CN1199421A - Method and chemical compounds for modifying polymers - Google Patents

Abstract

The invention relates to methods and compositions for improving the fluid, electrical or strength properties of a polymer by binding an effector moiety to the polymer via a protein. The invention particularly relates to improving the properties of paper by binding thereto a moiety capable of conferring a property such as improved wet strength, dry strength or sizing, via a protein such as a cellulase capable of binding to cellulose in the paper.

Description

Methods and compounds for improving polymers

1. Technical field

The present invention relates to methods and compounds for improving the physical properties of polymers. In particular, the present invention relates to methods and compounds for improving the physical properties of polymers by combining them with a compound which imparts improved fluidic, electrical or strength properties to the polymer, hereinafter referred to as an "effector moiety".

2. Background of the invention

Polymers and materials containing polymers are ubiquitous in everyday life. Natural polymers include, for example, proteins (including keratin, which is the major constituent in hair), starches, pectins, guar gum, chitin, lignin, agar, alginates, and polysaccharides such as cellulose and hemicellulose (including xylan, mannose and arabinose). Common forms of cellulose are, for example, products based on wood fibers or fibers from annual crops (e.g. hemp, wheat straw, rice, flax, jute), such as paper, and can be fibers, yarns, threads or various textile and Cotton products in the form of nonwoven fabrics or textile products. Xylose is the main component of xylan, a substance also called hemicellulose found in grasses, cereal plants, straw, chaff and wood. Starch is present in seeds, fruits, leaves, bulbs, etc.

The physical properties of polymers and materials comprising polymers can be improved by a variety of chemical and physical treatments. These chemical and physical treatments can be used to improve the structure of the polymer itself or to improve the bulk properties of the material comprising the polymer.

For example, the bulk properties of a polymer-containing material can be improved by incorporating into the material treating agents such as wet strength enhancers, dry strength enhancers, or other compounds that modify the physical properties of the material. Typically, the mixing of these compounds and materials does not allow the compound to be tightly bound to the polymer, thus encountering wastage of the compound and problems with leaching of the compound from the material, resulting in unstable properties of the material. Leakage of the compound can be reduced by a charge balancing procedure in which the ionic charge of the compound is equal and opposite to that of the polymer-containing material. In practical systems, however, the charges of the two components vary widely and need to be carefully and often controlled. The modified effect of the compound also relies on covalent binding to the polymer in order to obtain the correct modified effect. Additionally, accelerators are required to facilitate the incorporation of certain chemicals and materials.

In addition, the compound can be applied to the surface of the material by, for example, dipping or printing. Also typically, the compounds are not bound to the surface of the material, and problems with diffusion of the compounds from the intended application site are also encountered.

Various non-covalent binding interactions are known, such as those between an antibody and an antigen and between biotin and avidin or streptavidin. Typically, enzymes that alter the enzyme substrate also rely on non-covalent interactions with the enzyme substrate to function.

Such enzymes include enzymes that degrade polymers such as proteases, keratinases, chitinases, ligninases, agarases, alginases, xylanases, mannanases, amylases, cellulases, and hemicelluloses prime enzyme. For example, cellulase and hemicellulase can cleave sugar or polysaccharide molecules from cellulose and hemicellulose, respectively, and amylase can cleave glucose from starch.

The interaction between cellulose and cellulase proteins, especially those bound to cellulose fibers in a catalytically active manner, has been described and reviewed (Cellulase: Beguin, Annu. Rev. Microbiol ., 44, 219-248, 1990; Cellulases and xylanases: Gilbert and Hazelwood, Journal of General Microbiology, 139, 187-194, 1993). This class of enzymes includes cellulases and hemicellulases that contain functionally distinct protein domains. Specifically, the catalytically active domain is structurally distinct from the cellulose-binding domain. These domains have evolutionarily conserved sequences that are quite similar for all proteins of this class (Gilkes et al., Microbiological Reviews, 303-315, June 1991).

The binding domain and active site domain on this type of protein can be separated by proteolysis. Isolated bonding domains have been found to retain bonding capability (Van Tilbeurgh et al., FEBS Letters, 204(2), 223-227, August 1986). The use of the cellulose-binding domains of cellulase enzymes has been proposed as a means of roughening the surface textures of cellulose supports, and the use of cellulase active sites as a polishing treatment for these surface textures has been proposed (International Patent Application WO93/05226).

Many bonding domains have been characterized at the genetic level (Ohmiya et al., Microbial Utilization of Renewal Resource, 8, 162-181, 1993) and have been subcloned to generate novel fusion proteins (Kilburn et al., published International Patent Application WO90/00609; Ong et al., Enzyme Microb. Technol, 13, 59-65, January 1991; Shoseyov et al., Published International Patent Application WO94/24158). Some of these fusion proteins have been used as anchor proteins in some special applications. These proteins are used as aids in protein purification by binding fusion proteins to cellulose carrier materials used in protein purification methods (Kilburn et al., U.S. Patent 5,137,819; Greenwood et al., Biotechnology and Bioengineering, 44, 1295-1305, 1994). The ability of fusion proteins to be immobilized on cellulose supports has been suggested as an immobilization method for enzyme bioreactors (Ong et al., Bio/Technology, 7, 604-607, June 1989; Le et al., Enzyme Microb. Technol. , 16, 496-500, June 1994), and as a method of attaching chemical "tags" to cellulose supports (International Patent Application WO93/21331).

3. Brief description of the invention

The present invention provides a method of treating polymers such that at least one property selected from fluidic, electrical and strength properties is improved, comprising binding effector fragments to said polymers via protein linkages for obtaining said improvements Preferably, said effector fragment is different from said protein linkage, and said protein linkage is different from said polymer, said effector fragment and said protein linkage are present in amounts effective to obtain said improvement.

It should be recognized that a polymer may comprise polymeric molecules or a polymeric material comprising polymeric molecules. Furthermore effector fragment and protein linkage refer to at least one effector fragment and at least one protein linkage, respectively. The present invention therefore includes a method of treating a polymer such that at least one property selected from fluidic, electrical and strength properties is improved, comprising linking at least one effector A fragment is bound to at least one of said polymers, said at least one effector fragment is different from said at least one protein linkage, said at least one protein linkage is different from said at least one polymer, said at least An effector fragment and said at least one protein linkage are present in amounts effective to obtain said improvement.

According to another aspect of the present invention, the present invention provides a method of treating polymers such that at least one property selected from fluidic, electrical and strength properties is improved, comprising effector fragments and A protein is contacted with said polymer, said effector fragment is different from said protein, said protein is different from said polymer, said effector fragment and said protein are present in amounts effective to obtain said improvement. The present invention comprises a method of treating a polymer so that at least one property selected from fluidic, electrical and strength properties is improved, comprising combining at least one effector fragment and at least one protein for obtaining said improvement with At least one of said polymers is contacted, said at least one effector fragment is different from said at least one protein, said at least one protein is different from said at least one polymer, said at least one effector fragment is and said at least one protein is present in an effective amount to obtain said improvement.

According to another aspect of the present invention, the present invention provides a chemical composition comprising:

a) an effector fragment; and

b) a protein capable of binding said effector fragment to a polymer;

Where the effector fragment is different from the protein, the composition results in improved at least one property of the polymer selected from fluidic, electrical and strength properties.

The invention also provides compositions of material comprising a polymer to which an effector fragment is bound by a protein linkage, said effector fragment being different from said protein linkage, wherein said effector fragment and said Protein linkage is present in an effective amount such that at least one property selected from the group consisting of fluidic, electrical and strength properties of the polymer is improved.

According to another aspect of the present invention, the present invention provides a method of treating paper or paper's constituent fibers so that at least one property selected from the group consisting of fluid, electrical and strength properties is improved, comprising the method for obtaining said improvement at least one protein linkage binds at least one effector fragment to said paper or paper constituent fibers, said at least one effector fragment being different from said at least one protein linkage, said at least one protein linkage being different In said paper or constituent fibers of paper, said at least one effector fragment and said at least one protein linkage are present in amounts effective to obtain said improvement. 4. Detailed Description of the Invention

The present invention provides methods and compounds for improving the fluidic, electrical, and/or strength properties of polymers or materials comprising polymers by incorporation onto the polymer of effector fragments that impart desired properties.

The term polymer includes polymer-containing materials. A polymer-containing material may consist entirely of polymers or may comprise polymers compounded with other ingredients.

A polymer may include any polymer having any number of monomer units. Preferably, the polymer comprises a natural polymer or a chemically modified derivative thereof. For example, natural polymers may include proteins such as keratin, or polysaccharides such as starch, pectin, guar gum, chitin, lignin, agar, alginate. Preferably, the polymer comprises polysaccharides. Polysaccharides may include any polysaccharide, such as mannose, xylose, cellulose or hemicellulose, preferably cellulose. Cellulose-containing materials include eg wood fiber or annual crop fiber (eg hemp, wheat straw, rice, flax, jute) based materials such as paper. Additionally the material may also include cotton in the form of fibres, threads or woven and non-woven fabrics, cloth or tissue. Preferably, said material comprises paper.

The present invention can be used to improve any fluid, electrical or strength properties of polymers. Improved polymer properties include wet and dry strength, sizing properties, hydrophobicity, stain and stain repellency, fluid permeability, oleophobicity and hydrophobicity, conductance and resistance, capacitance, pH and biometallicity.

Proteins employed in the present invention may include any protein that can bind to a polymer. Preferably, the protein is polymer-binding with a dissociation constant (Kd) of less than 1×10 ⁻³ M. The term "protein" here includes peptides, oligopeptides and polypeptides, as well as protein residues, protein-containing substances, amino acid chains and molecules containing peptide bonds. Sometimes protein refers to protein residues as the context requires (eg, when the protein is bound to another molecule). The term "protein linkage" refers to the protein or protein residue by which the effector fragment is linked to the polymer. Proteins may include natural proteins, or fragments thereof, or modified proteins produced by chemical modification or synthesis, or by genetically modified genes encoding expressed proteins. The term "modified protein" herein includes chemical analogs of proteins that can be bound to polymers. Examples of proteins that can be bound to polymers are well known and include those selected from the group consisting of cellulase, hemicellulase, mannanase, xylanase, protease, keratinase, chitinase, ligninase, agarose Enzymes, alginase and amylase. For example, various cellulases are known that act by binding to cellulose. Examples of such cellulases are those that can be isolated from bacteria such as Cellulomonas fimi and molds such as Trichoderma viride, Aspergillus niger, Penicillium funiculosum, Trichoderma reesei and Humicola insolens , which are commercially available from Sigma Chemical Sigma-Aldrich Company Ltd., Novo Nordisk A/S, BDH Ltd. or ICN Biomedicals Ltd. In addition, proteins produced by recombinant DNA techniques as disclosed in International Patent Application WO94/24158 may also be used. Typically a cellulase comprises a cellulase binding domain and a cellulase activity domain. The present invention may employ whole cellulase enzymes or fragments thereof which bind to cellulose. The cellulase binding domain is obtained by treating the whole cellulase with a protease such as papain. Both exo-cellulases and endo-cellulases can be employed in the present invention.

Preferably, the protein includes a native enzyme that can bind the polymer. More preferably, the catalytic activity is deactivated for the paper. The catalytic activity of an enzyme can be inactivated, for example, by linking effector fragments or by cross-linking the enzyme. Enzymatic cross-linking can be performed using suitable protein cross-linking agents such as dialdehydes such as glutaraldehyde. Preferably, the protein comprises an inactivated native cellulase.

The effector fragment may be attached to the polymer-binding protein in any convenient manner. For example, the effector fragment can be directly covalently linked to the protein through the effector fragment and an appropriate reactive functional group on the protein. The identification of appropriate reactive functional groups and, if necessary, their chemical modification to facilitate covalent linkage is within the level of ordinary skill in the art. Examples of covalent bond formation include carboxyl and amine groups to form amide bonds via carbodiimide or dimethylformamide activation of carboxyl groups.

The effector fragment may be bound to any suitable portion of the polymer binding protein. The effector fragment can be bound to the protein N-terminus of the polymer-binding protein, eg, via the N-terminal amino group. Alternatively, it can also bind to the C-terminus of the protein, for example via the C-terminal carboxyl group. In addition, the effector fragment is bound to the protein by optionally present functional groups, for example in the amino acid chain of the protein or in its side chains or introduced into the protein for binding to the effector fragment. For example, the effector moiety can be passed through the sulfhydryl group of cystine, the hydroxyl group of serine or threonine, the amino group of lysine or arginine, the amide group of asparagine or glutamine, the aspartic acid or carboxyl group in glutamic acid or aryl or heteroaryl group in phenylalanine, tyrosine, tryptophan or histidine, or derivatives thereof. The effector fragment can be attached to the protein via a linker. A linker may include, for example, a bifunctional molecule that can react with a protein reactive site and a reactive site of an effector fragment to link the protein and effector fragment. It may be advantageous to introduce the linker as a spacer between the protein and the effector fragment, allowing the two substances to be sufficiently separated in space so as not to sterically affect the respective activity. Linkers are also advantageous in providing suitable functional groups for linking effector fragments and proteins.

Additionally, or also as part of a linker, the effector fragment can be attached to the protein by a non-covalent binding pair of molecules. Examples of such molecular non-covalent binding pairs include biotin and avidin, streptavidin or neutralite.

Therefore, one possibility is that the effector fragment is covalently linked to streptavidin, while the polymer binding protein is covalently linked to biotin. Binding of these components facilitates the binding of the streptavidin and biotin moieties of each component, thereby linking the effector fragment to the polymer-binding protein. It will be appreciated that the effector fragment-streptavidin component may be mixed with the protein-biotin component either before or after the protein component is bound to the polymer. It should also be recognized that the effector fragment can be covalently linked to biotin, whereas the protein can be covalently linked to avidin, streptavidin, or neutralite

It is recognized that there may be more than one type of effector segment attached to the polymer. Two or more types of effector fragments may be employed to enhance individual effects or to provide two or more effects simultaneously.

It will be appreciated that typically the effector fragment can be associated with the polymer binding protein either before or after binding of the polymer binding protein to the polymer. The methods of the invention may comprise contacting a conjugate of an effector fragment and a protein with a polymer, or may comprise contacting an effector fragment with a conjugate of a protein and a polymer. Furthermore, coupling of effector fragments to proteins and coupling of proteins to polymers can be accomplished in situ in a one-step process.

The precise nature of the manner in which the effector fragment is bound to the protein linkage and the protein linkage is bound to the polymer is not defined by the present invention. Linkage can be through chemical bonds, such as covalent bonds, or through non-covalent physical relationships, linkages, associations, attractions, or affinities.

Effector fragments may comprise any fragment that imparts the desired physical property. An effector fragment may comprise an atom, molecule or compound, or residues thereof, which impart a desired physical property. In one embodiment the effector moiety may include a compound that imparts a desired physical property. For example, the treating agent may include wet strength enhancers such as aldehydes such as glutaraldehyde or dialdehyde starch, or cationic derivatives thereof, polyamide resins, polyacrylamide copolyglyoxal, glyoxyl polyacrylamide, polyethyleneimine , polyamine epichlorohydrin polymers, polyamidoamine epichlorohydrin polymers, polymers of urea-formaldehyde and melamine-formaldehyde, synthetic latex, formaldehyde-modified proteins or other polymers used to enhance paper wet strength; Dry strength enhancers such as starch, anionic or cationic starch, polyacrylamide, amphoteric, anionic or cationic polyacrylamide copolymer, anionic or cationic guar gum, locust bean gum, or cationic or anionic modifications thereof, polyvinyl alcohol, Carboxymethyl cellulose; sizing agents such as abietic acids including abietic acid, adducted abietic acids including saponified fumaric gum rosin adducts, abietic acid derivatives including tall oil, fatty acids including myristic acid, palmitic acid or Stearic acid; other hydrophobic agents include alkenyl succinic anhydride (ASA) or 2-oxetanone (2-oxetanone) compounds such as alkyl or alkynyl ketene dimers or polymers (AKD) or ASA or derivatives of AKD, gums, adducted gums, wood or tall oil rosin, linear or branched saturated or unsaturated carboxylic acids with a chain length of about 4 to 30 carbon atoms, alkyl groups derived from carboxylic acids Dimers of ketenes, alkyl succinic anhydrides with a chain length of about 4 to 30 carbon atoms, perfluorinated or partially fluorinated carboxylic acids or derivatives thereof, alkylketene dimers, perfluorinated or partially fluorinated Alkyl succinic anhydrides; stain or stain repellents; oleophobic or hydrophobic agents such as fluorinated compounds including fluorinated fatty acids or fluorinated derivatives of ASA or AKD; softening agents such as treatments that can break hydrogen bonds in fibers including surfactants , detergents, fatty amides or enzyme preparations such as expansin (McQueen-Mason et al., Proc.Natl.Acad.Sci.USA, 91, 6574-6578 (July 1995)); provide conductivity reagents such as metals; can provide rigidity agents that can provide absorbency; agents that can provide hydrophilicity; agents that can improve density; metallating agents; agents that can improve pH, such as buffering agents (for example, to enhance resistance to acid degradation).

In another embodiment, the effector fragment may include a cross-linker or backbone former or residue thereof, which itself may be used to modify the physical properties of the polymer, or may be used to modify the properties of the protein thereby improving the properties of the polymer, or Can be used to introduce other formulations that modify the physical properties of polymers. Examples of preferred crosslinking backbone forming agents include dialdehydes such as glutaraldehyde. For example dialdehydes such as glutaraldehyde can form the backbone of cellulase-derived proteins. The cellulase/glutaraldehyde backbone imparts improved wet and dry strength to the paper, sizing the paper and/or other agents such as _TiO2 or _CaCO3 can be introduced.

A detailed review of compounds used in papermaking is given by Robert et al. (Paper Chemistry, Chapman Hall New York, 1991), the entire contents of which are hereby incorporated by reference. The reference specifically reviews retention aids, wet strength additives, dry strength additives, sizing agents and fillers.

The term "paper" as used herein refers to any bonded sheet or web material comprising an interwoven network of cellulose comprising fibers of vegetable origin optionally mixed with various proportions of vegetable, mineral, Animal or synthetic fibers, and optionally mixed with various proportions of fine particles of inorganic materials, such as oxides, carbonates and sulfates of metal elements. The term "paper" includes sheets or paperboard having a web weight greater than 200 g/ ^m2 .

Vegetable sources of cellulose include wood, straw, bagasse, stipa, bamboo, canafes, grasses, jute, ramie, hemp, cotton, flax. Coarse plant-derived cellulose is processed into pulp from which paper is made either mechanically, chemically, or both. According to the classification of pulp preparation and purification methods, cellulose-containing pulp can be divided into mechanical pulp, chemical mechanical and preheated wood chip chemical groundwood pulp, semi-chemical pulp, high-yield chemical pulp, and full chemical pulp (see "Pulp Pulp and paper, Chemistry and Chemical Technology", Third Edition, Volume 1, Pages 164, 165, edited by James P. Cassay, ISBN 0-471-03175-5 (V. 1)).

Effector fragments may be added to the polymer at any suitable stage of polymer or polymer-containing material manufacture and processing.

If the effector segment is applied to paper, the effector segment may be added at the pulp stage or at any stage during the formation of the wet pulp skeleton or during the extrusion and rolling of the skeleton to form paper. Alternatively, the effector fragments may be added to the finished paper product, for example, by dipping the paper into a bath containing reagents for attaching the effector fragments or by applying suitable spraying, smearing, brushing, coating or Printing attaches the effector segments to the formed paper.

If the effector fragment is applied to cotton, the effector fragment can be added at any stage of cotton processing. It can be attached to cotton, silk, yarn or woven or non-woven cloth or fabric. For example, dipping the material into a bath containing reagents for attaching the effector moieties or attaching the effector moieties using suitable spraying, painting, brushing, coating or printing methods.

By selecting the timing of attachment of the effector fragments during fabrication of the polymer or polymer-containing material, the distribution of the effector fragments within the polymeric material or substantially localized on the surface of the material can be controlled.

In cases where the effector fragments are used to improve the bulk properties of the material, it is advantageous to ensure a uniform distribution of the effector fragments in the material. Therefore, effector fragments should be linked at an early stage of manufacture. For example when the effector fragments are used to improve the bulk properties of paper, the effector fragments should be added at the pulp stage during the papermaking process.

Where the effector fragment is used to modify the surface properties of a material, the effector fragment can be substantially confined at the surface level of the material, with the attendant benefit of reducing the amount of effector fragment required. Therefore, preferably, the effector fragment should be added at a late stage of manufacture. For example, when the effector fragments are used to improve the surface properties of paper, the effector fragments should be applied to the surface of the paper during the papermaking process.

One or two flat surfaces of the paper are coated with effector segments, depending on the application requirements. For example treating both surfaces of the paper with effector segments comprising wet strength agents, leaving one or more of its edges untreated, facilitates the preparation of sandwich structures in which poor wet strength properties and good The liquid absorbent paper layer is sandwiched between two paper layers with good wet strength properties. Such a structure can transfer liquid through the capillary action of its middle layer, which is particularly useful in the manufacture of dipped diagnostic test strips.

A particular aspect of the invention is the ability to reversibly improve the physical properties of polymers or polymer-containing materials. Conventional methods of manipulating polymers to impart specific physical properties are often irreversible. Furthermore, conventional processing methods often render the polymer unsuitable for regeneration. For paper recycling, it is more difficult or impossible to repulp the paper if it is treated with conventional wet strength enhancers. The invention provides methods for releasing effector fragments and regenerating materials. For example, the effector fragment can be released from the polymer-containing material by protease treatment that cleaves the protein to which the effector fragment is attached; alternatively, the effector fragment can be attached to the polymer by a selectively cleavable linker. Proteins; crosslinkers such as aldehyde-substituted starches can be cleaved by amylases.

Another benefit of the present invention is that the desired physical properties are imparted to the material substantially immediately. In conventional treatments for imparting wet strength to paper, several weeks of heat treatment and aging are required.

The present invention will be described below with reference to figures and examples. In the picture:

Figure 1 shows the effect of cellulase concentration on wet strength imparted by glutaraldehyde crosslinked cellulase;

Figure 2 shows the effect of glutaraldehyde concentration on wet strength imparted by glutaraldehyde crosslinked cellulase;

Figure 3 shows the effect of pH on wet strength imparted by glutaraldehyde-crosslinked cellulase;

Figure 4 shows the effect of temperature on wet strength imparted by glutaraldehyde crosslinked cellulase;

Figure 5 shows the effect of incubation time on wet strength imparted by glutaraldehyde-crosslinked cellulase;

Figure 6 shows the effect of pre-incubation time on the wet strength imparted by glutaraldehyde-crosslinked cellulase;

Figure 7 shows the effect of glutaraldehyde crosslinked cellulase on the wet strength of paper made from different wood pulps.

It will be appreciated that the following are examples only and that modifications of detail may be made within the scope of the invention.

experiment

Principles and operation of effector-fragment linkage

The following defined protocol represents the characterized technique for attaching effector fragments to cellulose using cellulases as biobridging agents. Preparation of starting materials

A 1/3 concentration of phosphate buffered saline (1/3 PBS) was used. The following is the recipe for 1/3PBS:

200 liters of deionized or demineralized water (DEMI water)

197g anhydrous sodium dihydrogen phosphate (NaH ₂ PO ₄ )

767g anhydrous disodium hydrogen phosphate (Na ₂ HPO ₄ )

389g sodium chloride (NaCl)

Anhydrous materials are not required, but for hydrous salts the stated weights should be deducted from the "water of crystallization".

The cellulase used is derived from mold and can be obtained in the form of aqueous solution or freeze-dried powder. Penicillium funiculum

Cellulase from Penicillium funiculosa (Sigma Aldrich Co. Ltd., Poole, Dorset, U.K.) is available as a brown powder and should be stored below 0°C.

When used as an additive to handsheets, the cellulase was first made into a 20% total solids solution in 1/3 PBS. Place 200 g of dry enzyme preparation in a large shallow beaker. Then 800g 1/3 PBS was slowly added thereto. Stir the mixture slowly with a glass rod. Do not stir the solution vigorously to disperse the powder as it will denature the enzyme. Any block of enzyme preparation can be broken gently with a glass rod. If the enzyme solution is prepared the day before use, store it at 4°C. Trichoderma reesei

Cellulases derived from Trichoderma reesei are available in powder form from Sigma Aldrich Co. Ltd., Poole, Dorset, U.K. or in aqueous solution from Novo Nordisk A/S, Bagsvaerd, Denmark. When the powder is used, the method and procedure herein for preparing the aqueous solution of Penicillium funiculosum is also used.

The cellulase solution is added to the raw material based on the total protein content in the enzyme solution (eg 10 parts dry protein per 100 parts dry fiber). Use Coomassie Brilliant Blue G250 dye (Sedmak and Grassberg (Analytical Biochemistry, 79,544-552 (1997)) to make albumen staining, measure the total protein content of the cellulase solution that makes with UV absorption (=620nm). 1, fiber Sulfase-bound fiber test

A sample of cellulose (typically between 25-500 mg, often 100 mg), eg microcrystalline cellulose (Avicel, SigmaCell) or unsized pulp is weighed into a series of test tubes/flasks.

A solution of cellulase (typically 200-600 mg protein per ml in 3 ml buffer) was added to each tube. At the beginning of the binding assay the exact concentration of the added protein is first measured using the assay developed by Sedmak and Grassberg (Analytical Biochemistry, 79, 544-552 (1977)).

Shake the tube at the desired temperature (typically between 4°C-30°C, usually room temperature) for a period of time (typically 1-90 min, usually between 5-15 min). Samples (0.5-1 ml) were then taken for testing.

Samples were centrifuged in 1 ml Eppendorf tubes for 5 minutes using a bench-top microcentrifuge and the supernatant was retained for determination of the concentration of residual protein (unbound cellulase) in the supernatant.

The amount of celluloprotease bound to the cellulose mass was expressed by subtracting the supernatant protein concentration from the initial protein concentration.

Bovine serum albumin (BSA) was used as a control in the experiment.

Results are expressed as the amount of cellulose-bound protein as a percentage of added protein or as a percentage of protein/cellulose (% w/w) as a percentage of cellulose-bound protein. 2. Using chemiluminescence to visualize the linkage of effector fragments. Preparation of cellulase for ECL detection system 1. Biotinylation of cellulase

A solution (1 mg/ml) of biotinamido N-hydroxysuccinimide ester (B _cap NHS) in N,N-dimethylformamide (DMF) was prepared. A solution of cellulase (77 mg/ml) was prepared in distilled water.

1 ml of the cellulase solution was added to 1 ml of the B _cap NHS solution, and the mixture was incubated at room temperature for 2.5 hours with shaking. Then the reaction solution was fully dialyzed with 500ml 1/3 PBS buffer solution (PBS, pH7.5: Na ₂ HPO ₄ , 11.5g; NaH ₂ PO ₄ , 2.96g; NaCl, 5.84g diluted to 1L with distilled water) for 1 hour. 2. Binding of biotinylated cellulase to the paper [application of cellulase to the surface of the paper]

Put a piece of unsized paper, usually 2 cm ² , in a petri dish with shaking at 4°C, and ^biotinylated Cellulase (10 ml) was incubated together for 45 minutes to 2 hours. Experiments using PBS containing Tween 20 (0.1% vv ^-1 ) were also performed. 3. Incorporation of biotinylated cellulase into pulp and subsequent paper sheet preparation [Applying cellulase to paper substrate]

The pulp was incubated with biotinylated cellulase in 1/3 PBS containing Tween 20 (0.1% vv ^-1 ) for 45 minutes at room temperature with shaking. Paper sheets were made from pulp-biotinylated cellulase using a papermaking screen. Remove the paper sheets from the strainer, roll up and dry overnight. 4. Binding of HRP-labeled streptavidin to biotinylated cellulase

Conjugation of HRP-labeled streptavidin and biotinylation of cellulase was carried out according to the manufacturer's recommendations (Amersham Led., Amersham, U.K.; Whitehaead, T.P. et al., Clin. Chem. 26, 1531-1546, 1997). ECL detection.

The paper was incubated with milk powder (4% wv ^-1 ) in PBS for 45 minutes with shaking at 4°C or room temperature to mask non-characteristic binding of the HRP streptavidin conjugate. The discs were then washed 3 x 3 minutes with 0.5% (wv ^-1 ) milk powder in 1/3 PBS containing Tween 20 (0.1% vv ^-1 ). Discard the solution and replace with fresh solution after each wash.

Horseradish peroxidase (HRP)-streptavidin conjugate was made 1:1000 using 0.5% (wv ⁻¹ ) milk powder in 1/3 PBS containing Tween 20 (0.1% vv ⁻¹ ). substance solution. An appropriate volume (2 to 10 ml) of solution was added to cover the disc, followed by incubation at 4°C for 45 minutes with shaking.

Discs were washed 3×5 min with 0.5% (wv ⁻¹ ) milk powder in 1/3 PBS containing Tween 20 (0.1% vv ⁻¹ ). Discard the solution and replace with fresh solution after each wash. Discs were then washed 3 x 5 min with 1/3 PBS. Discard the solution and replace with fresh solution after each wash.

The cellulose bound to the cellulase-biotin-HRP-streptavidin conjugate can then be visualized by ECL or quantified by OPD. 3. Enhanced chemiluminescence (ECL) method

This method needs to be carried out in a photographic darkroom.

Amersham ECL detection reagent 1+2 was mixed in equal volume (amount of about 0.13ml/cm ² paper). Excess buffer is then removed from the paper and detection reagent is added to completely cover the paper surface.

Incubate the paper at room temperature for exactly 1 minute without agitation. Then remove the detection reagent and sandwich the paper between two pieces of toilet paper to blot the excess reagent. The blotted paper is then transferred to a piece of adhesive film and wrapped to ensure removal of any air pockets.

Place the paper in a membrane cassette to minimize the delay between incubating the paper and exposing the paper to the upper membrane. The film was carefully placed on top of the paper, ensuring that the film was not moved during the exposure and the film was exposed for 15 seconds. The first film was then removed and immediately replaced with a second film of paper which was then exposed for 1 minute.

The film was then immediately developed to reveal the result. Additional films can be exposed for 1 to 60 minutes if desired. 4. Quantitative detection of effector fragments bound to cellulose (OPD) method

Dissolve 1 OPD tablet (60mg; o-phenylenediamine dihydrochloride, Sigma Chemicals, UK) in 150ml of 0.06M phosphate-citrate buffer (0.2M Na ₂ HPO ₄ , 121.5ml; 0.1M citric acid 121.5 ml was diluted to 500 ml with distilled water, and the pH was adjusted to 5.0) to prepare substrate buffer, resulting in a final OPD concentration of 0.4 mg ml ^-1 . Note that this reagent is photosensitive. Add 10 μl of fresh 30% _H2O2 per ₂₅ ml of substrate buffer saline just before use.

Paper samples containing biotinylated cellulase were placed in 50ml Falcon tubes. Add 25ml of prepared substrate buffer to the test tube and shake at room temperature for 30 seconds to 20 minutes, usually 5 to 15 minutes, then add 1ml 3M H ₂ SO ₄ to stop the reaction. Absorbance was then measured at 492 nm, and to calculate the concentration of biotinylated cellulase on or in the paper, reference was made to a standard curve of OD ₄₉₁ versus biotinylated cellulase concentration. 5. Using carbodiimide to link paper effector fragments to enzyme peptide chains

Carbodiimides react with carboxyl groups to form activated carboxyl groups. The amino group is then attacked to activate the carboxyl group to form a peptide bond. This chemical reaction is used to attach a paper effector fragment containing a free carboxyl group to an amino group on a peptide.

The chemistry of carbodiimides used to link paper effector fragments to cellulases was based on conventional methods (Hoare et al. J. Biol. Chem, 242(10), 2447-2453, 1967).

In the method described below, cellulase was coupled with abietic acid.

Cellulase (21 mg ml ^-1 ) was dissolved in distilled water and abietic acid (100 mg) was dissolved in 25 ml 10% (vv ^-1 ) methanol. Add 0.5ml 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide-HCl (WS-CDI; 63mg ml ^-1 ) to 1.0ml abietic acid solution, and wash with HCl (0.1N) Adjust the pH to 4.5 ± 0.5. The mixture was then stirred at room temperature (5 min). 2ml of cellulase solution was added and the mixture was stirred (16hr) at room temperature.

The reaction was terminated by addition of sodium acetate and excess abietic acid (0.1 M; pH 5.0), and WS-CDI was removed by complete dialysis in phosphate buffer.

The abietic acid is then bridged to the cellulose using a coupled cellulase as described. 6. Demonstration of wet tensile strength properties by glutaraldehyde-crosslinked cellulase

Experiments have been performed on the application of glutaraldehyde-crosslinked cellulases to unsized pulps and demonstrated that they can impart wet strength properties to paper sheets.

Unsized pulp was prepared as follows: 10 g of unsized paper was cut into ¹ cm squares and macerated in 100 ml of distilled water for 3 minutes in a domestic herbal mill (CH100, Kenwood Ltd. UK).

2.15 g of unsized pulp (10% wv ^-1 ), containing 0.2 g of cellulose, was taken and the following additions were made: 1. 10 ml 1/3 strength phosphate buffered saline (PBS), pH 7.0 as a reference. 2. 10ml 1/3 PBS3 containing T.reesei cellulase (2mg ml ^-1 ), 10ml 1/3 PBS4a containing glutaraldehyde (25μl ml ^-1 ), 10ml containing T.reesei cellulase (2mg ml ^-1 ) and glutaraldehyde (25 μl ml ⁻¹ ) in 1/3 PBS, which were co-incubated at room temperature for 1 hour before adding the pulp. 4b. 10 ml of 1/3 PBS containing T. reesei cellulase (2 mg ml ⁻¹ ) and glutaraldehyde (25 μl ml ⁻¹ ) was added directly to the pulp.

All samples were incubated for 1 hour at room temperature on a horizontal shaker before being made into disks.

To prepare the test paper, the volume was increased to 100ml with distilled water, and the paper (6cm ² ) was prepared using the papermaking apparatus designed in the laboratory in the following manner: Pour the pulp suspension (0.2% WV ^-1 ) into a filter with Screened plastic filter box. A vacuum is applied for a few seconds to form the pulp into a sheet supported by a screen. The filter screen is removed from the instrument, the paper is sandwiched between the second nylon screen and absorbed with absorbent paper. The sheets are carefully removed from the forming screen, flattened by rollers and dried.

Determination of wet strength according to the following method; A. Stability of paper in water

A sample (1.5 cm ² ) of each measuring paper piece was placed in a Universal bottle and 25 ml of distilled water were added respectively. Shake the tube and check periodically for signs of loss of sample integrity.

The results are listed in Table 1.

Table 1 Detection of the stability of paper samples in water sample number Repeat Sample No. Condition after shaking in water initial damage total damage 1 12 disintegration disintegration -- -- 2 12 disintegration disintegration -- -- 3 12 disintegration disintegration -- -- 4a 12 Complete and complete ＜18h＜18h 36h36h 4b 12 Complete and complete ＞8d＞8d ＞8d＞8d In Table 1, "-" refers to B, paper strength that is not applicable to the experiment

A sample (4 cm x 1 cm) of each test paper sheet was taken and 25 μl of distilled water was dropped onto the middle of the paper to ensure even distribution. The paper is hung in two sleeved clips and a container is mounted on the bottom clip. Add water to the container and weigh the amount of water needed to tear the paper.

The results are presented in Table 2 and demonstrate that paper samples prepared with glutaraldehyde crosslinked cellulase had the greatest wet tensile strength.

Table 2 Determination of Paper Strength sample number Repeat Sample No. Add weight (g) 1 12 27.4343.48 2 12 ＜22.00＜22.00 3 12 <22.0034.63 4a 12 66.3449.96 4b 12 >77.3364.20

The wet strength of the paper samples was retested using BSA as a control to assess the specificity of the bridging protein action. Paper samples were prepared as follows: 1. 10 ml 1/3 strength phosphate buffered saline (PBS), pH 7.02, 10 ml 1/3 PBS containing T. reesei cellulase (2 mg ml ⁻¹ ) 3, 10 ml containing glutaraldehyde (25μl ml ^-1 ) in 1/3 PBS4, 10ml containing BSA (2mg ml ^-1 in 1/3 PBS)5, 10ml containing T. reesei cellulase (2mg ml ^-1 ) and glutaraldehyde (25μl ml ^{-1 1} ) of 1/3 PBS was added to the pulp and incubated on a horizontal shaker at room temperature for 1 hour before disk preparation. 6. 10 ml of 1/3 PBS containing BSA (2 mg ml ^-1 ) and glutaraldehyde (25 μl ml ^-1 ) was added to the pulp and incubated on a horizontal shaker at room temperature for 1 hour before disc preparation.

Add the paper sample and 25ml of water to a 50ml Universal bottle and vortex with a laboratory mixer until the paper sample is completely disintegrated. The results are listed in Table 3.

Table 3 Demonstration of paper stability when vortexed in water sample number Repeat Sample No. Time required for complete disintegration (seconds) 1 12 <5<5 2 12 <5<5 3 12 <5<5 4 12 <5<5 5 12 ＞1020＞1080 6 12 1010

While the paper samples prepared using crosslinked BSA (sample 6) showed an increase in wet tensile strength compared to the control, it was 100 times smaller than that of glutaraldehyde crosslinked cellulase.

To determine the optimum conditions for the glutaraldehyde/cellulase treatment to improve wet strength, the following parameters were varied sequentially. For this work, the control parameters are set as follows: add cellulase (2mg ml ^-1 ) and glutaraldehyde (0.6% vv ^-1 ) to the pulp respectively; suspend the pulp in the buffer solution (pH7.0) at 25°C . The mixture was incubated for 60 minutes prior to disk preparation. The following parameters were changed in turn: cellulase (0.5 to 8mg ml ^-1 ); glutaraldehyde (0.1 to 2.5vv ^-1 ); pH (5.0 to 10.0); temperature 25°C, 37°C and 45°C; incubation time (5 to 120 minutes); pre-incubation time of cellulase and glutaraldehyde (15 to 60 minutes).

All sheets were dried overnight at room temperature prior to wet tensile strength determination. The results are presented in Figures 1 to 6. 7. Demonstration of glutaraldehyde crosslinked cellulase (GCC)-wet tensile strength performance with different types of pulp

Application of the GCC-wet strength composition to paper made from different types of pulp: groundwood pulp (GWP), chemically groundwood pulp from preheated chips (CTMP), hardwood pulp (HWP), softwood pulp (SWP) and unsized pulp (W-LP; 70% HW: 30% SW). Pulps were prepared conventionally, of course the GWP and CTMP pulps were soaked in water overnight before mixing to facilitate fiber dispersion.

The pulp was treated with PBS buffer (10 ml); or PBS buffer (10 ml) + cellulase (20 ml) + glutaraldehyde (0.6% vv ⁻¹ ). Pulp samples were then made into test sheets (6 cm ² ) as described prior to wet tensile strength testing. The results are listed in Table 4 and represented graphically in FIG. 7 .

The results showed improved wet tensile strength for all tested pulps. The final strength of sheets prepared with GWP or CTMP was greater than those prepared with HWP, SWP and W-LP. However, the GCC composition produced a greater percentage increase in wet tensile strength in the HWP and SWP samples.

Table 4: Wet tensile strength of sheets made of different pulps pulp Approach Vortex Mixing (sec) ¹ Wet tensile strength (g) Ground wood pulp Buffer Cellulase Glutaraldehyde Cellulase + Glutaraldehyde 135120315＞1200 105.445.094.5249 CTMP Buffer Cellulase Glutaraldehyde Cellulase + Glutaraldehyde 480420420＞1200 78.58871.5242.5 HWP Buffer Cellulase Glutaraldehyde Cellulase + Glutaraldehyde <5<10<10>300 <15.3<15.3<15.3119 SWP Buffer Cellulase Glutaraldehyde Cellulase + Glutaraldehyde <5<5<5>300 <15.3<15.3<15.3207 W-LP Buffer Cellulase Glutaraldehyde Cellulase + Glutaraldehyde ＜5NDND＞300 <15.3NDND193

Time required for complete destruction 8. Demonstration of reversibility of wet tensile strength endowed by glutaraldehyde-crosslinked cellulase

To demonstrate the reversibility of the wet tensile strength imparted by glutaraldehyde-crosslinked cellulase, the following protease solutions were prepared using the following enzyme preparation supplied by Sigma Chemical-Aldrich Company Ltd., Fancy Road, Poole, Dorset, BH177NH: Ficin (4 μl ^ml solution in pH 6.5 PBS buffer); papain (5 μl ^ml solution in pH 6.5 PBS buffer); proteinase K ( ^2.8 mg ml solution in pH 8.0 PBS buffer ); α-chymotrypsin (1.0 mg ml ⁻¹ solution in PBS buffer, pH 8.0).

Discs (1.5 x 1.5 cm) made of glutaraldehyde-crosslinked cellulase-enhanced unsized pulp were taken and incubated according to the treatments listed in Table 5.

Table 5 wet strength reversibility treatment method sample number Approach 1 Ficin (10ml) + PBS buffer (pH 6.5) 2 Papain (10ml) + 10ml PBS buffer (pH 6.5) 3 Proteinase K (1ml) + 19ml PBS buffer (pH 8.0) 4 α-chymotrypsin (1ml) + 19ml PBS buffer (pH 8.0) 5 Ficin (10ml) + Papain (10ml) 6 Proteinase K (1ml) + α-chymotrypsin (1ml) + 18ml PBS buffer (pH 8.0) 7 Ficin (10ml) + Papain (10ml) + Proteinase K (1ml) + α-Chymotrypsin (1ml) 8 0.2M phosphate buffer pH 6.5 (20ml) 9 0.2M phosphate buffer pH 8.0 (20ml)

Samples were incubated in a horizontal shaker at 70 rpm at 30°C. Samples were tested at 4 hours and 20 hours and after 20 hours vortexed for 10 seconds to see if the paper remained intact. The experiment was repeated and the results are listed in Table 6.

Table 6 Determination of paper damage in different treatments Approach Incubation time (h)4 20 After an additional 10 sec vortex mixing Ficin XX XXXX XXXXXXXX Papain XXXX XXXXXXXX ------ Proteinase K XXXX XXXX XXXXXXXX α-chymotrypsin 00 XXXX XXXXXXXX Papain + Ficin XXXX XXXXXXXX ------ α-chymotrypsin + proteinase K XX XXXX XXXXXXXX All 4 proteases XXXX XXXXXXXX ---XXXX Control phosphate buffer (pH 6.5) 00 00 00 Control phosphate buffer (pH 8.0) 00 00 00

Qualitative observations are expressed on an arbitrary scale from 0 to XXXX, where 0 represents no visible damage to the paper, XXXX represents complete damage to the paper; ---- represents the loss of paper integrity before testing. 9. Determining the effect of protease treatment on the reproducibility of paper wet strength enhanced by glutaraldehyde-crosslinked cellulase

Paper made with glutaraldehyde crosslinked cellulase wet strength enhancement of unsized pulp (0.2 g) or pulp not treated with any wet strength enhancer (0.2 g) was subjected to a series of treatments.

Treatment 1 Paper sheets (0.2 g) made of pulp enhanced with glutaraldehyde cross-linked cellulase wet strength were cut into 1 cm x 1 cm squares and mixed with 20 ml of 0.2 m phosphate buffer containing proteinase K (14 mg) (pH 8.0) placed in a Petri dish. The samples were incubated for 2 hours at 37°C with shaking (60 rpm). The discs were then removed and dipped in phosphate buffer (pH 8.0), then placed into a Universal bottle containing 20 ml of fresh phosphate buffer (pH 8.0). Vortex the sample to macerate the paper. Any fibers remaining in the dish from the initial incubation were collected by centrifugation at 6,000 rpm, washed with phosphate buffered saline (pH 8.0) and added to the macerated samples in Universal bottles. 2 ml of T. reesei cellulase (10 mg ml ^-1 ) and 0.5 ml of glutaraldehyde solution (25%) were added and the samples were incubated at 25°C for 60 minutes. This sample was then used to prepare new paper sheets.

Treatment 2 Unsized paper sheets (0.2 g) prepared from pulp made with only PBS without any wet strength enhancer and 10 ml of 1/3 strength PBS were placed in a Universal bottle. The samples were vortex mixed to macerate the paper and the resulting pulp was used to prepare new sheets.

Treatment 3 Sheets (0.4 g) made of glutaraldehyde cross-linked cellulase wet strength enhanced pulp and 30 ml 1/3 strength PBS were placed in a mixer for maceration. The resulting pulp is removed and made into new paper sheets.

Treatment 4 Sheets (0.2 g) made of glutaraldehyde crosslinked cellulase wet strength enhanced pulp were cut into ¹ cm2 pieces and macerated with 30 ml of 1/3 strength PBS in a mixer. 2 mg of T. reesei cellulase (10 mg ml ^-1 ) and 0.5 ml of glutaraldehyde solution (25%) were added and the samples were incubated at 25°C for 60 minutes. This sample was then used to prepare new paper sheets.

Each disc was dried overnight at room temperature before breaking a 1 ^cm2 sample in water using a vortex mixer to test its integrity. The paper test results are listed in Table 7.

Table 7: Integrity of recycled paper method vortex mixing (time, sec) Description of paper condition 1 300 paper shredded into three pieces 2 5 paper completely disintegrated 3 50 lots of small pieces 4 345 small holes in the paper

The results showed that the GCC-containing pulp retained some wet tensile strength properties when made into new sheets; the pulp made by protease treatment, as opposed to physical damage, produced stronger paper when regenerated, and continued addition of GCC allowed Recycled paper sheets achieve the best wet tensile strength properties. 10. Demonstration of improved wet strength, dry strength and sizing properties of paper after glutaraldehyde and cellulase treatment

Experiments were performed to determine the effect of cellulase (protein ligand) and glutaraldehyde (effector fragment) on wet strength, dry strength and sizing properties of paper. The following materials and general protocol were used in the experiments: Cellulase

A water-based Trichoderma reesei cellulase preparation ("Cellulast 1.5 L", supplied by Novo Nordisk Bioindustry S.A. 9 2017 Nanterre Cedex, France) was used. glutaraldehyde

A 25% aqueous solution of glutaraldehyde, commercially available from Merck Ltd (Poole, Dorset, U.K.), was used in the following examples. Raw material preparation:

Unless otherwise specified, the furnish used was a blend of ECF bleached hardwood and softwood pulp (ratio 70:30 HW/SW). Stocks were prepared with 1/3 PBS and no filler added. The process is as follows:

280 g of bleached hardwood pulp and 120 g of bleached softwood pulp were added to 18 liters of 1/3 PBS. Stir vigorously to disperse the fibers. Then transfer the raw materials to the beater and beat until the homogeneity value is 25oSR (usually 30 to 35 minutes). Then add 1/3 PBS to adjust the final concentration of the stock to 2%. Addition/incubation of additives

The cellulase solution and glutaraldehyde solution were added to the viscous (2% strength) stock. Two liters of material (containing 40 g of fiber) were placed in a metal tank and stirred at the lowest possible speed to cause slow movement of the material. Vigorous agitation should be avoided as this can cause enzyme denaturation during the incubation period. The starting material was at room temperature (20 to 25°C). The cellulase solution was added to the feedstock first (avoid any splashing or splashing of the solution) and one minute after adding the enzyme, the aqueous glutaraldehyde solution was then added.

The incubation time for the additives was 15 minutes from the time the enzyme was added. Movement of ingredients may be easier/faster during incubation. If the change is obvious, the stirrer speed should be reduced as much as possible.

When the 15 minute incubation period is complete, add the viscous material to the dispenser. Dispenser

Thin the viscous material in the dispenser to a concentration of 0.25% with DEMI water only. The ingredients are mixed using the usual agitation speed in the batcher. Formation of handwritten paper

The white water tank was filled with DEMI water for handsheet formation. The handsheets are formed into wires and put into the mold, and one liter of raw material from the batcher is added to the plate and frame box, and water from the white water tank is added at the same time. The contents of the plate and frame box were stirred (up and down five times) with a perforated stirrer. When the fifth pull is complete, a stirrer is placed on the surface of the water to aid in the movement of the water in the still box. The water is then pumped back into the white water tank so that an incipient body is formed.

Depending on how vigorously the agitation was performed some foaming may occur in the plate and frame box. Foam can continue to exist after the initial wet body is formed, and can be very profuse. If you continue to pump for a few seconds after the water is removed to remove the air from the billet, some of the foam will disappear. Pressing and drying of handsheets

The green body and handsheet wires are removed from the mold and pressed. The moisture content of the pressed paper should be 70%. The pressed sheets are then dried in an electrically heated drum dryer. The surface temperature of the drying machine is between 60° C. and 105° C., and the rotating speed of the drying machine is such that the pressed paper sheet is in contact with the hot surface for 35 to 180 seconds. The final moisture content of the sheet should be between 4 and 7% (typically 5%).

If the moisture content of the sheet after pressing is less than 70%, the sheet may then stick to the surface of the drum dryer when the conditions described are applied. This may be due to uneven pressure across the width of the sheet. This should be avoided during operation.

When the surface temperature of the can dryer is less than 105°C but 70°C or higher, longer contact times are required to achieve a final moisture content of 5% for the handsheet.

If the surface temperature of the drum dryer is lower than 70°C, it is necessary to further increase the contact time or increase the initial pressure on the wet billet to remove more water or both. It is possible to reduce the moisture content of the pressed sheet to less than 60%. test

Paper aging and testing was carried out according to the methods listed in "Tappi Test Methods" TAPPI Publishing, Technology Park Atlanta, PO BOX 105113, Atlanta GA 30348, USA, ISBN 0-89852-200-5 (Volumes 1 and 2). Method T456om-87 defines the wet tensile burst strength of paper and paperboard; T4940m-81 defines the tensile burst strength of paper and paperboard; HST (Hercules sizing test) is defined as the sizing test of paper with ink resistance T530pm-83 Glue test; Cobb test defined with T441om-84.

A series of experiments were performed in which cellulose concentration, glutaraldehyde concentration, drying time and temperature, aging time and temperature were varied individually. The results are listed in the following table, where:

"Natural aging" refers to storage at 23°C±1°C and relative humidity of 50.0±2% for a specific period of time defined in T402om-83;

"Oven aging" refers to treatment at 80°C for 30 minutes;

"Standard drying conditions" means drying at 105°C for 35 seconds.

Wet and dry tensile strength were tested by methods T456om-87 and T4940m-81 respectively, and the ratio of wet tensile strength to dry tensile strength was expressed as a percentage. These data are presented in the table, where the higher the value the better the wet strength. The sizing effect was measured by HST (Hercules Sizing Test) (TAPPI method T530om-83) and the data are reported in seconds. The larger the value, the better the sizing effect. The resulting HST value is preferably greater than 20 seconds, more preferably greater than 120 seconds, even more preferably greater than 200 seconds. A Cobb test can also be used (TAPPI method T441om-90). Data are reported in units of grams/ ^m2 . "Fully saturated" means that the paper is not sized at all. The lower the Cobb value, the better the sizing effect. The resulting Cobb value is preferably less than 30 g/m ² , more preferably less than 21 g/m ² . Wet strength and sizing properties of cellulase/glutaraldehyde systems in handsheets dried under standard conditions "% wet strength after 24 hours of natural aging" protein on fiber 0% 5% 10% Glutaraldehyde on Fiber 10% 0.26 1.63 2.64 20% 0.80 3.44 6.24 40% 0.97 5.00 8.86 Control: -0.25% Kymene SLX = 4.57% "% wet strength after 2 weeks of natural aging" protein on fiber 0% 5% 10% Glutaraldehyde on Fiber 10% 0.94 1.97 3.01 20% 0.99 3.57 6.06 40% 1.05 5.56 10.30 Control: -0.25% Kymene SLX = 9.01% "HST (sec) after 24 hours of natural aging" protein on fiber 0% 5% 10% Glutaraldehyde on Fiber 10% 1 51 81 20% 1 89 128 40% 1 108 159 Control: -0.25% Kymene SLX = 1s "HST (seconds) after 2 weeks of natural aging" protein on fiber 0% 5% 10% Glutaraldehyde on Fiber 10% 1 66 125 20% 1 86 163 40% 1 120 132 Control: -0.25% Kymene SLX = 1s "HST after oven aging (seconds)" protein on fiber 0% 5% 10% Glutaraldehyde on Fiber 10% 1 60 108 20% 1 101 165 40% 1 108 149 Control: -0.25% Kymene SLX = 1s "Cobb (gsm) after 24 hours of natural aging" protein on fiber 0% 5% 10% Glutaraldehyde on Fiber 10% fully saturated 39.0 25.3 20% fully saturated 24.7 22.3 40% fully saturated 24.4 21.6 Control: -0.25% Kymene SLX = fully saturated "Cobb (gsm) after 2 weeks of natural aging" protein on fiber 0% 5% 10% Glutaraldehyde on Fiber 10% fully saturated 35.3 21.1 20% fully saturated 24.1 20.4 40% fully saturated 23.1 20.3 Control: -0.25% Kymene SLX = fully saturated "Oven Aged Cobb (gsm)" protein on fiber 0% 5% 10% Glutaraldehyde on Fiber 10% fully saturated 35.0 22.8 20% fully saturated 29.2 20.7 40% fully saturated 24.5 20.3 Control: -0.25% Kymene SLX = wet strength and sizing performance of the cellulase/glutaraldehyde system in fully saturated handsheets dried under variable conditions "% wet strength after 24 hours of natural aging" Protein/Glutaraldehyde on Fiber 5%/20% 10%/40% Drying conditions (surface temperature/contact time) 23℃/overnight 0.65 4.13 60℃/120s 2.00 5.65 70℃/180s 2.54 6.50 105℃/35s 2.86 6.97 Control: -0.25% Kymene SLX (105°C/35s) = 6.03% "% wet strength after oven aging" Protein/Glutaraldehyde on Fiber 5%/20% 10%/40% Drying conditions (surface temperature/contact time) 23℃/overnight 2.14 5.66 60℃/120s 3.04 6.11 70℃/180s 3.25 7.28 105℃/35s 2.99 8.82 Control: -0.25% Kymene SLX (105°C/35s) = 11.38% "HST (seconds) after 24 hours of natural aging" Protein/Glutaraldehyde on Fiber 5%/20% 10%/40% Drying conditions (surface temperature/contact time) 23℃/overnight 3 3 60℃/120s 51 39 70℃/180s 154 184 105℃/35s 83 208 Control: -0.25% Kymene SLX (105°C/35s) = 1s "HST after 2 weeks of natural aging (seconds)" Protein/Glutaraldehyde on Fiber 5%/20% 10%/40% Drying conditions (surface temperature/contact time) 23℃/overnight 3 2 60℃/120s 55 56 70℃/180s 158 221 105℃/35s 82 231 Control: -0.25% Kymene SLX (105°C/35s) = 1s "HST after oven aging (seconds)" Protein/Glutaraldehyde on Fiber 5%/20% 10%/40% Drying conditions (surface temperature/contact time) 23℃/overnight 2 2 60℃/120s 56 42 70℃/180s 134 193 105℃/35s 96 157 Control: -0.25% Kymene SLX (105°C/35s) = 1s "Cobb (gsm) after 24 hours of natural aging" Protein/Glutaraldehyde on Fiber 5%/20% 10%/40% Drying conditions (surface temperature/contact time) 23℃/overnight fully saturated fully saturated 60℃/120s 26.8 26.9 70℃/180s 21.9 19.6 105℃/35s 28.0 21.1 Control: -0.25% Kymene SLX (105°C/35s) = fully saturated "Cobb (gsm) after 2 weeks of natural aging" Protein/Glutaraldehyde on Fiber 5%/20% 10%/40% Drying conditions (surface temperature/contact time) 23℃/overnight fully saturated fully saturated 60℃/120s 25.6 25.8 70℃/180s 20.4 20.3 105℃/35s 22.7 19.3 Control: -0.25% Kymene SLX (105°C/35s) = fully saturated "Oven Aged Cobb (gsm)" Protein/Glutaraldehyde on Fiber 5%/20% 10%/40% Drying conditions (surface temperature/contact time) 23℃/overnight fully saturated fully saturated 60℃/120s 26.3 26.3 70℃/180s 21.5 20.6 105℃/35s 22.9 20.6 Control: -0.25% Kymene SLX (105°C/35s) = fully saturated wet/dry strength and sizing performance of the cellulase/glutaraldehyde system in handsheets dried under two-stage aging conditions "48 hours natural % wet strength after aging" Drying conditions (surface temperature/contact time) % wet strength experiment number Drying condition A drying condition B 1 40℃/60s 70℃/180s 4.66 a 55℃/60s 70℃/180s 3.73 b 40℃/180s 70℃/180s 5.29 ab 55℃/180s 70℃/180s 2.61 c 40℃/60s 105℃/35s 5.57 ac 55℃/60s 105℃/35s 5.58 bc 40℃/180s 105℃/35s 6.21 abc 55℃/180s 105℃/35s 5.01 Control: -0.25% Kymene SLX (105°C/35s) = 8.48% "% wet strength after oven aging" Drying conditions (surface temperature/contact time) % wet strength experiment number Drying condition A drying condition B 1 40℃/60s 70℃/180s 5.38 a 55℃/60s 70℃/180s 4.56 b 40℃/180s 70℃/180s 5.82 ab 55℃/180s 70℃/180s 2.84 c 40℃/60s 105℃/35s 5.17 ac 55℃/60s 105℃/35s 5.49 bc 40℃/180s 105℃/35s 6.03 abc 55℃/180s 105℃/35s 5.53 Control: -0.25% Kymene SLX (105°C/35s) = 12.53% "Dry strength after 48 hours of natural aging" Drying conditions (surface temperature/contact time) Dry strength/kNm ^-1 experiment number Drying condition A drying condition B 1 40℃/60s 70℃/180s 5.40 a 55℃/60s 70℃/180s 5.12 b 40℃/180s 70℃/180s 5.43 ab 55℃/180s 70℃/180s 4.77 c 40℃/60s 105℃/35s 5.09 ac 55℃/60s 105℃/35s 5.50 bc 40℃/180s 105℃/35s 5.13 abc 55℃/180s 105℃/35s 5.27 Control: - Blank (105°C/35s) = 4.28kNm ^-1 ; 0.25% Kymene SLX (105°C/35s) = 4.41kNm ^-1 "Dry strength after oven aging" Drying conditions (surface temperature/contact time) Dry strength/kNm ^-1 experiment number Drying condition A drying condition B 1 40℃/60s 70℃/180s 5.24 a 55℃/60s 70℃/180s 4.65 b 40℃/180s 70℃/180s 5.11 ab 55℃/180s 70℃/180s 4.75 c 40℃/60s 105℃/35s 5.09 ac 55℃/60s 105℃/35s 5.18 bc 40℃/180s 105℃/35s 5.46 abc 55℃/180s 105℃/35s 4.78 Control: - Blank (105°C/35s) = 3.93kNm ^-1 ; 0.25% Kymene SLX (105°C/35s) = 4.64kNm ^-1 "HST after 48 hours of natural aging" Drying conditions (surface temperature/contact time) HST/sec experiment number Drying condition A drying condition B 1 40℃/60s 70℃/180s 277 a 55℃/60s 70℃/180s 216 b 40℃/180s 70℃/180s 243 ab 55℃/180s 70℃/180s 169 c 40℃/60s 105℃/35s 258 ac 55℃/60s 105℃/35s 274 bc 40℃/180s 105℃/35s 310 abc 55℃/180s 105℃/35s 195 Control: -0.25% Kymene SLX (105°C/35s) = 1s "HST after 2 weeks of natural aging" Drying conditions (surface temperature/contact time) HST/sec experiment number Drying condition A drying condition B 1 40℃/60s 70℃/180s 304 a 55℃/60s 70℃/180s 241 b 40℃/180s 70℃/180s 190 ab 55℃/180s 70℃/180s 178 c 40℃/60s 105℃/35s 239 ac 55℃/60s 105℃/35s 251 bc 40℃/180s 105℃/35s 290 abc 55℃/180s 105℃/35s 171 Control: -0.25% Kymene SLX (105°C/35s) = 1s "HST after oven aging" Drying conditions (surface temperature/contact time) HST/sec experiment number Drying condition A drying condition B 1 40℃/60s 70℃/180s 314 a 55℃/60s 70℃/180s 205 b 40℃/180s 70℃/180s 242 ab 55℃/180s 70℃/180s 149 c 40℃/60s 105℃/35s 212 ac 55℃/60s 105℃/35s 275 bc 40℃/180s 105℃/35s 308 abc 55℃/180s 105℃/35s 210 Control: -0.25% Kymene SLX (105°C/35s) = 1s "Cobb after 48 hours of natural aging" Drying conditions (surface temperature/contact time) Cobb/gsm experiment number Drying condition A drying condition B 1 40℃/60s 70℃/180s 21.7 a 55℃/60s 70℃/180s 21.9 b 40℃/180s 70℃/180s 22.0 ab 55℃/180s 70℃/180s 25.1 c 40℃/60s 105℃/35s 20.7 ac 55℃/60s 105℃/35s 20.1 bc 40℃/180s 105℃/35s 22.1 abc 55℃/180s 105℃/35s 25.9 Control: -0.25% Kymene SLX (105°C/35s) = fully saturated "Cobb after 2 weeks of natural aging" Drying conditions (surface temperature/contact time) Cobb/gsm experiment number Drying condition A drying condition B 1 40℃/60s 70℃/180s 20.8 a 55℃/60s 70℃/180s 19.2 b 40℃/180s 70℃/180s 28.2 ab 55℃/180s 70℃/180s 21.2 c 40℃/60s 105℃/35s 22.1 ac 55℃/60s 105℃/35s 20.6 bc 40℃/180s 105℃/35s 21.7 abc 55℃/180s 105℃/35s 21.9 Control: -0.25% Kymene SLX (105°C/35s) = fully saturated "Oven Aged Cobb" Drying conditions (surface temperature/contact time) Cobb/gsm experiment number Drying condition A drying condition B 1 40℃/60s 70℃/180s 21.7 a 55℃/60s 70℃/180s 20.4 b 40℃/180s 70℃/180s 22.3 ab 55℃/180s 70℃/180s 22.3 c 40℃/60s 105℃/35s 20.5 ac 55℃/60s 105℃/35s 20.4 bc 40℃/180s 105℃/35s 20.9 abc 55℃/180s 105℃/35s 22.3 Control: -0.25% Kymene SLX (105°C/35s) = full saturation Comparison of the effects of different drying procedures on the wet strength and sizing properties of the cellulase/glutaraldehyde system "Effect of different drying procedures on wet strength (oven internal aging data)” additive Drying conditions (surface temperature/contact time) % wet strength Drying condition A drying condition B 5% protein/20% glutaraldehyde 105℃/35s - 2.99 5% protein/20% glutaraldehyde 70℃/180s - 3.25 5% protein/20% glutaraldehyde 40℃/60s 105℃/35s 5.17 5% protein/20% glutaraldehyde 55℃/180s 70℃/180s 5.49 0.25% Kymene SLX 105℃/35s - 12.53 "Effect of different drying protocols on HST (oven aging data)" additive Drying conditions (surface temperature/contact time) HST/sec Drying condition A drying condition B 5% protein/20% glutaraldehyde 105℃/35s - 96 5% protein/20% glutaraldehyde 70℃/180s - 134 5% protein/20% glutaraldehyde 40℃/60s 105℃/35s 212 5% protein/20% glutaraldehyde 55℃/180s 70℃/180s 149 0.25% Kymene SLX 105℃/35s - 1 "Effect of different drying protocols on Cobb (oven aging data)" additive Drying conditions (surface temperature/contact time) Cobb/gsm Drying condition A drying condition B 5% protein/20% glutaraldehyde 105℃/35s - 22.9 5% protein/20% glutaraldehyde 70℃/180s - 21.5 5% protein/20% glutaraldehyde 40℃/60s 105℃/35s 20.5 5% protein/20% glutaraldehyde 55℃/180s 70℃/180s 22.3 0.25% Kymene SLX 105℃/35s - fully saturated

Taken together, the data demonstrate that cellulase/glutaraldehyde treatment of paper results in improved wet strength, dry strength and sizing properties of the paper. 11. Proof of biometallization of paper

Biometallization of paper is demonstrated. The technique is based on the affinity of streptavidin for biotin. The biotin label is attached to the cellulase, which is then attached to gold particle-labeled streptavidin.

The pulp was incubated in 1/3 PBS buffer (pH 7.4) containing Tween 20 (0.1% vv ^-1 ) for 45 minutes in the greenhouse with shaking. Paper sheets (6 cm ² ) were made, rolled up and dried overnight at room temperature.

Paper samples (1.5 cm ² ) containing biotinylated cellulase and controls without biotinylated cellulase were incubated with 5% (wv ⁻¹ ) BSA in 10 mM PBS pH 7.4 at room temperature with shaking. Incubate for 30 minutes.

Auroprobe BLplus-labeled streptavidin conjugate and accelerator (Amersham Ltd., Amersham, U.K.) were used according to the manufacturer's recommendations for attaching and developing gold particles (Fostel et al., Chromosoma, 90, 254, (1984 ); Hutchinson et al., J. Cell Biol., 95, 609, (1982)). The accelerator solution coated the gold particles with silver to produce an orange/brown color, demonstrating the presence of the metal. Control discs without biotinylated cellulase did not appear orange/brown and thus were not metal coated. 12. Capacitance measurement of biometallized paper

The capacitance of the biometallized paper was compared to that of a control paper to determine if the gold-labeled cellulase altered the paper's capacitance properties.

Paper sheets were prepared from control pulps containing cellulase, gold-labeled cellulase, enhanced gold-labeled cellulase, and no cellulase. Sandwich the paper between two metal plates attached to the capacitance measuring instrument. Place the metal plates in a rack to ensure a constant and reproducible distance between the metal plates.

Calculate the capacitance (C) using the following formula $C=\frac{\mathrm{\&epsiv;o\&epsiv;rA}}{d}$ where d = the distance between the two plates

A = area

εo = constant

εr = relative permittivity

The measurements obtained showed that the presence of gold-labeled cellulase increased the capacitance of the disc. The measurement results are listed in Table 7.

Table 7: Capacitance Measurements sample Capacitance (pF) Machine Calibration (Control) 10.00 Paper without cellulase 10.97 Paper + Cellulase 10.65 Paper + gold-labeled cellulase 13.86 13. Proof that amylase binds starch

Two amylases were characterized by HPLC: α-amylase from Aspergillus oryzae (crude preparation type X-A) and amyloglucoside from Aspergillus niger (from Sigma Aldrich Co. Ltd., Poole, Dorset , obtained by the United Kingdom). The main catalytic peak for each formulation was determined using the amyloglucose release assay. The binding efficiency of each protein was determined against a variety of starches in the evaluation using BSA as a control.

Add 32mg ml ^-1 (dry weight) of α-amylase solution into 0.1M PBS (pH 7.0). Inject 100 μl of sample into HPLC, use Bio-Sil SEL gel permeation column, eluent is 0.1M phosphate buffer, flow rate is 1ml min ^-1 . The eluate (1 ml) was collected and tested for reducing sugars released from the starch suspension using a standard microtiter test (glucose).

Glucose and cellobiose in the samples were detected by the following quantitative tests. Assays were performed on microtiter plates at room temperature. Reagent ingredients: 10 μl phenol reagent (0.128M phenol in 0.1M phosphate buffer pH 7.0) 10 μl aminopyrine reagent (19.7mM 4-aminophenazone in 0.1M phosphate buffer pH 7.0) 10 μl peroxidase 0.1M Phosphate buffer pH7.0 (800Eu/ml) 10μl glucose oxidase 0.1M phosphate buffer pH7.0 (250Eu/ml) 60μl 0.1M phosphate buffer pH7.0

These reagent components are mixed and added to the wells of the microtiter plate. A 100 [mu]l sample was added followed by an excess of substrate (starch). A red color indicates the presence of amylase.

The HPLC curve of amyloglucosidase can also be made in the same way. Amyloglucosidase was determined to be a liquid preparation containing approximately 262 mg ml ^-1 protein using the Coomassie blue technique. Dilute it with 0.1MPBS (pH7.0) to the original concentration of 0.007, take 100μl HPLC sample and monitor at 230nm 0.1AUS. 1 ml of the eluate was collected and assayed for reducing sugars released from the starch suspension as described.

The ability of alpha-amylase and amyloglucosidase to bind conventional starch in suspension was evaluated. Starch (0.2 g; Roquette) was added to 9 ml 0.1 M PBS (pH 7.0) and 1 ml α-amylase solution (9.5 mg ml ⁻¹ as determined by Coomassie blue) was added. Incubate for 20 minutes on a shaker.

The sample was centrifuged at 13000 rpm for 5 minutes, and 100 μl of the sample was injected into the HPLC column. The peak height of bound alpha-amylase at 20 minutes was compared to the T=0 sample. The percent binding of the enzyme was thus calculated. Cationic starch was also used to determine the binding rate of amyloglucosidase. BSA was used as a control in the same method. The final concentration of BSA used was 0.2% (WV ^-1 ) in 0.1 M PBS.

The results of the binding experiments are listed in the table below. starch binding curve enzyme substrate % binding rate α-amylase starch 32 amyloglucosidase starch 27 amyloglucosidase cationic starch 45 BSA starch 7 BSA cationic starch 6

The results show that both alpha-amylase and amyloglucoside bind specifically to starch and cationic starch, and are therefore suitable as protein linkages for effector fragment-bound starch.

Claims (36)

1, a kind of method of handling polymkeric substance, make at least a character that is selected from fluid, electricity and intensive property be improved, it comprises connecting by the albumen that is used to obtain described improvement the effector fragment is combined in described polymkeric substance, described effector fragment is different from described albumen and connects, described albumen connects and is different from described polymkeric substance, and described effector fragment and described albumen connect by the significant quantity that obtains described improvement and exists.

2, the method for processing polymkeric substance as claimed in claim 1 can make the fluid dipped type of described polymkeric substance improve.

3, the method for processing polymkeric substance as claimed in claim 1 can make the sizability of described polymkeric substance be improved.

4, the method for processing polymkeric substance as claimed in claim 1 can make the conductivity of described polymkeric substance be improved.

5, the method for processing polymkeric substance as claimed in claim 1 can make the metallicity of described polymkeric substance be improved.

6, the method for processing polymkeric substance as claimed in claim 1 can make the wet tenacity performance of described polymkeric substance be improved.

7, the method for processing polymkeric substance as claimed in claim 1 can make the dry strength performance of described polymkeric substance be improved.

8, any method of claim as described above, wherein said polymkeric substance is a polysaccharide.

9, method as claimed in claim 8, wherein said polymkeric substance are Mierocrystalline cellulose.

10, any method of claim as described above, wherein said polymkeric substance is the composition fiber of paper or paper.

11, any method of claim as described above, wherein said albumen connects and comprises natural enzyme or its fragment.

12, method as claimed in claim 1, wherein said albumen connect and comprise and be selected from cellulase, hemicellulase, mannase, zytase, proteolytic enzyme, M-Zyme, chitinase, lignoenzyme, agarase, algin enzyme and diastatic enzyme or its fragment.

13, any method of claim as described above, wherein said albumen connects and comprises polysaccharidase and fragment thereof.

14, as the method for claim 13, wherein said albumen connects and comprises cellulase and fragment thereof.

15, as the method for claim 14, wherein said albumen connects the cellulose binding domain that comprises cellulase.

16, any method of claim as described above, wherein said effector fragment is connected in described albumen by connecting agent.

17, as the method for claim 16, wherein said connection agent comprise non-altogether bond close right.

18, as the method for one of claim 1 to 16, wherein said effector fragment is covalently bonded in described albumen and connects.

19, any method of claim as described above, wherein said effector fragment can be from the described polymkeric substance optionally cracking get off.

20, a kind of chemical substance composition comprises:

A) effector fragment; With

B) can be attached to protein on the polymkeric substance to described effector fragment;

Wherein said effector fragment is different from described protein, and described composition can make at least a character that is selected from fluid, electricity and intensive property of described polymkeric substance be improved.

21, a kind of material compositions, it comprises by the albumen connection combination segmental polymkeric substance of effector thereon, described effector fragment is different from described albumen and connects, wherein said effector fragment and described albumen connect by significant quantity and exist, and at least a character that is selected from fluid, electricity and intensive property of described polymkeric substance is improved.

22, a kind of method of handling polymkeric substance, make at least a character that is selected from fluid, electricity and intensive property be improved, contact comprising the albumen that makes polymkeric substance and effector fragment and be used to obtain described improvement, described effector fragment is different from described albumen and connects, and be different from described polymkeric substance, described albumen is different from described polymkeric substance, and described effector fragment and described albumen exist by the significant quantity that obtains described improvement.

23,, comprise the step that described effector fragment and described proteinic conjugate are contacted with described polymkeric substance as the method for claim 22.

24,, comprise the step that described effector fragment is contacted with the conjugate of described protein and described polymkeric substance as the method for claim 22.

25, a kind of method of handling polymkeric substance, make at least a character that is selected from fluid, electricity and intensive property be improved, comprising connecting at least a effector fragment is combined at least a described polymkeric substance by at least a albumen that is used to obtain described improvement, described at least a effector fragment is different from described at least a albumen and connects, described at least a albumen connects and is different from described at least a polymkeric substance, and described at least a effector fragment and described at least a albumen connect by the significant quantity that obtains described improvement and exists.

26, a kind of method of handling polymkeric substance, make and be selected from fluid, at least a character of electricity and intensive property is improved, comprising at least a effector fragment is contacted with at least a described polymkeric substance with at least a protein that is used to obtain described improvement, described at least a effector fragment is different from described at least a protein, and be different from described at least a polymkeric substance, described at least a protein is different from described at least a polymkeric substance, and described at least a effector fragment and described at least a protein exist by the significant quantity that obtains described improvement.

27, the method of the composition fiber of a kind of treatment paper or paper, make and be selected from fluid, at least a character of electricity and intensive property is improved, comprising connecting the composition fiber that at least a effector fragment is combined in described paper or paper by at least a albumen that is used to obtain described improvement, described at least a effector fragment is different from described at least a albumen and connects, described at least a albumen connects the composition fiber that is different from described paper or paper, and described at least a effector fragment and described at least a albumen connect by the significant quantity that obtains described improvement and exists.

28, the method for the composition fiber of a kind of treatment paper or paper, at least a character that is selected from fluid, electricity and intensive property is improved, comprising connecting the composition fiber that the effector fragment is combined in described paper or paper by the albumen that is used to obtain described improvement, described effector fragment is different from described albumen and connects, described albumen connects the composition fiber that is different from described paper or paper, and described effector fragment and described albumen connect by the significant quantity that obtains described improvement and exists.

29, as the method for claim 28, wherein said effector fragment can be given the wet tenacity that described paper improves.

30, as the method for claim 28, wherein said effector fragment can be given the dry strength that described paper improves.

31, as the method for claim 28, wherein said effector fragment can be given the sizability that described paper improves.

32, as the method for claim 28, wherein said effector fragment is a linking agent.

33, as the method for claim 32, wherein said effector fragment is a dialdehyde crosslinking agent.

34, as the method for claim 33, wherein said effector fragment is a glutaraldehyde.

35, as the method for one of claim 28 to 34, it is cellulase that wherein said albumen connects.

36, effector fragment and proteolytic enzyme are in a kind of purposes of handling in the method that at least a character that is selected from fluid, electricity and intensive property that polymkeric substance makes described polymkeric substance is improved.

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