FIELD OF THE INVENTION

[0001] This application, claims the benefit of OS provisional .application. umber ^'

61/71 1,269, fiied 9 October 2012, the entire contents of which are hereby incorporated by reference.

[0002] The present invention relates to a cellulase composition comprising niono- compon.ent - endo-celluiases; eationic fixatives and/or ndniomc detackifying polymers; cellulase protein stabilizers:; and cellulase enhancers, The present invention also relates to the use of a cellulase composition to improve dry strength properties of a paper product by treating cellulosic fibers in pul furnish by using the eehulase composition at an endo- cellulase activity of from about: 5 ECU to about 2500 ECU pe kilogram (kg) of dr fiber prior to mechanical refining in a papermaking process,

BACKGROUND OF THE INVENTION

[0003] Cellulase can be used to modify the cellulose surface of cellulosic fibers enhancin the efficiency of mechanical. refinin of wood fiber saving refining energ in

papermaking. While the combined, action of the celiuiase treatment followed by mechanical refining of cellulosic fiber helps in fibri.Ilating the fiber, many commercial cellulases also contain specific cellulase activities that are capable of defibrillating cellulosic fiber by hydroly^mg the fib.nii.ated area on the fiber surface. This action of celiuiase is detrimental for paper dry strength properties as the fihrillated area i needed for better fiber to fiber interaction in a paper product upon drying and providing beiter dry strength. In addition, those specific cellulase activities mentioned above may be capable of hydro! zing small cellulosic fiber debris or fine particles. While this property of cellulase can help reduce pulp ^" Viscosity and improve pulp drainage; it can also cause fiber loss with increased chemical oxygen demand (COD) in paper production. It is. not mechanistically clear how a cellulase product can be applied to a papermakin ^■ process for impro vin dry strengt properties of a paper product

I [0004] Cellulase is generally referred to as an enzyme composition derived from a microorganism fungi or bacteria that can catalyze the hydrolysis of β-l, 4-glyeo.sidic bonds of a cellulose molecule or its derivatives. As shown in Table L en io-celMases, exo-ceflulases- and ceilobiase celiulases are three types of specific celiulases that have distinctive activity that is different from each other towards specific cellulose molecules. The three types of celiulases are physically, chemically and enzymatieally different Among them, end ^' o-celiulase or β-glucanase randomly ydroiyzes internal: amorphous anomalies within crystalline cellulose, yielding high oligosaccharides or shortened cellulose polysaccharides. Exo-cellulases or exq-cel iobiohydrola-se (CBH1 or CBH2) release oligosaccharides of a degree of polymerization (DP) of 2 to 4 from the reducing end or non -reducing end of a cellulose polymer. Ceilobiase or β-glucosidase has. no activity towards cellulose polymer or oligosaccharides but catalyzes the hydrolysis of ceilobiase to glucose. Celiulases are used in a variety of industries and are produced in large scale from various species such as. Trichoderma, tiumicol , I ^' hermomyces, Bacillus, etc., via genetic enzyme engineering,

^' [0005] ^' To determine endo-cellulase activit in a cellulase product, a water soluble Cellulose derivative such as carboxymetfiyl cellulose (CMC) or hydroxyethyl cellulose (HEC). is conventionally used as a substrate and the reducing sugar released b the enzyme is measured by a dmitrosalicylic acid (DNS) method. The exo-eellulase activity may be distinguished from, the endo-cellulase activity by using water insoluble cellulose such as cellulose filter paper or wood fiber as a substrate and the reducing sugar released from the insoluble fiber is then, determined by the DNS method mentioned above. The ceilobiase activity in a cellulase product is usually determined using eellobiose as a substrate, and the amount of glucose released is assayed usin a glucose oxidase (GO) method.

Table I. Classification of. Cellulase

[0006] A cellulase derived from microorganisms may contain all three types of celiulases. While such a product can work synergistically to attack crystalline cellulose and convert it to small sugars, and eventually to glucose, it is not preferred for use in paperaiaking applications to improve paper dry strength, The endo-cellulase activity in the cellulase product attacks the amorphous anomalies within the crystalline cellulose and disrupts the crystalline structure. This enhances: the efficiency of mechanical refining in fibriliating eeliiiiosic fiber and helps improve dry strength of a paper. However, the exo-cel!iilase activity ¹ that exists in the cellulase product may defibril!ate the cellulosic fiber and generate cellulose fines'. In theory exo-cellulase activity may help improve pulp drainage via defibrillation, but it could also have -a negative effect on paper dry strength properties. Not all celMases are effective for paper strength applications and some can actually hull the dry strength properties.

[0007] A cellulase. derived from a .microorganism, may have multiple components with more than one endo-cellulase and exo-celiobiohydmlase. For example, a eeiluiase from Trichoderma longibrachiaium can have two CBH components, CBH I and CBH IT, and three endo-cellulase components, EG I, EG II and EG ILL A mono -component cellulase can be produced by the cloning of a specific eeiluiase DMA sequence encoding the single cellulase and expressed in a host organism. In other words, a mono-component endo- cellulase is a single endo-cellulase component essentially free of other ee!lulase such as exo-celiulases and β-glueosidase . that usually exist in a cellulase product produced by a conventional microorganism, Single endo-cellulases can be used in the present Invention for improving dry strength of a paper product in papermakihg.

[0008] U.S. Patent. No. 51.69497, No, 5423946, No. 67701.70, No. 6939437, and U.S. Patent Appl, No. 20.1 10168344, disclose that, a cellulase product can be used to improve drainage of a wood pulp when used in combination with cationic polymers. However, the references are silent on how those combinations affect paper dry strength, which specific celluiases may be used in the application or how the cellulase dosage affects _: the performance for paper dry strength.

[0009] ^' U.S. Patent No.- 5507914 (the "914 patent), describes a process, for enhancing pulp freeness and also paper strength using a combination of a eeiluiase with a cationic polymer. The. '914 patent teaches a dosage level of 0.05-0.25% cellulase based on the dry pulp was used. This is equivalent to about 2500 ECU/kg to about ί 2500 ECU/ kg dry fiber based on the present invention. Our studies indicate that at these higher addition levels, dry 'Strength properties are negatively impacted.

[0010] U.S. Patent No. 6635146 (the T46 patent), discloses a method of treating papermaking wood fibers using a one or more, truncated hydrolytic enzyme in. amounts of 5,000 ECU to 200,000 ECU per kilogram of fiber.

[00111 U.S.. Patent Appl. No. 20020084046 (the Ό46 ap lication), describes a process for making paper by adding an enzymatic material to a storing stage that is subsequent to the pulping or refining stage for a paper product having improved softness, bulk and absorbenc while maintaining strength,

[0012] General literature teaches that cellulase activity may be improved in an enzyme assay when used in. combination with anionic and non-ionic surfactants. The possible mechanism i that the surfactants reduce cellulase- adsorption to -non-cellulose

components such as ligniii, free cellulase for the eeliulosic- substrate and aid i thermal, stability of the cellulase protein. Tween 20 and Tween 80 are two examples of such surfactants. Polyethylene glycol and its surfactant derivatives may als help improve cellulase activity in ceJiuiase- assays. However, little information is available in public on using combination of cellulase and surfactants in papermaking application and how those combinations would affect specific activities of the three different types of cellulases,

[0013J U.S, Paten t Appl No. 20040038841 discloses a cellulase formulation produced from nonionic surfactants together with endo-glueanases derived from Zygomycetes, which can be used in the treatment of fabrics.

[0014] Japanese Patent No. 5507615 discloses a polyvinyl alcohol and

poiy(vinylpyrrolidone) i a cellulase formulation to enhance cellulase activity,

[0015] The publications, listed, above -are all incorporated herein b reference.

DETAILED DESCRIPTION OF THE INVENTION

10016] The present in ention relates to a cellulase composition for papermaking comprising: cellulase; contaminant. control polymer(s), wherein the contaminant control, polymer can be- cationic fixative polymer{s), detackifying polymer(s), and mixtures thereof; cellulase protein stabilizer: and cellulase. -enhancer. [0017] The eellulase composition of the present, invention exhibits improved eellulase activity and storage stability over h original eellulase.

[0018] la another embodiment, the present invention relates to the use of a eellulase composition to improve dry strength propertie of a paper product by treating. cellulosic- fibers in a pulp, stock or furnish with a eellulase composition prior to mechanical refining in a apermakmg process. Mechanical refining of cellulosic plant substances (e.g., wood) is used in the papemiakin process to generate pulp, the basis and raw material for making paper products. Pulp is generated by removing cellulose fibers from, their wood matrix. This -can be accomplished by using chemicals, heat, and pressure, e.g. chemical pulping, or mechanical energy, heat, and pressure-, e.g. mechanical pulping. Additionally, individual pulp fibers can be liberated from recycled fiber or dry finished pulp, e.g.

market, pulp, through application of mechanical energy while slurrying in water. This resulting material can be termed a pulp, pul slurry, stock or furnish, which terms are used interchangeably and are understood to mean a suspension of -cellulosic fiber either before- or after mechanical refining. Mechanical refining as used herein refers to treatment of a pulp slurry largely made-up of individual pulp fibers rotating between metal bar --containing discs in a stock refiner. This mechanical action develops fibriliaied rnici'Ostrueture on the- surface of individual fibers, which allows better bonding to- each other upon sheet consolidation and drying. This typ of refiner is a common unit operation in paper mills,

[(1019] Dependent upon the type of paper or paperboard being produced, a ..papermaker will refine the pulp to a desired freeness. "Freeness" refers to the measurement. of water drainage from pul or the ability of a pulp and water mixture to release or retain water or drainage. Pulps having greater freeness values are characterized as being faster draining, coarser pulps. Freeness is typically reported as Canadian Standard Freeness (CSF).

Freeness is dependent upon both the mechanical properties of the refiner and the physical properties of the wood chips. An operator may vary the parameters, of the refiner to attain a freeness target. The target or desired freeness is dependent upon the grade of paper or paperboard being produced. [0020] -CeJlulases used in the present invention are available from any one of several en¾yme producers. They can be either mono-component or multiple-component cellulase products. A mono-component eiido-celluiase is a cellulase product essentially free of exo-cellulases and eei!obiase. Examples of mono-component endo-cellulase include, but is not limited to, FiberCare® R and FiberCare® U from Novozymes (Bagsvaerd, Denmark), Optimase® CX S6L from DuPont Industrial Biosciences (Palo Alto, CA, USA) and EcoRilp® R from AB Enzymes (Fort Mill, SC, USA). Examples of multi- component celiulases include, but are not limited to, FiberCare© D, Celiuclast® L5L from Novozymes and Optimase® CX 40L from DuPont Industrial Biosciences.

[0021] The en do-cell uiase s, exo-eel!u!ases and cell obiase ceilulases are known in the ait to act synergistieally toward cellulosic fibers converting them to glucose. In

papermaking, cellulosic fiber may be modified by a specific endo-cellulas with minimal effect on fibe length. It is generally accepted that paper dry strength lies primari ly in the bonds between the cellulose fibers and fiber length. -Similar to mechanical refining, fiber fibrillation by endo-eellulases creates larger surface area with strong inter- fiber interaction, resulting in lower permeability of the paper product and improved paper dry strength and stiffness. A multi-component cellulase- product derived from a

microorganism may be employed in this in vention:. However, if the cellulase contains a significant amount of exo-cellulases, that could function in defiboi!ating the cellulosic fiber thereby having a negative effect on paper dry strength, Endo-cellulases and mono- component cellulases that are free of any exo-cellulases can be used for improving dry strength properties of a paper product. It should be noted that a multi-component cellulase may exhibit higher cellulase activity in the DNS cellulase assay as described in the. experimental section, and it could be more effective than a mono-component cellulase for treating wood pulp to Improve pulp drainage.

[0022] The contaminant control polymef(s) of the present cellulase composition may contain one or more papermaking detacktfying polymer(s) including, for example, nonionic and anionic cletaekifiers, hydropliobicaliy end-capped polyethylene glycol), poly(vinyl alcohol-vinyl acetate), whey protein, soy protein, hydrophobic/hydrophilie block copolymers, and hydrophobically modified ^" hydrox.ye.ihyl cellulose (HEC).

Commercially available nonionic cletaekifiers are available from Ashland Inc,

Wilmington, DE, USA. among others, Nonionic detackifiers include., but are not limited to, DeTac® DC779F, DeTac® DC3970, and DeTac® DC7225. Anionic detackifiers such as, DeTac® DC720 are also envisioned, in addition to the ability of stabilizing and enhancing endo-cel!u!ase activity, the detackifiers of the present cellulase composition also provide benefits of controlling pitch and stick ies deposits in a papemiaking process.

[0023] The contaminant control polymer-(s) of the. present cellul ase composition, may also be one or more papemiaking cationic fixativ polyraer(s), for example,

poly(DAf)MAC) (poly(diall ldimethylammonfarn chloride), poly(DMA-EPi-EDA) (dimethylmnme-epicM condensation polymers), cationic poly(acrylamide), GPAM (glyoxylated po!yacrylaimde), poly(ethyleneimine), epichlorohydrin (EPi)-reaeted pal (amidoamine , poiy(vinylarmne), hydrophobica!ly modified cationic polymers such -as, alkylated, polyethyleneimine (PEI), alkylated poly(lysme), alkylated homo- and co-polymers of vinylamine, -alkylated

poly (aminoami.de), alkylated polyaeiylamide, copolymers of vinylamine- containing amino groups with hydrophobic ^■ monomers, copolymer's of dimethyl diallyl ammonium chloride with hydrophobic monomers, copolymers of acryiate containing amino groups with hydrophobic monomers, and alkylated amino containing natural and modified polysaccharides, alkylated cationic proteins and mixtures thereof, C8-C10 alkyi .glycidyl ether modified- poly(aminpaniide), cationic natural products, and amphoteric polymers having a specific cationic unit and an anionic unit such as amphoteric acryl amide polymer formed from both anionic and cationic monomers, the amphoteric vinykmide polyme formed from both anionic and cationic monomers, an amphoteric dimethyl diallyl ammonium chloride derivative, poly(acry!amide-co-acrylic acid-co-dimethyl ally! ammonium chloride copolymer), poly(ac-iylic acid-co-dimethyl diallyl. ammonium chloride copolymer), amphoteric starch, amphoteric polysaccharides, amphoteric polymeric- micropartjcle polymer, and mixtures thereof. Cationic fixative polymers for the use in the present invention are commercially available from Ashland Inc,

Wilmington, DE, USA, among others, -and include, for example, Zenix DC® 7429, Zenix© DC7479, Hercobond© 6363, Hercobond® 6350 and DeTac® DC786C. The cationic fixative polymers and contaminant control detackifiers- can be used separately or together in the cellulase- composition. Furthermore, a separate cationic polymer product with contaminant control properties can be applied to a papemiaking system in conjunction with the present cellulase composition to improve overall ^' performance. [0024] Additionally, other additives used in the papermaking process can be used in conjunction with the present cellulase composition including, for example, cationie papermaking additives suc as, dry strength additives, wet strength additives, ilocculants, retention aids, and drainage aids. These cationie papermaking additives may possess fixative properties for anionic components in a papermaking process,

[0025] The present cellulase composition also contains eelkilase protein stabilizers including, for example, propylene glycol, glycerol, ethylene glycol, sugar, sorbitol, lactic acid,, glucose, galactose, maitodexlrin, oligosaccharides, com syrup, and inorganic salts such as,. sodium and potassium chloride; a pH buffer system such as, sodium or potassium phosphates, sodium citric acid, tris(.hydro.xymethyl)methylamine (Tris), 4-2- hydroxyethyl- 1 npiperazineethanesuifo ic acid (H PES), piperazin.e-N,N-bis(2- ethanesulfonic acid), 2 2-(N-morpholino.)ethanesi.d.fon ^' ie- acid, and protein ligands such -as, glucose and N- cetyl -D-glucosamine, and other protein stabilizers that are well known in the art to stabilize a protein tertiary structure and help maintain enzyme activity.,

[00263 The cellulase composition of the present invention may also contain one or more metal ion salts that enhance cellulase stability and activity. Metal ion -salts include, for example, calcium chloride;, zinc chloride and magnesium chloride.

[0027] In one embodiment the cellulase composition is a mono-component endo- eelluJase and the contaminant control polymer is a polyvinyl acetate-co-vinyl alcohol), hydrophobically end -capped polyethylene glycol detackifier or a mixture thereof; the cellulase stabilizer is propylene glycol, glycerol, sorbitol or mixtures thereof; and the enhancer i calcium chloride.

[0028] In yet another embodiment, -the -cellulase composition is a mono-component endo-celiulase; the contaminant control polymer(s) is a ca ionie fixative po!yrner(s) such as, polyiTJADM AC), poly(DMA-EPI-EDA), hydrophobically modified cationie fixative or .mixtures: thereof; the cellulase stabilizer is propylene glycol, . glycerol, sorbitol or mixtures thereof; and the cellulase enhancer is calcium c loride.

[0029] The ratio of the four main components in the cellulase .composition can be changed in a specific range to provide optimized cellulase activity and protein stability under specific pH, ionic strength and temperature conditions. The ratio may also affect its ceijuiase efficiency of treating ceUulosic fibers for paper dry ^■ strength applications and the performance of the paperinaking contaminant control polymers in a papermaking system. The celluiase composition of the present invention is an. aqueous formulation containing up to about 95% water and from about.5% to about 50% oilier non-aqueous components.

[00301 In one embodiment, the celluiase composition has an active concentration of a mono-component endo-cellulase of from, about 2 wt. % to about 80 wt, % of the total composition on an active basis; can be about 3 wt. % to about 40 wt. % Of the total composition on an acti ve basis; and -may be from .about 5 wt. % to about 25 wt. % of the total composition on an active basis; the contaminant, control polymer concentratio can be from about 2 wt. % to about 50 wL % on an active basis; can be about 5% to about 40 wt. % on an active basis; and may be 10 wt. % to 20 wt. % on an active basis; the protein stabilizer content can be from about 0.1 wt, % to about 50. wt. % on a non-aqueous or dry basts; can be from about 5 wt. % to about 40. wt. % on dry basis; and may be from about 10 wt. % to about 30 wt. % on dry basis. The celluiase enhancer can be from: 0.1 wt, % to about 0.5 wt. % on dry basis; can be from 0.001 wt. % to 0.25 wt. % on dry basis; and may be from about 0,005 w ^' t, % to about 0,1 wt. % on dry basis.

[0031] I another embodiment, the celluiase composition has an active concentration of a muJti-cornponent celluiase of from about 2 wt. % to about 80 wt. %. of the total composition on an acti ve basis; can be about 3 wt. % to about 40 wt. % of the total composition on an active basis; and may be from about 5 wt. % to about 25 wt. % of the total composition on. an. active basis; the contaminant control polymer concentration can be from about 2 wt. % to. about 50 wt, % on .an active basis; can be about 5% to about 40 wt. % on an active basis; and may be 10 wt. % to 20 wt. % on an active basis; the protein stabilizer content can be from about 0,1 wt. % to about 50 wt. % on a non-aqueous or dry basis; can be from about 5 wt. % to about 40 wt. % on dry basis; and may be from about 10 wt % to about 30 wt. % on dr basis. The celluiase enhancer can be from 0,1 wt % to about 0.5 wt, on dry basis; can be from 0.001 wt, % to 0.25 wt. % on dry basis; and may be from about 0.005. wt. % to about QJ wt. % on dry basis. [0032] The active percentages of the contaminant control polymer, the protein stabilizer and cellulase enhancer in the cellulase composition are defined as non-aqueous parts of these polymers or chemicals in the cellulase composition. The active weight percentage of the endo-cellulase or cellulase active in the cellulase composition is based on the assumption that the original cellulase is 100% active as it is obtained from a commercial source.

[0033] The pH of the cellulase composition of the present invention affects the stability of the protein stabilizer and activity of the cellulase enzyme. The proper pH prevents protein denaturation that can result in deactivation of the cellulase. The pH of the present cellulase composition can be in the range of from about 3 to about 10; can be in the range of from about 4 to about 8, and may be in the range of from about 5 to about 7,

Typically, in a process of producing the present cellulase composition, the contaminant control polymer can be mixed with the protein stabilize and the cellulase enhancer in water for about 5 to about 30 minutes at room temperature followed by the addition of the mono-component endo-cellulase product. The four components can be added together in a random sequence prior to introduction into the papermaking furnish of the papennaking. process. The pH of the cellulase composition can be adjusted with an acid or an alkali if needed after the composition becomes homogenous in appearance, A buffer system may also be used to control the pH of the cellulase composition in a specific range.

[0034] The cellulase composition of the present invention exhibited improved cellulase activity relative to the cellulase activity of a conventional composition. The present cellulase composition also had better cellulase storage stability and better physical storage stability relati ve to the original cellulase* particularly .at higher temperatures of about 50°C or higher. The term "improved cellulase storage stability ¹¹ means that the present cellulase composition after being stored for a period of time at a certain temperature and subjected to the. same standard test conditions as the conventional cellulase, exhibits a lower reduction in cellulase activity compared with that of the original cellulase, The term "good physical stability" means that the cellulase composition- has maintained desired physical properties in appearance, homogeneity and light color with no

deteriorated odor. [0035] For the cethjlases intended to be used in the present cellulase compositions, the celfulase activity including endo-cellulase (ECU) activity, exo-cellobjohydrolases and β- glueosidases activity were -tested using standard methods as described in Table I. The endo-cellulase (ECU) activity of the original cellulase- measured by DNS ^' assay, as described in the- experimental section, is in the range of. from about 500 ECU/g to about 20000 ECU/g; can be from about 1000 ECU/g to about 15000 ECU/g; and may be from about 2000 ECU/g to about. 10000 ECU/g, The cellulase activity can ^' vary with specific batches of cellulase products, and the materials from different commercial sources. The endo-cellulase activity of the cellulase composition of the present invention is normally in the range of from about 25 ECU/g to about 10000 ECU/ g; can be from about 50 ECU/g to about 5000 ECU/g; and may be from about 100 ECU/g to about 3000 ECU/g. The cellulase activity of the cellulase composition, may be evaluated under specific pH and temperature cofldiHons with different cellulase substrates as needed. The activities of the cellulase composition oF the present invention and the original cellulase with respec t to producing reducing sugar from a water soluble cellulose derivative and the reducing sugar from a. water insoluble cellulosic fiber were compared to -determine the -selectivity of the cellulase as. art endo-celiulase towards a fiber, The present cellulase composition as a specific endo-cellulase produces higher reducing sugars from a water soluble cellulose derivative and lower reducing sugars - from a water insoluble .cellulosic fiber than the original cellulase composition.- Optionally, ceilobiase activity in a cellulase product may be determined using a glucose oxidase (GO) method to measure glucose generated from cellobiose by the cellulase product and compared with that of a known endo-cellulase. The lower the ceilobiase and exo-cellulase activity, the more pure the cellulase composition it is as an endo-cellulase product

[0036J The present cellulase compositions may be used in papermaking processing for treating all types of cellulosic fibers including bleached and unbleached virgin fiber, mechanical fiber and rec cled fiber, and can be used for virgin fiber and good quality recycled fiber in paper mills that use refiners. The modification of the surface of cellulosic fibers by the present cellulase composition results in a reduction of energy consumption of the mechanical refiner. To evaluate the effectiveness of a cellulase composition on the cellulosic fiber in a practical application in papermaking, one should be able to observe the same refining efficiency with lower refiner energy, improved dry strength properties of the paper product and the change in drainages of the pulp slurry before and after the refiner, in general, a -combination of an increased freeness or drainage in the pre-refining pulp -and a decrease or unchanged freeness of the post- refining pulp is an indication of effective- treatment by the eeilulase composition,

[0037] One embodiment of the present invention is- the process of making a paper product wherein a cellulosic fiber in. an aqueous suspension that is being -agitated is treated with a. eeilulase composition comprising ¾ mono-component encfo-eel!uiase; contaminant control polyrner(s) such as, detackifiers and/or cailoMc fixative- poiymer(s), or .mixtures thereof eeilulase protein stabilizer; and eeilulase enhancer and the eeilulase activity is from, between about.5 ECU and about 2500 ECU per kg of dry fiber at a teniperature of f om about 20°C to about 70°C and a pH of from about 4 to about and wherein the eeilulase composition is in contact with the- cellulosic fiber for at least 10 minutes prior to the cellulosic fiber being refined by a refiner and forming and dryin the fiber into a desired product.

[0038] Another embodiment of the present invention is: the process of making- a paper product wherein a cellulosic fiber in _. an aqueous suspension that is being agitated is treated with a eeilulase composition comprising a multi-component eeilulase;

contaminant control polymer(s) such as, detackifiers and/or cationic fixative polymer(s), or mixtures thereof; eeilulase protein stabilizer; and eeilulase enhancer and the eeilulase activity is from between about 5 ECU and about 25Q0 ECU per kg of dry fiber at a temperature of from about 20°C to about 70°C and a H of from about 4 to about 9 and wherein the. eeilulase composition, is in contact with the ceil ulosie fiber -for -at least .10 .minutes prior to the cellulosic fiber being refined by a refiner and forming and drying the fiber into a desired, product,

[00.39] The mono-component endo-cellulase. and the eeilulase composition of the present invention can be used for paper dry strength applications in a specific endo-cellulase activity dosage range. Overdosing with, a eeilulase composition..may cause damage to the cellulosic fiber by shortening the fiber length, resulting in reduced bond strength. The dosage of an endo-cellulase needs be controlled -at a level that it will not defibril!ale the fiber too much and pot shorten the fiber length. Surprisingly, it was found that the present eeilulase composition made with noniomc- detackifiers had. little or no negative effect on dry strength properties, such as the Mullen Burst test, in an overdose situation, i:2 However, when a detackifier was used with the original eellulase composition a decrease in Mullen Burst was observed. This indicates that the present cellufase -composition is much more tolerable in a practical -application when the paper furnish is accidently overdosed, due to situations such as, paper machine shutdowns or other unexpected events in a pape mill,

[0040] The eellulase composition of the present invention made from a multi-component eellulase containing majorly endo-celiitlase activity may be also used, for paper dry strength applications. It should be noted that treating virgin or recycled fiber with this composition could generate more cellulosic fines than a mono -component endo -eellulase composition does at the same overall eellulase active due to the presence of exo~cellulase components. Further the multi-component eellulase composition may be- more prone to hurt strength property when it is overdosed,

[0041] Another embodiment relates to a process of making paper products by treati ng cellulose fiber in an aqueous solution that i agitated during contact with the -eellulase composition comprising _. at least about 5 ECU of eellulase activity per kg of cellulosic dry fiber.

[0042] Another embodiment relates to a process of making a paper product by treating cellulose fibers in an aqueous suspension with a eellulase composition. A eellulase composition according to the- resent invention is added to a paper furnish that is undergoing agitation. The eellulase composition comprising an amount not to exceed about 2500 ECU of cefiulase activity per kg of cellulosic dr fiber; can be from about 20 ECU to about 2000 ECU of ceifuiase acti vity per kg of cellulosic. dry liber; and may be about 50 ECU/kg to about 1500 ECU of eellulase activity per kg of cellulose dr fiber.

[0043] The pH in the process of making a paper product with the present eellulase composition is at least about pB 3 but not to exceed a pH of about 9; the pfl can be from about 4 to about 8,5; and may be from about 4.5 to. about 8. Contact time of ^' the eellulase composition with cellulosic fiber is at least, about 10 minutes and ean.be up to about 5 hours.; can be from, about 0.2 to about 3 hours; and may be from about: 0.3 hours to about

2 hours. Temperature is at least 10°C bu not higher than about 70°C; can be- from about

23°C to about 60°C; and may be in the range of from about 30°C to about 50°C. The pulp Slurry o furnish temperature in a papermaking system varies with paper machines and specific paper grades. Therefore, it is often expected that, the cellulase composition has higher activity in a paperniaking system that has higher stock temperature. Th •selectivity or specificity with regard to the endo-celiuiase activity vs.. e o-cel!ulase activity of a specific cellulase composition of the present, invention may also change in paper mills that have different system stock H,

[0044] In yet another embodiment, a method of improving the drainage of a celiulosie fiber in. a papenriaidng process is provided. A eeliuiase composition is: provided containing cellulase, contaminant control polymer(s), and mixtures thereof, eeliuiase protein stabilizer(s); and cellulase euhaucerfs), wherein the cellulase composition is added to a pulp slurry in an amount in eeliuiase activity ranging from about 5 EOU/kg to about 2,500 ECll kg dry wood fiber,

[0945] In the present process the cellulase composition may be used to treat virgin celiulosie fiber, for example, softwood bleached kraft (SWBK), hardwood bleached kraft (HWBK), or a mixture thereof. The present eeliuiase composition can also be used to treat recycled fiber. In a lab setting, the treatment can be conducted under effective agitation at about 50 °C for about §0 minutes.. The treated celiulosie fiber is then subjected to a laboratory refiner such, as a PFI mill or valley beater to a desired freeness. The refined pulp is then used to prepare a paper product, such as, haiidsheets at a specific basis weight. Paper dry strength properties such as Mullen Burst, Dry Tensile, etc... are tested and the data normalized, based on the basis weight over a blank (the fiber has not been treated with a eeliuiase .composition) and a control using the original cellulase. In addition to improving dry strength, the present cellulase composition may be used to treat, virgin or recycled fiber io improve drainage and retention with or without, mechanical refining. The present cellulase composition may also be applied to celiulosie fiber after refining and prior to the paper product being formed,

[0046] Contaminant control polymers such as detaeklfiers or catiomc fixative polymers are generally used in a papermaking process for cleaning contaminants from celiulosie fibers and paper machine surfaces. One advantage of blending a contaminant control polymer such as, a nonionic and anionic detacldfier and or cationic fixative polymer into the eeliuiase composition is. to help remove stickies adhered on the surface of celiulosie fibers and allow better access of the endo-cellu!ase to the fiber. The cationic fixative polymer may also nteract with the anionic group on the fibers: surface thus interrupting hydrogen bonding between cellulosic fibers in the crystalline structure. Additionally, the cationic fixative, polymer may help the cellulase penetrate into the fiber wall.

[0047] Treating a recycled pulp containing stickies and pitches with the present cellulase composition improved pulp drainage and cellulase efficiency towards the cellulosic fiber. In some cases, the mono-component endo -cellulase and contaminant control polymers had a synergistic effect providing improved paper dry strength properties. When a contaminant control polymer was introduced into the present celiu ^' lase compositions, better fiber retention was observed than was seen with the original cellulase.

Additionally, the present cellulase ·. composition would be expected to have a positive- effect on the chemical oxygen demand (COD) reduction in a paper mill. The contaminant, control polymers are compatible to the endo-ceilulase of the present, invention and forms homogenous and stable aqueous compositions with the cellulases.

[0048] The present cellulase composition may be used in combination with other paperroaking performance additives including cationic, anionic, amphoteric, non-ionic synthetic compounds, and natural, polymers. Examples of compound suitable for use with the present .ceEuIase composition include, but are not. limited to, dry strength additives such as. starch, starch derivatives, polyacrylamide derivatives, guar,

poly(vinylamine); wet strength additives such as, polyethyleneirnine, urea formaldehyde resin, epichlorohydnn reacted poiy(aminoamide), starch aldehyde, GPAM; flocculants; coagul nts; drainage aids;, retention aids; sizing agents; adhesives; debonders; crepiiig adhesives; plasticizers; and modifiers. Individual components of any of the above combinations may be applied together or sequentially in paperroaking. Additionally, individual components of an of the above combinations may be blended together prior to use.

[0049] in another embodiment, the cellulase composition is combined with a

poly(vinylamiiie) derivative improving pulp freeness and enhancing dry strength properties of a. paper product. Poly(vinylanune) interacts with the cellulosic fiber that is already treated by cellulase and refined by mechanic refining via fJ peculation to preserve the fibnilated cellulose structure and improve pulp drainage. Cellulosic fiber may be attacked by the impurity of exo-cellulase activity in an endo-cellulase product, resulting in producin fiber debris or cellulose fine particles and causing a reduction in total or fine fiber retention in a papermaking process. It was found that a cationic papermaking •additive with a high cationic charge density such as, a pGly(vlnyiamine), could be used in a combi ation with the present cellulase composition to maintain good total fiber retention,

[0050] The present cellulase composition can be present in or introduced into a pulper during the pulping stage, or brought into contact at any stock storage chest, high consistency chest or other holdin tank. It can also be added into the paper machine white water or, alternatively, can be applied in the water treatment loops of virgin or recycling mills to treat wood fiber. However, addition of the cellulase composition should be at least 10 minutes before the mechanical refiner, allowing contact time of the cellulase composition with the cellulosic fiber. Effective agitation or mixing is needed if the cellulase. is to have an effective action on the fiber. Pulp consistency also contributes to the effectiveness of the treatment by the cellulase composition. High pulp consistency reduces mass-transfer efficiency, resulting in. non-uniform interactions between the cellulase and fiber. Low pulp consistency decreases the concentration of the cellulase in the pulp at a fixed eellulase/dry fiber ratio and reduces cellulase efficiency, hi general, the pulp consistency of the cellulose fiber treated by the cellulase compositio is at least about 0.3% and should not exceed about IQ%. The pulp consistency can be in the range of from about i.% to about 5%; and may be. in the range of from about 2% to about 4%.

[00511 Treating the pulp slurry using a combination of the present cellulase composition with one or more other enzymes may achieve an enhanced performance in pul drainage and dry strength properties of a paper product. Such enzymes typically include hydrolases such as, hemiceilulases, amylases, proteases, lipases, esterases, and peetinas.es; lyases such as, pectate lyase, Additionally, other enzymes may be used in combination with the present cellulase composition. Other enzymes include oxidoreductases, such as, laccase,. lignio oxidase, glucose oxidase, and peroxidases. These enzymes can be used in any form, such as liquid, gel or solid form, Individual enzymes or any combinations of different enzymes may be applied together with, the present cellulase composition, or applied sequentially before or after the addition of the present cellulase composition. Individual enzymes may be also blended together with the present cellulase composition to form a blended composit on prior to use,

[0052] The following examples further illustrate the present invention and are not intended to be in any way limiting to the scope of the invention as claimed.

CELLULASE ASSAYS

Reducing Sugar Estimation by D.initrosalicylic Acid (DNS) torEndo-celluIase Activity

[0053] The endo-cellulase activity assay was performed using 1 % carboxymethyi cellulose (CMC, M7F, Ashland, Wilmington DE, USA) as the substrate, in 0.1. Molar (M), pH 7.0 sodium phosphate buffer. The. reducing sugar was determined using a dinitrosaiicylic acid (DNS) method,, in which dinitrosaiicylic acid is reduced to 3-amino- 5-nitrosa.licylie acid under alkaline conditions producing a color that is then measured spectrome-tricaO at a UV absorbance of 540 nm, Glucose was the standard for the calibration. One endo-1, 4-P-glucanase unit . ECU) is defined as the amount of cellulase producing one mieromolar (pmol) of reducing sugars as- glucose from CMC in. one second at pH 7.0.

[0054] In a typical example, 0,2 grams ^' (g) of a 0, 1 % solution of the cellulase composition

(equivalent to approximately 0,1 ECU to 0.15 ECU of cellulase) was added to 1.8 g of a CMC solution (1.0%, pH 7 _f 0) in a.. test tube. The mixture was incubated with shaking at 5Q°C for 10 minutes, after which, 3 milliliters (ml) DNS reagent (freshly prepared according to Miller, G, L, 1959, Analytical Chemistry 31, p. 426), was added to the mixture and the resulting mixture heated in boiling water for exactly 5 minutes. The solution in the test tube wa cooled to room temperature and UV absorbance. at 540 nm was measured. The standard curve (UV 540 ftm vs. glucose concentration) was established simultaneously using 0,1%: glucose with the same DNS test reagents.

[0055] In general, the endo-cellulase activity (ECU) of the present cellulase composition was in the range of from about 60 ECU/g to about 3600 ECU/g cellulase solution using the above assay under the specific conditions.

Measurement of CMC Viscosity Reduction to Determine Relative Endo-cellulase .Activity

[0056] This method was used to determine- relative endo-cellulase activity in percentage of the present cellulase composition compared with the original cellulase. In this method, a viscous solution of carboxymethyl cellulose. (CMC, M7F) was incubated at 40 °C with a sample of -cellulase composition. The degradation of CMC resulted .in reduced viscosity of the solution. To be accurate, trie final viscosity should be measured at least 40% and not exceed 60% of the original viscosity. The degree of the decrease in the viscosity is proportional to the endo-cellulase activity., The viscosity of a CMC solution · containing the original, cellulose- and a CMC solution containing the present cellulase composition were measured using a DV-E orDV-II Viscometer (Brookfield Viscosity Lab,

Middleboro, MA) at a selected spindle (number 3) and speed (30 rpm). The units are in centipoises (ops).

[0057] As. an example, 60 grams of- CMC solution (2,6% in 0.1 M sodium phosphate buffer at pH 7.0, wit Brookfield viscosity around 1500 cps) wa prepared and the viscosity was measured (Vo-sample). The solution was heated to 40°C and maintained at 40°C for 5 minutes and a small, amount of cellulase (equivalent to approximately 1 ECU to 2 ECU of cellulase.) as a 1.0% solution, in 0.1 M sodium phosphate buffer at pH 7.0 was added, The resulting mixture was incubated with agitation at 40°C for 10 minutes and the mixture was cooled to 23°C and the viscosity measured (Ve-sample). The same analysis was conducted with the original cellulase with the same batch of the CMC ^• solution as used, with the present cellulase composition. The viscosity of the starting solution and the end solution, were- measured as Vo-standard and Ve-standard

respectively. The relative eel lulase activity of the sample was calculated as (Vo-sample - Ve-sarnple)* 10G / (Vo-standard -- Ve-standard),

Relative Exo-eeliuiase Activity using the Dinitrosriicyiic Acid (DNS ) Method

[0058] An amount of the- present cellulase composition (equivalent to approximately 2 ECU/g to 3 ECU/g dry fiber) was added to -celiulosic fiber suspended in water at pH- 7.0 forming a pulp slurry. The resulting slurry was incubated at 50°C for 8 hours. The pulp was filtered off and the reducing sugar Content in the filtrate was determined by the DNS method described previously. One milliliter (ml) of the filtrate was incubated, with 4 ml. DNS reagent in boiling water for exactly 5 minutes.. The sample was cooled to room temperature and the- U V absorbance at 540 nm measured, A standard, curve was established simultaneously using the DNS test method referred to above and a 0.1% glucose solution at varying concentrations. PROTEIN ASSAY

[0059] Protein concentration of the present celiulase compositions were determined using a Bio-Rad ^' Protein Assay Method, which is a dye-binding assay based on a method developed by M. M. Bradford (see Bradford M.M., "A rapid and sensitive method of determining microgram quantities of protein utilizing the principle of protei n-dye binding", Analytical Biochemistry 7:2:248-254, 1976). An acidic dye reagent is added to a protein solution and the UV absorbance of the solution was measured at 595 nm ith a UV spectrometer. Comparison of these results, to .the bovine serum albumin (BSA) standard curve provides a relative measurement of protein concentration. A Bio-Rad protein assay reagent was obtained from Bio-Rad Laboratories. As a standard procedure, the dye reagent was freshly prepared by diluting 1 part of the Bio-Rad protein assay dye reagent with 4 parts of water. Fi ve dilutions of BSA standard were prepared in a li ear range from 0.2 milligram per milliliter (mg ml) to 0.9 mg/ml. In the test, 100 microliters (μΐ) of the BSA dilutions and the protein sample of an unknown concentration were pipetted into test tubes and 5 ml of the diluted dye reagent was added to the protein sample. The mixtures in the test tubes were, vortexed and incubated at room temperature for 10 minutes, and the UV absorbance was measured at 595 nrn.

[0.060] The protein assay was used to measure protein content as a percentage of the celiulase composition and the specific celiulase activity was. determined. In general, the protein concentration in weight percentage of the present -celiulase composition, was in the range of from about 0,02% to about 1 %,

Example 1, Formulating the celiulase composition

[0061] This example illustrates a general method of preparing the present, celiulase composition using an. endo-ceiliilase or a multi-component celiulase; a contaminant, control polymer, a celiulase protein stabilizer; and a celiulase enhancer.

[0062] A homogenous solution was prepared by sequentially adding a contaminant control polymer, a celiulase protein stabilizer and a celiulase enhancer to a desired amount of water at. a temperature of about 20°C with constant stirring forming a homogenous solution. A solution of celiulase was slowly added to the homogenous solution over a 20 minute time period at a temperature not exceeding 28°C resulting in mixtures according to the Examples found i Table Π. The temperature of each mixture was taken to ^' 20°C and agitated for 20 minutes. The pH of each mixture was then adjusted to 6 using HCl or MaOH as needed, to obtain a homogenous and transparent celiulase composition. The active percentages of the contaminant control polymer, the protein stabilizer and celiulase enhancer in the present celiulase composition are defined as non -aqueous parts of these polymers- or chemicals in the celiulase- composition. The active weight percentage of the endo-ceilulase or celiulase -active in the present celiulase composition is based on the assumption that the original celiulase is 100% active as it is obtained from a commercial source. The Bio-Rad protein assay was occasionally performed to determine the protein concentration of the cel iulase composition and to verify the active percentage of the original celiulase in- the celiulase composition.

Example 2. Celiulase Activity of the Celiulase Compositions

[0063] Example 2, demonstrates improvements in endo-ceiiulase activity of the present celiulase compositions compared with the original celiulase compositions. In thi experiment, a .mono-component endo-ceJiulase in the form of PiberCare^R and a multi- component celiulase in the form of PiberCare^ D were used,

[0064] The contaminant control polymers used for the- celiulase compositions are all commercially available from Ashland Inc, Wilmington, DE, USA. Cationic fixative polymers used in the experiment included Zenix® 0C7429 and Zenix® DC7 79. The hydrophobieaily modified cationic fixative was DeTae® DC786C, and nonionic papermaking detackiflers DeT'ae® DC779F and DeTae® DC397Q were also used.

[0065] The mono-component endo-cel !ulase used in the present celiulase compositions (Example 2-3 to Example 2-9} was also used in Comparative Example- 1 and Examples 2- 1 to 2-2, as shown in Table 11. Additionally, all of the celiulase compositions used in this study were prepared fresh and tested after one day stored at room temperature. Results as summarized in Table II, indicate that the contaminant control polymers enhanced the action of celiulase activity to ward CMC substrate.

[0066] The muiti -component celiulase used in the present celiulase- compositions (Example 2-10 to Example 2-11 ) was also used in Comparative Example 2, as shown in Table II. The results indicate, that the contaminant control polymers, Zenix® DC7429 and DeTae® DC3970, enhanced the action of celiulase activity toward CMC substrate, Table 1ΐ. CeOulase Activity of the Cellulase Compositions

[0067] Table IT, also Illustrates improved eftd.o-eellulase activity of the present cellulase composition containin a small amount of calcium chloride (Example 2-1) vs. the same composition without calcium chloride (Example 2-2).

Example 3. Cellulase Stability of the Cellulase Compositions

[0068] Example 3, demonstrates that the present cellulase compositions formulated with papexmaking cpntaminattt control polymers were more stable than the original cellulase compositions in endo-cellulase activity after storage. The relative endo -cellulase activity of the present cellulase composition was determined as percentage of. the original cellulase after stored at 50 °C for 46 days and a CMC viscosity reduction method was used to test relative activity as described below.. [0069] The relative activity of a conventional endo-cel iuiase composition stored in a refrigerator (Comparative Example 1 at 4°C was measured and used as a control reference as 100% active, it should be noted that all the assays were performed using the same cellulase active. It: should also be noted that the difference between cellulase activity vs. cellulase active in a cellulase composition is that the term "cellulase activity" is referred to as the cellulase activity as measured by the DNS and CMC viscosity reduction assays while the "cellulase active" is referred to by the weight percentage of a commercial cellulase product in the cellulase composition, and a commercial or conventional or original cellulase is usually considered 100% acti ve as it is.

Table III. Cellulase Stability of Cellulase compositions

[0070] As shown in Table III, the present cellulase composition (Example 2-3, 2-5, 2-6, 2-7 and 2-8) retained ..more than 81% of the original cellulase activit after being stored at 50 °C for 46 ^' days. The cellulase composition in the absence of a papermaking contaminant control polymer had an activity of only 54% of the conventional

composition. Two cellulase compositions (Example 2-3 and 2-5} formulated with Zenix® DC7429 and Zenix DC® 7479 exhibited more than 90% of the original cellulase activity and were more active than the original cellulase (Comparative Example 1) after storage. [0071] Celluiase can undergo protein denaturation and deactivation quickly at higher temperatures of 50°C or higher. Therefore, shelf-life of a eelln!ase ptOdiiet is one factor to. consider for large-scale industrial applications, particularl during hot summer months. The present celluiase compositions have shown improved. stability at high temperatures. Physical stability was also .monitored and it was observed that the present celluiase compositions listed in Table Hi remained homogenous and transparent without sedimentation: or any color and odor development over 46.days.

Example 4; Dry Strength of Handsheets Made from Virgin Fiber

[0072] Example 4, demonstrates improvement i dry strength properties of handsheet made from a virgin fiber that had .been treated by the present celluiase compositio vs. the fiber treated by the original celluiase.. Softwood bleached kraft (S ^' WBK was pulped in water at. 3% consistency and then, treated with both the present celluiase compositions and the original cellulases. The original celluiase was used as a control -and was used at the same dosage of the celluiase active at 50 °C for. 1 hour under effective agitation as the celluiase composition of the present invention. The celluiase active dosage of the control at 0.1 % vs. dry fiber was equivalent to approximately 750 ECU per kg of dry pulp. The treated.:SWBK pulp was then blended with hardwood bleached kraft (HWB ^' K) pulp furnish that had been made down to 3% consistency at a 30/70 (SWBK HWBK) weight ratio. The resulting virgin fiber pulp had a freeness of 530 Canadian Standard Freeness (CSF) and was refined to 480-490 CSF by a laboratory valley beater using TAPPI Test Method 200 sp-01.

[0073] Paper handsheets having a basis weight of 25 IbJSOOO sq. ft. were made on a Noble and Wood handsheet machine at pH 7,0. The Handsheets were wet pressed to 33% solids and dried on a drum drie at 240°F for 1 minute giving a moisture content of 3% to 5%, Dry tensile (TAPFT Test Method T494, om-01) and Mullen Burst (TAPPI Test- Method T403) were determined. The dry strength properties of the handsheet made with the present celluiase compositions were compared with handsheets made with the original celluiase in the absence of the contaminant control polymers (Example 2-2, as a control). Dry tensi le and Mulien Burst properties of the handsheets can be seen in Table IV and are expressed as % versus the control. Table IV. Dry Strength Peifomtanc.es of the Cellulage Compositions

[0074] Results of Example 4, show thai the cellulase composition of the present invention (Example 2-3, 2-7, and 2-9) improved dry strength, performance in both Mullen Burst and Dry Tensile strength of the haiidsbeeis when compared with the control (Example 2-2). Separate experiments indicated that contaminant control polymers used alone with a cellulase, had no benefit in paper dry strength.

Example 5, Effect of Cellulase. Dosage on Paper Dry Strength Properties

[0075] Mullen Burst of a paper product can vary with treatment conditions and fiber quality. This may be explained by the hypothesis that Mullen Burst is a combination of different paper properties, combining fiber length and inter-fiber bond ng; It was found that fiber length within a paper product suffered when the wood pulp was treated with a cellulase composition before refining.

[0076] Example 5, demonstrates the dosage effect of a cellulase composition on Mullen Burst as compared with the original cellulase. Example 5, also provides a. comparison of the Mullen Burst- of a handsheet made with a mono-component endo-cel. ulase vs. a multi- component cellulase on the dosage effect on a paper product A 30/70 w/w ratio mix of S WBK/HWBK was pulped in water at 3% consistency forming a suspension or slurry. The temperature of the suspension as adjusted to 5Q°C. and treated with a. cellulase composition at a dosage of 500 ECU to 5500 ECU per kg of dry pul and agitated for 1 hour. The resulting treated pulp was refined to between about 400 CSF and about 480 CSF by a PFI mill using TAPP1 Test. Method T-248. Paper handsheets having a 25 Ib./3000 sq. ft. basis weight were prepared on a Noble and Wood handsheet machine at pH 7 using the same method as described in Example 3. Mullen Burst of the handsheets made witii the present cellulase composition (Example 2-7) were compared with comparative Examples I and 2), expressed as percentage versus the blank wiihoni. any cellulase treatment, of the virgin fiber before refining.

Table V, Cellulase Dosage Effect of Present Cellulase Compositions vs. Original ccllula.se

Compositions on Mullen Burst of Handsheets

[0077] As shown in Table V, handsheets made using the original cellulase. (Comparative Example 1) show a tendency of decreasing Mullen Burst, from a 14% increase to a 3% increase as the cellulase dosage increased from 500 ECU/kg dry fiber to 5000 BCU/kg dry fiber. This tendency in Mullen Burst property is not observed with the cellulase composition of the present, invention (Example 2-7), which had a 14% increase in Mullen Burst, at a dosage of 5490 ECU/kg fiber and a .20% increase in Mullen Burst at 1091 ECU kg fiber. ^' The handsheets made from a multi -component cellulase (Comparative Example 2) contained a significant amount of exo-celluiase activity and had a 16 increase, in Mullen: Burst at 1 ,250 ECU/kg fiber over the fiber treated with the. original cellulase. However, when overdosed, at 5,000 ECU/kg fiber with the present

composition, Mullen Burst, was nly 87% of the- mono-component celiulase control (comparative Example 2) at comparative dosages.

[0078] Example 5, indicate that both the selection of the cellulase type and management of the cellulase activity dosage play a role in. paper dry strength application. Overdosing a conventional muM-component cellulase to eellulosie fiber can result in shortened fiber length and reduced dry strength properties. This i particularly true of the Mulle Burst of a paper product, due to the action of the e o-cellobiohydrolase activity that exists in the product. Overdosing a mono-component endo-eellula _. se to the fiber might cancel out the improvement, in paper dry- strength properties that is achievable at a lowe and proper celiulase activity dosage, in a practical situation the celiulase concentration can build up unexpectedly hig in a papermaking system if the white water is recycled in a closed system, or the paper machine is shut down for cleaning and other maintenance.

Additionally, Example 5, shows that handslieets made usin the present celiulase- composition at high celiulase dosages, had no. negative effect on Mullen _. Burst.

Example- 6. Dry Strength and Drainage Performance, of the Celiulase Composition on Recycled Fiber

[0079] Example 6. demonstrates impro vement in dry strength propertie of the handsheet made from recycled, fiber treated with both the present eeilulase composition and the original celiulase. Recycled fiber from 100% recycled medium was pulped at 3% consistency and treated with the celiulase compositions of the present invention and the original celiulase as a: control. Celiulase active dosages of 0.02% based on dry pulp were used. The treatment was conducted at.50°C for .1 hour under effective agitation. The resulting pulp was refined by a laboratory valley beater using TAPPI Test Method 200 sp- 01, for 6 minutes under the same conditions. The freeness was measured before and after the refining. Paper handslieets of 80 lb,/3000 sq. ft. basis; weight were prepared on a Noble and Wood handsheet machine at pH 7,0. The handsheets were wet pressed to 33% solids and dried on a drum drier at 240 °F for 1 minute to give 3-5% moisture. Dry Tensile (TAPPI Test Method T 494 om-01) and Ring Crush (TAPPI Test. Method T8.22 om-02) of the handslieets were determined. The Dry Tensile and Ring Crush properties were normalized and expressed as % versus that from the Blank.

Table VI. Dry Strength Performances of the Present Celiulase Compositions versus Ori inal Celiulase Compositions on High Basis Weight Recycled Paper

[0080] The results as shown in Table VT demonstrate that the cell lase composition of the present. invention (Example 2-9) provided an 8% improvement in Rin Crush and equivalent performance in Dry Tensile relative to the original cellulase (Comparative Example 1). There was almost a 40 CSF freeness improvement of the recycled fiber furnish when the fiber was treated with the cellulase composition of the present invention (Example 2-9) over the fiber thai was _: treated with the original cellulase after mechanical refining, .Additionally, an alternate -eellttlase composition according to the present invention (Example 2-8) gave a 30 CSF freeness improvement to {he post: refining furnish.

Example 7. Recycled Fiber Pulp Drainage

[0081] E m le 7 demonstrates improvement in pulp drainage by treating recycled fiber with the present Gellulase compositions over cehuiosie fibe treated with the original cellulase. Recycled pulp slurry was made using 100% recycled medium at 3.3% consistency, The temperature of the slurry was adjusted, to 50°C and treated with a cellulase composition at a dosage of 0.03% cellulase active based on dry fiber, and the treated slurry agitated for 1 hour. The efficiency in drainage of the present cellulase: composition was compared with that of the original cellulase and a blank (having no cellulase treatment), using a _. vacuum drainage test (VDT) as described belo w. The comparison in drainage efficiency was also conducted in the presence of a eationi.c poly(vinylamine), Hercobond ^® 6350 (Ashland Inc, Wilmington, DE, USA), at 0,2% based on the dry pulp. The results are summarized in Table VII.

[0082] A vacuum drainage test (VDT) setup is similar to a Buchner funnel test, consisting of a 300-rnl magnetic Gelman filter funnel, a 250-ml graduated cylinder, a quick disconnect ^" , a water trap, and a vacuum pump with, a vacuum gauge and regulator. ^• The VDT test was conducted by first setting the vacuum to 10 inches Hg and placing the funnel on the graduated cylinder. Two hundred fifty grams of 0.5 wt. % of pulp stock was charged into a beaker and the cationie polymer Bercobond^ 6350 was added to the stock while being agitated by an overhead. mixer. The stock was then poured into the filter funnel and the vacuum pump was turned on while simultaneously starting a stopwatch. The drainage efficacy is reported a the time (seconds) required to obtain 230 ml of filtrate. The shorter the time the better the pulp drainage. Table VII, Improvement in Recycled Fiber Drainage by the Cellulase Compositions

[0083] As shown in 'Fable VIL the recycled pulp treated with the present cellulase composition (Example 7-1 and 7-2) resulted in improved drainage with reduced VDT time of 67.7 seconds and 69.0 seconds respectively, .. compared to 72,4 seconds when the recycled pulp was treated with the original, cellulase (Comparative .Example 1) and 7.9.5 seconds fo the blank. The contaminant control polymers (honionic detaeldfiers)

(Comparative Example 4 and 5) did not reduce the VDT time when used alone. Example 7,. suggests _, synergistic effect of the mono-corriponent endo-cellulase and nonionic detackifiers for improving drainage of a recycled fiber furnish.

[0084] The combination of the present cellulase composition and catiorde

poly(viny!aiTiine) Hercobond© 6350 (Example 7-4) further reduced the VDT time to 49.6 seconds while the combination of the original cellulase and Hercobond© 6350 (Example 7-3) reduced the VDT time to 5.6,2, which was about 6 -7 seconds longer tha Example 7- 4, These drainage test results further illustrates that the present cellulase composition provides for increased pulp drainage rates when other cafcionic papermaj ng additives are also used. Example 8. Paper Dry Strength Using a Combination of the Cellul se Composition, and

Folyfvinylarmne)

[008-5] Example 8, demonstrates, improved dr strength performance of the present cellulase composition over the original cellulase when the cellulase composition was used in combination with the poly(vinylamine) Hereobond® 6350, A sample of 100% recycled fiber was pulped to a 3% consistency. The- resulting pulp slurry was treated with cellulase compositions at a dosage o 0.2% based on dry fiber for 1 hour at 50 °C The resulting treated slurry was then refined using a valley beater for 3 minutes using TAPPI Test. Method 200- sp-OL Handsheets of 50 lb./ 3000 sq. ft. were prepared .using the cellulase treated slurry with addition of 0.2% active Hereobond® 6350 based on the dry pulp using the methods described in the previous examples. Experiments were conducted using both the present cellulase composition and the. original cellulase at the same cellulase active dosage. Dry Tensile of the handsheets were tested. Additionally, STF.I short span compression strength was- tested using TAPPI Method T-815. These dry strength properties are expressed as % versus the control without cellulase -and

Hereobond© 6350.

Table VIIL Dry Strength Performances of Combination of Cellulase composition and

Poly(viny!amine) on Recycled Paper

[0086] The data in Table VOL, indicates that th present cellulase composition (Example 7-5) provides greater improvement in Dry Tensile (1 .13%) and STFl (127%), when used in combination with Hereobond® 6350, than the fiber treated with the original cellulase (Comparative Example 1) under the same treatment conditions. Example 8. also demonstrates the differentiating performance of the present cellulase composition vs. the original cellulase in paper dry strength application. E ample 9. Fixative and Retention Propertie Using the Cellulase Compositions

[0087] Example 9, demonstrates lowe turbidity of wood pulp obtained by treating the fiber with the present cellulase composition relative to that b the conventional cellulase, indicating potentially bette fiber retention or fixative properties with the present cellulase composition. A virgin fiber mix 30/70 w/w SWBK/HW.BK, was pulped to a 3,3% consistency and the resulting pulp slurry treated with the cellulase composition of the present, invention and the original ceilulases. Treatment was done at the same cellulase active at 50°C for 1 hour and a pH 7.0 ^■ usin effective agitation. The treated pul was cooled to about 25°C and refined to between about 480 CSF and about. 420 CSF by a valley beater using TAPPI Test Method 200 sp-0L The treated pulp slurry was added to a Brittjar with ^' Whatman 541 filter paper and stirred for 5 minutes at room temperature using a mechanical, stirrer at 1000 rpni. The pulp was filtered under vacuum and 150 ml of filtrate was collected. A turbidity meter was used to measure turbidity of the filtrate as for a¾in attenuation unit (FAU). The lower the FAU number, the better the fixative property or retention the pulp has. The turbidity data (FAU) is summarized In Table IX, and the fixative properties of the present cellulase compositions and the original ceilulases are also expressed as percentage, turbidity of the blank handsheet (the handsheet made with untreated fiber) shown in the last column of Table IX. The lower the percentage, the better fixative properties and retention the handsheet has.

Table IX, Reduced Turbidity of Filtrate from Virgin Fiber Treated by Cellulase

Com ositions

[0088] As shown in Table IX, the pul slurry treated with the present cellulase compositions (Example 9-1 to 9-3) provides filtrates having 20-30%. lower turbidity than when treated with the original cellulase (Comparative- Example 1), These results indicate that the present cellulase compositions provide better fiber retention than the original ceilulases.