Stitching enzymes for modifying fibers of an aqueous pulp

Abstract

A method of treating a fibrous pulp, such as a papermaking pulp, is provided. The fibrous pulp includes fibers having one or more of an aldehyde group, an aromatic group, a carboxyl group, a hydroxyl group, or a ketone group, present as surface terminated groups or end groups, and optionally one or more of an amino group, a halogen group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group, and one or more polymers having an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group. The method involves combining, with the fibrous pulp, a stitching enzyme configured to enzymatically, covalently bond together, the fibers and / or to enzymatically, covalently bond together the fibers and the one or more polymers. The method can further involve combining the pulp with an alpha-glucan enzyme that is designed to enzymatically, covalently bond monomers together to form the one or more polymers. A reactive fiber pulp is also provided that includes fibers, such as papermaking fibers, having an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group, one or more polymers having an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group, a stitching enzyme, and optionally a sizing enzyme.

Description

STITCHING ENZYMES FOR MODIFYING FIBERS OF AN AQUEOUS PULP BACKGROUND OF THE INVENTION

[0001] This application claims the benefit under 35 U.S.C. §119(e) of prior U.S. Provisional Patent Application No. 63 / 709,536 filed October 21, 2024, which is incorporated in its entirety by reference herein.

[0002] For many applications involving paper, paperboard, fiber board, fiber mat, and other fibrous products, there is a need to make the fibers used in the product more resistant to water penetration. Current sizing methods to make fiber more resistant to water penetration include the use of alkenyl succinic anhydride (ASA), alkyl ketene dimer (AKD), rosin, and starch. Methods using starch involve applying a starch coating that neither is not covalently attached to individual fibers nor provides much hydrophobicity to the surface of the fibrous product. While ASA and AKD sizing can provide more hydrophobicity to the surface of the fiber mat, both methods have drawbacks when it comes to paper-making processes.

[0003] AKD is derived from fatty acids that are waxy solids and only melt at high temperatures. AKD must be applied in the form of an emulsion when at normal ambient temperatures, to maintain a liquid that is capable of being pumped and distributed onto a fiber mat. Sizing with AKD requires a three-step process. First, the emulsion particles need to be spread onto the surface of a paper or other fiber mat. Second, a chemical reaction must take place between the hydroxyl groups on the cellulose surface and the alkylated ketene dimers. Third, the process is completed by the elimination of water to prevent unwanted hydrolysis of the AKD. AKD sizing is difficult to control, and, because of the organic nature of the emulsion, AKD sizing can cause agglomeration on the surface of the fiber mat as well as deposit issues on the felt of a paper mill.

[0004] The very nature of the process for making ASA requires a lengthy and not so environmentally friendly process involving distillation and excessive temperatures of greaterthan 200 °C. Not only is the preparation of the chemical unfavorable, but the reaction can produce unwanted side reactions. Similar to AKD, ASA emulsions are not easy to stabilize and can create sticky deposits as a result of hydrolyzed ASA.

[0005] With the increased demand to use environmentally sustainable materials such as wood / non-wood / fiber based molded packaging over the use of plastics, there is a need to create sizing, that is, hydrophobicity on the surface of fibers, so that a resulting fibrous product can meet the same performance requirements of plastic. There is also a need for renewable solutions for bottles. There is also a need for materials other than plastics, to provide environmentally friendly packages.

[0006] The pulp and paper industry consumes ten to three hundred cubic meters of water per ton of production. To be more sustainable, the water used in the mill must be processed and recycled. White water is a slurry of fine particles that drains to the pit below the paper machine. White water is processed using various methods, including by removing fines that are recycling into the system. As a result, a continuous loss or incomplete recovery of fibers happens due to multiple repetitive treatment and wash steps. This in turn results in loss of capital and material.

[0007] Physical recovery of fines from white water is typically performed using membrane filtration, chemical coagulation, evaporation, oxidative treatment, or by biological or enzymatic degradation of fibers. These methods result in fouling, low volumetric output, continuous clogging of membranes that require replacement, poor dewatering, and salt buildup, and make removal of degraded products difficult.

[0008] Membrane filters separate materials from liquid based on size. Ceramic membrane filters sand polymer filters that are often used cause a buildup of filtrates resulting in a clogged or fouled filter. Such filters cannot withstand prolonged exposure to high temperatures. Throughput can be greatly affected if fines cause continuous fouling of the filter. Retentionaids and wet end additives are sometimes affected by the high mass of polymeric materials and by byproducts arising from the pulp.

[0009] Chemical coagulation techniques involve the addition of aluminum sulfate, poly aluminum chloride, and other cationic polymers. These coagulants agglomerate suspended particles into larger-size units that do not block pores completely. However, these coagulants are not effective by themselves and are typically added along with retention aids.

[0010] Flotation is another common method of treating white water that uses air bubbles to lift solid materials. The solid materials can then be removed physically and added back to the paper machine system. Dissolved air flotation (DAF) units are commonly effective for white water clarification, however, the mechanical units come with the drawbacks of capital investment and continuous maintenance.

[0011] Biological or degrading enzymatic treatment of white water is effective to reduce biological oxygen demand (BOD) of process water or target specific substrates in the white water. Nevertheless, they solve only certain problems in the white water and pose disadvantages. The disadvantages include salt buildup, deaerated water, a loss of brightness,turbidity issues, and presence of live microbes, and other problems causing concerns associated with recycling the white water back into a system.

[0012] Finally, oxidation may be effective in degrading recalcitrant compounds such as dyes and surfactants but can also produce larger fragments from other components that can clog membrane filters.

[0013] A need exists for a method and system that overcome the problems associated with the foregoing treatment methods, while providing increased recyclability of fines and fibers.SUMMARY OF THE PRESENT INVENTION

[0014] A feature of the present invention is to provide a fibrous products made of fibers that have been surface-modified to improve the ability to retain or prevent barrier crossing of liquids, oils, waxy solids, waxy-like substances, and gases.

[0015] Another feature of the present invention is to provide methods for the enzymatic covalent attachment, to a fiber, of a strengthening agent that provides improved strength to the fiber.

[0016] Another feature of the present invention is to provide methods for the enzymatic covalent attachment, to a fiber, of a sizing agent that provides improved and permanent surface sizing.

[0017] Another feature of the present invention is to provide methods for the enzymatic covalent attachment of a strengthening agent to fibers, to provide improved wet strength to fibers used in the pulp and paper industry.

[0018] Another feature of the present invention is to provide a method of modifying a fibrous pulp by combining at least one xyloglucan endotransglucosylase (XET) enzyme with a fibrous pulp under temperature and pH conditions such that XET enzymatic activity occurs. The fibrous pulp can comprise an aqueous suspension of cellulosic fibers and hemicellulose fibers,and the at least one XET enzyme can be included in the aqueous suspension in an amount such that the at least one XET enzyme stitches together various parts.

[0019] Another feature of the present invention is to stitch together cellulosic fibers, hemicellulose fibers, or a combination thereof, to form a modified fibrous pulp comprising stitched-together fibers.

[0020] Another feature of the present invention is to provide a reactive aqueous suspension comprising cellulose fibers, hemicellulose fibers, or a combination thereof, and at least one xyloglucan endotransglucosylase (XET) enzyme.

[0021] Another feature of the present invention is to provide a reaction product of a reactive aqueous suspension that comprises cellulose fibers, hemicellulose fibers, or a combination thereof, and at least one xyloglucan endotransglucosylase (XET) enzyme.

[0022] Additional features and advantages of the present invention will be set forth in part in the description that follows, and in part will be apparent from the description, or can be learned by practice of the present invention. The objectives and other advantages of the present invention will be realized and attained by means of the formulations, elements, and combinations particularly pointed out in the description and appended claims.

[0023] To achieve these and other advantages and in accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention, in part, relates to a method of increasing fiber surface sizing by enzymatically reacting a surface sizing agent with a fiber, for example, with a cellulosic fiber. It has been found, according to the present invention, that such methods of sizing can also impart increased fiber strength and fibrous product strength in a product made form the fiber.

[0024] The present invention further relates to methods whereby enzymes are used to achieve functionalization of the surface of a fiber through an enzymatic covalent linkage of a biopolymer, polymer, or other type of fiber treating or coating agent, to enhance the surface sizing of the fibers.

[0025] The present invention further relates to methods whereby fibers are treated with fiber treatment agents through reactions that are driven by enzymes rather than by traditional chemistry. Enzymes are considered an environmentally friendly and sustainable option as compared to AKD and ASA based chemistry.

[0026] It has been found that, according to the present invention, improved and permanent sizing, and improved fiber strength and fibrous product strength, can be obtained by reacting fibers in a fiber pulp with certain enzymes referred to herein as sizing enzymes.

[0027] It has been found that, according to the present invention, improved and permanent sizing, and improved fiber strength and fibrous product strength, can be obtained by reacting fibers in a fiber pulp with certain enzymes, such as alpha-glucan enzymes.

[0028] It has been found that, according to the present invention, improved and permanent sizing, and improved fiber strength and fibrous product strength, can be obtained by reacting fibers with sizing enzymes alone, with alpha-glucan enzymes alone, with sizing enzymes in combination with members of the family of xyloglucan endotransglucosylase / hydrolase (XET / XTH) enzymes, or with alpha-glucan enzymes in combination with members of the family of xyloglucan endotransglucosylase / hydrolase (XET / XTH) enzymes. Endotransglucosylase / hydrolase (XET / XTH) has demonstrated the ability to “stitch” two ends of a cellulose-like material together, covalently.

[0029] The present invention further relates to methods whereby enzymes are used to achieve increases in the strength of fibers by j oining together smaller pieces of fibers to form longer pieces and by building continual chains through functionalizing the surface of the fiber.

[0030] The present invention can be used to reinvigorate the strength of old corrugated containerboard and recycled fibers. The strength of fibers and products made from such materials is constantly dwindling due to repeated recycling that causes the fibers to shorten. The fiber sources are constantly recycled into short fines that do not contribute the same amount of strengthas freshly pulped virgin kraft from softwoods. The methods of the present invention can be used to reconstruct longer fibers from such shortened fibers, while imparting to the longer fibers strength like that observed in fibers of virgin kraft from softwoods.

[0031] The present invention further relates to methods whereby raw fibrous pulp, or a treated fibrous pulp, is treated with a combination of a sizing enzyme, an XTH enzyme, an XET enzyme, and a degrading enzyme such as a cellulase or hemicellulase, for example, a degrading enzyme that is known and useful for pulp and paper making applications.

[0032] The present invention further relates to methods whereby a fibrous pulp is treated sequentially with a first degrading enzyme, then with a second degrading enzyme, then with both a sizing enzyme and a stitching enzyme, to form a treated pulp for paper making applications.

[0033] The present invention further relates to a paper or paperboard product comprising paper or paperboard made from the treated fibers and pulp as described herein.

[0034] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide a further explanation of the present invention, as claimed.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 is a schematic diagram illustrating enzymatic sizing and stitching reactions between a biopolymer and cellulosic fibers, using an alpha-glucan enzyme and a stitching enzyme that differs from the alpha-glucan enzyme.

[0036] FIG. 2 is a schematic diagram illustrating enzymatic sizing and stitching reactions between a polymeric moiety treatment agent and cellulosic fibers, using only an alpha-glucan enzyme.

[0037] FIG. 3 is a schematic diagram illustrating enzymatic sizing and stitching reactions between a polymeric moiety treatment agent and cellulosic fibers, using an alpha-glucan enzyme and a stitching enzyme that differs from the alpha-glucan enzyme.

[0038] FIG. 4 is a schematic diagram illustrating enzymatic sizing and stitching reactions between a polymeric moiety treatment agent and cellulosic fibers, using an alpha-glucan enzyme and a stitching enzyme that differs from the alpha-glucan enzyme.

[0039] FIG. 5 is a schematic diagram illustrating sequential enzymatic reactions including the degradation of a cellulosic fiber using a hemi cellulase to form a degraded pulp, further degradation of the degraded pulp using a cellulase to form a treated pulp, and then sizing and stitching reactions between a polymeric moiety treatment agent and cellulosic fibers in the treated pulp, wherein the sizing and stitching reactions use an alpha-glucan enzyme and a stitching enzyme that differs from the alpha-glucan enzyme.

[0040] FIG. 6 is a schematic illustration of an open white-water system in which white water can be treated by an XET enzyme, in accordance with the present invention.

[0041] FIG. 7 is a schematic illustration of a closed white-water system in which white water can be treated by an XET enzyme, in accordance with the present invention.

[0042] FIG. 8 is a schematic illustration of an enzymatic reaction in accordance with the present invention, wherein an XET enzyme stitches together fibers to form stitched fibers.DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0043] A reactive fiber pulp is provided that comprises fibers (which are referred to at times as starting fibers), one or more polymers, and a sizing enzyme, for example, an alpha-glucan enzyme. The reactive fiber pulp can also comprise a stitching enzyme. The sizing enzyme andthe stitching enzyme can be the same enzyme. The stitching enzyme can be the same as, or different than, the sizing enzyme.

[0044] The fibers (e.g., starting fibers) can have glucose-terminated surface groups or end groups. The fibers can have one or more chemical end groups or terminated surface groups. The chemical end groups or terminated surface groups can be one or more of an aldehyde group, an aromatic group, a carboxyl group, a hydroxyl group, or a ketone group, or any combinations thereof. The fibers can be chemically modified to have other non-naturally occurring groups or end groups or chemical groups (e.g., one or more of an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group, or any combinations thereof). Different types of fibers can be used, where one type of fiber has one or more different chemical groups compared to a second type of fiber that can be optionally present (e.g., one fiber type can have an aldehyde group and a second type of fiber can have a carboxyl group). The fibers can be used in combination with other additional types of fibers, such as modified fibers and / or reactive fibers. The additional fibers can have one or more of an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group, or any combinations thereof.

[0045] The one or more polymers can have glucose-terminated end groups. The one or more polymers can have one or more chemical end groups or terminated surface groups. The chemical end groups or terminated surface groups can be one or more of an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group, or any combinations thereof. Different types of polymers can be used, where one type of polymer has one or more different chemical groups compared to a second type of polymer that can beoptionally present (e.g., one polymer type can have an aldehyde group and a second type of polymer can have a carboxyl group). The one or more polymers can be one or more sizing polymers, as that term is understood in the art. Examples include, but are not limited to, hydrophobic agents or synthetic hydrophobic agents like Alkyl Ketene Dimer (AKD) and Alkyl Succinic Anhydride (ASA) for internal sizing, and polymeric film-formers like polyvinyl alcohol (PVA), styrene-acrylate copolymers, and polyurethanes for surface sizing.

[0046] The sizing enzyme can be designed to enzymatically, covalently bond together a plurality of monomers, to form the one or more polymers.

[0047] The stitching enzyme can be configured to enzymatically, covalently bond the fibers to the one or more polymers. For instance, the fibers can be bonded to the one or more polymers at the glucose-terminated surface groups of the fibers and at the glucose-terminated end groups of the one or more polymers. The fibers can be bonded to the one or more polymers via one or more glycolytic bonds (e.g., a glycosylation reaction). The fibers can be bonded to the one or more polymers by the stitching enzyme such that the end group or terminated group of the fibers are bonded to the end group or terminated group of the one or more polymers (e.g., via an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group, or any combinations thereof.)

[0048] For purposes of the present invention, the raw source of the fibers utilized herein are sourced from paper-making pulps and / or paper-making pulp fibers. The pulp or pulp fibers utilized herein are not considered ‘dissolving pulps’ as that term is understood in the industry. The pulp or pulp fibers utilized herein preferably have a purity of cellulose of up to 90 wt% based on the weight of the pulp (e.g., from 50 wt% to 90 wt%, from 55 wt% to 90 wt%, from 60 wt% to 90 wt%, from 65 wt% to 90 wt%, from 70 wt% to 90 wt%, from 75 wt% to 90 wt%, from 75 wt% to 85 wt%, from 60 wt% to 85 wt% or any range based upon any two valuesdescribed herein.) The pulp or pulp fibers utilized herein preferably have a hemicellulose content of above 10 wt%, such as from 11 wt% to 25 wt%, or from 12 wt% to 25 wt%, or from 15 wt% to 25 wt%, or from 20 wt% to 25 wt%, or from 11 wt% to 20 wt%, or any range based upon any two values described herein. The pulp or pulp fibers utilized herein as a starting pulp fiber (e.g., precursor fibrous pulp, cellulosic fibers, hardwood fibers, softwood fibers, Kraft pulp, Kraft pulp fibers, fiber sources) is a fiber or pulp with these one or more characteristics.

[0049] For purposes of the present invention, the source of fibers utilized herein can be chemically treated pulp or pulp fibers (e.g., kraft treated, bleached treated and the like). The fibers can optionally be chemically modified and may have other chemical groups present that are not naturally present in the raw source of fiber. The fiber can be considered a cellulose fiber used in paper- or package-making.

[0050] The present invention, in parts, is directed to a reactive fiber pulp comprising: a) fibers (e.g., pulp fibers as described herein) having an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group or any combinations thereof; b) one or more polymers having an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group; c) a sizing enzyme that is designed to enzymatically, covalently bond monomers to form the one or more polymers; and a stitching enzyme configured to enzymatically, covalently bond the fibers to the one or more polymers (e.g., via glycosylation or via glycolytic bonding).

[0051] The sizing enzyme and the stitching enzyme can be different from one another. The sizing enzyme and stitching enzyme can be the same enzyme used for dual purposes (e.g., sizing and stitching).

[0052] The sizing enzyme and / or the stitching enzyme can be an XTH enzyme. The enzyme (e.g., XTH enzyme) can be from the genome of Arabidopsis thaliana, from the genome of Populus tremula, and / or an enzyme (e.g., alpha-glucan enzyme) from the genome Leuconostoc citreum or any combinations thereof.

[0053] The stitching enzyme can comprise a xyloglucan endotransglucosylase / hydrolase (XTH) enzyme. The stitching enzyme can comprise a xyloglucan endotransglucosylase / hydrolase (XTH) enzyme, a xyloglucan endotransglucosylase (XET) enzyme, a xyloglucan endohydrolase (XEH) enzyme, or a combination thereof.

[0054] The sizing enzyme and the stitching enzyme can be different enzymes and the stitching enzyme can comprise an XTH enzyme from a tree. The sizing enzyme and the stitching enzyme can be different enzymes and the stitching enzyme can comprise an XTH enzyme from the genome of Arabidopsis thaliana, an XTH enzyme from the genome of Populus tremula, an XET enzyme from Vigna angularis, or a combination thereof. The stitching enzyme can comprise, for example, AtXTH22, AtXTH3, PtXTH3, VaXET16, or the like, or a combination thereof.

[0055] The one or more polymers can be or include one or more biopolymers. The one or more biopolymers can comprise polycarbohydrates, for example, one or more polysaccharides. During an enzymatic reaction of the reactive fiber pulp, the stitching enzyme can enzymatically, covalently bond together: (1) the fibers; (2) one or more polymers; or (3) both the fibers and the one or more polymers.

[0056] The reactive fiber pulp can comprise the starting fibers in the form of short fibers (e.g., at least a portion thereof). Each of the short fibers can comprise a respective length, first and second ends that are opposite one another, and a cellulosic terminal end group at, at least one of the first and second ends. During an enzymatic reaction of the reactive fiber pulp, the stitching enzyme can enzymatically, covalently bond the cellulosic terminal end group of a firstone of the short fibers to the cellulosic terminal end group of a second one of the short fibers. The stitching enzyme can enzymatically, covalently bond together short fibers to form a longer fiber. The stitching enzyme can enzymatically, covalently bond together many short fibers to form a longer fiber, for example, three or more short fibers, four or more short fibers, five or more short fibers, ten or more short fibers, 100 or more short fibers, or the like.

[0057] The reactive fiber pulp can further comprise at least one degrading enzyme, for example, a hemicellulase, a cellulase, or both a hemicellulase and a cellulase. The reactive fiber pulp can comprise one or more of xylanase, mannanase, lipase, lignocellulase, endoglucosidase, and beta-glucosidase. The reactive fiber pulp can further comprise at least one degrading enzyme and short fibers.

[0058] The present invention also provides a reaction product resulting from an enzymatic reaction of a reactive fiber pulp as described herein. The reaction product can be a result of an enzymatic reaction of a reactive fiber pulp comprising the fibers and a cellulosic terminal end group at, at least one end thereof, and a stitching enzyme that enzymatically, covalently bonds the cellulosic terminal end group of a first one of the fibers to the cellulosic terminal end group of a second one of the fibers.

[0059] The present invention also provides a method of treating a fibrous pulp, for example, a fibrous pulp comprising the starting fibers, and the one or more polymers. The method can comprise combining, with the fibrous pulp, (i) a sizing enzyme, for example, an alpha-glucan enzyme, that is designed to enzymatically, covalently bond monomers together to form the one or more polymers, and (ii) a stitching enzyme configured to enzymatically, covalently bond the fibers to the one or more polymers. The combining can occur under conditions such that the stitching enzyme enzymatically, covalently bonds together the starting fibers to the one or more polymers The sizing enzyme and the stitching enzyme can be the same enzyme. The sizing enzyme and the stitching enzyme can be different enzymes.

[0060] A method is provided whereby a reactive fiber pulp, comprising short fibers (i.e., short starting fibers), a sizing enzyme, and a stitching enzyme, is enzymatically reacted. The short fibers can have glucose-terminated surface groups or the one or more of the chemical end groups or surface groups identified above.

[0061] Each of the short fibers can have a respective length, first and second ends that are opposite one another, and a cellulosic terminal end group at, at least one of the first and second ends. During the enzymatic reaction, the stitching enzyme enzymatically, covalently bonds the cellulosic terminal end group of a first one of the short fibers to the cellulosic terminal end group of a second one of the short fibers, to form a longer fiber. The method can comprise, prior to combining of the fibrous pulp with the sizing enzyme and the stitching enzyme, first forming the fibrous pulp.

[0062] Forming a fibrous pulp can comprise combining a first precursor fibrous pulp with a degradation enzyme, for example, a cellulase, a hemicellulase, or a combination thereof. The first precursor fibrous pulp can comprise cellulosic material. The first precursor fibrous pulp and the degradation enzyme can be combined under conditions resulting in the enzymatic degradation of the cellulosic material, to form the fibrous pulp. Prior to combining the fibrous pulp with the sizing enzyme and the stitching enzyme, the fibrous pulp can be washed or otherwise treated to remove at least some, or all, of the degradation enzyme.

[0063] The method can involve enzymatically reacting a fibrous pulp that comprises hemicellulose and cellulose. The method can comprise combining the fibrous pulp with a hemicellulase and a cellulase under conditions resulting in the hemicellulolysis and cellulolysis of the hemicellulose and cellulose, respectively. The combining of the fibrous pulp with the hemicellulase and the cellulase can occur before the fibrous pulp is combined with the sizing enzyme and the stitching enzyme. The method can comprise combining of the fibrous pulp with the hemicellulase and the cellulase at the same time that the fibrous pulp is combined withthe sizing enzyme and the stitching enzyme. The method can comprise combining the fibrous pulp with the hemicellulase and the cellulase after the fibrous pulp is combined with the sizing enzyme and the stitching enzyme.

[0064] Prior to the combining of the fibrous pulp with the sizing enzyme and the stitching enzyme, the method can first comprise forming the fibrous pulp. Forming the fibrous pulp can comprise combining a precursor fibrous pulp, comprising hemicellulose and cellulose, with a hemicellulase and a cellulase. The precursor fibrous pulp, the hemicellulase, and the cellulase can be combined under conditions resulting in the hemicellulolysis and cellulolysis of the hemicellulose and cellulose, respectively.

[0065] Forming the fibrous pulp can comprise combining a first precursor fibrous pulp with a hemicellulase, wherein the first precursor fibrous pulp comprises hemicellulose and the first precursor fibrous pulp and the hemicellulase are combined under conditions resulting in the hemicellulolysis of the hemicellulose. The result can be the formation of a second precursor fibrous pulp that comprises cellulose.

[0066] The method can then entail combining the second precursor fibrous pulp with a cellulase under conditions resulting in the cellulolysis of the cellulose, and the formation of a fibrous pulp. Prior to combining the second precursor fibrous pulp with the cellulase, the second precursor fibrous pulp can be washed or otherwise treated to remove at least some, or all, of the hemicellulase.

[0067] Prior to combining the fibrous pulp with the sizing enzyme and the stitching enzyme, the fibrous pulp can be washed or otherwise treated to remove at least some, or all, of the cellulase. Prior to the combining of the fibrous pulp with the sizing enzyme and the stitching enzyme, the fibrous pulp can be washed or otherwise treated to remove at least 90% (wt%) of the cellulase. The method can use a stitching enzyme that differs from the sizing enzyme, and the stitching enzyme can comprise xyloglucan endotransglucosylase / hydrolase.

[0068] The present invention also provides a method of improving wet strength of a fibrous product. The method can involve carrying out an enzymatic reaction of a reactive fiber pulp, as described herein, to form a treated fibrous pulp, and forming a fibrous product from the treated fibrous pulp. As a result of the method, the fibrous product can exhibit a wet strength, and the wet strength of the fibrous product can be greater than the wet strength of a fibrous product made from the same fibrous pulp but which has not been combined with the sizing enzyme and the stitching enzyme.

[0069] The present invention also provides a method of improving the strength of fibers in a fiber or fibrous pulp. The method can comprise carrying out an enzymatic reaction of a reactive fiber pulp, as described herein, to form a treated fibrous pulp that includes treated fibers The treated fibers can exhibit a strength and the strength of the treated fibers can be greater than the strength of fibers in the same fibrous pulp, but which have not been combined with the sizing enzyme and the stitching enzyme.

[0070] The present invention also provides a method of permanently sizing fibers in a fibrous pulp. The fibrous pulp can comprise the fibers as described herein (i.e., the starting fibers), and the one or more sizing polymers. The method can comprise combining, with the fibrous pulp, (i) a sizing enzyme that is designed to enzymatically, covalently bond monomers together to form the one or more polymers, and (ii) a stitching enzyme configured to enzymatically, covalently bond the fibers to the one or more sizing polymers (e.g., via a glycosylation reaction or via glycolytic bonding). The combining can be made to occur under conditions such that the stitching enzyme enzymatically, covalently bonds together the fibers to the one or more sizing polymers to form a treated fibrous pulp comprising permanently sized fibers.

[0071] According to the present invention, a paper or paperboard product is also provided and can comprise paper or paperboard that is made from a treated fibrous pulp producedaccording to one or more of the methods described herein. A paper or paperboard product is also provided that is made from the enzymatically reacted product of a reactive fiber pulp as described herein.

[0072] According to the present invention, a method of modifying a fibrous pulp is also provided and involves combining at least one xyloglucan endotransglucosylase (XET) enzyme with a fibrous pulp under temperature and pH conditions such that XET enzymatic activity occurs.

[0073] The fibrous pulp is sourced from paper-making pulps and / or paper-making pulp fibers. The pulp or pulp fibers utilized herein are not considered ‘dissolving pulps’ as that term is understood in the industry. The pulp or pulp fibers utilized herein preferably have a purity of cellulose of up to 90 wt% based on the weight of the pulp (eg., from 50 wt% to 90 wt%, from 55 wt% to 90 wt%, from 60 wt% to 90 wt%, from 65 wt% to 90 wt%, from 70 wt% to 90 wt%, from 75 wt% to 90 wt%, from 75 wt% to 85 wt%, from 60 wt% to 85 wt% or any range based upon any two values described herein.) The pulp or pulp fibers utilized herein preferably have a hemicellulose content of above 10 wt%, such as from 11 wt% to 25 wt%, or from 12 wt% to 25 wt%, or from 15 wt% to 25 wt%, or from 20 wt% to 25 wt%, or from 11 wt% to 20 wt%, or any range based upon any two values described herein. The pulp or pulp fibers utilized herein as a starting pulp fiber (e.g., precursor fibrous pulp, cellulosic fibers, hardwood fibers, softwood fibers, Kraft pulp, Kraft pulp fibers, fiber sources) is a fiber or pulp with these one or more characteristics.

[0074] The fibrous pulp can comprise an aqueous suspension of cellulosic fibers and hemicellulose fibers. The at least one XET enzyme can be included in the aqueous suspension in an amount such that the at least one XET enzyme stitches at least a portion of the cellulosic fibers together. The at least one XET enzyme can be included in the aqueous suspension in an amount such that the at least one XET enzyme stitches at least a portion of the hemicellulosefibers together. The at least one XET enzyme can be included in the aqueous suspension in an amount such that the at least one XET enzyme stitches at least a portion of the cellulosic fibers and at least a portion of the hemicellulose fibers together. The result is a modified fibrous pulp comprising stitched together fibers. The modified fibrous pulp can comprise an aqueous fibrous pulp useful for manufacturing a paper product.

[0075] The method can utilize an aqueous suspension that is a by-product of manufacturing a paper product. The aqueous suspension can comprise white water. The white water can be part of an open white-water system. The white water can be part of a closed white-water system. The method can further comprise recovering the stitched fibers.

[0076] The amount of the at least one XET enzyme can be from 0.01 kg / ton to 1 kg / ton based on the dried weight of the cellulosic and hemicellulose fibers in the aqueous suspension. The method can comprise maintaining the pH of the aqueous suspension to be from 2 to 8, for example, the pH of the aqueous suspension can be adjusted to be from 4 to 6. The method can involve buffering the aqueous suspension to an optimal pH range such that XET activity occurs. Buffering the aqueous suspension can comprise adding to the aqueous suspension at least one of a sodium acetate buffer, a phosphate buffer, and a citrate buffer. The temperature conditions of the aqueous suspension can include a temperature of from 10 °C to 75 °C, for example, a temperature of from 15 °C to 50 °C. This can comprise heating or cooling the aqueous suspension to attain an optimal temperature range for XET activity to occur.

[0077] The majority (by number) of the cellulosic fibers and the majority (by number) of the hemicellulose fibers can be fibers having lengths of greater than or equal to 0.5 mm. The majority of the cellulosic fibers and the majority of the hemicellulose fibers can be fines that are less than 0.5 mm in length. The cellulosic fibers and the hemicellulose fibers can comprise a combination of fines that are less than 0.5 mm in length and fibers that are greater than orequal to 0.5 mm in length. The majority (by number) of the resulting stitched fibers can be from 0.1 mm to 5 mm in length.

[0078] The method can further comprise combining modified oligosaccharides with the aqueous suspension. The modified oligosaccharides can be oligosaccharides bonded to 2-aminopyridine.

[0079] The XET enzyme can comprise an XTH enzyme from the genome of Arabidopsis thaliana, an XTH enzyme from the genome of Populus tremula, or a combination thereof. The XET enzyme can comprise AtXTH22, AtXTH3, PtXTH3, VaXET16, or a combination thereof.

[0080] A reactive aqueous suspension is also provided that comprises cellulosic fibers, hemicellulose fibers, and at least one xyloglucan endotransglucosylase (XET) enzyme. The at least one XET enzyme can be present in an amount such that, under enzymatic reaction conditions, the XET enzyme stitches together at least a portion of the cellulosic fibers. The at least one XET enzyme can be present in an amount such that, under enzymatic reaction conditions, the XET enzyme stitches together at least a portion of the hemicellulose fibers. The at least one XET enzyme can be present in an amount such that, under enzymatic reaction conditions, the XET enzyme stitches together at least a portion of the cellulosic fibers and a portion of the hemicellulose fibers.

[0081] The reactive aqueous suspension can be at a temperature and a pH at which XET activity occurs. The pH of the reactive aqueous suspension can be from 2 to 8, for example, a pH of from 4 to 6. The reactive aqueous suspension can be at a temperature of from 10 °C to 50 °C. The temperature can be from 20 °C to 40 °C.

[0082] In the reactive aqueous suspension, the cellulosic fibers and the hemicellulose fibers can be part of an aqueous fibrous pulp. The cellulosic fibers and the hemicellulose fibers can be components of white water. In the reactive aqueous suspension, the amount of the at least one XET enzyme can be from 0.01 kg / ton to 1 kg / ton based on the dried weight of the cellulosicand hemicellulose fibers in the reactive aqueous suspension. In the reactive aqueous suspension, the majority of the cellulosic fibers and the majority of the hemicellulose fibers can be fibers having lengths greater than or equal to 0.5 mm. In the reactive aqueous suspension, the majority (by number) of the cellulosic fibers and the majority (by number) of the hemicellulose fibers can be fines that are less than 0.5 mm in length. The cellulosic fibers and the hemicellulose fibers can comprise a combination of fines that are less than 0.5 mm in length and fibers that are greater than or equal to 0.5 mm in length.

[0083] The XET enzyme in the reactive aqueous suspension can comprise an XET enzyme from the genome of Arabidopsis thaliana, an XET enzyme from the genome of Populus tremula, an XET enzyme from Vigna angularis, or a combination thereof. The XET enzyme can comprise AtXTH22, AtXTH3, PtXTH3, VaXET16, or a combination thereof

[0084] A reaction product is also provided that results from an enzymatic reaction of the reactive aqueous suspension that comprises cellulosic fibers, hemicellulose fibers, and at least one xyloglucan endotransglucosylase (XET) enzyme. The reaction product can comprise stitched fibers having a length of from 0.1 mm to 5 mm.

[0085] Regarding the suspensions and methods comprising and using sizing enzymes, exemplary sizing enzymes can be those designed to convert sucrose into a polysaccharide. The sizing enzyme can be an alpha-glucan enzyme. The sizing enzyme can be a non-naturally occurring enzyme. The sizing enzyme can be created in a laboratory. The sizing enzyme can be synthetic. The alpha-glucan enzymes used in the production of the biopolymers of International Flavors & Fragrances Inc, of New York, New York, and DuPont Industrial Biosciences USA, LLC of Wilmington, Delaware, can be utilized. In an exemplary application, the enzymatic polymerization driven by the alpha-glucan enzymes can be designed to work at ambient temperature and pressure to convert aqueous solutions of plant-based sugar into a polysaccharide. The polysaccharide can then be separated. The sizing enzyme, for example,alpha-glucan enzyme, can enable an enzymatic polymerization process that can precisely control the linking together of sugar molecules, for example, glucose molecules.

[0086] The sizing enzyme and / or the stitching enzyme can be one or alpha-glucan enzymes. The enzyme(s) can be designed enzymatic biomaterials (DEB) enzymes such as those used in the production of Nuvolve®, AURIST AGC, and Lyrature biopolymers.

[0087] The present invention further relates to methods whereby enzymes are used to enzymatically treat fibers and comprise one or more enzymes as described in U.S. Patent Application Publication No. US 2016 / 0348035A1 to Berlin et al., and U.S. Patent Application Publication No. US 2016 / 0333292 Al to Berlin et al., both of which are incorporated herein in their entireties by reference.

[0088] According to the present invention, by using sizing enzymes, biomaterials with a wide range of polymer lengths and derivatization can be formed and covalently bonded to fibers, for example, in a fiber pulp for paper making. Examples of biopolymers or other biobased products created by alpha-glucan enzymes and that can be incorporated into the fibers of the fiber pulp include Nuvolve® (biopolymers for packaging), AURIST AGC (hair care product biopolymers), and Lyrature (bio-degradable detergent biopolymers), each of which is available from International Flavors & Fragrances Inc. NUVOLVE is a registered trademark of DuPont Industrial Biosciences USA, LLC of Wilmington, Delaware.

[0089] According to various embodiments, the sizing enzyme used according to the present invention can be an enzyme that naturally, or that has been designed to, covalently bond an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group terminated polymer or biopolymer to one or more aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate groups on the surface of the fiber, to createcovalent linkages via enzymatic processing. The glucose terminated polymers and / or biopolymers can be of a sustainable nature.

[0090] Examples of the alpha-glucan include, but are not limited to, beta 1-3, beta 1-4, beta 1-6, or combinations thereof. Stitching enzymes such as alpha-glucan enzymes, XET, XTH enzymes, can be used to facilitate sizing through the attachment of any polymer to the fiber. The XET enzymes described herein can be used as a sizing enzyme.

[0091] The use of any particular enzyme or type of enzyme, can be selected or designed based on the target treatment agent and / or polymeric moiety desired to be covalently attached to the fiber, and based on the fiber end groups and / or fiber surface groups of the fibers to which the target treatment agent and / or polymeric moiety is to be attached.

[0092] The sizing enzyme can be one or more enzymes. The sizing enzyme can be combination of enzymes. The sizing enzyme can be one or more of a hydrolase, an oxidoreductase, a lyase, a transferase, an isomerase, a ligase, and a glycosidase.

[0093] Exemplary hydrolases that can be used as the sizing enzyme include lipases, esterases, cellulases, and proteases. These enzymes can be used to catalyze the hydrolysis of chemical bonds, breaking down larger molecules into smaller ones. They are often used to create biodegradable materials and modify surface properties.

[0094] Exemplary oxidoreductases include laccases, peroxidases, and tyrosinases. One or more oxidoreductase can be used to catalyze oxidation-reduction reactions, for cross-linking, and / or for modifying polymers to enhance mechanical properties.

[0095] Exemplary lyases that can be used include deoxyribose-phosphate aldolase. One or more lyases can be used to catalyze the breaking of various chemical bonds by means other than hydrolysis and oxidation, and for forming new structures.

[0096] Exemplary transferases that can be used include glycosyltransferases and transaminases. One or more transferases can be used to catalyze the transfer of functionalgroups from one molecule to another. These enzymes can be used to modify a material to add a specific functional group.

[0097] Exemplary isomerases that can be used include glucose isomerase and protein disulfide isomerase. One or more isomerases can be used to catalyze the rearrangement of atoms within a molecule and to modify the internal structure of a material without altering its overall composition. In papermaking, this can lead to changes in the physical properties of the material, such as its crystallinity or thermal stability.

[0098] Exemplary ligases that can be used include DNA ligase and protein ligase. One or more ligases can be used to catalyze the joining together of two molecules, to create polymers, and to cross-linking materials.

[0099] Exemplary glycosidases that can be used include chitosanase and amylase. One or more glycosidases can be used to break down glycosidic bonds in carbohydrates and to create biomaterials that degrade in response to biological signals.

[0100] Enzymes such as laccases or peroxidases can be used to create textiles with improved dye fixation, durability, or self-cleaning properties. Enzymes such as esterases can be used for biodegradable packaging materials.

[0101] According to various embodiments, longer chained glucose terminated biopolymers can be combined with one or more glucose-terminated ends of one or more fiber source to create covalent linkages via enzymatic processing. The longer chained glucose terminated biopolymers can be of a sustainable nature.

[0102] According to various embodiments, shorter chained glucose terminated biopolymers can be combined with one or more glucose-terminated ends of one or more fiber sources to create covalent linkages via enzymatic processing. The shorter chained glucose terminated biopolymers can be of a sustainable nature.

[0103] The present methods can involve utilizing the mechanisms of a degrading enzyme, such as a cellulase or a hemicellulase, together with a sizing enzyme and / or an XTH enzyme. Exemplary cellulase enzymes include those that can be used in the production of biofuel, cellulases that break down cellulose into glucose for use in producing ethanol, and FIBERCARE U cellulase enzymes available from Novozymes A / S, part of Novonesis Group, of Bagsvaerd, Denmark. FIBERCARE is a registered trademark of Novozymes A / S.

[0104] Other exemplary cellulase enzymes include those that can be used in the production of pulp and paper and that create more exposed fiber surface area with more glucose-terminated functionality. Cellulase enzymes can be used that have reactive functionality that is coordinated with a sizing enzyme and / or with an XTH enzyme to provide more surface-active sites available for further enzymatic linkages. Processing with degrading enzymes can comprise one or more rinsing steps, one or more washing steps, diluting steps, temperature treatment steps, or the like. The further processing can be used to reduce, or eliminate, unwanted reactions.

[0105] The compositional ratios of the pulp constituents can be modified to provide, for example, various amounts of sizing agent. The compositional ratios of the pulp constituents can be modified to provide, for example, various degrees of hydrophobicity. The order of processing steps can be selected or modified to provide a desired outcome, for example, the order of enzymatic reactions. Any useful order can be used, and the enzymatic treatments can be simultaneous or sequential, as desired. Treatment steps can include, or consist of, treatment with a sizing enzyme, an XTH enzyme, a cellulase enzyme, a xylanase enzyme, a mannanase enzyme, a lignocellulase enzyme, a lipase enzyme, or any combination thereof. The treatment step can result in a covalent bond, a permanent attachment of a treatment agent, and / or a modification to the surface of a fiber.

[0106] In an example, the method can involve first treating fibers in a fiber pulp with a high concentration of xylanase and mannanase to break down hemicelluloses and to provide amore amorphous or crystalline cellulose. The xylanase and mannanase can be added or combined in the presence of one or more stitching enzymes to covalently attach ends of shorter fibers together, enabling a stronger paper product made from the fiber pulp.

[0107] In another example, the method can involve first treating fibers in a fiber pulp with a high concentration of cellulase, for example, consisting of or including beta-glucosidase, to drive the enzymatic reaction from cellulose to glucose-based sugars. This can be carried out in the presence of, or prior to exposure to, one or more stitching enzymes. The method can increase the rate at which longer chained fibers are created. The method can involve simultaneously eliminating the cellulase or beta-glucosidase enzymes that break down fibers.

[0108] In cases where the fiber source is such that it is extremely difficult for enzymes to gain access to the surface, a combination of different enzymes can be used. A combination of different enzymes can be used, for example, when the fiber source includes old corrugated containerboard (OCC) and / or cardboard boxes. The combination can include lipase, lignocellulose, a combination thereof, or the like. The combination can include one or more stitching enzymes, for example, to enable stitching of shorter chained fibers, to enable the creation of favorable chemical moieties for covalent attachment, or a combination thereof.

[0109] A combination including an XTH enzyme and a sizing enzyme can be used, for example, in applications for improving surface sizing, improving internal sizing, or both. The combination can act to attach or randomly attach functionalized chemical moieties. The functionalized chemical moieties can be attached within shorter fragments of fiber, to the ends of shorter fragments of fiber, and / or together on the surface of the fibers.

[0110] The present invention provides a combination of enzymatic treatments carried out in an order to achieve increases in fiber strength and sizing, by joining smaller pieces of fiber together into longer pieces and functionalizing the surface of the fiber with enzymatic covalent linkages to a treatment agent. The treatment agent can be, for example, a sizing agent. Thetreatment agent can comprise, for example, a biopolymer, a polymer, a sizing agent, or a treatment agent to enhance the surface sizing of the fibers. The treatment can be driven by enzymes rather than through the use of traditional chemistry.[OHl] The combined enzymatic reactions can be used with hardwood bleached fibers, hardwood unbleached fibers, or the like, which do not yield much strength but can contribute to other desirable properties. The resultant fibers and products made therefrom can be used to form an environmentally friendly bottle instead of wasting short fibers.

[0112] The combined enzymatic reactions can be used with hardwood short fiber sources, into a strength producing fiber source, by using a combination of a sizing enzyme, for example, an alpha-glucan enzyme, and an XTH enzyme to lengthen short hardwood fibers into longer hardwood fibers. This enables hardwoods, for example, eucalyptus, oak, and others, to be used as packaging material. The methods can use fibers from eucalyptus trees, for more sustainable production. Eucalyptus trees can favorable be used because they grow fast and grow well within densely packed areas. Eucalyptus trees are thus sustainable and implementing them in the present invention increases the use of sustainable fiber sources.

[0113] The present invention provides many advantages. Different fiber sources can be used together. Fibers from a softwood and fibers from a hardwood can be combined together. Papermakers can custom products to have a desired strength and feel. Products can be made, for example, having strong fibers yet a soft feel on the surface. The porosity of resultant products can be customized.

[0114] Modifications of the surface and internal aspects of a fiber can be modified easily through covalent interactions with an enzyme or combination of enzymes, and oligosaccharide-based chemical moieties.

[0115] The present invention further relates to a reactive aqueous suspension. The reactive aqueous suspension includes cellulosic fibers, hemicellulose fibers, and at least one xyloglucanendotransglucosylase (XET) enzyme. The at least one XET enzyme is present in an amount such that, under enzymatic reaction conditions, the XET enzyme stitches together at least a portion of the cellulosic fibers, stitches together at least a portion of the hemicellulose fibers, stitches together at least a portion of the cellulosic fibers and a portion of the hemicellulose fibers, or a combination thereof.

[0116] The present invention further relates to a method of modifying a fibrous pulp. The method includes the step of combining at least one XET enzyme with a fibrous pulp under temperature and pH conditions such that XET enzymatic activity occurs. The fibrous pulp can comprise an aqueous suspension of cellulosic fibers and hemicellulose fibers. The at least one XET enzyme is included in the aqueous suspension in an amount such that the at least one XET enzyme stitches together at least a portion of the cellulosic fibers, stitches together at least a portion of the hemicellulose fibers, stitches together at least a portion of the cellulosic fibers and at least a portion of the hemicellulose fibers, or a combination thereof, to form a modified fibrous pulp comprising stitched fibers.

[0117] The phrase “cellulosic fibers” is defined herein as fibers that are primarily derived from cellulose. Cellulosic fibers can be obtained from wood, cotton, hemp, recycled paper, and the like. The phrase “hemicellulose fibers” are defined herein as fibers that are heteropolysaccharides formed by more than one type of monomer, such as five or six monomeric residues. Examples of hemicellulose fibers include glucose, galactose, xylose, arabinose, mannose, glucuronic acid, and the like. Hemicellulose fibers are embedded in the cell walls of plants and bind with pectin to cellulose to form a network of cross-linked fibers. The phrase “aqueous suspension” is defined herein as a suspension of cellulosic fibers, hemicellulose fibers, and water or other liquids.

[0118] The at least one XET enzyme is an enzyme that is part of XTH (Xyloglucan Endotransglucosylase / Hydrolase). In nature, XET enzymes play an important role inremodeling of plant cell walls. XET enzymes are particularly involved in the endotransglucosylation process, where the enzyme catalyzes the cutting and rejoining of xyloglucan molecules. The xyloglucan molecule can be or comprise a hemicellulosic polysaccharide that interacts with cellulose microfibrils in the cell wall. XET enzymes do not exhibit hydrolase activity, meaning the enzyme does not break down xyloglucan into smaller sugar units.

[0119] The present invention can use any XET enzyme that possesses suitable enzyme activity at a pH and temperature. The source of the XET enzyme used is not critical in the present invention. Accordingly, the XET enzyme can be obtained from any source such as a plant source, a microorganism source, or an animal source.

[0120] As an option, the XET enzyme can be obtained from a plant source. The XET enzyme can be obtained from cotyledons of the Brassicaceae family, such as from the genus Arabidopsis, and in particular, Arabidopsis thaliana. As an option, the XET enzyme can be obtained from cotyledons of the Salicaceae family, such as from the genus Populus in particular, Populus termula. As an option, the XET enzyme can be obtained from cotyledons of the Fabaceae family, such as from the genus Phaseolus, in particular, Phaseolus aureus. As an option, the XET enzyme can be obtained from cotyledons of the Fabaceae family, such as from the genus Vigna, in particular, Vigna angularis. The monocotyledons can be non-graminaceous monocotyledons and liliaceous monocotyledons.

[0121] The XET enzymes can also be extracted from moss and liverwort, as described in Fry et al., 1992, Biochem. J. 282: 821-828, which is incorporated herein by reference in its entirety. For example, the XET enzymes can be obtained from cotyledons, i.e., a dicotyledon or a monocotyledon, in particular a dicotyledon selected from the group consisting of azuki beans, canola, cauliflowers, cotton, poplar or hybrid aspen, potatoes, rapes, soybeans, sunflowers, thalecress, tobacco, and tomatoes, or a monocotyledon selected from the groupconsisting of wheat, rice, com, and sugar cane. See, for example, WO 2003 / 033813 and WO 97 / 23683, which are incorporated herein by reference in their entirety.

[0122] As an option, the XET enzyme can be derived from Carica papaya, Cucumis sativus, Daucus carota, Festuca pratensis, Glycine max, Hordeum vulgare, Lycopersicon esculentum, Medicago truncatula, Oryza sativa, Sagittaria pygmaea, Sorghum bicolor, Vigna angularis, Zea mays, or the like.

[0123] The XET enzyme can be extracted from plants. Suitable methods for extracting the XET enzyme from plants are described Fry et al., 1992, Biochem. J. 282: 821-828; Sulova et al., 1998, Biochem. J. 330: 1475-1480; Sulova et al., 1995, Anal. Biochem. 229: 80-85; WO 95 / 13384; WO 97 / 23683; or EP 562836, each of which are incorporated herein in their entirety.

[0124] The XET enzyme can include AtXTH22, AtXTH3 , PtXTH3 , VaXET 16, any of the XET enzymes derived from the plants listed herein as well as plants, microorganisms, or animals not listed herein, or any combination thereof.

[0125] The XET enzyme can also be produced by cultivation of a transformed host organism containing the appropriate genetic information from a plant, microorganism, or animal. Transformants can be prepared and cultivated by methods known in the art.

[0126] As an option, a nucleic acid construct can be constructed to comprise a gene encoding the XET enzyme operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. The gene can be manipulated in a variety of ways to provide for expression of the XET enzyme. Techniques for preparing the nucleic acid construct are described in U.S. Patent Application Publication No. US20160348035A1 to Berlin et al. and in U.S. Patent Application Publication No. US20160333292A1 to Berlin et al., both of which publications are incorporated herein by reference in their entireties.

[0127] As described herein, the at least one XET enzyme is included in the aqueous suspension in an amount such that the at least one XET enzyme stitches at least a portion of the cellulosic fibers together, stitches at least a portion of the hemicellulose fibers together, stitches at least a portion of the cellulosic fibers and at least a portion of the hemicellulose fibers together, or a combination thereof, to form a modified fibrous pulp having stitched fibers.

[0128] The XET enzyme can be prepared as an aqueous solution prior to combining the XET enzyme with the aqueous fibrous pulp. The aqueous solution can include the XET enzyme mixed with a fluid medium, such as water. The XET enzyme and the fluid medium can be mixed together using conventional mixing techniques, such as a mixer, blender, stirrer, and / or an open vessel.

[0129] The XET enzyme aqueous solution can be prepared as masterbatches for dilution at a later time or the desirable concentration can be made at the same time that the composition is prepared. The XET enzyme aqueous solution can be prepared on-site or off-site. The XET enzyme aqueous solution can be formed immediately prior to the introduction into the papermaking process or sheet making process, or the compositions can be prepared beforehand, such as before use, minutes before use, hours before use, or days or weeks or months before use, for example, within about 2 to 3 weeks of usage.

[0130] The XET enzyme can be added to the aqueous fibrous pulp in an amount, for example, of from 0.01 kg / ton to 1 kg / ton, from 0.05 kg / ton to 0.95 kg / ton, from 0.10 kg / ton to 0.90 kg / ton, from 0.15 kg / ton to 0.85 kg / ton, from 0.20 kg / ton to 0.80 kg / ton, from 0.25 kg / ton to 0.75 kg / ton, from 0.30 kg / ton to 0.70 kg / ton, from 0.35 kg / ton to 0.65 kg / ton, from 0.40 kg / ton to 0.60 kg / ton, from 0.50 kg / ton to 0.55 kg / ton, based on the dried weight of the cellulosic and hemicellulose fibers in the aqueous suspension, or any range based upon any two values described herein.

[0131] The pH and / or the temperature of the combined XET enzyme and aqueous fibrous pulp can be controlled to activate the XET enzymatic activity and to deactivate the XET enzymatic activity. The pH and / or temperature required for XET enzymatic activity can differ between different XET enzymes. The pH and / or temperature that is optimal for the XET enzyme to stitch cellulose to cellulose, or to stitch cellulose to hemicellulose, or to stitch hemicellulose to hemicellulose, can differ. The pH and / or temperature of the combined XET enzyme and aqueous fibrous pulp can be adjusted based on a desired outcome of stitched fibers. For example, the XET enzyme and aqueous fibrous pulp combination can initially be at a pH and temperature that is optimal for the XET enzyme to stitch cellulose to cellulose. After a period of time, the pH and temperature can be adjusted to be optimal for the XET enzyme to stitch cellulose to hemicellulose, or the like. Accordingly, the aqueous suspension can be adjusted to an optimal pH and / or temperature, or to an optimal pH range and / or temperature range, such that a specific XET activity occurs. The specific XET activity can include a cellulose-cellulose bonding activity, a cellulose-hemicellulose bonding activity, a hemicellulose-hemicellulose bonding activity, or a combination thereof. The pH and / or temperature adjustment can affect the specific XET activity of one XET enzyme. If more than one XET enzyme is used, the pH and / or temperature adjustment can be used to activate a first of the XET enzymes while not activating a second of the XET enzymes, enabling a specific XET activity to occur. For example, the XET enzyme(s) and aqueous fibrous pulp combination can initially be at a pH and / or temperature that is optimal for the first XET enzyme to stitch cellulose to cellulose. After a period of time, the pH and / or temperature can be adjusted to be optimal for the second XET enzyme to stitch cellulose to hemicellulose, or to stitch hemicellulose to hemicellulose, or the like. If more than one XET enzyme is used, the pH and / or temperature adjustment can be used to activate a first and a second of the XET enzymes,or to deactivate a first and a second of the XET enzymes, thus enabling a specific sequence of XET activities to occur.

[0132] The pH of the combined XET enzyme and aqueous fibrous pulp for optimal XET activity to occur can be from 2.0 to 8.0, from 3.0 to 7.0, from 4.0 to 6.0, from 5.0 to 5.5, or any range based upon any two values described herein. The pH of the combined XET enzyme and aqueous fibrous pulp can be adjusted by buffering the combination with at least one of a sodium acetate buffer, a phosphate buffer, a citrate buffer, or the like.

[0133] The temperature of the combined XET enzyme and aqueous fibrous pulp for optimal XET activity to occur can be from 10 °C to 75 °C, from 15 °C to 70 °C, from 20 °C to 65 °C, from 25 °C to 60 °C, from 30 °C to 55 °C, from 35 °C to 50 °C, from 40 °C to 45 °C, or any range based upon any two values described herein. The temperature of the combined XET enzyme and aqueous fibrous pulp can be adjusted by heating or cooling methods that are known in the art.

[0134] As an option, the XET enzyme can be added to an aqueous fibrous pulp used for manufacturing a paper product. The XET enzyme is added to the aqueous fibrous pulp to form a modified fibrous pulp having the stitched fibers. The modified fibrous pulp can be further processed and formed into a paper or paperboard having a dry strength greater than paper or paperboard formed from an aqueous fibrous pulp without the stitched fibers.

[0135] The present invention can be practiced on conventional papermaking machines with modifications that can be easily made in view of the present invention. The present invention can be practiced, for example, on a wet end assembly of a conventional papermaking machine with modifications that can be easily made in view of the present invention. The present invention can employ many different types of papermaking pulp or combinations thereof.

[0136] The aqueous fibrous pulp can be treated with the XET enzyme at any location in the papermaking system before formation of the paperweb on the wire, e.g., an addition pointprior to the headbox in the system. The methods of the present invention can be practiced on any pulp related applications, including, for example, where pulps are treated and dewatered. The methods can be practiced, for example, on conventional paper making machines (such as a Fourdrinier type paper machine), for example, on wet end assemblies of paper making machines, with modifications that can be made in view of the present invention. On the wet end, a paper machine can include, for example, a pulp tank, a blend chest, a stuff box, a white water silo, a fan pump, a screen, and a head box. The paper machine can optionally include one or more refiners. The XET enzyme can be added to the aqueous fibrous pulp at or between any of these machine components. The XET enzyme can be added to the aqueous fibrous pulp at a suitable point that allows sufficient time for the enzyme to act on the pulp fibers.

[0137] In general, the present application is useful in (and e g., the XET enzyme can be added to) many different types of papermaking pulp, stock, or combinations of pulps or stocks. For example, the pulp can contain virgin pulp and / or recycled pulp, such as virgin sulfite pulp, broke pulp, Kraft pulp, soda pulp, thermomechanical pulp (TMP), alkaline peroxide mechanical pulp (APMP), chemithermomechanical pulp (CTMP), chemimechanical pulp (CMP), groundwood pulp (GP), mixtures of such pulps, and the like. The Kraft pulp can be, for example, a hardwood kraft pulp, a softwood kraft pulp, or combinations thereof. The recycled pulp can be or include wastepaper, old corrugated containers (OCC), and other used paper products and materials. For example, there are a variety of mechanical pulping methods to which this invention can be applied. Thermomechanical pulp (TMP) uses a combination of heated wood chips and mechanical processes. Stone Groundwood (SGW) grinds or macerates the wood chips. Chemithermomechanical pulp (CTMP) uses a variety of chemicals, heat, and grinding techniques to produce pulp. Different types of pulp are used to produce different types of paper, although many papers can use a combination or “blend” of several different types of pulp including or not including recycled / recovered paper.

[0138] Any desired paper product can be produced, for example, 130, 165, and 190 g / m2Kraft UM paper. 117MI, 117NO, 150UH, 165UH, 175UH, and 190UH papers can be produced. Paper and paperboard made using the methods of the present invention are marked by their enhanced dry strength. Dry strength can be measured using any suitable technique and apparatus. For example, a ring crush test (RCT) and / or a corrugating medium test (CMT) can be employed. Industry protocols that can be used include, for example, Ring Crush of Paperboard (TAPPI T818 cm-97), Ring Crush of Paperboard (Rigid Support Method (TAPPI T822), Short Span Compressive Strength of Paperboard (TAPPI T826), and Flat Crush of Corrugating Medium (CMT test) (T809 om-93). Examples of other strength tests include, for example, an internal bond test (Scott bond test), a burst test (Mullen test), a dry or wet tensile test, and the like. The use of stitched fibers can increase dry strength by at least 1.0%, at least 5.0%, at least 8.0%, at least 10%, at least 12%, at least 15%, at least 20%, at least 25%, or at least 50% compared to the absence of the stitched fibers.

[0139] The present invention can be used to recycle fibers and fines. For example, the aqueous suspension can be a by-product of manufacturing a paper product, such as white water. In the papermaking process, "white water" refers to the water that is drained from the paper pulp during the sheet-forming process on the paper machine. White water contains fine fibers (fines), fillers, and other additives that do not get incorporated into the paper sheet and are instead carried away with the water. When the aqueous suspension is white water, the present invention includes a step of recovering the stitched fibers from the modified fibrous pulp. The recovered stitched fibers can then be recycled by incorporating the recovered stitched fibers in an aqueous fibrous pulp useful for manufacturing a paper product, as described herein.

[0140] With reference to the drawings, FIG. 1 is a schematic diagram illustrating enzymatic sizing and stitching reactions between a biopolymer 104 and cellulosic fibers 106, using an alphaglucan enzyme 102 and a stitching enzyme 108 that differs from alpha-glucan enzyme 102,according to various embodiments of the present invention. In the reaction depicted, biopolymer 104 can be, for example, an oligosaccharide with a hydrophobic end group that is enzymatically, covalently bonded to the surface of fiber 106. An exemplary biopolymer is the Nuvolve® biopolymer of International Flavors & Fragrances Inc and DuPont Industrial Biosciences USA, LLC. Simultaneously with the surface attachment, stitching enzyme 108 stitches together different sections or lengths of fiber 106. A treated, elongated fiber results. A different sizing enzyme can be used in place of the alpha-glucan enzyme.

[0141] FIG. 2 is a schematic diagram illustrating enzymatic sizing and stitching reactions between a polymeric moiety 204 and cellulosic fibers 206, using only alpha-glucan enzyme 202. The same alpha-glucan enzyme 202 is used for stitching together ends of short fibers 206 to form longer ones, and for surface treating fibers 206 by effecting the enzymatic covalent attachment of the polymeric moiety treatment agent 204 onto surfaces of fibers 206. The polymeric moiety treatment agent can be a sizing agent, a biopolymer, a polymer, a hydrophobic agent, or the like. The treatment agent can be any type of polymeric moiety exhibiting a desirable functionalization and that can be attached by alpha-glucan enzyme 202. Alpha-glucan enzyme 202 can be a designed enzymatic biomaterials enzyme used to create the biopolymers of International Flavors & Fragrances Inc. A different sizing enzyme can be used in place of the alpha-glucan enzyme.

[0142] FIG. 3 is a schematic diagram illustrating enzymatic sizing and stitching reactions between a polymeric moiety treatment agent 304 and cellulosic fibers 306, using alpha-glucan enzyme 302 and a stitching enzyme 308 that differs from alpha-glucan enzyme 302. The same alpha-glucan enzyme 302 can be used for stitching together ends of short fibers 306 to form longer ones, and for surface treating fibers 306 by effecting the enzymatic covalent attachment of treatment agent 304 onto surfaces of fibers 306. The same stitching enzyme 308 can be used for stitching together ends of short fibers 306 to form longer ones, and for surface treating fibers 306 by effecting the enzymatic covalent attachment of treatment agent 304 onto surfaces of fibers 306.Polymeric moiety treatment agent 304 can be a sizing agent, a biopolymer, a polymer, a hydrophobic agent, or the like. The treatment agent can be any type of polymeric moiety exhibiting a desirable functionalization and that can be reacted by, that is, utilized by, alpha-glucan enzyme 302, stitching enzyme 308, or both. A different sizing enzyme can be used in place of the alpha-glucan enzyme.

[0143] As shown in FIG. 3, at 312, the fibers to be stitched together can already be sized or otherwise have and agent such as treatment agent 304 attached thereto, before they are stitched together. Arrow 320 points to a stitching enzyme reaction using a stitching enzyme 308 and stitching a fiber that already has a treatment agent 304 attached thereto.

[0144] FIG. 4 is a schematic diagram illustrating enzymatic sizing and stitching reactions between a polymeric moiety treatment agent 404 and cellulosic fibers 406, using alpha-glucan enzyme 402 and a stitching enzyme 408 that differs from alpha-glucan enzyme 402. The reactive mixture depicted also comprises a cellulase enzyme 430 and a hemicellulase enzyme 440. Degradation of fibers 406 occurs simultaneously with the stitching together of, and surface treatment of, fibers 406. Compositional ratios, washing, rinsing, and / or other treatments or treatment cycles can be used to control the reaction. Cellulase enzyme 430 can be, for example, an endo-glucosidase, a beta-glucosidase, or the like. Hemicellulase enzyme 440 can be, for example, xylanase, mannanase, or the like. Polymeric moiety treatment agent 404 can be, for example, a sizing agent, a biopolymer, a polymer, a hydrophobic agent, or the like. The treatment agent can be any type of polymeric moiety exhibiting a desirable functionalization and that can be reacted by, that is, utilized by, alpha-glucan enzyme 402, stitching enzyme 408, or both. A different sizing enzyme can be used in place of the alpha-glucan enzyme.

[0145] FIG. 5 is a schematic diagram illustrating sequential enzymatic reactions. To the left of the figure, the diagram depicts the degradation of cellulosic fibers 506 using a hemicellulase 540 to form a degraded pulp. In a next step, as shown at the end of the first arrow, the degradedpulp including fibers 506 is then degraded further with a cellulase 530 to form a treated pulp. Subsequently, as shown at the end of the second arrow and to the right of the diagram, sizing and stitching reactions take place between a polymeric moiety treatment agent 504 and cellulosic fibers 506 in the treated pulp. The sizing and stitching reactions use alpha-glucan enzyme 502 and a stitching enzyme 508 that differs from alpha-glucan enzyme 502. Washing, rinsing, neutralizing, or other treatment steps can be taken between the reaction steps depicted. Polymeric moiety treatment agent 504 can be, for example, a sizing agent, a biopolymer, a polymer, a hydrophobic agent, or the like. The treatment agent can be any type of polymeric moiety exhibiting a desirable functionalization and that can be reacted by, that is, utilized by, alpha-glucan enzyme 502, stitching enzyme 508, or both. A different sizing enzyme can be used in place of the alpha-glucan enzyme.

[0146] FIG. 6 illustrates a method for treating white water with an XET enzyme, wherein the white water can be part of an open white-water system 600. An alpha-glucan enzyme can be omitted and / or a different sizing enzyme can be used, in the method depicted. An open white-water system in the papermaking process refers to a system where the white water drained from the pulp during the paper formation stage is not fully recycled or reused within the paper mill. This system results in significant water consumption in paper mill as the water is used once and discharged as an effluent. This approach leads to high water consumption and is most common for producing printing and writing papers. For open-white water system 600, white water 602 having fines 604 is stored in a tank 606. XET enzyme 608 is then added to tank 606. White water 602 and XET enzyme 608 are brought to an optimal pH and temperature for XET enzymatic activity to occur. Fines 604 are then stitched together by the XET enzyme 608 to produce stitched fibers 610 within white water 602. White water 602 and XET enzyme 608 can be stored in tank 606 for a prolonged period of time, such as weeks or months. Stitched fibers 610 can be collected from the tank 606 and added as broke to an aqueous fibrous pulp mixture.

[0147] The white water treated by the XET enzyme can be part of a closed white-water system 700, as illustrated in FIG. 7. An alpha-glucan enzyme can be omitted and / or a different sizing enzyme can be used, in the method depicted. Closed-water systems continuously recirculate water during the process of papermaking and thus reduce freshwater consumption and minimize wastewater discharge. Closed-water systems typically have less chemical consumption, lower losses of fiber, fines, and fillers, and a reduced cost of heating white water. This type of system is often used in board or packaging paper grades. Closed white-water system 700 can include a pulper (not shown), a fourdrinier tray 702 carrying short fines, and a tank 704 that contains an aqueous solution including the XET enzyme 706. Short fines of the white water are delivered from fourdrinier tray 702 to the tank 704, where fines are stitched together by the XET enzyme 708 to produce stitched fibers. Stitched fibers are then reintroduced to the pulper. Closed white-water system 700, where the water is continuously recycled, can continuously inhabit enzyme and can acquire increased length of fibers present as well as potential strength to the paper. The continuous presence of enzyme in the system, unless denatured, can reduce expense as well as continuously stitch fibers together, resulting in a strengthened paper product.

[0148] Prior to stitching, the aqueous fibrous pulp used for manufacturing a paper product, as described herein, can include a majority of the cellulosic fibers and a majority of the hemicellulose fibers having lengths of greater than or equal to 0.5 mm. Prior to stitching, the white water can include a majority of the cellulosic fibers and the majority of the hemicellulose fibers that are fines less than 0.5 mm in length. Prior to stitching, the aqueous fibrous pulp can include cellulosic fibers and hemicellulose fibers that are a combination of fines less than 0.5 mm in length and fibers that are greater than or equal to 0.5 mm in length.

[0149] As illustrated in FIG. 8, XET enzyme 800 stitches fibers 802 together to form stitched fibers 804. An alpha-glucan enzyme can be omitted and / or a different sizing enzymecan be used. The length of the stitched fibers can be from 0.1 mm to 5 mm, from 0.5 mm to 4.5 mm, from 1.0 mm to 4.0 mm, from 1.5 mm to 3.5 mm, from 2.0 mm to 3.0 mm, from 2.2 mm to 2.8 mm, from 2.4 mm to 2.6 mm, from 2.5 mm to 2.7 mm, from 2.6 mm to 2.9 mm, from 2.8 mm to 3.2 mm, or any range based upon any two values described herein.

[0150] The present invention can also be utilized for combining chemically modified substrates that can aid in analysis of the kind of oligosaccharides present in the aqueous fibrous pulp. In order to confirm the activity of the XET enzymes, oligosaccharides (2-6 sugar residues) can be coupled with 2-aminopyridine. The resulting derivative is fluorescent and carries a positive charge that can be detected using reverse phase High-Performance Liquid Chromatography (HPLC). Detection of these compounds is sensitive and has a lower limit of 0.003 Pico moles. The modified substrate is then added to the aqueous fibrous pulp along with XET enzymes, which results in the fibers of the aqueous fibrous pulp being stitched to the modified substrate with 2-aminopyridine. This modification can aid in determining the type of substrate present in the aqueous fibrous pulp. For example, a modified cello-oligosaccharide carrying 2-aminopyridine can bind to cellulose and can provide information on whether the aqueous fibrous pulp contains cellulose or not.

[0151] The present invention includes the following aspects / embodiments / features in any order and / or in any combination:1. The present invention relates, in part, to a reactive fiber pulp comprising: a) fibers having one or more of an aldehyde group, an aromatic group, a carboxyl group, a hydroxyl group, or a ketone group, present as surface terminated groups or end groups, and optionally one or more of an amino group, a halogen group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group; b) one or more polymers having an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group or any combinations thereof; c) a sizingenzyme that is designed to enzymatically, covalently bond monomers to form the one or more polymers; and a stitching enzyme configured to enzymatically, covalently bond the fibers to the one or more polymers.2. The reactive fiber pulp of any preceding or following embodiment / feature / aspect, wherein the sizing enzyme is an alpha-glucan enzyme, and the alpha-glucan enzyme and the stitching enzyme are the same enzyme.3. The reactive fiber pulp of any preceding or following embodiment / feature / aspect, wherein the sizing enzyme and the stitching enzyme are different enzymes and the stitching enzyme comprises a xyloglucan endotransglucosylase / hydrolase (XTH) enzyme, a xyloglucan endotransglucosylase (XET) enzyme, a xyloglucan endohydrolase (XEH) enzyme, or any combinations thereof.4. The reactive fiber pulp of any preceding or following embodiment / feature / aspect, wherein the sizing enzyme and the stitching enzyme are different enzymes and the stitching enzyme comprises an XTH enzyme from the genome of Arabidopsis thaliana an XTH enzyme from the genome of Populus tremulc, or a combination thereof.5. The reactive fiber pulp of any preceding or following embodiment / feature / aspect, wherein the sizing enzyme and the stitching enzyme are different enzymes and the stitching enzyme comprises AtXTH22, AtXTH3, PtXTH3, VaXET16, or a combination thereof.6. The reactive fiber pulp of any preceding or following embodiment / feature / aspect, wherein the one or more polymers comprise one or more biopolymers.7. The reactive fiber pulp of any preceding or following embodiment / feature / aspect, wherein the one or more biopolymers comprise polycarbohydrates.8. The reactive fiber pulp of any preceding or following embodiment / feature / aspect, wherein, during an enzymatic reaction, the stitching enzyme enzymatically, covalently bondstogether: (1) the fibers; (2) the one or more polymers; or (3) both the fibers and the one or more polymers.9. The reactive fiber pulp of any preceding or following embodiment / feature / aspect, wherein:the fibers comprise short fibers;each of the short fibers comprises a respective length, first and second ends that are opposite one another, and a cellulosic terminal end group at, at least one of the first and second ends; andduring an enzymatic reaction, the stitching enzyme enzymatically, covalently bonds the cellulosic terminal end group of a first one of the short fibers to the cellulosic terminal end group of a second one of the short fibers.10. The reactive fiber pulp of any preceding or following embodiment / feature / aspect, further comprising a hemicellulase and a cellulase.11. The reactive fiber pulp of any preceding or following embodiment / feature / aspect, further comprising one or more of xylanase, mannanase, lipase, lignocellulase, endo-glucosidase, and beta-glucosidase.12. A reaction product resulting from an enzymatic reaction of the reactive fiber pulp of any of preceding embodiment / feature / aspect numbered 1-8.13. A reaction product resulting from an enzymatic reaction of the reactive fiber pulp of any preceding embodiment / feature / aspect numbered 9-11.14. The present invention further relates to a method of treating a fibrous pulp, the fibrous pulp comprising fibers having one or more of an aldehyde group, an aromatic group, a carboxyl group, a hydroxyl group, or a ketone group, present as surface terminated groups or end groups, and optionally one or more of an amino group, a halogen group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group; and one or more polymers having an aldehyde group, anamino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group, or any combinations thereof, wherein the method comprises:combining, with the fibrous pulp, (i) a sizing enzyme that is designed to enzymatically, covalently bond monomers together to form the one or more polymers, and (ii) a stitching enzyme configured to enzymatically, covalently bond the fibers to the one or more polymers, wherein the combining occurs under conditions such that the stitching enzyme enzymatically, covalently bonds together the fibers to the one or more polymers.15. The method of any preceding or following embodiment / feature / aspect, wherein the sizing enzyme is an alpha-glucan enzyme, and the alpha-glucan enzyme and the stitching enzyme are the same enzyme.16. The method of any preceding or following embodiment / feature / aspect, wherein: the fibers comprise short fibers;each of the short fibers comprises a respective length, first and second ends that are opposite one another, and a cellulosic terminal end group at, at least one of the first and second ends; andthe stitching enzyme enzymatically, covalently bonds the cellulosic terminal end group of a first one of the short fibers to the cellulosic terminal end group of a second one of the short fibers.17. The method of any preceding or following embodiment / feature / aspect, wherein, prior to the combining of the fibrous pulp with the sizing enzyme and the stitching enzyme, the method first comprises forming the fibrous pulp, and the forming of the fibrous pulp comprises:combining a first precursor fibrous pulp with a degradation enzyme, wherein the first precursor fibrous pulp comprises cellulosic material, the first precursor fibrous pulp and thedegradation enzyme are combined under conditions resulting in the enzymatic degradation of the cellulosic material, and the fibrous pulp is formed.18. The method of any preceding or following embodiment / feature / aspect, wherein, prior to the combining of the fibrous pulp with the sizing enzyme and the stitching enzyme, the fibrous pulp is washed to remove at least some of the degradation enzyme.19. The method of any preceding or following embodiment / feature / aspect, wherein the fibrous pulp further comprises hemicellulose and cellulose, and the method further comprises:combining the fibrous pulp with a hemicellulase and a cellulase under conditions resulting in the hemicellulolysis and cellulolysis of the hemicellulose and cellulose, respectively.20. The method of any preceding or following embodiment / feature / aspect, wherein the combining of the fibrous pulp with the hemicellulase and the cellulase occurs before the fibrous pulp is combined with the sizing enzyme and the stitching enzyme.21. The method of any preceding or following embodiment / feature / aspect, wherein the combining of the fibrous pulp with the hemicellulase and the cellulase occurs at the same time that the fibrous pulp is combined with the sizing enzyme and the stitching enzyme.22. The method of any preceding or following embodiment / feature / aspect, wherein the combining of the fibrous pulp with the hemicellulase and the cellulase occurs after the fibrous pulp is combined with the sizing enzyme and the stitching enzyme.23. The method of any preceding or following embodiment / feature / aspect, wherein, prior to the combining of the fibrous pulp with the sizing enzyme and the stitching enzyme, the method first comprises forming the fibrous pulp, and the forming of the fibrous pulp comprises:combining a precursor fibrous pulp with a hemi cellulase and a cellulase, the precursor fibrous pulp comprising hemicellulose and cellulose,wherein the precursor fibrous pulp, the hemicellulase, and the cellulase are combined under conditions resulting in the hemicellulolysis and cellulolysis of the hemicellulose and cellulose, respectively.24. The method of any preceding or following embodiment / feature / aspect, wherein, prior to the combining of the fibrous pulp with the sizing enzyme and the stitching enzyme, the method first comprises forming the fibrous pulp, and the forming of the fibrous pulp comprises:combining a first precursor fibrous pulp with a hemicellulase, wherein the first precursor fibrous pulp comprises hemicellulose, the first precursor fibrous pulp and the hemicellulase are combined under conditions resulting in the hemicellulolysis of the hemicellulose, and a second precursor fibrous pulp that comprises cellulose, is formed; andcombining the second precursor fibrous pulp with a cellulase, wherein the second precursor fibrous pulp and the cellulase are combined under conditions resulting in the cellulolysis of the cellulose, and the fibrous pulp is formed.25. The method of any preceding or following embodiment / feature / aspect, wherein, prior to the combining of the second precursor fibrous pulp with the cellulase, the second precursor fibrous pulp is washed to remove at least some of the hemicellulase.26. The method of any preceding or following embodiment / feature / aspect, wherein, prior to the combining of the fibrous pulp with the sizing enzyme and the stitching enzyme, the fibrous pulp is washed to remove at least some of the cellulase.27. The method of any preceding or following embodiment / feature / aspect, wherein, prior to the combining of the fibrous pulp with the sizing enzyme and the stitching enzyme, the fibrous pulp is washed to remove at least 90% of the cellulase.28. The method of any preceding or following embodiment / feature / aspect, wherein the stitching enzyme differs from the sizing enzyme and comprises xyloglucan endotransglucosylase / hydrolase.29. The present invention further relates to a method of improving wet strength of a fibrous product, comprising:carrying out the method of any preceding or following embodiment / feature / aspect, to form a treated fibrous pulp; andforming a fibrous product from the treated fibrous pulp, whereinthe fibrous product exhibits a wet strength, and the wet strength of the fibrous product is greater than the wet strength of a fibrous product made from the same fibrous pulp but which has not been combined with the sizing enzyme and the stitching enzyme.30. The present invention further relates to a method of improving strength of fibers in fibrous pulp, comprising:carrying out the method of any preceding or following embodiment / feature / aspect, to form treated fibers and a treated fibrous pulp, whereinthe treated fibers exhibit a strength, and the strength of the treated fibers is greater than the strength of fibers in the fibrous pulp, which have not been combined with the sizing enzyme and the stitching enzyme.31. The present invention further relates to a method of permanently sizing fibers in a fibrous pulp, the fibrous pulp comprising fibers having one or more of an aldehyde group, an aromatic group, a carboxyl group, a hydroxyl group, or a ketone group, present as surface terminated groups or end groups, and optionally one or more of an amino group, a halogen group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group; b) one or more sizing polymers having an aldehyde group, an amino group, an aromatic group, a carboxyl group, a halogen group, a hydroxyl group, a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or a sulfonate group or any combinations thereof; wherein the method comprises:combining, with the fibrous pulp, (i) a sizing enzyme, for example, an alpha-glucan enzyme, that is designed to enzymatically, covalently bond monomers together to form the one ormore polymers, and (ii) a stitching enzyme configured to enzymatically, covalently bond the fibers to the one or more sizing polymers, whereinthe combining occurs under conditions such that the stitching enzyme enzymatically, covalently bonds together the fibers to the one or more sizing polymers, to form a treated fibrous pulp comprising permanently sized fibers.32. The present invention further relates to a method of modifying a fibrous pulp, comprising:combining at least one xyloglucan endotransglucosylase (XET) enzyme with a fibrous pulp under temperature and pH conditions such that XET enzymatic activity occurs, wherein the fibrous pulp comprises an aqueous suspension of cellulosic fibers and hemicellulose fibers, andthe at least one XET enzyme is included in the aqueous suspension in an amount such that the at least one XET enzyme stitches at least a portion of the cellulosic fibers together, stitches at least a portion of the hemicellulose fibers together, stitches at least a portion of the cellulosic fibers and at least a portion of the hemicellulose fibers together, or a combination thereof, to form a modified fibrous pulp comprising stitched fibers.33. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the modified fibrous pulp comprises an aqueous fibrous pulp useful for manufacturing a paper product.34. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the aqueous suspension is a by-product of manufacturing a paper product comprising white water, and the method further comprises recovering the stitched fibers.35. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the white water is part of an open white-water system.36. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the white water is part of a closed white-water system.37. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the amount of the at least one XET enzyme is from 0.01 kg / ton to 1 kg / ton based on the dried weight of the cellulosic and hemicellulose fibers in the aqueous suspension.38. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the method comprises maintaining the pH of the aqueous suspension to be from 2 to 8.39. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the pH of the aqueous suspension is adjusted to be from 4 to 6.40. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, further comprising buffering the aqueous suspension to an optimal pH range such that XET activity occurs.41. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein buffering the aqueous suspension comprises adding to the aqueous suspension at least one of a sodium acetate buffer, a phosphate buffer, and a citrate buffer.42. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the temperature conditions of the aqueous suspension comprise a temperature of from 10 °C to 75 °C.43. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the temperature conditions of the aqueous suspension comprise a temperature of from 15 °C to 50 °C.44. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, further comprising heating or cooling the aqueous suspension to attain an optimal temperature range for XET activity to occur.45. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the majority of the cellulosic fibers and the majority of the hemicellulose fibers are fibers having lengths of greater than or equal to 0.5 mm.46. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the majority of the cellulosic fibers and the majority of the hemicellulose fibers are fines that are less than 0.5 mm in length.47. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the cellulosic fibers and the hemicellulose fibers are a combination of fines that are less than 0.5 mm in length and fibers that are greater than or equal to 0.5 mm in length.48. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the majority of the stitched fibers are from 0.1 mm to 5 mm in length.49. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, further comprising combining modified oligosaccharides with the aqueous suspension.50. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the modified oligosaccharides are oligosaccharides bonded to 2-aminopyridine.51. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the XET enzyme comprises an XTH enzyme from thegenome of Arabidopsis thaliana, an XTH enzyme from the genome of Populus tremula, an XET enzyme from Vigna angularis, or a combination thereof.52. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, wherein the XET enzyme comprises AtXTH22, AtXTH3, PtXTH3, VaXET16 or a combination thereof.53. The present invention further relates to a reactive aqueous suspension comprising cellulosic fibers, hemicellulose fibers, and at least one xyloglucan endotransglucosylase (XET) enzyme, wherein the at least one XET enzyme is present in an amount such that, under enzymatic reaction conditions, the XET enzyme stitches together at least a portion of the cellulosic fibers, stitches together at least a portion of the hemicellulose fibers, stitches together at least a portion of the cellulosic fibers and a portion of the hemicellulose fibers, or a combination thereof.54. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the reactive aqueous suspension is at a temperature and a pH at which XET activity occurs.55. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the pH is from 2 to 8.56. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the pH is from 4 to 6.57. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the temperature is from 10 °C to 50 °C.58. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the temperature is from 20 °C to 40 °C.59. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the cellulosic fibers and the hemicellulose fibers are part of an aqueous fibrous pulp.60. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the cellulosic fibers and the hemicellulose fibers are part of white water.61. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the amount of the at least one XET enzyme in the reactive aqueous suspension is from 0.01 kg / ton to 1 kg / ton based on the dried weight of the cellulosic and hemicellulose fibers in the reactive aqueous suspension.62. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the majority of the cellulosic fibers and the majority of the hemicellulose fibers are fibers having lengths greater than or equal to 0.5 mm.63. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the majority of the cellulosic fibers and the majority of the hemicellulose fibers are fines that are less than 0.5 mm in length.64. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the cellulosic fibers and the hemicellulose fibers comprise a combination of fines that are less than 0.5 mm in length and fibers that are greater than or equal to 0.5 mm in length.65. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the XET enzyme comprises an XET enzyme from the genome of Arabidopsis thaliana, an XET enzyme from the genome of Populus tremula, an XET enzyme from Vigna angular is, or a combination thereof.66. The reactive aqueous suspension according to any preceding or following embodiment / feature / aspect, wherein the XET enzyme comprises AtXTH22, AtXTH3, PtXTH3, VaXET16 or a combination thereof.67. The present invention further relates to a reaction product resulting from an enzymatic reaction of the reactive aqueous suspension according to any preceding or following embodiment / feature / aspect.68. The reaction product according to the preceding embodiment / feature / aspect, comprising stitched fibers having a length of from 0.1 mm to 5 mm.69. The method of modifying a fibrous pulp according to any preceding or following embodiment / feature / aspect, further comprising buffering the aqueous suspension to an optimal pH range such that a specific XET activity occurs, wherein the specific XET activity comprises a cellulose-cellulose bonding activity, a cellulose-hemicellulose bonding activity, a hemicellulose-hemicellulose bonding activity, or a combination thereof.

[0152] The present invention can include any combination of these various features or embodiments above and / or below as set forth in sentences and / or paragraphs. Any combination of disclosed features herein is considered part of the present invention and no limitation is intended with respect to combinable features.

[0153] Applicants specifically incorporate the entire contents of all cited references in this disclosure. Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within therange. It is not intended that the scope of the invention be limited to the specific values recited when defining a range.

[0154] It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments of the present invention without departing from the spirit or scope of the present invention. Thus, it is intended that the present invention covers other modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims

WHAT IS CLAIMED IS1. A method of modifying a fibrous pulp, comprising:combining at least one xyloglucan endotransglucosylase (XET) enzyme with a fibrous pulp under temperature and pH conditions such that XET enzymatic activity occurs, wherein the fibrous pulp comprises an aqueous suspension of cellulosic fibers and hemicellulose fibers, andthe at least one XET enzyme is included in the aqueous suspension in an amount such that the at least one XET enzyme stitches at least a portion of the cellulosic fibers together, stitches at least a portion of the hemicellulose fibers together, stitches at least a portion of the cellulosic fibers and at least a portion of the hemicellulose fibers together, or a combination thereof, to form a modified fibrous pulp comprising stitched fibers.

2. The method of claim 1, wherein the modified fibrous pulp comprises an aqueous fibrous pulp useful for manufacturing a paper product.

3. The method of claim 1, wherein the aqueous suspension is a by-product of manufacturing a paper product comprising white water, and the method further comprises recovering the stitched fibers.

4. The method of claim 3, wherein the white water is part of an open white-water system.

5. The method of claim 3, wherein the white water is part of a closed white-water system.

6. The method of claim 1, wherein the amount of the at least one XET enzyme is from 0.01 kg / ton to 1 kg / ton based on the dried weight of the cellulosic and hemicellulose fibers in the aqueous suspension.

7. The method of claim 1, wherein the method comprises maintaining the pH of the aqueous suspension to be from 2 to 8.

8. The method of claim 1, wherein the pH of the aqueous suspension is adjusted to be from 4 to 6.

9. The method of claim 1, further comprising buffering the aqueous suspension to an optimal pH range such that a specific XET activity occurs, wherein the specific XET activity comprises a cellulose-cellulose bonding activity, a cellulose-hemicellulose bonding activity, a hemicellulose-hemicellulose bonding activity, or a combination thereof.

10. The method of claim 9, wherein buffering the aqueous suspension comprises adding to the aqueous suspension at least one of a sodium acetate buffer, a phosphate buffer, and a citrate buffer.

11. The method of claim 1, wherein the temperature conditions of the aqueous suspension comprise a temperature of from 10 °C to 75 °C.

12. The method of claim 1, wherein the temperature conditions of the aqueous suspension comprise a temperature of from 15 °C to 50 °C.

13. The method of claim 1, further comprising heating or cooling the aqueous suspension to attain an optimal temperature range for XET activity to occur.

14. The method of claim 1, wherein the majority of the cellulosic fibers and the majority of the hemicellulose fibers are fibers having lengths of greater than or equal to 0.5 mm.

15. The method of claim 1, wherein the majority of the cellulosic fibers and the majority of the hemicellulose fibers are fines that are less than 0.5 mm in length.

16. The method of claim 1, wherein the cellulosic fibers and the hemicellulose fibers are a combination of fines that are less than 0.5 mm in length and fibers that are greater than or equal to 0.5 mm in length.

17. The method of claim 1, wherein the majority of the stitched fibers are from 0.1 mm to 5 mm in length.

18. The method of claim 1, further comprising combining modified oligosaccharides with the aqueous suspension.

19. The method of claim 18, wherein the modified oligosaccharides are oligosaccharides bonded to 2-aminopyridine.

20. The method of claim 1, wherein the XET enzyme comprises an XTH enzyme from the genome of Arabidopsis thaliana, an XTH enzyme from the genome of Populus tremula, an XET enzyme from Vigna angularis, or a combination thereof.

21. The method of claim 1, wherein the XET enzyme comprises AtXTH22, AtXTH3, PtXTH3, VaXET16 or a combination thereof.

22. A reactive aqueous suspension comprising cellulosic fibers, hemicellulose fibers, and at least one xyloglucan endotransglucosylase (XET) enzyme, wherein the at least one XET enzyme is present in an amount such that, under enzymatic reaction conditions, the XET enzyme stitches together at least a portion of the cellulosic fibers, stitches together at least a portion of the hemicellulose fibers, stitches together at least a portion of the cellulosic fibers and a portion of the hemicellulose fibers, or a combination thereof.

23. The reactive aqueous suspension of claim 22, wherein the reactive aqueous suspension is at a temperature and a pH at which XET activity occurs.

24. The reactive aqueous suspension of claim 23, wherein the pH is from 2 to 8.

25. The reactive aqueous suspension of claim 23, wherein the pH is from 4 to 6.

26. The reactive aqueous suspension of claim 23, wherein the temperature is from 10 °C to 50 °C.

27. The reactive aqueous suspension of claim 22, wherein the temperature is from 20 °C to 40 °C.

28. The reactive aqueous suspension of claim 22, wherein the cellulosic fibers and the hemicellulose fibers are part of an aqueous fibrous pulp.

29. The reactive aqueous suspension of claim 22, wherein the cellulosic fibers and the hemicellulose fibers are part of white water.

30. The reactive aqueous suspension of claim 22, wherein the amount of the at least one XET enzyme in the reactive aqueous suspension is from 0.01 kg / ton to 1 kg / ton based on the dried weight of the cellulosic and hemicellulose fibers in the reactive aqueous suspension.

31. The reactive aqueous suspension of claim 22, wherein the majority of the cellulosic fibers and the majority of the hemicellulose fibers are fibers having lengths greater than or equal to 0.5 mm.

32. The reactive aqueous suspension of claim 22, wherein the majority of the cellulosic fibers and the majority of the hemicellulose fibers are fines that are less than 0.5 mm in length.

33. The reactive aqueous suspension of claim 22, wherein the cellulosic fibers and the hemicellulose fibers comprise a combination of fines that are less than 0.5 mm in length and fibers that are greater than or equal to 0.5 mm in length.

34. The reactive aqueous suspension of claim 22, wherein the XET enzyme comprises an XET enzyme from the genome of Arabidopsis thaliana, an XET enzyme from the genome of Populus tremula., an XET enzyme from Vigna angularis, or a combination thereof.

35. The reactive aqueous suspension of claim 22, wherein the XET enzyme comprises AtXTH22, AtXTH3, PtXTH3, VaXETI 6 or a combination thereof.

36. A reaction product resulting from an enzymatic reaction of the reactive aqueous suspension of claim 22.

37. The reaction product of claim 36, comprising stitched fibers having a length of from 0.1 mm to 5 mm.

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