WO2013165568A1

WO2013165568A1 - Pulp fiber modification using expansin or swollenin in combinations with one or more enzymes

Info

Publication number: WO2013165568A1
Application number: PCT/US2013/030555
Authority: WO
Inventors: Jianhau MA; James G. Tausche
Original assignee: Enzymatic Deinking Technologies, Llc
Priority date: 2012-05-01
Filing date: 2013-03-12
Publication date: 2013-11-07

Abstract

Methods for the modification of pulp fibers, such as recycled and/or virgin fibers, to increase the pulp drainage and/or strength and/or increase the rate of water absorption of resulting handsheets and/or paper products are described herein. The methods involve treating pulp with expansin and/or swollenin in combination with an enzyme composition containing one or more hemicellulases, one or more pectinases, and combinations thereof. In one embodiment, the enzyme composition further contains one or more cellulases, for example, one or more endo-glucanases. The expansin and/or swollenin can be applied simultaneously with the enzyme composition in the same formulation or different formulations.

Description

PULP FIBER MODIFICATION USING EXPANSION OR SWOLLENIN IN COMBINATIONS WITH ONE OR MORE

ENZYMES

FIELD OF THE INVENTION

This invention is in the field of treatments for papermaking pulp fibers, particularly enzyme-based treatments, to improve the physical and chemical properties of the fibers and paper products prepared from the treated fibers.

BACKGROUND OF THE INVENTION

Paper, cardboard, linerboard, corrugated paperboard, tissue, towels, corrugated containers, and boxes are made from processed plant fibers. Harvested plant and/or plant materials go through different pulping or cooking processes to produce pulp fibers, such as chemical pulping, mechanical pulping and semi mechanical pulping.

The individual pulp fibers generally contain three major components: cellulose, hemicellulose, and lignin. The cellulose chains within each fiber are physically associated with imbedded hemicellulose and pectin chains, and chemically bonded to each other through covalent bonds and non- covalent bonds, such as hydrogen bonding, to form a matrix. It is this matrix that provides rigidity to the fiber. The physical interwoven fiber structure, the covalent bonding, and the hydrogen bonding within fibers and between fibers play a critical role in the properties of paper products prepared from the fibers.

In addition to the pulping processes, bleaching and refining have been widely used to impact the physical and chemical properties of the fibers. Papermaking additives have also been used to impact the bonding between fibers and formation of sheets to improve the physical and chemical properties of paper products, such as wet/dry strength, softness, water absorbance and printing ability.

Expansins is a class proteins found in the cells of various plants. Expansin is believed to promote cell expansion and thus cell growth by allowing slippage or movement of the cellulose, pectin, and/or hemicellulose chains within the plant fibers. Expansins have been shown to weaken the hydrogen bonds between paper fibers of recycled paper, including commercial papers, such as coated papers from magazines and catalogs, which can be difficult to recycle. U.S. Patent No. 5,990,283 to Cosgrove et al. describes the effect of expansin on the mechanical properties of pure cellulose filter paper.

The combination of expansin and whole cellulase has been alleged to completely hydrolyze cellulosic biomass in order to facilitate conversion of biomass into sugars using cellulases for ethanol production (see U.S. Patent Application Publication No. 201 1/0136182 to Huang et al, U.S. Patent

Application Publication No. 2010/0173386 to Kim et al., and U.S. Patent No. 6,326,470 to Cosgrove). However, such methods are not applicable to fiber modification for pulp and paper production since in biomass conversion, cellulose, the major component of fibers, is completely degraded to individual sugar molecules, leaving no intact fibers for pulp and

papermaking.

In addition to disrupting the bonds between fibers, the fibers themselves must be modified in order to obtain the desirable chemical and physical properties of the resulting paper for a given application. In order to modify the fibers, treatments, such as chemical and/or enzymatic treatments must be able to access the fibers in order to chemically modify the structure of the fibers.

Therefore, there exists a need for improved methods for modifying pulp fibers in order to obtain desirable physical and chemical properties for a given application.

Therefore, it is an object of the invention to provide methods for modifying pulp fibers in order to obtain desirable physical and chemical properties for a given application.

SUMMARY OF THE INVENTION

Methods for the modification of pulp fibers, such as recycled and/or virgin fibers, to increase drainage and/or strength and/or increase the rate of water absorption are described herein. The methods involve treating pulp with expansin and/or swollenin in combination with an enzyme composition containing one or more hemicellulases, one or more pectinases, and combinations thereof. In one embodiment, the enzyme composition further contains one or more cellulases, for example, one or more endo-glucanases.

The expansin and/or swollenin can be applied simultaneously with the enzyme composition in the same formulation or different formulations. "Simultaneous administration", as used herein, means administered at the same time or substantially the same time, i.e., within 30 second, one minute, two minutes, three minutes, four minutes, or five minutes. Alternatively, the expansin and/or swollenin and the enzyme composition can be applied sequentially. For example, the expansin and/or swollenin can be applied first followed by the enzyme composition. In some embodiments, expansin and/or swollenin is administered first and the enzyme compositions is administered at least five minutes after the administration of the

expansin/swollenin, e.g., greater than 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 45, 60 minutes or longer after the administration of the expansin and/or swollenin.

In one embodiment, the enzyme formulation contains expansin and/or swollenin at a concentration of about 0.01 to about 1000 mg per 100 g oven dried ("OD") fiber, preferably from about 0.2 to about 400 mg per 100 g OD fiber, more preferably from about 0,2 to about 250 mg per 100 g of OD fiber, most preferably from about 0.2 to about 100 mg per 100 g of OD fiber. In some embodiments, the concentration of expansin and/or swollenin is about 0.25 mg per 100 g OD fiber.

In one embodiment, the concentration of expansin and/or swollenin is as described above and the concentration of the enzymes in the enzyme composition is from about 5 to about 600 enzyme activity units per 100 g OD fiber, more preferably from about 20 to about 200 enzyme activity units per 100 g OD fiber. The protein content and enzyme units can be determined as described below.

The expansin/swollenin and/or enzyme composition can contain one or more additives, for example, to stabilize the expansin/swollenin and/or enzyme composition. Suitable classes of additives include, but are not limited to, chemical additives, such as surfactants, stabilizers, polymers, biocides, and combinations thereof.

The pulp to be treated can be derived from any of a number of sources. Pulp can be made from wood, fiber crops and/or other non-wood sources such as bamboo, reed, wheat straw or rice straw or grass. Wood pulp comes from softwood and hardwood trees. Softwood trees include spruce, pine, fir, larch and hemlock. Hardwoods include eucalyptus, aspen and birch. The pulp can be virgin pulp or recycled pulp.

The point of the process in the pulp mill or deink plant or a tissue/paper mill at which the formulation containing expansin and/or swollenin and the enzyme composition is applied is dependent on a variety of factors such as (1) the incubation time of the formulation with the pulp; (2) the amount of time between the treatment and paper or tissue making; (3) the water loops in the pulp mill or deink plant. The incubation time of the formulation with the pulp stock can vary. The pulp stock is treated with the formulation for at least 1, 2, 3, 4, or 5 minutes. In some embodiments, the pulp is treated for longer than five minutes, such as at least 5 minutes, at least 10 minutes, or at least 15 minutes. The pulp is treated for a period of time and in an amount and under conditions necessary for fiber modification.

The enzyme treatments described herein are typically effective at temperatures of from 10°C to 90°C, preferably from about 25°C to 60°C. However, the temperature range can vary depending on the nature of the enzyme and the optimal activity range for each enzyme. Moreover, stabilizers can be added to the one or more both of the enzyme formulations that allow the enzyme to be active at higher temperature, e.g., 60-90°C. This can be advantageous since the pulping process often operates in this temperature range and sometime must be cooled to a lower temperature before addition of enzyme in order to avoid denaturing the enzyme.

The pH of the pulp stock can generally be from about 3.5 to about 9.5 for most enzymes, more preferably from about 4.5 to 8.0. The pH of the stock can be adjusted using pH modifiers such as alum or aluminates, certain acids, carbon dioxide, and various alkalis such as sodium hydroxide.

Expansin and/or swollenin can be applied at the same temperature and/or pH or a different temperature and/or pH as the enzyme formulation. The enzyme formulation is typically administrated to the wet pulp stock with a consistency of 0.1% to 35%, preferably 0.5% to 15%, and more preferably 1.0% to 10%.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB are graphs showing handsheet density (g/cm³) as a function of treatment (blank, enzyme composition alone, expansion alone, and expansin plus enzyme composition). Figures 1C and ID are graphs showing tensile strength (N-rn/g) as a function of treatment (blank, enzyme composition alone, expansion alone, and expansin plus enzyme composition) with different bleached Kraft softwood pulps.

Figure 2A is a graph showing handsheet density (g/cm³) as a function of treatment (blank, enzyme composition alone, expansion alone, and expansin plus enzyme composition). Figures 2B and 2C are graphs showing tear strength (mN-m²/g) as a function of treatment (blank, enzyme composition alone, expansion alone, and expansin plus enzyme composition) with different Eucalyptus pulps.

Figure 3 is a graph showing tensile strength (N-rn/g) as a function of treatment (blank, enzyme composition alone, expansion alone, and expansin plus enzyme composition) with bleached Kraft mixed hardwood pulp.

Figure 4A is graph showing pulp freeness as a function of treatment (blank, enzyme composition alone, expansion alone, and expansin plus enzyme composition). Figure 4B is a graph showing tear strength

(mN-m²/g) as a function of treatment (blank, enzyme composition alone, expansion alone, and expansin plus enzyme composition). Figure 4C is a graph showing water absorption rate (seconds) as a function of treatment (blank, enzyme composition alone, expansion alone, and expansin plus enzyme composition) with bleached spruce sulfite pulp.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

"Mechanical pulp" refers to pulp produced by reducing pulpwood logs and chips into fiber components by the use of mechanical energy, containing stone ground wood pulp, pressurized ground wood pulp and thermomechanical pulp. "Stone ground wood pulp" or "SGW" as used herein, refers to pulp which is produced by grinding wood into relatively short fibers with stone grinding. This pulp is used mainly in newsprint and wood-containing papers, such as lightweight coated (LWC) and super-calendered (SC) papers.

"Pressurized groundwood pulp" or "PGW" refers to pulp produced by a stone grinder where the whole grinder casing is pressurized and increased shower water temperature is used.

"Thermomechanical pulp" or "TMP" as used herein, refers to pulp that is produced in a thermo-mechanical process where wood chips or sawdust are softened by steam before entering a pressurized refiner. TMP generally has the same end-uses as stone groundwood pulp.

"Semi-chemical pulp" as used herein, refers to pulp produced by a combination of some chemicals (less than those used in Kraft pulping) and unpressurized mechanical processes. A variety of this pulp with pretreated chips at a temperature over 100°C followed by refining at atmospheric pressure is called "semichemical mechanical pulp" or "SCMP". This pulp has properties suitable for corrugated medium for container board production.

"Chemo-Thermomechanical Pulp" or "CTMP" as used herein, refers to mechanical pulp produced by treating wood chips with chemicals (usually sodium sulfite) and steam before mechanical defiberization. CTMP typically uses less chemicals compared to Kraft pulping.

"Chemical pulp", as used herein, refers to pulp produced by the treatment of wood chips or sawdust with chemicals to liberate the cellulose fibers by removing the binding agents such as lignin, hemicellulose, and wood extractive, such as resins. Sulphite and Sulphate or Kraft pulping are two major types of chemical pulping. Kraft is the predominant pulping process in chemical pulp production.

"Recycled pulp" or "recycled fibers" refers to fiber component of a paper or paperboard furnish that is derived from recycled paper and paperboard or wastepaper.

The term "effective amount" refers to any amount which results in a predetermined or desired outcome. For example, an effective amount of an enzyme formulation intended to enhance strength means the amount of formulation which is effective to increase fiber strength properties in the final sheet compared to pulps not treated with the same formulation under the same conditions.

The term "fiber modification" refers to any alteration or modification of the pulp fibers as a result of treatment with expansin/swollenin and an enzyme composition. The modification can either be a direct or indirect result of the treatment with the formulation.

II. Formulations

Methods for modifying papermaking pulp fibers to improve the physical and chemical properties of the pulp fibers, as well as the physical and chemical properties of the paper products made from the fibers, are described herein. The pulp fibers are treated with expansin and/or swollenin in combination with an enzyme composition containing one or more hemicellulases, one or more pectinases, or combinations thereof. In some embodiments, the enzyme composition further contains one or more cellulases, for example, endo-glucanase.

A. Expansin/swollenin

Expansin/swollenin weakens the hydrogen bonding between polymer chains resulting in temporary slippage between cellulose and hemicellulose polymer chains and hemicellulose and pectin polymer chains, and allows more accessibility for hemicellulases and pectinases, and optionally cellulases, which can further degrade hemicellulose and pectin/pectate chains resulting in permanent structure loosening without weakening the strength of the cellulose chains.

The enzymes do not reduce the length of the fibers, but rather alter the properties of the matrix which can result in changes in the chemical and physical properties of the fibers, such as flexibility, softness, and water absorbance. The changes in the structure of the fibers can also result in changes in refining behavior of the pulp prepared from the treated fibers. Flexible fibers with better softness and water absorbance may result from the reduction in the crystallinity of the fibers due to the reduction in the hydrogen bonding within amorphous areas and degradation of crosslinked polymers, such as hemicellulose and pectin with crystalline and/or amorphous areas of the fiber.

Treatment of expansin/swollenin with hemicellulase and/or pectinase, and optionally celluase, results in significant drainage and strength improvement, better water absorption, and higher bulk which are desirable for tissue and towel production.

1. Expansin

Expansin of either plant origin or microbial origin induces cell wall extension without detectable hydrolytic break down of cell walls. Alpha- expansin is responsible for cell wall disassembly and cell separation and beta-expansin is responsible cell wall loosening action. In contrast to conventional enzymatic theories of wall loosening by cellulases, expansin weakens the hydrogen bonding between molecules of cellulose and glucomannan, cellulose and xyloglucan, xyloglucan and xyloglucan, and xyloglucan and pectin/pectate, allowing the cellulose and hemicellulose chains to slide against each other. Expansin movement may be confined to lateral diffusion along the surfaces of cellulose microfibril, which enables the expansin to search the microfibril surface, locally loosening up the cellulose/hemicellulose matrix.

Expansin can be isolated from a variety of plant sources, such as fiber crops and cereals (i.e., oat, corn, maize, barley, etc.), stem and leaf vegetables (i.e., broccoli, cabbage, celery, etc.), fruit and seed vegetables (i.e., tomato, cucumber, pea, radish, etc.), and forest and ornamental crops (i.e., cotton, zephyr lily, etc.). Expansin can also be isolated from snails and their feces. Expansin also includes variants of expansin, provided the variant or variants exhibits equivalent or similar activity to the naturally occurring protein.

Expansin shows no cellulases activity or protease activity, but enhances the hydrolysis of crystalline cellulose by cellulases by making the fiber or microfibril surface more accessible to enzymatic actions by cellulases, hemicellulases and pectin degrading enzymes such as pectinase, pectate lyase, and pectin lyase. 2. Swollenin

Swollenin is a group of proteins or polypeptides of microbial origin, which have the ability to facilitate weakening of filter paper and swelling of cotton fibers without having any cellulolytic activity or protease activity. It is more convenient to define swollenin loosely in terms of the expansin protein described in McQueen-Mason et al, Plant Cell, Vol. 4, pp. 1425- 1433(1992).

Swollenin has distinct properties with a low level of identity to plant expansin. Swollenin generally has a higher molecular weight and has cellulolytic enzyme activity enhancing capability.

Expansin/swollenin content is measured using the Micro Lowry Total Protein Kit with Peterson's modification using Sigma product TP0300 and L 3540 (St. Louis, Missouri, USA). Swollenins and their sources are described in U.S. Patent No. 6,967,246 to Swanson et al, which is incorporated herein by reference.

3. Variants

In addition to the many natural variants of expansins and swollenin proteins found in plants, microbes, and other organisms, non-naturally occurring variants of these proteins may be used in the disclosed

compositions and methods.

Expansins have been identified in a wide variety of land plants, from angiosperms and gymnosperms to ferns and mosses. The model plant Arabidopsis thaliana contains around 26 different a-expansin genes and 6 β- expansin genes. Expansins proteins from different families share only 20- 40% identity with each other (Sampedro J and Cosgrove DJ. Genome Biol. 2005 6(12):242). In addition, some proteins in bacteria and fungi are known to have distant sequence similarity to plant expansins. For example,

Swollenin is a Trichoderma reesei protein with sequence similarity to the plant expansins that exhibits disruption activity on cellulosic materials. The crystal structure of the YOAJ protein from bacterium Bacillus subtilis is also very similar to the structure of plant expansins, despite the low sequence similarity. Proteins related to YOAJ were found in diverse species of plant pathogenic bacteria. Some animals can also produce a functional expansin, such the plant-parasitic nematode Globodera rostochiensis, which uses it to loosen cell walls when invading its host plant. Therefore, in some embodiments the disclosed expansin/swollenin is a naturally occurring expansin/swollenin protein isolated from a plant, bacteria, nematode, or other organisms known to express a functional expansin.

In other embodiments, the expansin/swollenin is a polypeptide having an amino acid sequence with at least 80%, 85%, 90%, 95%, or 99% sequence identity to a naturally occurring expansin/swollenin that weakens the hydrogen bonding between polymer chains resulting in temporary slippage between cellulose and hemicellulose polymer chains and hemicellulose and pectin polymer chains. In preferred embodiments, these variants contain conservative amino acid substitutions. Suitable variants may also contain amino acid additions or deletions that do not affect protein function. These variants may be identified using routine screening methods.

B. Enzyme composition

Expansin and/or swollenin is administered in combination with an enzyme composition containing one or more hemicellulases, one or more pectinases, or combinations thereof. In some embodiments, the enzyme composition further contains one or more cellulases.

1. Hemicellulases

Hemicellulases are a category of enzymes that include enzymes capable of hydro lyzing hemicellulose polymers to shorter oligomers.

Examples of hemicellulases include, but are not limited to, xylanase, gamanase, arabinase, and mannanase.

For determining xylanase activity, the substrate employed is azurine- crosslinked wheat arabinoxylan. Endo-l,4-B-D-xylanases hydro lyze the xylan backbone, releasing water soluble dyed fragments. The amount of released dyed fragments can be directly related to enzyme activity. The Xylazyme AX tablets are ordered from Megazyme International Ireland Ltd., Bray Business Park, Bray, Co. Wicklow, Ireland. The absorbance of these dyed fragments can be detected spectrophotometrically at 590 nm. A quantitative determination of enzyme activity is made by testing a known standard of xylanase at multiple dilution rates to obtain a standard curve. The standard curve correlates the enzyme activity of a known standard xylanase with absorbance. Novozymes Pulpzyme® HC has a standard activity unit of 40,000 XU/ml.

Gamanase breaks down galactomannans, such as bean gum, to reducing sugars which are measured with the DNS method. Locust bean gum at 0.1% (w/w) is used as the gamanase substrate. Enzyme is diluted with pH 6.8 50 mM phosphate buffer, added to the bean gum solution and incubated at 40°C water bath for 20 minutes. Shake the samples well every 5-10 minutes and stop the reaction using 0.10 ml of 1M a₂C0₃ solution, and then transfer 0.5 ml from each of the sample test tubes to glass test tubes to react with DNS solution and measure the absorbance at 575 nm. One unit of gamanase activity (GU/ml) is defined as the amount of enzyme generating 1 μιηοΐε of reducing sugar released from 0.1% locust bean gum at pH 6.8 and 40°C per minute.

2. Pectinases

Pectinases are a class of loosely defined enzymes which hydrolyze pectin and pectate materials in plant cells. Pectinases include, but are not limited to, pectinase, pectin lyase, pectate lyase and pectin esterase.

Pectinases hydrolyze the bond of polygalacturonic acid polymers, while pectin/pectate lyases cleave the glycosidic linkages between galacturonic acid monomers in pectin/pectate by trans-elimination reaction and generate unsaturated oligomers in the non-reducing ends. Most commercially available pectinase or pectin/pectate lyases are mixtures of these enzymes.

Pectinases break down 1.0% pectin solution (Sigma P-9135) with 50 nM acetate buffer at pH 5.0 resulting in galacturonic acid (BioChemika 48280). The concentration of pectin in the solution is determined by measuring the content of galacturonic acid. The treatment of pectin solution with sulfuric acid will develop a color in the presence of carbazole (Sigma C-5132), which measured at 520 nm, is proportional to the total pectin concentration. One unit of pectinase activity is the amount of pectinase needed to produce one μιηοΐε of galacturonic acid at pH 5.0, temperature of 40°C in 50 mM acetate buffer per minute. 3. Cellulases

Cellulases are a well classified category of enzymes and include enzymes capable of hydrolyzing cellulose polymers to shorter oligomers and/or glucose. The majority of industrial cellulases are obtained from cellulolytic organisms such as fungi and bacteria with three components as exo-cellobiohydrolases, endo-glucanases and beta-glucosidases. Endo- glucanase is an enzyme acting mainly on the amorphous parts of cellulose fibers to randomly hydro lyze internal -l,4-gluccosidic bonds of a cellulose chain. Cellobiohydrolase (CBH) or exo-glucanase hydrolyzes cellulose chains to cellobiose from the reducing or non-reducing ends of cellulose chains, while β -glucosidase or β-D-glucoside glucohydrolase acts on cellobiose to D-glucose units. These three components work synergistically to break down cellulose into cellobiose and glucose.

Most commercial cellulase mixtures, also referred as whole cellulase, used for biomass conversion contain these three enzyme components, which are essential for complete cellulose hydrolysis to individual sugar molecules. These types of whole cellulases are not suitable for fiber modification due to their aggressive hydrolyzing capability, while pure endo-glucanase randomly breaks accessible cellulose chains at amorphous area showing great potential for fiber modification. In one embodiment, the cellulase is an endo- glucanase, free of cellobiohydrolase (CBH) or exo-glucanase and β - glucosidase or β-D-glucoside glucohydrolase, which allows limited breaking of β-(1,4) linkages for desired fiber modification and minimizes aggressive cellulose hydrolysis to form glucose.

Endo-β- Ι, 4-glucanases (EC 3.2.1. 4) attack β-(1,4) linkages in amorphous areas of cellulose fibers. Carboxymethylcellulose (CMC), and phosphoric acid-swollen cellulose are water soluble cellulose derivatives compared to wood fibers with high crystallinity. The method described herein to measure endo- β-l, 4-glucanase activity relies on degradation of CMC chains, which causes a measurable reduction in the viscosity of the CMC gum solution. The chemicals used to determine cellulase activity include 0.30% of sodium carboxymethyl cellulose (CMC) gum, AQUALON® CMC 7 (Hercules Incorporated, Wilmington, DE) in pH 5.2 500 mM acetate buffer, and CELLULAST® 1.5 L cellulase (Novozymes A/B, Denmark) as a standard cellulase with an activity of 20000 CMC Units/ml. A diluted enzyme sample or filtrate sample is mixed with 10.0 ml of gum solution for 10 minutes at 40°C water bath and the viscosity is measured using Fisher Brand Glass Ubbelohde Viscometer Tubes. The reduction in viscosity is converted to CMC units based on the standard cellulase CELLULAST® 1.5L.

C. Expansin/enzyme formulations

The formulations for the treatment of papermaking pulp and fibers include expansin and/or swollenin and an enzyme composition containing one or more hemicellulases, one or more pectinases, and combinations thereof. In some embodiments, the enzyme composition further contains one or more cellulases.

In one embodiment, the enzyme formulation contains expansin and/or swollenin at a concentration of about 0.01 to about 1000 mg per 100 g oven dried ("OD") fiber, preferably from about 0.2 to about 400 mg per 100 g OD fiber. In some embodiments, the concentration of expansin and/or swollenin is about 0.25 mg per 100 g OD fiber.

In one embodiment, the concentration of expansin and/or swollenin is as described above and the concentration of the enzymes in the enzyme composition is from about 5 to about 600 enzyme activity units per 100 g OD fiber, more preferably from about 20 to about 200 enzyme activity units per 100 g OD fiber. The protein content and enzyme units can be determined as described above.

The expansin/swollenin and/or enzyme composition can contain one or more additives. Suitable classes of additives include, but are not limited to, chemical additives, such as surfactants, stabilizers, polymers, biocides and combinations thereof. Suitable surfactant dispersants include, but are not limited to, primary and branched alkoxylates, fatty acid alkoxylates, phosphate esters and their alkoxylates, alkylphenol alkoxylates, block copolymers of ethylene and propylene oxide, alkanesulfonates,

olefinsulfonates, fatty amine alkoxylates, glyceride alkoxylates, glycerol ester alkoxylates, sorbitan ester alkoxylates, polyethylene glycol esters, polyalkylene glycols, polyacrylic acids, sodium polyacrylate, acrylic acid copolymer, acrylate copolymer, acrylic crosslinked copolymer, and their derivatives; maleic acid and acrylic acid or acrylate copolymer, maleic acid/olefin copolymer, and their derivatives; methycellulose, ethyl cellulose and their derivatives; polyvinyl alcohol/polyvinyl acetate copolymers, polyvinyl pyrrolidone, and their derivatives, cationic polymers, and combinations thereof.

Suitable cationic polymer is selected from the group consisting of, but not limited to, epichlorohydrin/dimethylamine polymers (EPI-DMA) and cross-linked solutions thereof, polydiallyl dimethyl ammonium chloride (DADMAC), polyethylenimine (PEI), hydrophobically modified

polyethylenimine, polyamines, resin amines, polyacrylamide,

DADMAC/acrylamide copolymers, and ionene polymers.

The one or more additives can be used for a variety of functions, such as stabilizing the expansin/swollenin and/or enzyme composition.

III. Methods for treating pulp

The formulations described herein (expansin/swollenin + enzyme composition) can be used to modify pulp fibers to improve the physical and chemical properties of the pulp, as well as the paper products prepared from the pulp.

The expansin and/or swollenin can be administered simultaneously with the enzyme composition. For example, the expansin and/or swollenin can be administered in the same formulation as the enzyme composition or in separate formulations (e.g., expansin/swollenin in one formulation and the enzyme composition in another formulation). "Simultaneous

administration", as used herein, means administered at the same time or substantially the same time, i.e., within 30 second, one minute, two minutes, three minutes, four minutes, or five minutes.

In other embodiments, expansin and/or swollenin and the enzyme composition are administered sequentially. In some embodiments, expansin and/or swollenin is administered first and the enzyme compositions is administered at least five minutes after the administration of the expansin/swollenin, e.g., greater than 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 45, 60 minutes or longer.

A. Pulp type

The pulp and pulp fibers can be derived from any of a number of sources. Pulp can be made from wood, fiber crops and/or other non-wood sources such as wheat straw or rice straw or grass. Wood pulp comes from softwood and hardwood trees. Softwood trees include spruce, pine, fir, larch and hemlock. Hardwoods include eucalyptus, aspen and birch. One source of fiber that is widely used due to its lower cost and ready availability is eucalyptus pulp (e.g., South American, Australian, or Iberian varieties). Application of enzymatic treatments to eucalyptus pulps is especially beneficial due to the lower strength aspects of this short fiber, the fiber's lower cost, and the relative significant improvement from enzymatic treatment in terms of drainage and strength.

The pulp can be virgin pulp or recycled pulp. Virgin pulp is pulp that has never been made into a final paper product, while recycled pulp refers to pulp that was recycled from waste paper such as through a deinking or recycling process.

Commercial market pulps were obtained from different parts of the world from different tree species with different cooking and bleaching chemistries. Five widely used commercial pulps are used as followings: bleached loblolly pine Kraft pulp, mixed hardwood Kraft pulp, Oxygen delignified ECF Kraft pulps of spruce, Eucalyptus globulus, and Eucalyptus grandis, and bleached spruce sulfite pulp.

B. Timing/location of application

The point of the process in the pulp mill or deink plant or a tissue/paper mill at which the expansin and/or swollenin and enzyme formulation is applied is dependent on a variety of factors such as (1) the incubation time of the enzyme with the pulp; (2) the amount of time between the enzyme treatment and paper or tissue making; (3) the water loops in the pulp mill or deink plant. The incubation time of the expansin and/or swollenin and the enzyme formulation with the pulp stock can vary. The pulp stock is treated with expansin and/or swollenin and the enzyme formulation for at least 1, 2, 3, 4, or 5 minutes or longer. In some embodiments, the pulp is treated for at least 5 minutes, at least 10 minutes, or at least 15 minutes. The treatment time for the expansin and/or swollenin and the enzyme formulation can be the same of different. The pulp is enzymatically treated for a period of time and in an amount and under conditions resulting in fiber modification.

The enzyme treatment can be performed in tanks in the pulp and paper mills before the forming section of tissue or paper products. Other equipment stages in which the methods can be performed include, but are not limited to, repulper, dump chests, bleach towers, feed tanks, high density towers, silo water, white water tanks, machine chests, fan pump and head box where a minimum of 1 minute of contact time occurs.

C. Temperature, H, and consistency

It is well known in the art that enzyme activity is temperature and pH dependent. The enzyme treatments described herein are typically effective at temperatures of from 10°C to 90°C, preferably from about 25°C to 60°C. However, the temperature range can vary depending on the nature of the enzyme and the expansin proteins used and the optimal activity range for each expansin/swollenin and enzyme.

The enzyme formulation is typically administrated to the wet pulp stock with a consistency of 0.1% to 35%, preferably 0.5% to 15%, and more preferably 1.0% to 10%.

D. Pulp strength and water absorption rate

Determination of pulp strength and water absorption rate

The enzymatically modified fibers can result in increased strength in the final paper product. Fiber strength can be determined using many different measures which conform to the strength needs of the paper grade. Certain grades such as tissue focus on machine direction and cross direction tensile strength targets. Fine printing and writing grades also require tensile strength standards but with additional focus on surface printing ability. Packaging grades require a broad assortment of strength parameters including tensile, tear, Mullen burst, ring crush, STFI/SCT test, Concora, plybond, sizing test or box crush tests with finished products. Each of these tests is conducted according to industry standard testing equipment and methods. Increased paper strength is any increase in paper strength over the standard or usual paper strengths achieved relative to untreated paper or paper treated with a method such as starch, chemical processing or refining. Paper strength can be measured using TAPPI test methods T494 (tensile strength), T403 (burst strength), T414 (tear strength), T432-CM-99 (water absorption rate), and other standardized industry procedures.

In some embodiments, an "effective amount of expansin/swollenin and enzyme composition" means that the tensile strength and/or tear strength ofhandsheets increase by about at least 10%, 1 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25% or greater compared to the control (no treatment), enzyme treatment only, or expansin treatment only, wherein the sheets are prepared from pulp treated at a temperature from about 35°C to about 60°C, such as 35°C or 45°C. In some embodiments, the pulp is treated at 45°C and pH 6.5 for 45 minutes.

In some embodiments, an "effective amount of expansin/swollenin and enzyme composition" means that water absorption rate ofhandsheets decrease by at least 5%, 6%, 7%, 8%, 9%, 10%, 1 1%, 12%, 13%, 14%, 15%, or greater compared to the control, pulp treated with enzyme only, or pulp treated with expansin only.

Determination of pulp drainage

Pulp drainage is determined by the rate and degree to which water drains from a pulp stock. Two of the most widely used tests are the

Canadian Standard Freeness test and the European Schopper-Riegler test. One liter of 0.3% or 0.2% consistency pulp stock suspension, respectively, is drained over a conical object and the quantity of water which passes through an overflow tube is monitored. A quick draining stock creates a large overflow and results in a high number of ml of filtrate passing over the cone as calculated by a Canadian Standard Freeness test. Alternate methods of tracking drainage use vacuum systems whereby the time rate and maximum degree of water removal from a pulp suspension are tracked. Improved drainage on a paper machine is noted by changes in flat box vacuums or couch roll vacuums or the position of dry lines on a Fourdrinier forming table. Additionally, improvements in drainage may be noted in reductions in final sheet moisture with all other factors (e.g., machine speed, vacuums, sheet base weight, drying section temperature) remaining constant.

Increased pulp drainage is any increase in pulp drainage over the standard pulp drainage achieved without enzyme treatment as described herein considering the consistency and temperature of the suspension while the drainage is conducted.

As shown in the examples below, pulp samples treated with expansin alone showed little effect on freeness (Figure 4A) compared to the control (no treatment at all). Regarding handsheet density, treatment with expansin resulted in handsheets having the essentially the same density as the control. In contrast, handsheets prepared from pulp treated with expansin and enzymes showed a significant decrease in density but significantly higher tensile strengths and tear strengths compared to handsheets prepared from pulp treated only with expansin. The lowered density should significantly impact the tissue bulk resulting in softer tissue and towels with better water absorption.

Expansin-only treated pulp exhibited essentially the same tensile strength and tear strength as the control. The increase in tensile strength and tear strength for treatment with expansin and enzymes was more than additive for pulp treated with enzyme alone and expansin alone.

Finally, handsheets prepared from pulp treated with expansin and enzyme exhibited significantly faster water absorption, in some substantially more than additive compared to pulp treated with enzyme alone and expansin alone.

Treatments were performed on pulp fibers. Freeness of the pulp/fibers was measured while tear and tensile strength was measured on handsheets prepared from treated or untreated fibers as described in the Examples. Examples

Example 1. Extraction of expansin protein from native plant cell walls

Plant expansin proteins were extracted from sprouts of mungbean, soybean and cucumber. Seeds from each of the plant sources were soaked in water in the dark for about 5-7 days, and then exposed to light until the hypocotyls were enlarged. A 1 cm section of hypocotyl of the seedlings was cut off and collected for expansin extraction. The native expansin proteins from mungbean, soybean and cucumber sprouts were extracted as follows:

1. At least 25 grams of hypocotyl plant tissue from the growing region of the sprouts was collected.

2. A homogenization buffer solution was prepared containing 25 mM HEPES, 2 mM sodium metabisulfite and 2 mM EDTA with pH adjusted to pH 7.0 with NaOH. Triton X-100 at 0.10% can be included in the buffer for high yield of expansin protein. However, any resulting foaming should be washed out well to prevent protein denaturation.

3. The plant tissue was added to a chilled blender with buffer solution in an amount about ten times the weight of plant tissue. The mixture was homogenized for 20 to 60 seconds at medium-high speed for complete cell wall disruption.

4. The tissue pulp was collected in a filter bag of 70μιη pore size. The liquid was squeezed out and the tissue pulp washed with chilled distilled water or homogenization buffer solution until the washing filtrate was clear. All liquids were then squeezed out of the tissue pulp.

5. The tissue pulp collected in step 4 was transferred into an extraction buffer solution with 1 M NaCl, 25 mM HEPES, 2 mM EDTA and 2 mM sodium metabisulfite at pH 7.0 to make a loose slurry, having a consistency like wet oatmeal. The ratio of extraction buffer solution to tissue pulp was about 3: 1 by weight. 6. The tissue slurry was cooled to 4°C and agitated with a gentle motion for one hour. The use of stir bars was generally avoided as they can get stuck and often generate foaming leading to expansin protein denaturation. 7. The tissue pulp was removed from the extraction buffer solution with a filter bag of 70 μιη pore size. The extraction buffer solution with expansin protein was collected. This procedure can be repeated as necessary to ensure a high yield of expansin protein extraction.

8. Ammonium sulfate was added to the extraction buffer solution

containing the expansin protein. The mixture was cooled and allowed to sit for one hour to induce protein crystallization. The extraction buffer solution was decanted. The isolated protein crystals were stored in a freezer until ready for use.

9. Prior to use, the sample of expansin protein was diluted with

extraction buffer solution to a concentration of 10 mg/ml.

Example 2. The effect of expansin with hemicellulase and endo- glucanase on softwood pulps

A sample of commercial pulp was soaked in water for 4 hours following disintegration with a British Disintegrator for 20,000 Revolutions at 3.5%. The stock was conditioned to the desired temperature and pH with enzyme blends with or without expansin protein. The treatment temperature was 45°C, pH was 5.5 to 6.5, and treatment time was 45 minutes with continuous mixing in a water bath. The treated stock was then diluted to about 0.3% for drainage measurement and standard hand sheets were prepared for physical strength tests.

Similar procedures were used for experiments with different expansin proteins and enzymes. Bleached softwood Kraft pulps were treated with expansin protein from mungbean sprouts and cellulase or cellulase and hemicellulase at different treatment temperatures. The results are shown in Table 1. Table 1. Effect of expansin with cellulase and cellulase/hemicellulase on bleached softwood Kraft pulps (Enzyme A is an endo-glucanase, and Enzyme B is an enzyme blend of an endo-glucanase and a hemicellulase)

The results are shown in Figures 1A-1D. Expansin protein alone doesn't appear to impact freeness or strength of the hand sheets, but the combination of expansin protein with cellulase and hemicellulase showed significant improvement in freeness and strength of resulting handsheets.

Also, the combined treatment of expansin protein with either Enzyme A or B lowered the hand sheet density significantly. The lowered density should significantly impact the tissue bulk resulting in softer tissue and towels with better water absorption.

Example 3. Effect of expansin with pectinase and endo-glucanase on softwood pulps

Similar experiments were conducted at similar conditions using expansin proteins from two different sources with pulps from Eucalyptus globulus and Eucalyptus grandis. The results are shown in Table 2.

Table 2. Effect of enzyme and expansin protein on two types of pulps from Eucalyptus (Enzyme C is an enzyme blend of an endo-glucanase and a pectinase)

The results are shown in Figures 2A-2C. Expansin proteins from soybean sprouts showed less impact on freeness of stock or the strength of resulting hand sheets; while expansin protein from cucumber showed increased handsheet burst and tensile strength with no changes in stock freeness. However, expansin protein from soybean in combination with Enzyme C increased the swelling of fiber more relative to expansin from soybean and cucumber with decreased sheet density.

Example 4. Effect of expansin with hemicellulase and pectinase on bleached mixed hardwood Kraft pulp

Bleached mixed hardwood Kraft pulp was treated with expansin from cucumber sprouts with hemicellulase and pectinase under similar conditions as previous experiments. The results are shown in Table 3.

Table 3. Effect of expansin with hemicellulase and pectinase on drainage and water absorption of mixed hardwood pulp (Enzyme D is an enzyme blend of a hemicellulase and a pectinase)

The results are shown in Figure 3A. Figure 3A shows the tensile strength of hand sheets prepared from untreated pulp, Enzyme D only (without expansin), cucumber expansin only, and cucumber expansin in combination with Enzyme D. Based on the results, expansin protein from cucumber sprouts lowered the stock freeness slightly with no significant impact on tear, burst and tensile strength of the resulting hand sheets. In contrast, the combination of expansin protein with Enzyme D showed significant improvement of both dry and wet tensile strength along with better water absorption rate and higher bulk of the hand sheets. Expansin protein with hemicellulase and pectinase appear to loosen fiber structure resulting in swelling of fiber with better water absorption and improved strength for tissue/towel production.

Example 5. Effect of expansin with cellulase, hemicellulase and pectinase on bleached spruce sulfite pulp

Bleached spruce sulfite pulp was treated at a consistency of 3.5% at pH 6.5, and a temperature of 50°C for 50 minutes with and without expansin and an enzyme blend of cellulase, hemicellulase and pectinase. The results are shown in Table 4.

Table 4. Effect of expansin with cellulase, hemicellulase and pectinase on bleach spruce sulfite pulp (Enzyme E is an enzyme blend of an glucanase, a hemicellulase, and a pectinase)

The results are shown in Figures 4A-4C. Figure 4A is a graph showing pulp freeness for untreated pulp, pulp treated with expansin alone, pulp treated with Enzyme E alone, and pulp treated with expansin in combination with Enzyme E. Expansin protein from mungbean sprouts showed little impact on freeness and strength of resulting handsheets;

however, expansin protein together with enzymes showed significant improvement on handsheet tear strength and water absorption rate along with improved bulk (Figures 4B and 4C).

Claims

We claim:

1. A method for treating papermaking pulp, the method comprising applying to the papermaking pulp an effective amount of expansin, swollenin, or combinations thereof in combination with an effective amount of an enzyme composition comprising one or more hemicellulases, one or more pectinases, or combinations thereof.

2. The method of claim 1, wherein the method comprises applying expansin to the pulp.

3. The method of claim 1 or 2, wherein the expansin is alpha- and/or beta-expansin.

4. The method of any one of claims 1-3, wherein the method comprises applying swollenin to the pulp.

5. The method of any one of claims 1-4, wherein the enzyme composition further comprises one or more cellulases.

6. The method of any one of claims 1-5, wherein the one or more cellulases consist of endo-glucanase.

7. The method of any one of claims 1-6, wherein the one or more hemicellulases comprise xylanaase, gamanase, mannanase, arabinase, and combinations thereof.

8. The method of any one of claims 1-7, wherein the one or more pectinases comprise pectinase, pectin lyase, pectate lyase, pectin esterase, and combinations thereof.

9. The method of any one of claims 1-8, wherein the papermaking pulp is derived from wood, fiber crops, or non-wood sources.

10. The method of claim 9, wherein the non-wood source is bamboo, reed, kneaf, wheat straw, rice straw or grass.

1 1. The method of any one of claims 1-10, wherein the papermaking pulp is virgin pulp or recycled pulp.

12. The method of any one of claims 1-11, wherein the enzyme composition is applied at a locations selected from the group consisting of storage tanks in the pulp and paper mills before the forming section of tissue or paper products, repulper, dump chests, bleach towers, feed tanks, high density towers, silo water, white water tanks, machine chests, fan pump, and head box.

13. The method of any one of claims 1-12, wherein the enzyme composition contacts the papermaking pulp for at least one minute, preferably one minute to five minutes, more preferably at least five minutes.

14. The method of any one of claims 1-13, wherein the enzyme composition is applied at a pH of 3.5-9.5, preferably 4.5-8.0, more preferably 4.5-6.0.

15. The method of any of claims 1-14, wherein the enzyme composition is applied to the papermaking pulp at a temperature of about 10°C to about 90°C, preferably from about 20°C to about 60°C, more preferably from about 30°C to about 60°C.

16. The method of any one of claims 1-15, wherein the concentration of expansin or swollenin is from about 0.01 to about 1000 mg per 100 g oven dried ("OD") fiber, preferably from about 0.2 to about 400 mg per 100 g OD fiber.

17. The method of any one of claims 1-16, wherein the enzyme composition is applied to papermaking pulp in the form of pulp stock having a consistency of from about 0.1% to about 35%, preferably from about 0.5% to about 15%, and more preferably from about 1.0% to about 10%.

18. A formulation for use in the method of any one claims 1-17.

19. A formulation comprising one or more expansins, one or more swollenins, or combinations thereof, and one or more hemicellulases.

20. A formulation comprising one or more expansins, one or more swollenins, or combinations thereof, and one or more pectinases.

21. A formulation comprising one or more expansins, one or more swollenins, or combinations thereof, and one or more endo-glucanases.

22. A formulation comprising one or more expansins, one or more swollenins, or combinations thereof, one or more hemicellulases, and one or more pectinases.

23. A formulation comprising one or more expansins, one or more swollenins, or combinations thereof, one or more hemicellulases, and one or more endo-glucanases.

24. A formulation comprising one or more expansins, one or more swollenins, or combinations thereof, one or more pectinases, and one or more endo-glucanases.