlorine dioxide (D-stage), oxygen (O-stage), hydrogen peroxide (P-stage), ozone (Z-stage) and peracetic acid (CH 3 C0 3 H; Paa- stage) or a reducing agent such as sodium dithionite (Y-stage).
• oxidizing agents such as chlorine (C-stage), chlorine dioxide (D-stage), oxygen (O-stage), hydrogen peroxide (P-stage), ozone (Z-stage) and peracetic acid (CH 3 C
• Cold Caustic Extraction A cold alkali extraction, also called Cold Caustic Extraction (CCE), is a method used to to remove short-chain noncellulosic carbohydrates (cellulose purification) that is based on physical effects such as swelling and solubilization.
• CCE Cold Caustic Extraction
• a CCE stage takes place at temperatures below 45°C and using very high NaOH dosage that, in the liquid phase, can reach values up to 100 g/L. Depending on the pulp consistency in use, this will determine the amount of NaOH per dry weight of pulp.
• Typical conditions for a CCE-stage can be 5-10% w/w NaOH in the liquid phase for at least 10 min.
• Hot Caustic Extraction HCE: the term“Hot Caustic Extraction” (HCE) is synonymous with“hot alkali extraction”. HCE is a method to remove short chain hemicellulose and amorphous cellulose in pulps.
• a hot caustic extraction (HCE)-stage is a purification process that is based on chemical reactions, in particular alkaline peeling of hemicelluloses, which is carried out at higher temperatures and lower NaOH concentration compared to CCE.
• ISO Brightness is defined in ISO 2470-1 (method for measuring ISO brightness of pulps, papers and boards), and it is the intrinsic radiance [reflectance] factor measured with a reflectometer having the characteristics described in ISO 2469.
• Pulp viscosity is measured by dissolving the pulp in a suitable cellulose solvent such as in cupri-ethylenediamine (CED) and measuring the solution viscosity. This measurement gives an indication of the average degree of polymerization of the cellulose. This property can be referred as intrinsic viscosity in ml_/g and measured according to ISO 5351 or as TAPPI viscosity in cP and measured according to TAPPI T 230.
• Unbleached or partially bleached or alkaline extracted kraft dissolving pulp is produced by a kraft based cooking process such as pre-hydrolysis kraft (PHK) cooking but not fully bleached and purified until becoming a commercial kraft dissolving pulp and thus it is not a finished product.
• a kraft based cooking process such as pre-hydrolysis kraft (PHK) cooking but not fully bleached and purified until becoming a commercial kraft dissolving pulp and thus it is not a finished product.
• PTK pre-hydrolysis kraft
• ISO brightness below 90% such as below 85%, such as below 80%, such as below 75%, such as below 70%, such as below 65%, such as below 60%, such as below 55%, such as below 50%, such as below 45%, such as below 40%, such as below 35%, and such as below 30%).
• Unbleached or partially bleached or alkaline extracted sulfite dissolving pulp is produced by a sulfite based cooking process but not fully bleached and purified until becoming a commercial sulfite dissolving pulp and thus it is not a finished product. Typically it has an ISO brightness below 90% (such as below 85%, such as below 80%, such as below 75%, such as below 70%, such as below 65%, such as below 60%, such as below 55%, such as below 50%, such as below 45%, such as below 40%, such as below 35%, and such as below 30%).
• ISO brightness below 90% such as below 85%, such as below 80%, such as below 75%, such as below 70%, such as below 65%, such as below 60%, such as below 55%, such as below 50%, such as below 45%, such as below 40%, such as below 35%, and such as below 30%).
• Bleached kraft dissolving pulp and bleached sulfite dissolving pulp is produced by a kraft dissolving pulp or a sulfite based cooking process but fully bleached and purified until becoming a commercial dissolving pulp. Typically it has an ISO brightness above 90% (such as above 91%, such as above 92%, such as above 93%, such as above 94%, such as above 95%, such as above 96%, such as above 97%, such as above 98%, such as above 99%, such as 100%).
• Sequence identity The relatedness between two amino acid sequences or between two nucleo tide sequences is described by the parameter“sequence identity”.
• the sequence identity between two amino acid sequenc es is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EM BOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
• the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) sub stitution matrix.
• the output of Needle labeled“longest identity” is used as the percent identity and is calculated as follows:
• the invention relates to a method for treating dissolving pulp, comprising a step of subjecting the dissolving pulp to a lytic polysaccharide monooxygenase (LPMO).
• LPMO lytic polysaccharide monooxygenase
• the method of the present invention further comprises a step of subjecting the dissolving pulp to a cellulase.
• the step of subjecting the dissolving pulp to a lytic polysaccharide monooxygenase and the step of subjecting the dissolving pulp to a cellulase are carried out simultaneously or sequentially in any order.
• a lytic polysaccharide monooxygenase is added to the dissolving pulp together with the cellulase.
• a lytic polysaccharide monooxygenase is added to the dissolving pulp before the addition of a cellulase.
• a lytic polysaccharide monooxygenase is added to the dissolving pulp after the addition of a cellulase.
• the present method of the present invention comprises a step of bleaching the dissolving pulp.
• the step of subjecting the dissolving pulp to a lytic polysaccharide monooxygenase and the step of bleaching the dissolving pulp are carried out simultaneously or sequentially in any order.
• the step of bleaching the dissolving pulp is performed using a chemical selected from the group consisting of CI0 2 , 0 2 , 0 3 , H 2 0 2 , CH CO H and NaOCI.
• the method of the present invention further comprises the step of Alkaline Extraction.
• Alkaline Extraction is an E, HCE or CCE stage.
• Special alkaline purification treatments such as HCE or CCE treatments can yield higher cellulose levels in sulfite and kraft processes.
• HCE is typically employed to further purify the pulp after the sulfite cooking.
• the method of the present invention further comprises a step of subjecting the dissolving pulp to a lytic polysaccharide monooxygenase and an electron donor thereof, preferably ascorbic acid, gallic acid, pyrogallol or cysteine.
• the electron donor can exist in the dissolving pulp to be treated. In one embodiment, no or a little amount of electron donor is added to the dissolving pulp. In another embodiment, an effective amount of electron donor is added to the dissolving pulp.
• the dissolving pulp is an unbleached, partially bleached, bleached or alkaline extracted dissolving pulp.
• the dissolving pulp is kraft pulp or sulfite pulp.
• the lytic polysaccharide monooxygenase added in the method of the present invention has a sequence identity of at least 60% [such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least
• the cellulase added in the method of the present invention has a sequence identity of at least 60% (such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 60% (such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least
• the lytic polysaccharide monooxygenase added in the method of the present invention comprises or consists of SEQ ID NO: 1 or the mature polypeptide thereof, or SEQ ID NO: 2 or the mature polypeptide thereof, or SEQ ID NO: 3 or the mature polypeptide thereof; or the cellulase comprises or consists of SEQ ID NO: 4, SEQ ID NO: 5 or the mature polypeptide thereof, SEQ ID NO: 6 or the mature polypeptide thereof, or SEQ ID NO: 7 or the mature polypeptide thereof.
• the lytic polysaccharide monooxygenase added in the method of the present invention comprises amino acids 19 to 226 of SEQ ID NO: 1 , or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof.
• the lytic polysaccharide monooxygenase added in the method of the present invention comprises amino acids amino acids 20 to 254 of SEQ ID NO: 2, or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof.
• the lytic polysaccharide monooxygenase added in the method of the present invention comprises amino acids 22 to 249 of SEQ ID NO: 3, or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof.
• the cellulase added in the method of the present invention comprises SEQ ID NO: 4 or the full length thereof, or a homologous sequence thereof, an allelic variant thereof, or a functional fragment thereof.
• the cellulase added in the method of the present invention comprises amino acids 22-305 of SEQ ID NO: 5 or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof.
• the cellulase added in the method of the present invention comprises amino acids 22 to 293 of SEQ ID NO: 6 or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof. In another preferred embodiment, the cellulase added in the method of the present invention comprises amino acids 19 to 409 of SEQ ID NO: 7 or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof.
• the concentration of the lytic polysaccharide monooxygenase added in the method of the present invention is preferably from 0.05 mg/kg oven dry pulp to 100000 mg/kg oven dry pulp such as a concentration selected from the group consisting of from 0.05 mg/kg oven dry pulp to 250 mg/kg oven dry pulp, from 0.25 mg/kg oven dry pulp to 1000 mg/kg oven dry pulp, from 0.25 mg/kg oven dry pulp to 2000 mg/kg oven dry pulp, from 1.0 mg/kg oven dry pulp to 5000 mg/kg oven dry pulp, from 5.0 mg/kg oven dry pulp to 10000 mg/kg oven dry pulp, from 10.0 mg/kg oven dry pulp to 15000 mg/kg oven dry pulp, from 15.0 mg/kg oven dry pulp to 20000 mg/kg oven dry pulp, from 20.0 mg/kg oven dry pulp to 30000 mg/kg oven dry pulp, from 30.0 mg/kg oven dry pulp to 40000 mg/kg oven dry pulp, from 40.0 mg/kg oven dry pulp to 60000 mg/kg oven dry pulp, from 60.0 mg/kg oven dry pulp
• the concentration of the cellulase added in the present invention is from 0.05 mg/kg oven dry pulp to 100 mg/kg oven dry pulp such as a concentration selected from the group consisting of from 0.05 mg/kg oven dry pulp to 80.0 mg/kg oven dry pulp, from 0.25 mg/kg oven dry pulp to
• the method according to the invention results in an improved viscosity control, thereby allowing the reduction in the production of dissolving pulp outside final viscosity specification/target, typically more than 50% (such as more than 60% or more than 70%) reduction in the production of off-grade dissolving pulp with respect to viscosity.
• the method according to the invention results in increased reactivity of the kraft and/or sulfite dissolving pulp, particularly the kraft dissolving pulp having an increased reactivity of at least 10% (such as at least 20% or at least 30%).
• the method results in reduced viscosity and/or improved viscosity control in the dissolving pulp production process; and/or the method results in increased reactivity of the dissolving pulp, preferably increased Fock’s reactivity related to the viscose making process and therefore allowing savings in CS 2 and thus reducing costs and environmental impact; and/or the method results in increased content of oxidized groups of the dissolving pulp.
• This increase in oxidized groups can increase the reactivity of the dissolving pulp not only in terms of fiber swelling and chemical accessibility but also considering that more anchor points (carbonyl and/or carboxyl groups) in the cellulose will be available for subsequent derivatization processes in the production of cellulose derivatives.
• the method of the present invention further comprises subjecting the dissolving pulp to a xylanase and/or a mannanase and/or a lipase and/or laccase and/or peroxidase.
• a dissolving pulp made by the method described above is also part of the invention.
• a textile fiber or a derivatized cellulose made of the dissolving pulp described above is also part of the invention.
• the invention also relates to use of a lytic polysaccharide monooxygenase for treatment of dissolving pulp.
• lytic polysaccharide monooxygenase means an enzyme that oxidizes sp(3) carbons in polysaccharides such as chitin, cellulose, and starch in the presence of an external electron donor and, as currently hypothesized, utilizes copper at the active site to activate molecular oxygen.
• enzymes belong to Auxiliary Activity families AA9, AA10, AA1 1 , AA13, AA14 and AA15 as defined in the database of carbohydrate active enzymes (http://www.cazy.org/).
• the LPMO comprises the following motifs:
• x is any amino acid
• x(4,5) is any four or five contiguous amino acids
• x(4) is any four contiguous amino acids.
• the LPMO comprising the above-noted motifs may further comprise:
• x is any amino acid
• x(1 ,2) is any one or two contiguous amino acids
• x(3) is any three contiguous amino acids
• x(2) is any two contiguous amino acids.
• the LPMO further comprises H-x(1 ,2)-G-P-x(3)-[YW]-[AILMV] In another preferred aspect, the LPMO further comprises [EQ]-x-Y-x(2)-C-x-[EHQN]-[FILV]-x-[ILV] In another preferred aspect, the LPMO further comprises H-x(1 ,2)-G-P-x(3)-[YW]-[AILMV] and [EQ]-x-Y-x(2)-C-x-[EHQN]-[FILV]-x-[ILV]
• the LPMO comprises the following motif:
• x is any amino acid
• x(4,5) is any 4 or 5 contiguous amino acids
• x(3) is any 3 contiguous amino acids.
• the accepted IUPAC single letter amino acid abbreviation is employed.
• the LPMO comprises an amino acid sequence that has a sequence identity to the mature polypeptide of SEQ ID NO: 1 ( Thielavia terrestris), SEQ ID NO: 2 ( Lentinus similis), SEQ ID NO: 3 ( Thermoascus aurantiacus), SEQ ID NO: 8 ( Thielavia terrestris), SEQ ID NO: 9 ( Thielavia terrestris), SEQ ID NO: 10 ( Thielavia terrestris), SEQ ID NO: 1 1 ( Thielavia terrestris), SEQ ID NO: 12 ( Thielavia terrestris), SEQ ID NO: 13 ( Thielavia terrestris), SEQ ID NO: 14 ( Trichoderma reesei), SEQ ID NO: 15 ( Myceliophthora thermophila), SEQ ID NO: 16 ( Myceli - ophthora thermophila), SEQ ID NO: 17 ( Myceliophthora thermophila), SEQ ID NO: 18 ( My
• the LPMO is an artificial variant comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:
• SEQ ID NO: 23 SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:
• SEQ ID NO: 34 SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38; or a homologous sequence thereof, an allelic variant thereof, or a functional fragment thereof.
• amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
• conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine).
• Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York.
• the most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly.
• amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered.
• amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
• Essential amino acids in a parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081 -1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for cellulolytic enhancing activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708.
• the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64.
• the identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to the parent polypeptide.
• Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
• Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
• Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
• SEQ ID NO: 22 SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:
• SEQ ID NO: 27 SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32,
• SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
• the LPMO is used in the presence of a soluble activating divalent metal cation as described in WO 2008/151043, e.g., copper sulfate.
• the LPMO is used in the presence of an electron donor thereof.
• the electron donor can exist in the dissolving pulp to be treated. In one embodiment, no or a little amount of electron donor can be added to the dissolving pulp. In another embodiment, an effective amount of electron donor can be added to the dissolving pulp.
• the electron donor can be a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, or a sulfur-containing compound.
• the dioxy compound may include any suitable compound containing two or more oxygen atoms.
• the dioxy compounds contain a substituted aryl moiety as described herein.
• the dioxy compounds may comprise one or more (several) hydroxyl and/or hydroxyl derivatives, but also include substituted aryl moieties lacking hydroxyl and hydroxyl derivatives.
• Non-limiting examples of dioxy compounds include pyrocatechol or catechol; caffeic acid; 3,4- dihydroxybenzoic acid; 4-tert-butyl-5-methoxy-1 ,2-benzenediol; ascorbic acid, pyrogallol; gallic acid; methyl-3, 4, 5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone; 2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid; 4-chloro-1 ,2-benzenediol; 4-nitro-1 ,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1 ,4-diol; (croconic acid; 1 ,3-propanediol; tartaric acid; 2,4-pentanediol; 3-ethyoxy-1 ,2-propanediol; 2,4,4’- trihydroxybenzophenone; cis
• the bicyclic compound may include any suitable substituted fused ring system as described herein.
• the compounds may comprise one or more (several) additional rings, and are not limited to a specific number of rings unless otherwise stated.
• the bicyclic compound is a flavonoid.
• the bicyclic compound is an optionally subsituted isoflavonoid.
• the bicyclic compound is an optionally substituted flavylium ion, such as an optionally substituted anthocyanidin or optionally substituted anthocyanin, or derivative thereof.
• Non-limiting examples of bicyclic compounds include epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.
• the heterocyclic compound may be any suitable compound, such as an optionally substituted aromatic or non-aromatic ring comprising a heteroatom, as described herein.
• the heterocyclic is a compound comprising an optionally substituted heterocycloalkyl moiety or an optionally substituted heteroaryl moiety.
• the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted 5- membered heterocycloalkyl or an optionally substituted 5-membered heteroaryl moiety.
• the optionally substituted heterocycloalkyl or optionally substituted heteroaryl moiety is an optionally substituted moiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl, dimethylhydantoin, pyrazinyl,
• the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted furanyl.
• heterocyclic compounds include (1 ,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one; 4-hydroxy-5-methyl-3- furanone; 5-hydroxy-2(5H)-furanone; [1 ,2-dihydroxyethyl]furan-2,3,4(5H)-trione; a-hydroxy-y- butyrolactone; ribonic g-lactone; aldohexuronicaldohexuronic acid g-lactone; gluconic acid d- lactone; 4-hydroxycoumarin; dihydrobenzofuran; 5-(hydroxymethyl)furfural; furoin; 2(5H)- furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one;
• the nitrogen-containing compound may be any suitable compound with one or more nitrogen atoms.
• the nitrogen-containing compound comprises an amine, imine, hydroxylamine, or nitroxide moiety.
• nitrogen-containing compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol; 1 ,2-benzenediamine; 2,2,6,6-tetramethyl-1 -piperidinyloxy; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8- tetrahydropterine; and maleamic acid; or a salt or solvate thereof.
• the quinone compound may be any suitable compound comprising a quinone moiety as described herein.
• Non-limiting examples of quinone compounds include 1 ,4-benzoquinone; 1 ,4- naphthoquinone; 2-hydroxy-1 ,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1 ,4-benzoquinone or coenzyme Q 0 ; 2, 3, 5, 6-tetramethyl-1 ,4-benzoquinone or duroquinone; 1 ,4- dihydroxyanthraquinone; 3-hydroxy-1 -methyl-5, 6-indolinedione or adrenochrome; 4-tert-butyl-5- methoxy-1 ,2-benzoquinone; pyrroloquinoline quinone; or a salt or solvate thereof.
• the sulfur-containing compound may be any suitable compound comprising one or more sulfur atoms.
• the sulfur-containing comprises a moiety selected from thionyl, thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid, and sulfonic ester.
• Non-limiting examples of sulfur-containing compounds include ethanethiol; 2-propanethiol; 2-propene-1 -thiol; 2-mercaptoethanesulfonic acid; benzenethiol; benzene-1 ,2-dithiol; cysteine; methionine; glutathione; cystine; or a salt or solvate thereof.
• Cellulases or cellulolytic enzymes are enzymes involved in hydrolysis of cellulose.
• cellobiohydrolase (1 ,4-p-D-glucan cellobiohydrolase, EC 3.2.1 .91 , e.g., cellobiohydrolase I and cellobiohydrolase II
• endo-b-1 ,4-glucanase endo-1 ,4-p-D-glucan 4- glucanohydrolase, EC 3.2.1 .4
• b-glucosidase EC 3.2.1 .21
• cellobiohydrolase I is defined herein as a cellulose 1 ,4-beta-cellobiosidase (also referred to as exo-glucanase, exo-cellobiohydrolase or 1 ,4-beta-cellobiohydrolase) activity, as defined in the enzyme class EC 3.2.1 .91 , which catalyzes the hydrolysis of 1 ,4-beta-D-glucosidic linkages in cellulose and cellotetraose, by the release of cellobiose from the non-reducing ends of the chains.
• the definition of the term“cellobiohydrolase II activity” is identical, except that cellobiohydrolase II attacks from the reducing ends of the chains.
• Endoglucanases (EC No. 3.2.1 .4) catalyses endo hydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxy methyl cellulose and hydroxy ethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3 glucans such as cereal beta-D-glucans or xyloglucans and other plant material containing cellulosic parts.
• the authorized name is endo-1 ,4- beta -D- glucan 4-glucano hydrolase, but the abbreviated term endoglucanase is used in the present specification.
• the cellulase used in the present invention is an endoglucanase.
• the cellulases may comprise a carbohydrate-binding module (CBM) which enhances the binding of the enzyme to a cellulose-containing fiber and increases the efficacy of the catalytic active part of the enzyme.
• CBM is defined as contiguous amino acid sequence within a carbohydrate-active enzyme with a discreet fold having carbohydrate-binding activity.
• the cellulases may be a preparation as defined in WO 2008/151079 , which is hereby incorporated by reference.
• the cellulase preparation may further comprise a beta-glucosidase, such as the fusion protein disclosed in US 60/832,51 1 .
• the cellulase preparation also comprises a CBH II, preferably Thielavia terrestris cellobiohydrolase II CEL6A.
• the cellulase preparation also comprises a cellulase enzymes preparation, preferably the one derived from Trichoderma reesei.
• Cellulases are synthesized by a large number of microorganisms which include fungi, actinomycetes, myxobacteria and true bacteria but also by plants. Especially endoglucanases of a wide variety of specificities have been identified.
• the cellulase activity may, in a preferred embodiment, be derived from a fungal source, such as a strain of the genus Trichoderma, preferably a strain of Trichoderma reeser, a strain of the genus Humicola, such as a strain of Humicola insolens ; or a strain of Chrysosporium, preferably a strain of Chrysosporium lucknowense or a strain of the genus Thielavia, preferably Thielavia terrestris.
• a fungal source such as a strain of the genus Trichoderma, preferably a strain of Trichoderma reeser, a strain of the genus Humicola, such as a strain of Humicola insolens ; or a strain of Chrysosporium, preferably a strain of Chrysosporium lucknowense or a strain of the genus Thielavia, preferably Thielavia terrestris.
• the cellulase comprises an amino acid sequence that has a sequence identity to SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 7 of at least 50%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.
• the cellulase is an artificial variant comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 7; or a homologous sequence thereof, or an allelic variant thereof, or a functional fragment thereof.
• the total number of amino acid substitutions, deletions and/or insertions of SEQ ID NO: 4, the mature polypeptide of SEQ ID NO: 5, the mature polypeptide of SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 7 is not more than 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
• Fungi and bacteria produce a spectrum of cellulolytic enzymes (cellulases) which, on the basis of sequence similarities (hydrophobic cluster analysis), can be classified into different families of glycosyl hydrolases [Henrissat B & Bairoch A; Biochem. J. 1993 293 781 -788]. At present are known cellulases belonging to the families 5, 6, 7, 8, 9, 10, 12, 26, 44, 45, 48, 60, and 61 of glycosyl hydrolases.
• Any enzyme having xylanase, mannanase, lipase, laccase, and/or peroxidase activity can be used as additional enzymes in the use and process of the invention.
• the additional enzymes and a lytic polysaccharide monooxygenase are added simultaneously or sequentially in any order. Below some non-limiting examples are listed of such additional enzymes.
• the enzymes written in capitals are commercial enzymes available from Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark.
• the activity of any of those additional enzymes can be analyzed using any method known in the art for the enzyme in question, including the methods mentioned in the references cited.
• xylanase is the PULPZYME HC hemicellulase.
• mannanases are the Trichoderma reesei endo-beta-mannanases described in Stahlbrand et al, J. Biotechnol. 29 (1993), 229-242.
• lipase An example of a lipase is the RESINASE A2X lipase.
• An example of a xylanase is the PULPZYME HC hemicellulase.
• peroxidases, and laccases are disclosed in EP 730641 ; WO 01/98469; EP 719337; EP 765394; EP 767836; EP 7631 15; and EP 788547.
• acceptors e.g., oxygen or hydrogen peroxide
• enhancers, mediators and/or activators such compounds should be considered to be included.
• enhancers and mediators are disclosed in EP 705327; WO 98/56899; EP 677102; EP 781328; and EP 707637.
• the temperature for the method of the present invention is typically from 20°C to 100°C such as a temperature interval selected from the group consisting of from 20°C to 30°C, from 30°C to 40°C, from 40°C to 50°C, from 50°C to 60°C, from 60°C to 70°C, from 70°C to 80°C, from 80°C to 90°C, from 90°C to 100°C, or any combination of these intervals.
• the incubation time used for the mehod of the present invention is typically from 1 minute to 60 hours such as a time interval selected from the group consisting of from 1 minute to 15 minutes, from 15 minutes to 30 minutes, from 30 minutes to 45 minutes, from 45 minutes to 60 minutes, from 1 hour to 3 hours, from 3 hours to 6 hours, from 6 hours to 10 hours, from 10 hours to 12 hours, from 12 hours to 15 hours, from 15 hours to 20 hours, from 20 hours to 22 hours, from 22 hours to 25 hours, from 25 hours to 30 hours, from 30 hours to 40 hours, from 40 hours to 50 hours, from 50 hours to 60 hours, or any combination of these time intervals.
• the dissolving pulp used in the present invention can be wood pulp coming e.g. from softwood trees (such as spruce, pine, fir, larch and hemlock) and/or hardwoods (such as eucalyptus, aspen and birch) or other plant sources such as bamboo.
• softwood trees such as spruce, pine, fir, larch and hemlock
• hardwoods such as eucalyptus, aspen and birch
• other plant sources such as bamboo.
• the dissolving pulp is selected from the group consisting of dissolving hardwood pulp and dissolving softwood pulp, or a mixture thereof.
• the invention relates in one embodiment to dissolving pulp made by the method according to the invention.
• the invention relates in one embodiment to a kraft dissolving pulp or a sulfite dissolving pulp made by the method according to the invention.
• the invention further relates to use of the dissolving pulp according to the invention for production of textile fibers.
• the dissolving pulp produced may be used in the manufacture of regenerated cellulose such as viscose, rayon, lyocell and modal fibers.
• the method of the present invention can be performed in the presence of one or more surfactants such as one or more anionic surfactants and/or one or more nonionic surfactants and/or one or more cationic surfactants.
• surfactants can in one embodiment include poly(alkylene glycol)-based surfactants, ethoxylated dialkylphenols, ethoxylated dialkylphenols, ethoxylated alcohols and/or silicone based surfactants.
• poly(alkylene glycol)-based surfactant examples include polyethylene glycol) alkyl ester, poly(ethylene glycol) alkyl ether, ethylene oxide/propylene oxide homo- and copolymers, or polyethylene oxide- co-propylene oxide) alkyl esters or ethers.
• Other examples include ethoxylated derivatives of primary alcohols, such as dodecanol, secondary alcohols, polypropylene oxide], derivatives thereof, tridecylalcohol ethoxylated phosphate ester, and the like.
• anionic surfactant materials useful in the practice of the invention comprise sodium alpha-sulfo methyl laurate, (which may include some alpha-sulfo ethyl laurate) for example as commercially available under the trade name ALPHA-STEPTM-ML40; sodium xylene sulfonate, for example as commercially available under the trade name STEPANATETM- X; triethanolammonium lauryl sulfate, for example as commercially available under the trade name STEPANOLTM-WAT; diosodium lauryl sulfosuccinate, for example as commercially available under the trade name STEPANTM-Mild SL3; further blends of various anionic surfactants may also be utilized, for example a 50%-50% or a 25%-75% blend of the aforesaid ALPHA-STEPTM and STEPANATETM materials, or a 20%-80% blend of the aforesaid ALPHA- STEPTM and STEPAN
• nonionic surfactant materials useful in the practice of the invention comprise cocodiethanolamide, such as commercially available under trade name NINOLTM- 1 1 CM; alkyl polyoxyalkylene glycol ethers, such as relatively high molecular weight butyl ethylenoxide-propylenoxide block copolymers commercially available under the trade name TOXIMULTM-8320 from the Stepan Company. Additional alkyl polyoxyalkylene glycol ethers may be selected, for example, as disclosed in U.S. Pat. No. 3,078,315. Blends of the various nonionic surfactants may also be utilized, for example a 50%-50% or a 25%-75% blend of the aforesaid NINOLTM and TOXIMULTM materials.
• anionic/nonionic surfactant blends useful in the practice of the invention include various mixtures of the above materials, for example a 50%-50% blends of the aforesaid ALPHA-STEPTM and NINOLTM materials or a 25%-75% blend of the aforesaid STEPANATETM and TOXIMULTM materials.
• the various anionic, nonionic and anionic/nonionic surfactant blends utilized in the practice of the invention have a solids or actives content up to about 100% by weight and preferably have an active content ranging from about 10% to about 80%.
• blends or other solids (active) content may also be utilized and these anionic surfactants, nonionic surfactants, and mixtures thereof may also be utilized with known pulping chemicals such as, for example, anthraquinone and derivatives thereof and/or other typical paper chemicals, such as caustics, defoamers and the like.
• the intrinsic viscosity of the pulp was measured according to ISO 5351 (International Organization for Standardization 5351 ).
• the pulp viscosity was measured by mViPr according to WO 201 1/107472 A9.
• aldehyde groups (CHO content) were measured spectrophotometrically according to the procedure described by Obolenskaya et al., “Determination of aldehyde groups in oxidized pulps,” Laboratory Manipulations in Wood and Cellulose Chemistry, Ecologia, Moscow, 21 1 -212, 1991 , which is based on the reaction of 2,3,5-triphenyltetrazolium chloride (TTC) with the aldehyde groups leading to the formation of formazan (red colorant).
• TTC 2,3,5-triphenyltetrazolium chloride
• the Fock’s reactivity is a measure of how much of a known amount of pulp is reacted with CS 2 as a small-scale simulation of the viscose making process and it was carried out at 9% NaOH.
• Example 1 LPMO treatment of unbleached hardwood kraft dissolving pulp
• Unbleached hardwood kraft dissolving pulp with a kappa number of 6.8 (TAPPI T 236 procedure), ISO brightness of 51% and an intrinsic viscosity of 1025 mL/g produced by a pre hydrolysis kraft pulping process and further treated with a cold-caustic extraction stage from a dissolving pulp production process was used.
• This pulp was treated with several LPMOs in a small-scale assay using 24 mg of oven-dry fiber at 0.4% consistency, 45°C, pH 5.0 (acetate buffer, 50 mM) for 20 hours.
• the enzyme treatment (denoted as X-stage) was done at a dosage of 5 mg EP (enzyme protein) / g odp (oven-dry pulp).
• the pulp suspension at 0.4% consistency was disintegrated with a magnetic bar in glass test tubes placed in a heating block at 45°C.
• Ascorbic acid, gallic acid or pyrogallol was added as electron donor to a final concentration of 1 mM in the suspension, followed by the addition of the LPMO enzyme to a final volume of 6 mL.
• the tubes were cooled down in ice and then 6 mL of cupri-ethylenediamine (CED) solution added to dissolve the fibers.
• CED cupri-ethylenediamine
• the mViPr pipette consists of a modified Gilson Concept C300 pipette equipped with a pressure sensor and Diamond D300 Gilson tips. Samples were kept at a constant temperature within ⁇ 0.1 °C. A volume of 200 mI_ dissolved pulp was aspirated and dispensed in and out of the pipette, respectively, while recording the pressure in the pipette headspace. A pipette speed of 4 was applied. Apirations were followed by a 2s delay, and dispensing was followed by a 5s delay. Each sample measurement consisted of 15 aspiration-dispensing cycles, and pressure results were average of 15 aspiration or dispensing pressures, respectively.
• the aspiration pressure results from the mViPr measurements are presented in Table 1. It can be seen that the LPMOs can reduce the average size of the cellulose which is expressed as the reduction in the solution viscosity as measured by the reduction in aspiration pressure.
• the performance of each LPMO is also dependent on the specific electron donor utilized. In addition, the electron donor itself also degrades cellulose when compared to the original pulp, particularly ascorbic acid. Tt LPMO is quite effective in reducing pulp viscosity with all electron donors.
• Example 2 LPMO treatment of bleached hardwood kraft dissolving pulp
• LPMOs can reduce the average size of the cellulose molecules as expressed by the reduction in the solution (dissolved pulp) viscosity, measured by the reduction in aspiration pressure. Higher reduction in bleached pulp viscosity compared to controls (no enzyme) were achieved when the LPMOs were used together with gallic acic and pyrogallol as compared to the ascorbic acid which also reduces signfificantly viscosity itself.
• Tt LPMO is a more robust LPMO regarding viscosity reduction with all the electron donors used.
• Example 3 Effect of LPMO treatment with/without endoglucanase at medium pulp consistency on bleached dissolving pulp viscosity, reactivity and CHO content
• Bleached hardwood kraft dissolving pulp produced by a pre-hydrolysis kraft pulping process of viscose-grade was used having an intrinsic viscosity of 512 mL/g.
• This pulp was treated with several LPMOs using gallic acid (1 mM) as electron donor at 1.5% consistency in Distek vessels (Distek model Symphony 7100) with heating and continuous overhead stirring. Once the pulps were disintegrated and at the temperature set-point of 45°C, gallic acid was added and then the enzyme (LPMO and/or endoglucanase).
• the enzyme treatment of the pulp took 25.5 hour at pH 5.0 (acetate buffer, 50 mM) using 2 mg EP/g odp of LPMO and 1.2 mg EP/kg odp of endoglucanase. After the enzyme treatment, the pulps were filtered and washed in three consecutive steps with 1 L of tap water. Part of the pulp sample was dried before measuring the CHO content and Fock’s reactivity and part of the sample was kept wet in the fridge to test for intrinsic viscosity.
• Example 4 Effect of combined LPMO and endoglucanase treatment on unbleached hardwood kraft dissolving pulp
• Example 2 The same unbleached hardwood kraft dissolving pulp of Example 1 was used and treated with several LPMOs using gallic acid (1 mM) as electron donor in combination with several endoglucanases in a small-scale assay using 24 mg of oven-dry fiber at 0.4% consistency, 50°C, pH 6.0 (phosphate buffer, 50 mM) for 20 hours.
• the LPMO treatments were done at dosages of 2.5 mg EP (enzyme protein) / g odp (high dosage) or 0.5 mg EP / g odp (low dosage).
• the endoglucanase treatments were done at dosages of 0.5 mg EP / kg odp.
• the assay and viscosity measurements were performed using the same conditions and procedures as described in Example 1 .

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