WO1997007203A1 - Methods 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 CHEMICAL COMPOUNDS FOR MODIFYING POLYMERS

1 Technical Field

The present invention relates to methods and chemical compounds for modifying the physical properties of a polymer. In particular, the present invention relates to methods and chemical compounds for modifying the physical properties of a polymer by binding to the polymer a chemical compound, hereinafter referred to as an "effector moiety", which confers on the polymer improved fluid, electrical or strength properties.

Background

Polymers and materials containing polymers are a ubiquitous feature of every day life. Naturally occurring polymers include, for example, proteins (including keratin, which is the principal component of wool) , starch, pectin, guar, chitin, lignin, agar, alginate, and polysaccharides such as cellulose and hemi-celluloses (including xylan, mannose and arabinose) . Cellulose is encountered in the form of, for example, wood fibre and annual crop fibre (for example, hemp, straw, rice, flax, jute) based products such as paper, and cotton, which may be in the form of fibres, yarns, threads or a variety of woven and non-woven textile or fabric products. Xylanose is the principal component of xylan, otherwise known as he i-cellulose which occurs in grasses, cereal, straw, grain husks and wood. Starch occurs in seeds, fruits, leaves, bulbs etc.

The physical properties of polymers and materials containing polymers may be modified by a variety of chemical and physical treatments. Such chemical and physical treatments may be directed at modification of the polymer structure itself or at modification of the bulk properties of the material containing the polymer. The bulk properties of a material containing a polymer may, for example, be modified by admixture to the material of agents such as wet strength agents, dry strength agents or other chemical compounds which modify the physical properties of the material. Admixture of such chemical compounds to the material typically does not bind the compounds strongly to the polymer and problems may therefore be experienced with wastage of the chemical compounds and with the compounds leaching out of the material, resulting in variations in the properties of the material. Leaching out of the chemical compound may be reduced by a charge balancing protocol in which the ionic charge of the chemical compound is made equal and opposite to that of the polymer- containing material. However, in practical systems, the charge on both components varies widely requiring careful and frequent control measures. The modifying effect of the chemical compound may also rely on covalent binding to the polymer in order to properly achieve a modifying effect. In addition, promoters may be required to facilitate binding of certain chemicals to the material.

Alternatively, the chemical compounds may be applied to the surface of the material by, for example, immersion or printing. Once again, however, the chemical compounds typically do not bind to the surface of the material and problems may be encountered with diffusion of the compounds away from the intended site of application.

A variety of non-covalent binding interactions are known; for example, the binding interaction between an antibody and an antigen and the binding interaction between biotin and avidin or streptavidin. Enzymes capable of modifying an enzyme substrate also typically rely on a non-covalent binding interaction with the enzyme substrate in order to function.

One such class of enzymes comprise enzymes which degrade polymers, for example proteinases, keratinases, chitinases, ligninases, agarases, alginases, xylanases, mannases- amylases, cellulases and he i-cellulases. For example, cellulases and hemi-cellulases cleave saccharide or polysaccharide molecules from cellulose and hemi-cellulose, respectively, and amylases cleave glucose from starch.

The interactions between cellulose and cellulase proteins, in particular those that bind to the cellulose fibres as a prerequisite to catalytic activity have 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 group of enzymes include cellulases and hemi¬ cellulases which compriεe functionally distinct protein domains. In particular, the domain responsible for catalytic activity is structurally distinct from the cellulose binding domain. These domains are evolutionarily conserved sequences which are very similar in all such proteins (Gilkes et al ., Microbiological Reviews, 303-315, June 1991) .

The binding domains of such proteins can be separated from the active-site domains by proteolysis. The isolated binding domains have been shown to retain binding capabilities (Van Tilbeurgh, et al . , FEBS Letters, 204(2) . 223-227, August 1986). Use of cellulose binding domains of cellulases has been proposed as a means of roughening the texture of the surface of cellulosic support, while use of cellulase active-site domains has been proposed as a means of smoothing the texture of such surfaces (International patent application WO93/05226) .

A number of binding domains have also been characterised at the genetic level (Ohmiya et al .,Microbial Utilisation of Renewal Resources, 8., 162-181, 1993) and have been subcloned to produce new fusion proteins (Kilburn et al . , Published International Patent Application WO90/00609; Ong et al . , Enzyme Microb. Technol, H, 59-65, January 1991; Shoseyov et al . , Published International Patent Application

W094/24158) . Some of these fusion proteins have then been used as anchor proteins for specific applications. Such proteins have been used as an aid to protein purification through adhesion of the fusion proteins to cellulosic support materials used in protein purification strategies (Kilburn et al . , United States Patent 5,137,819; Greenwood et al . , Biotechnology and Bioengineering, 4_4, 1295-1305, 1994) . The ability to immobilize fusion proteins onto cellulosic supports has also been suggested as a means of immobilization for enzyme bioreactors (Ong et al . , Bio/Technology, J, 604-607, June 1989; Le et al . Enzyme Microb. Technol., 35, 496-500, June 1994), and as a means of attaching a chemical "tag" to a cellulosic material (International Patent Application 093/21331) .

3 Summary of the Invention

According to the present invention there is provided a method of treating a polymer to achieve an improvement in at least one property selected from fluid, electrical and strength properties comprising binding an effector moiety to said polymer via a protein linkage for the purpose of achieving said improvement, said effector moiety being different from said protein linkage and said protein linkage being different from said polymer, said effector moiety and said protein linkage being present in an amount effective to achieve said improvement.

It will be appreciated that the polymer may comprise a polymeric molecule or a polymeric material comprising polymeric molecules. Furthermore, reference to an effector moiety and a protein linkage refers to at least one effector moiety and at least one protein linkage, respectively. Accordingly, the present invention encompasses a method of treating a polymer to achieve an improvement in at least one property selected from fluid, electrical and strength properties comprising binding at least one effector moiety to at least one polymer via at least one protein linkage for the purpose of achieving said improvement, said at least one effector moiety being different from said at least one protein linkage and said at least one protein linkage being different from said at least one polymer, said at least one effector moiety and said at least one protein linkage being present in an amount effective to achieve said improvement.

According to a further aspect of the invention, there is provided a method of treating a polymer to achieve an improvement in at least one property selected from fluid, electrical and strength properties comprising contacting said polymer with an effector moiety and a protein for the purpose of achieving said improvement, said effector moiety being different from said protein and also different from said polymer, and said protein being different from said polymer, and said effector moiety and said protein being present in an amount effective to achieve said improvement. The invention encompasses a method of treating a polymer to achieve an improvement in at least one property selected from fluid, electrical and strength properties comprising contacting at least one polymer with at least one effector moiety and at least one protein for the purpose of achieving said improvement, said at least one effector moiety being different from said at least one protein and also different from said at least one polymer, and said at least one protein being different from said at least one polymer, and said at least one effector moiety and said at least one protein being present in an amount effective to achieve said improvement.

According to a further aspect of the present invention there is provided a chemical composition comprising:

a) an effector moiety; and

b) a protein capable of binding said effector moiety to a polymer; wherein said effector moiety is different from said protein and wherein said composition is capable of achieving an improvement in at least one property selected from fluid, electrical and strength properties of said polymer. The invention further provides composition of matter comprising a polymer to which is bound an effector moiety via a protein linkage, said effector moiety being different from said protein linkage, wherein said effector moiety and said protein linkage are present in an amount effective to achieve an improvement in at leaεt one property selected from fluid, electrical and strength properties of said polymer.

According to a further aspect of the invention there is provided method of treating paper or the constituent fibres of paper to achieve an improvement in at least one property εelected from fluid, electrical and strength properties comprising binding at least one effector moiety to said paper or constituent fibres of paper via at least one protein linkage for the purpose of achieving said improvement, said at least one effector moiety being different from said at least one protein linkage and said at least one protein linkage being different from said paper or constituent fibres of paper, and said at least one effector moiety and said at least one protein linkage being present in an amount effective to achieve said improvement.

4 Detailed Description of the Invention ,

The present invention provides methods and chemical compounds for modifying the fluid, electrical and/or strength properties of a polymer or material containing a polymer by binding to the polymer an effector moiety capable of conferring the desired property.

The term polymer includes reference to materials containing a polymer. The polymer-containing material may consist exclusively of polymer or may comprise polymer in combination with other components.

The polymer may comprise any polymer of any number of monomeric units. Preferably, the polymer comprises a naturally occurring polymer or a chemically modified derivative thereof. The naturally occurring polymer may, for example, comprise a protein such as keratin, or a polysaccharide such as a starch, pectin, guar, chitin, lignin, agar, alginate. Preferably, the polymer comprises a polysaccharide. The polysaccharide may comprise any polysaccharide, for example, mannose, xylanose, cellulose or a hemi-cellulose, preferably cellulose. Materials comprising cellulose may comprise wood-fibre or annual crop fibre (for example, hemp, straw, rice, flax, jute) based material, such as paper. Alternatively, the material may comprise cotton in the form of fibre, thread or woven or non-woven textile, fabric or cotton-based paper. Preferably, the material comprises paper.

The present invention may be employed to modify any fluid, electrical or strength property of the polymer. Properties of the polymer that may be modified include wet strength and dry strength, sizing, hydrophobicity, dye resistance and stain resistance, fluid penetration, oil and water repellency, electrical conductivity and resistance, electrical capacitance, pH and biometallic properties.

The protein employed in the present invention may comprise any protein capable of binding to the polymer. Preferably, the protein is capable of binding the polymer with a dissociation constant of (Kd) less than 1 x 10^"3M. As used herein, the term "protein" includes peptide, oligopeptide and polypeptide, as well as protein residues, protein- containing species, chains of amino acids and molecules containing a peptide linkage. Where the context requires (*.r examnle, when protein is bonded to another molecule). reference to a protein means a protein residue. The term "protein linkage" refers to a protein or protein residue via which an effector moiety is bound to a polymer. The protein may comprise a naturally occurring protein, or fragment thereof or modified protein obtainable by chemical modification or synthesis or by expression of a genetically modified gene coding for the protein. As used herein the term "modified protein" includes chemical analogs of proteins capable of binding to a polymer. Examples of proteins capable of binding polymers are well known and include enzymes selected from the group comprising cellulases, hemi-cellulases, mannases, xylanases, proteinases, keratinases, chitinases, ligninases, agarases, alginaεes and amylases. For example, a variety of cellulases are known which are dependent upon binding to cellulose for their activity. Examples of such cellulases are those isolable from bacterial organisms such as Cellulomonas fimi and fungal organisms such as Trichoderma viride, Aspergillus niger, Penicillium funiculosum, Trichoderma reesei and Humicula insolens, available as commercial preparations from Sigma Chemical Sigma-Aldrich Company Ltd., Novo Nordisk A/S, BDH Ltd., or ICN Biomedicals Ltd. Alternatively, the protein may be produced by recombinant DNA techniques as disclosed in, for example, International Patent application W094/24158. Cellulases generally comprise a cellulase binding domain and a domain responsible for cellulase activity. The present invention may employ the cellulase as a whole or a fragment thereof capable of binding to cellulose. A cellulase binding domain may be obtained from whole cellulase by treatment with protease(s), such as papain. The present invention may employ an exo-cellulase or an endo-cellulase.

Preferably, the protein comprises a naturally occurring enzyme which is capable of binding to the polymer. More preferably, in respect of paper, the catalytic activity is deactivated. The catalytic activity of the enzyme may be deactivated by, for example, attachment of the effector moiety or cross-linking of the enzyme. Cross-linking of the enzyme may be achieved with any suitable protein cross¬ linking agent such as a dialdehyde such as glutaraldehyde. Preferably, the protein comprises a deactivated naturally occurring cellulase.

The effector moiety may be attached to the protein capable of binding to the polymer in any convenient manner. For example, the effector moiety may be covalently bonded directly to the^' protein, via suitable reactive functional groups in the effector moiety and protein. Recognition of suitable reactive functional groups and, if necessary, their chemical modification to facilitate covalent bonding are within the ability of a person of ordinary skill in the art. Examples of covalent bond formation include formation of an amide bond between a carboxyl group and an amine group, by means of carbodiimide or dimethyl formamide activation of the carboxyl group.

The effector moiety may be attached to any suitable part of the polymer binding protein. The effector moiety may be attached to the polymer binding protein at the N-terminal end of the protein, for example via the N-terminal amino group. Alternatively, it may be attached at the C-terminal end of the protein, for example via the C-terminal carboxyl group. Alternatively, the effector moiety may be attached to the protein via an alternative functional group present, for example, in the amino acid chain of the protein or in a side chain thereof or introduced into the protein for the purpose of attachment to the effector moiety. The effector moiety may, for example, be attached via a thiol group present in cysteine, a hydroxyl group present in serine or threonine, an amino group present in lysine or arginine, an amide group present in asparagine or glutamine, a carboxyl group present in aspartic acid or glutamic acid or an aromatic or heteroaromatic group present in phenylalanine, tyrosine, tryptophan or histidine, or derivatives thereof. The effector moiety may be attached to the protein via a linker. The linker may, for example, comprise a difunctional molecule capable of reacting with a reactive site of the protein and a reactive site of the effector moiety so as to link the protein and effector moiety. It may be advantageous to include such a linker as a spacer between the protein and effector moiety, so that the two species are sufficiently spaced apart so as not to interfere sterically with each other's activity. A linker may also be advantageous in providing suitable functional group with which to join the effector moiety and protein.

Alternatively, or as part of a linker, the effector moiety may be attached to the protein via a non-covalent binding pair of molecules. Examples of such non-covalent binding pairs of molecules include biotin and avidin, streptavidin or neutralite.

Accordingly, one possibility is that the effector moiety is covalently attached to streptavidin whilst the polymer binding protein is covalently attached to biotin. Combining these components facilitates binding of the streptavidin and biotin portions of each component and hence attachment of the effector moiety to the polymer binding protein. It will be appreciated that the effector-streptavidin component may be mixed with the protein-biotin component either before or after the protein component has been bound to the polymer. It will be further appreciated that alternatively the effector moiety may be covalently attached to biotin, whilst the protein is covalently attached to avidin, streptavidin, or neutralite.

It will be appreciated that more than one type of effector moiety may be attached to the polymer. Two or more types of effector moiety may be used in order to reinforce each other's effect or to provide two or more effects simultaneously. It will be appreciated that in general the effector moiety may be attached to the polymer binding protein either before or after the polymer binding protein is bound to the polymer. The method of the present invention may comprise contacting a conjugate of the effector moiety and the protein with the polymer, or may comprise contacting the effector moiety with a comjugate of the protein and polymer. Alternatively, attachment of the effector moiety to the protein and attachment of the protein to the polymer may be accomplished in situ in a one-step process.

The present invention is not limited as to the precise nature of the manner in which the effector moiety is bound to the protein linkage and the protein linkage is bound to the polymer. Binding may be by means of a chemical bond such as a covalent bond or by means of a non-covolent physical interrelation, tie, association, attraction or affinity.

The effector moiety may comprise any moiety capable of conferring a desired physical property. The effector moiety may comprise an atom, molecule or chemical compound or residue thereof capable of conferring the desired physical property. In one embodiment the effector moiety comprises a chemical compound capable of conferring a desired physical property. For example, the agent may comprise a wet strength agent such as an aldehyde eg glutaraldehyde or dialdehyde starch or its cationic derivative, polyamide resin, polyacrylamide copolymer glyoxal, glyoxylated polyacrylamide, polyethyleneimine, polyamineepichlorohydrin polymers, polyamidoamine epichlorohydrin polymers, urea formaldehyde and melamine formaldehyde polymers, synthetic latexes, formaldehyde modified proteins or other polymers used for the purpose of imparting wet strength to paper; a dry strength agent such as starch, anionic or cationic starch, polyacrylamide, amphoteric, cationic or anionic polyacrylamide copolymers, anionic or cationic guar, locust bean gum or anionic or cationic modifications thereof. polyvinyl alcohol, carboxymethyl cellulose; a sizing agent such as rosin acids including abietic acid, adducted rosin acids including saponified fumaric acid gum rosin adduct, derivatives of rosin acids including tall oil, fatty acids including myristic acid, palmitic acid or stearic acid, other hydrophobic agents including alkenyl succinic anhydride (ASA) or 2-oxetanone compounds such as alkyl or alkynyl ketene dimer or multimer (AKD) or derivatives of ASA or AKD, gum, adducted gum, wood or tall oil rosin, saturated or unsaturated carboxylic acids with linear or branched chain lengths of from about 4 carbon atoms chain length to 30 carbon atoms chain length, alkyl ketene dimers made from such carboxylic acids, alkyl succinic anhydride of chain length from about 4 carbon atoms to about 30 carbon atoms, fully or partially fluorinated carboxylic acids or alkyl ketene dimer derived therefrom, fully or partially fluorinated alkyl succinic anhydride; a dye resistance or stain resistance agent; an oil or water repellant agent such as fiuorochemical including a fluorinated fatty acid or fluorinated derivative of ASA or AKD; an agent capable of conferring softness such as an agent capable of disrupting cellulose hydrogen bonding including surfactants, detergents, fatty amides or enzymatic agents such as expansin (McQueen-Maεon et al . , Proc. Natl. Acad. Sci. USA, 9_i, 6574-6578 (July 1995)); an agent capable of conferring electrical conductivity εuch aε a metal; an agent capable of conferring stiffness; an agent capable of conferring absorbency; an agent capable of conferring hydrophilicity; an agent capable of modifying density; a metallising agent; an agent capable of modifying pH, such as a buffer (for example, to impart resiεtance to acid degradation) .

In another embodiment, the effector moiety may comprise a croεε-linking or matrix forming agent or reεidue thereof, which may itεelf εerve to modify the phyεical properties of the polymer, or may serve to modify the properties of the protein and hence the physical properties of the polymer, or may serve to entrap a further agent capable of modifying the physical properties of the polymer. Preferred examples of cross-linking matrix forming agents compriseε dialdehydes, such as glutaraldehyde. Dialdehydeε such as glutaraldehyde can for example form a matrix with a cellulase derived protein. The cellulase/glutaraldehyde matrix confers improved wet strength and dry strength on paper, sizes the paper and/or may entrap further agents such aε Ti0₂ or CaC0₃.

An extensive review of compounds useful in papermaking is provided by Roberts et al . (Paper Chemistry, Chapman Hall New York, 1991) the entire contents of which are incorporated herein by reference. This reference particularly reviews retention aids, wet strength additives, dry strength additives, sizing agents and fillers.

As used herein, the term "paper" refers to any material in the form of a coherent sheet or web, comprising an interlaced network of cellulose containing fibres derived from vegetable sourceε optionally mixed with fibreε from vegetable, mineral, animal or εynthetic sources in various proportions and optionally mixed with fine particles of inorganic materials such as oxides, carbonates and sulphates of metallic elements in various proportions. The term "paper" includes paperboard which refers to paper when the weight of the paper sheet or web is greater than 200g/m².

Vegetable sourceε of celluloεe include wood, straws, Bagasse, Esparto, Bamboo, Kanaf, Grass, Jute, Ramie, Hemp, Cotton, Flax. The crude vegetable derived cellulose is processed to form pulp, the material from which paper is made, either mechanically, chemically or both. Cellulose containing pulps may be described as mechanical, chemimechanical and chemithermomechanical, semi chemical, high yield chemical, full chemical (see "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.l)) according to the method of pulp preparation and purification.

The effector moiety may be attached to the polymer at any suitable stage in the manufacture and proceεεing of the polymer or material containing the polymer.

If the effector moiety iε to be applied to paper, it may be attached at the pulp εtage or at any εtage during the formation of the wet pulp matrix or during the preεsing and rolling of the matrix to form paper. Alternatively, the effector moiety may be attached to the formed paper product by immersing the paper in a bath containing the reagents for attaching the effector moiety or by any suitable spraying, spreading, brushing, coating or printing process.

If the effector moiety is to be attached to cotton, it may again be attached at any stage in the procesεing of the cotton fibre. It may be attached to cotton fibre, thread, yarn or to woven or non-woven cotton fabric or textiles. The effector moiety may be attached by immersing the material in a bath containing the reagents for attaching the effector moiety or by any suitable spraying, spreading, brushing, coating or printing process.

By choosing the point in the manufacture of the polymer or material containing the polymer at which the effector moiety is attached, control may be exercised as to whether the effector moiety is distributed throughout the polymer material or is substantially restricted to the surface levels of the material.

In cases where the effector moiety is directed at modifying the bulk propertieε of the material, it may be advantageous to ensure even distribution of the effector moiety uniformly throughout the material. Accordingly, the effector moiety should be attached at an early stage in the manufacture. For example in the manufacture of paper where the effector moiety is directed at modifying the bulk properties of the paper, the effector moiety should be applied at the pulp stage.

In cases where the effector moiety is directed at modifying the surface properties of the material, it may be sufficient to restrict the effector moiety to the surface levels of the material, with an attendant advantage in reducing the quantities of effector moiety required. Accordingly, the effector moiety should preferably be supplied at a late stage in the manufacture. For example, in the manufacture of paper, where the effector moiety is directed at modifying the surface propertieε of the paper the effector moiety should be applied to the paper εurface.

Depending on the application it may be deεirable to apply the effector moiety to one or both planar surfaces of the paper. Treating both surfaceε of the paper with for example an effector moiety compriεing a wet εtrength agent, whilst leaving one or more of the edges untreated, facilitates preparation of a sandwich εtructure, in which a layer of paper having poor wet strength propertieε but good liquid absorption properties is sandwiched between two layers of paper having good wet strength properties. Such a structure is capable of transporting liquids through its middle layer by capillary action and is particularly useful in the manufacture of dip-εtick type diagnostic asεayε.

A particular feature of the preεent invention concerns the ability to modify the physical properties of the polymer or material containing the polymer in a reversible manner. Conventional treatment of polymers to impart particular physical propertieε are often non-reverεible. Furthermore, the conventional treatmentε often render the polymer unεuitable for recycling. In connection with recycling paper, the repulping of paper is made more difficult and may be imposεible if the paper iε treated with conventional wet strength agents. The present invention lends itself to the

SUBSTITUTE SHEET RULE 26J provision of means to permit release of the effector moiety to permit recycling of the material. The effector moiety may, for example, be released from the polymer-containing material by treatment with a protease which cleaves the protein attaching the effector moiety to the polymer; alternatively, the effector moiety may be attached to the protein by means of a selectively cleavable linker; cross¬ linking agents such aε aldehyde-εubεtituted εtarch may be cleaved by amylase.

A further advantage of the present invention lies in the fact that the desired physical property is imparted essentially immediately to the material. In conventional_, treatments to impart wet strength to paper, heat treatment and curing over several weeks may be required.

The invention will now be described with reference to the following figures and examples. In the Figures:

Figure 1 shows the effect of cellulase concentration on glutaraldehyde cross-linked cellulase imparted wet strength.

Figure 2 shows the effect of glutaraldehyde concentration on glutaraldehyde cross-linked cellulase imparted wet strength;

Figure 3 shows the effect of pH on glutaraldehyde cross¬ linked cellulase imparted wet strength;

Figure 4 showε the effect of temperature on glutaraldehyde croεε-linked cellulaεe imparted wet strength;

Figure 5 εhows the effect of incubation time on glutaraldehyde crosε-linked cellulaεe imparted wet strength;

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

Figure 7 shows the effect of glutaraldehyde cross-linked cellulase on the wet strength of paper produced from different wood pulpε.

It will be appreciated that the following is by way of example only and modification of detail may be made within the scope of the invention.

EXPERIMENTAL

Principles and"Applications of Effector Moiety Attachment

The protocols defined below represent the techniques used to characterize the use of cellulase as a biobridging agent for the attachment of effector moieties to cellulose.

For initial stock preparation one-third strength Phosphate Buffered Saline (¹/₃ PBS) was used. The formulation for ¹/₃ PBS waε as follows:

200 litres of deionized or demineralized water

(DEMI water)

197g of anhydrous εodium dihydrogen phoεphate

(NaH₂P0₄)

767g of anhydrous disodium hydrogen phosphate (Na₂HP0₄)

389g of εodium chloride (NaCl)

Anhydrous materials are not essential but the above mentioned weights should be recalculated to take into account any "water of crystallization" in the hydrated salts.

The cellulases that have been used were derived from fungal sourceε and are available either as aqueous solutionε or freeze dried powderε.

Penicillium funiculoεum

Cellulaεe derived from Penicillium funiculoεum (Sigma Aldrich Co. Ltd., Poole, Dorset, U.K.) is available as a tan powder and should be stored at below 0 C.

When used as an additive for handsheets the cellulase was first be prepared as a 20% total solids solution in ²/₃ PBS. Into a large shallow beaker was placed 200g of the dry enzyme preparation. To this was then added slowly 800g of ₃ PBS. The mixture was gently stirred with a glasε rod. Vigorous agitation of the solution should NOT be used to disperse the powder as denaturing of the enzyme may occur. Any clumps of enzyme preparation may be broken up gently with the glass rod. If the cellulase solution is prepared the day before use then it should be stored at 4 C.

Trichoderma Reesei

Cellulase derived from Trichoderma Reesei is available either as freeze dried powder from Sigma Aldrich Co. Ltd. Poole, Dorset, U.K. or as an aqueous solution from Novo Nordisk A/S, Bagsvaerd, Denmark. When using the powder, the procedure and handling practiεes for preparation of the aqueous solution with Penicillium funiculosum apply here as well.

The cellulase solution was added to the stock on the basis of the total protein content of the enzyme solution (e.g. 10 parts of dry protein per 100 parts of dry fibre) . The total protein content of the prepared cellulase solution was determined by the UV absorbance ( = 620nm) of the protein stained with Coomassie Brilliant Blue G250 dye (Sedmak and Grasεberg (Analytical Bioche iεtry, 7_9, 544-552 (1977)).

1. To assay for the binding of the cellulase to cellulose

Samples (typically between 25 to 500 mg and normally 100 mg) of cellulose, such as microcrystalline cellulose (Avicel, SigmaCell) or water-leaf paper pulp, were weighed into a series of tubes/flasks. Cellulaεe εolutions (typically containing between 200-600 mg protein ml^-1 in 3 ml buffer) , were added to each tube. The exact concentration of protein added initially was experimentally determined at the start of the binding assay using the assay developed by Sedmak and Grassberg (Analytical Biochemistry, 79., 544-552 (1977)).

The tubes were shaken at the desired temperature (typically between 4°C and 30°C but usually at room temperature), for a period of time (typically 1 to 90 min, usually between 5 to 15 min). Samples (0.5 - 1ml) were then taken for assay.

The sampleε were centrifuged in a 1 ml Eppendorf tube using a bench-top microfuge for 5 min and the supernatant retained for determination of protein concentration remaining in the supernatant (unbound cellulaεe) .

The supernatant protein concentration was subtracted from the initial protein concentration thereby defining the amount of cellulase associated with the cellulose pellet.

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

The results were presented as either the amount of protein bound to the cellulose as a percentage of the protein added, or as the amount of protein bound to the celluloεe aε a percentage of the protein/celluloεe (%w/w) .

2. Visualization of the effector moiety attachment using chemiluminescence

Preparation of cβllulaεe for the ECL detection svstem

1. Biotinylation of cellulase

A solution of biotinamido N-hydrosuccinimide ester (B_capNHS) in N,N-dimethylformamide (DMF) was prepared (1 mg ml^-1) . A solution of cellulase was prepared in distilled water (77 mg ml^-1) .

lml of the cellulase solution was added to 1ml of the B_capNHS εolution and the mixture incubated for 2.5 h at room temperature with εhaking. The reaction was then exhaustively dialysed against 500 ml 1/3 PBS buffer (PBS, pH 7.5: Na₂HP0₄, 11.5g; NaH₂P0₄, 2.96g; NaCl, 5.84 g diluted to IL with distilled water) for 1 h.

2. Binding of the Biotinylated cellulase to paper sheets [Application of the cellulase to the surface of a paper sheet]

A water-leaf paper sheet, usually 2 cm² , was incubated with biotinylated cellulase at a range of concentrations between 0.05 to 100 μg ml^"1 protein in 1/3 PBS (10 ml) for 45 min to 2 h at 4°C in a shallow Petri-dish with shaking. Experiments using PBS containing Tween 20 (0.1% vv^-1) were also performed.

3. Binding of the biotinylated cellulase to paper pulp and subsequent production of a paper sheet [Application of the cellulase to the paper matrix]

Paper pulp was incubated with the biotinylated cellulase in 1/3 PBS containing Tween 20 (0.1% vv^-1) for 45 min at room temperature with shaking. A disc of paper was formed from the paper pulp-biotinylated cellulase using the paper making filter. The paper disc was removed from the filter, rolled and allowed to dry overnight.

4. Binding of HRP-labelled streptavidin to the biotinylated cellulase

Binding of HRP-labelled streptavidin and ECL detection of the biotinylated cellulase was subεequently performed according to the manufacturer'ε recommendations (Amersham Ltd., Amersham, U.K.; Whitehead, T.P. et al . , Clin. Chem. 2j5, 1531-1546, 1979).

The paper was incubated with milk powder (4% wv^"1) in PBS for 45 min at either 4°C or room temperature with shaking to block non-specific binding of the HRP- streptavidin conjugate. The paper was then washed, 3x 3 min, uεing 0.5% (wv^-1) milk powder in 1/3 PBS containing Tween 20 (0.1%vv^_1). The εolution was discarded and replaced after each wash.

The horseradiεh peroxidase (HRP) - εtreptavidin conjugate was prepared as a 1:1000 part solution using milk powder (0.5% wv^-1) made up in 1/3 PBS containing Tween 20 (0.1%vv^_1). A suitable volume (2 to 10 ml) was added to cover the paper sheet which waε then incubated for 45 min at room temperature with εhaking.

The paper was then washed 3x 5 min, in 1/3 PBS containing milk powder (0.5% wv^-1) and Tween 20 (0.1%w~ ¹) . The waεh εolution was discarded and replaced after each wash. The paper was then washed 3x 5 min using 1/3 PBS and again the wash solution was discarded and replaced after each wash.

The cellulose bound cellulase-biotin-HRP-streptavidin conjugate was then visualiεed by the ECL method or quantified uεing the OPD methodology.

3. Enhanced chemiluminescence (ECL) method

It is necessary to carry out this method in a photographic darkroom.

Amersham ECL Detection Reagents 1 + 2 were mixed together in equal volumes (required approximately 0.13 ml cm² paper). Excess buffer was then drained from the paper and the detection reagents added to completely cover the paper surface.

The paper was incubated for exactly 1 min at room temperature without agitation. The detection reagent was drained off and the paper was blotted between two pieces of tissue paper to remove excess reagent. The blotted paper was then transferred to a piece of cling film and wrapped securely to remove any air pockets.

The paper was placed in a film caεsette minimising the delay between incubating the paper and exposing it to the Hyperfilm. The film was carefully placed on top of the paper and the film exposed for 15 s ensuring that the film did not move during exposure. This first sheet of film was then removed and immediately replaced with a second film which was then exposed for 1 min.

The films were then immediately developed to visualize the results. If necessary further sheets of film can be exposed with exposures of 1 to 60 min.

4. The (OPD) method for quantification of effector moiety bound to cellulose

The substrate buffer was prepared by dissolving 1 OPD tablet (60 mg; o-phenylenediamine dihydrochloride, Sigma Chemicals, UK) in 150 ml 0.06 M phosphate-citrate buffer (0.2 M Na₂HP0₄, 121.5 ml; 0.1 M citric acid 121.5 ml made up to 500 ml distilled water and the pH adjusted to 5.0) to give a final OPD concentration of 0.4 mg ml._j. Note that thiε reagent is light senεitive. 10 μl of fresh 30% H₂0₂ per 25 ml of substrate buffer was added immediately prior to use.

The paper samples containing the biotinylated cellulase were placed into a 50 ml Falcon tube. 25 ml of the complete substrate buffer solution was added to the tube and shaken at room temperature for 30 ε to 20 min, and usually between 5 and 15 min, then the reaction was stopped by adding 1 ml of 3M H₂S0₄. The absorbence was then determined at 492 nm and reference was made to a standard curve of 0D₄₉ vs biotinylated cellulase concentration in order to calculate the concentration of biotinylated cellulase present on or in the paper.

5. The coupling of paper effector moieties to enzyme peptides using carbodiimide

Carbodiimides react with carboxylate groups to form activated carboxyls. Amino groups then attack these activated carboxyls to form covalent peptide bonds. This chemistry can be used to attach paper effector chemicals which contain free carboxyl groups to the amino groups on peptides.

The carbodiimide chemistry used in the linkage of a paper effect chemical to the cellulase was based on conventional methodology (Hoare et al . , J. Biol. Chem, 242(10) - 2447- 2453, 1967).

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

Cellulase (21 mg ml^-1) waε diεεolved in distilled water, and abietic acid (100 mg) was disεolved in 25 ml of 10% (w^-1) methanol. 0.5 ml 1-(3-dimethylamino propyl)-3-ethyl carbodiimide-HCl (WS-CDI; 63 mg ml^-1) was added to 1.0 ml abietic acid solution and the pH adjusted to pH 4.5 ± 0.5 using HCl (0.1 N) . The mixture was then stirred at room temperature (5 min) . 2 ml cellulase solution was then added and the mixture left at room temperature with stirring (16 h).

The reaction was then stopped by the addition of sodium acetate (0.1 M; pH 5.0) and excesε abietic acid and WS-CDI was removed by exhaustive dialysiε in phoεphate buffer. The coupled cellulase was then used to bridge the abietic acid onto cellulose as described above.

6. Demonstration of wet tensile strength properties using glutaraldehyde cross-linked cellulase.

The application of glutaraldehyde crosε-linked cellulase to water-leaf paper pulp has been performed and demonstrated to impart wet-strength properties to the paper sheet.

Water-leaf paper pulp slurry was produced in the following manner: 10 g water-leaf paper was cut into 1 cm² squares and macerated in a domestic herb mill (CH100, Kenwood Ltd. UK) for 3 min with 100 ml distilled water.

2.15 g of a water-leaf paper pulp slurry (10% wv""¹) containing 0.2 g cellulose was taken and the following additions were made:

1 10 ml of 1/3 strength phosphate buffered saline (PBS) , pH 7.0 as a negative control.

2 10 ml of 1/3 PBS containing T . reesei cellulase (2 mg ml^"1) 3 10 ml of 1/3 PBS containing glutaraldehyde (25 μl ml^-1) 4a 10 ml of 1/3 PBS containing T. reesei (2 mg ml^"1) and glutaraldehyde (25 μl ml^"1) incubated together for 1 h at room temperature prior to addition to the pulp 4b 10 ml of 1/3 PBS containing T. reesei cellulase (2 mg ml^"1) and glutaraldehyde (25 μl ml^"1) added directly to the pulp

All the samples were then incubated for 1 h at room temperature on an orbital shaker before production of the paper sheetε.

To produce the paper εheetε, the volume waε increased to 100 ml with distilled water and paper sheets (6 cm²) produced using a laboratory-designed paper making apparatus operated in the following manner: a suspension of paper pulp (0.2% wv^-1) was poured into a plastic filter holder which houses a fine nylon filter mesh. By applying a vacuum for a few seconds the pulp was formed into a paper εheet εupported by the meεh. The filter meεh was removed from the apparatus and the paper sheet εandwiched between a second nylon mesh and blotted between adsorbent paper towelε. The paper sheet was carefully removed from the paper-making meεh, flattened by rolling and allowed to dry overnight.

Wet-εtrength waε determined in the following ways;

A Paper stability in water

Samples from each test paper sheet (1.5 cm²) were placed in Universal bottles and 25 ml distilled water added to each one. The tubes were shaken and periodically examined for signs of losε of integrity of the paper sampleε.

The reεults are given in Table 1

Table 1 determination of the stability of the paper samples in water

Sample Replicate Condition Initial Total

Number Number after shaking Disruption Disruption shaking in H₂o 1 1 Disintegrated -

2 Disintegrated

2 1 Disintegrated -

2 Disintegrated -

3 1 Disintegrated 2 Disintegrated - -

4a 1 Intact <18 h 36 h 2 Intact <18 h 36 h

4b 1 Intact >8 d >8 d 2 Intact >8 d >8 d

In Table 1, "-" means not applicable. B Paper Strength

Samples from each test paper sheet (4 cm x 1 cm) were taken and 25 μl of distilled water was pipetted across the middle of the paper ensuring an even diεtribution. The paper was suεpended between two bull-dog clips and a container was secured to the bottom clip. Water was added to the container and the weight of water necesεary to cause the paper to tear was determined.

The results are given in Table 2 and illustrate that the sampleε prepared uεing glutaraldehyde croεε-linked cellulase demonstrated the greatest wet tensile strength.

Table 2 determination of paper strength

Sample Replicate Added Weight

Number Number (g) 1 27.43 2 43.48

1 <22.00 2 <22.00

1 <22.00 2 34.63

4a 1 66.34

2 49.96

4b 1 >77.33 2 64.20

The wet-strength of the paper samples were retested to include BSA controls to assess the specificity of action of the bridging protein. The paper samples were prepared aε follows

1 10 ml of 1/3 strength phosphate buffered saline (PBS) , pH7.0

2 10 ml of 1/3 PBS containing T. reesei cellulase (2 mg ml"¹)

3 10 ml of 1/3 PBS containing glutaraldehyde (25 μl ml^"1)

4 10 ml of 1/3 PBS containing BSA (2 mg ml^"1) 5 10 ml of 1/3 PBS containing T . reesei cellulase (2 mg ml^"1) and glutaraldehyde (25 μl ml^"1) added to the pulp and incubated for 1 h at room temperature on an orbital shaker before production of the paper sheets

6 10 ml of 1/3 PBS containing BSA (2 mg ml^"1) and glutaraldehyde (25 μl ml^"1) added to the pulp and incubated for 1 h at room temperature on an orbital shaker before production of the paper sheetε.

The paper samples were placed in 50 ml Universal bottles with 25 ml water and vortexed using a laboratory mixer until complete disintegration of the paper sampleε occurred. The results are given in Table 3. Table 3 Determination of the stability of the paper when vortex mixed in water.

Sample Replicate Time required until Number Number complete disintegration (sec)

1 <5 2 <5

1 <5 2 <5

1 <5 2 <5

1 <5 2 <5

1 >1020 2 >1080

1 10 2 10

Although samples prepared using cross-linked BSA (Sample 6) showed an increased wet tensile strength compared to the controls, this was 100 fold less than that of the glutaraldehyde crosε-linked cellulaεe.

To determine optimum conditions for glutaraldehyde/cellulase treatment of paper to improve wet-strength, the following parameters were varied in turn. For the purposeε of thiε work the control parameters were εet at: cellulaεe (2 mg ml^"1) ; glutaraldehyde (0.6% vv^-1) added εeparately to the pulp; pulp suspended in buffer (pH 7.0) at 25°C. The mix was incubated for 60 min before paper sheet formation. Each parameter was varied in turn as follows: cellulase (0.5 to 8 mg ml"¹); glutaraldehyde (0.1 to 2.5 vv^"1) ; pH (5.0 to 10.0); temperature 25°, 37° and 45°C; incubation time (5 to 120 min); and time of pre-incubation of the cellulase and glutaraldehyde (15 to 60 min) .

All the paper sheets were allowed to dry overnight at ambient temperature prior to wet tensile strength testing. The results are illustrated in Figure 1 to 6.

7. Demonstration of Glutaraldehyde Cross-linked Cellulase (GCC) -Wet Tensile Strength Properties with Different Pulp Types.

The GCC wet strength composition was applied to paper produced from different types of pulp: ground wood pulp (GWP) , chemo- thermo-mechanical pulp (CTMP) , hard wood pulp (HWP) , soft wood pulp (SWP) and water-leaf pulp (W-LP; 70% HW: 30% SW) . The pulps were prepared in the usual manner, however, GWP and CTMP pulps were soaked in water overnight before blending to promote dispersion of the fibres.

The pulps were treated with either PBS buffer (10 ml); PBS buffer (10 ml) + cellulase (20 mg) + glutaraldehyde (0.6% vv^"1) . Then paper sheets (6 cm² ) were prepared from the pulp sampleε aε deεcribed above before wet tenεile εtrength testing. The resultε are given in Table 4 and illustrated graphically in Figure 7.

The results indicate that there was an improvement in the wet tensile εtrength of all the pulps tested. The final strength of the paper sheets produced using either GWP or CTMP was greater than that of the HWP, SWP and W-LP. The GCC composition did however induce a greater percentage increase in tenεile εtrength in the HWP and SWP samples. Table 4: Wet Tensile Strength of Paper Sheets Produced from Different Pulps.

Pulp Treatment V o r t e X Wet Tensile m i x i n g 1 S t r e n g t h

(seconds) (g)

Ground Buffer 135 105.4 wood Cellulase 120 45. 0 Glutaraldehyde 315 94 .5 Cellulaεe + > 1200 249 Glutaraldehyde

CTMP Buffer 480 78.5 Cellulase 420 88

Glutaraldehyde 420 71.5 Cellulase + > 1200 242.5 Glutaraldehyde

HWP Buffer < 5 < 15.3 Cellulase < 10 < 15.3 Glutaraldehyde < 10 < 15.3 Cellulase + > 300 119 Glutaraldehyde

SWP Buffer < 5 < 15.3 Cellulase < 5 < 15.3 Glutaraldehyde < 5 < 15.3 Cellulase + > 300 207 Glutaraldehyde

W-LP Buffer < 5 < 15.3 Cellulase ND ND Glutaraldehyde ND ND Cellulase + > 300 193 Glutaraldehyde

Time needed to complete disruption 8. Demonstration of the reversibility of glutaraldehyde cross¬ linked cellulase imparted wet tensile strength

To determine the reversibility of wet strength imparted by the glutaraldehyde cross-linked cellulase the following protease solutions were prepared using commercial protease preparation supplied by Sigma Chemical Sig a-Aldrich Company Ltd., Fancy Road, Poole, Dorset, BH17 7NH: ficin (4μl ml^-1 solution in PBS buffer at pH 6.5); papain (5μl ml^"1 solution in PBS buffer at pH 6.5); Protease K (2.8 mg ml^-1 solution in PBS buffer pH 8.0); α-chymotrypsin (1.0 mg ml^"1 solution in PBS buffer at pH 8.0) .

Paper squareε (1.5 x 1.5 cm) prepared from water-leaf paper pulp strengthened with glutaraldehyde cross-linked cellulase were taken and incubated with the following treatments outlined in Table 5.

Table 5 Wet strength reversibility treatments

Sample No. Treatment

1 Ficin (10 ml) + PBS buffer (pH 6.5)

2 Papain (10 ml) + 10 ml PBS buffer (pH 6.5)

3 Protease K (1 ml) + 19 ml PBS buffer (pH 8.0)

4 α-chymotrypsin (1 ml) + 19 ml PBS buffer (pH 8.0)

5 Ficin (10 ml) + Papain (10 ml)

6 Protease K (1 ml) + α-chymortrypsin (1 ml) +

18 ml PBS buffer (pH 8.0)

7 Ficin (10 ml) + Papain (10 ml) + Proteaεe K (1 ml) + α-chymotrypεin (1 ml)

8 0.2 M Phoεphate buffer pH 6.5 (20 ml)

9 0.2 M Phoεphate buffer pH 8.0 (20 ml)

The εamples were incubated at 30°C on an orbital shaker at 70 rpm. The samples were examined after 4h and 2Oh and were vortex mixed for 10 sec after 20 h if the paper was still intact. The determinations were performed in duplicate and the resultε are given in Table 6.

Table 6 determination of paper disruption in the presence of various treatments

Treatment Incubation Time (h) After further 4 20 10 sec vortex

Ficin X XX xxxx X XX xxxx

Papain XX XXXX XX XXXX

Protease K XX XX xxxx XX XX xxxx

α-chymotrypsin 0 XX xxxx 0 XX xxxx

Papain + Ficin XX XXXX XX XXXX

α-chym + Prot K X XX xxxx

X XX xxxx

All 4 proteases XX xxxx XX xxx xxxx

C o n t r o l 0 0 0 Phosphate 0 0 0 buffer (pH 6.5)

0 0 0 C o n t r o l 0 0 0 Phosphate buffer (pH 8.0)

The key to the qualitative observationε is given as an arbitrary scale of 0 to XXXX where 0 repreεents no visible disruption of the paper and XXXX representε total paper diεruption; represents previous loεs of paper integrity.

9. To determine the effect of protease treatment on the recyclability of paper wet strengthened by glutaraldehyde cross-linked cellulase

Paper sheetε prepared either from water-leaf paper pulo (0.2 g) wet εtrengthened with glutaraldehyde croεs-linked cellulase or with pulp (-0.2 g) prepared without any wet strength agent were taken and subjected to a serieε of treatments.

Treatment 1 A paper sheet (0.2 g) made from pulp wet strengthened with glutaraldehyde cross-linked cellulase was cut into 1 cm x 1 cm squares which were placed in a petri-dish with 20 ml 0.2 m phoεphate buffer (pH 8.0) containing Proteaεe K (14 mg) . The εample waε incubated at 37°C for 2 h with εhaking (60 rpm) . The squares were then removed and dipped into phosphate buffer (pH 8.0) and placed in a universal bottle containing 20 ml fresh phosphate buffer (pH 8.0) . The sample was then vortex mixed to ascerate the paper. Any fibreε left in the petri- dishes after the original incubation were harvested by centrifugation at 6,000 rpm, washed with phosphate buffer (pH 8.0) and added to the macerated sample in the universal bottle. 2 ml T. reesei cellulase (10 mg ml^"1) and 0.5 ml glutaraldehyde solution (25%) were added and the sample waε incubated at 25°C for 60 min. The εample was then used to form a new sheet of paper.

Treatment 2 A water-leaf paper sheet (0.2 g) made from pulp prepared with PBS without any wet strength agent was placed in a universal bottle with 14 ml 1/3 strength PBS. The sample was vortex mixed to macerated the paper and the pulp was made into a fresh piece of paper.

Treatment 3 Squares of paper made from pulp wet strengthened with glutaraldehyde croεs-iinked cellulaεe (0.4 g) were aεcerated in a blender with 30 ml of 1/3 εtrength PBS. The resultant pulp was removed and made into a fresh square of paper.

Treatment 4 A glutaraldehyde cross-linked cellulase strengthened paper sheet (0.2g) was cut into 1 cm² pieces and mascerated in a blender in 30 ml of 1/3 strength PBS. 20 mg T. reesei cellulaεe (10 mg ml^"1) and 0.5 ml glutaraldehyde solution (25% vv^-1) were added. The sample was incubated on an orbital shaker for 60 min at room temperature, The pulp was uεed to prepare a new εheet of paper.

Each paper sheet was allowed to dry overnight at ambient temperature before testing the integrity of 1 cm² samples to destruction in water using the vortex mixer. The resultε of the paper determination are given in Table 7.

Table 7: Integrity of Recycled Paper

Strategy Vortex mix Description of (Time, s) paper

300 Paper broken into 3 pieces

Paper totally disintegrated

50 Lots of small fragments

345 Small hole in middle of paper

Theεe reεultε indicate that GCC - containing pulp, when made into a new paper sheet, retains some wet tensile strength properties; that pulp produced by protease treatment, as opposed to physical disruption, generates stronger paper when recycled and that the further addition of GCC imparts the best wet tensile strength properties to the recycled sheets.

10. Demonstration wet-strength, dry-strength and sizing in paper following treatment with glutaraldehyde and cellulase

Experimentε were performed to determine the effect of cellulase (protein ligand) and glutaraldehyde (effector moiety) 'on the wet strength, dry strength and sizing of paper. In the experimentε, the following materials and general protocols were employed:- Cellulase

An aqueous Trichoderma reesei cellulase preparation was employed ("Cellulast 1.5L" supplied by Novo

Nordisk Bioindustry S.A. 92017 Nanterre Cedex. France)

Glutaraldehyde

The glutaraldehyde used in the following examples was a 25% aqueous solution commercially available from Merck Ltd. (Poole, Dorset, U.K.)

Stock Preparation

Except where otherwise indicated, the furnish used was a blend of ECF bleached hardwood and softwood pulps (ratio of 70:30 HW/SW) . The stock waε prepared with ¹/₃ PBS and no fillerε were added. The procedure waε aε followε:

280g of bleached hardwood pulp and 120g of bleached εoftwood pulp were added to 18 litreε of ¹/₃ PBS. The fibres were dispersed by vigorous agitation. This stock was then transferred to the Hollander and beaten until a freeness value of 25oSR was attained (time taken was usually 30 to 35 minutes) .

The stock was then adjusted to a final consiεtency of 2% with further ^α/₃ PBS as neceεsary. Addition /Incubation of Additives

Both the cellulase solution and glutaraldehyde solution were added to the thick (2% consistency) stock. Two litres of the thick stock (containing 40g of fibre) was contained in a metal jug and stirred at the lowest possible speed to achieve a slow movement of the stock. Vigorous agitation should be avoided otherwise denaturing of the enzyme may occur during the incubation period. The sto^'ck was at ambient temperatures (20-25'C). The cellulase solution was added first to the stock (avoiding any splashing or splattering of the solution) . When one minute had elapsed from the addition of the enzyme, the aqueous glutaraldehyde was added.

The incubation time of the additives was fifteen minutes, starting from the end of enzyme addition. During this incubation period the movement of the stock may appear to become easier/faεter. If this is apparent then reduce the stirrer speed as much as posεible.

After the fifteen minute incubation period had elapεed the thick stock was then added to the proportioner.

Pro por tioner

The thick stock in the proportioner was then diluted to a consiεtency of 0.25% uεing DEMI water only. Normal agitation speeds in the proportioner were employed to mix the stock.

Handεheet Formation The white water box was filled with DEMI water for handsheet formation. With the handsheet forming wire in place in the mould asεembly, one litre of stock from the proportioner was added to the Deckle Box, together with water from the white water box. The contents of the Deckle Box were agitated with the perforated agitator (moved up and down five timeε) . After the fifth εtroke the agitator was reεted on the surface of the water to help dampen the motion of the water in the Deckle Box. The water was then pumped back to the white water box and the initial wet mat was formed.

Depending on how vigorous the agitation has been some foaming may occur in the Deckle Box. This foam may still persiεt after the initial wet mat iε formed and can be quite substantial. Some of this foam can be dispersed if the pump is kept on for a few seconds after the water has been removed so that air can be drawn through the mat.

Handεheet Pressing and Drying

The wet mat and handsheet wire were removed from the mould to the presε. The moisture content of the pressed sheet should be 70%. The pressed sheet was then dried on an electrically heated drum dryer. The surface temperature of the dryer was between 60'C and 105'C and the speed of the dryer was such that the pressed sheet was 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%, then the sheet may stick to the surface of the drum dryer when the above conditions are employed. This may occur because of nonuniform presε pressures being applied across the width of the sheet. Steps should be taken to avoid this.

When the surface temperature of the drum dryer is less than 105"C but is 70'C or higher, longer contact times are required in order for the handsheet to have a final moisture content of 5%.

If the surface temperature of the drum dryer is below 70^*C, it is necessary to extend the contact time further or increase the initial pressing on the wet mat to remove more water or to do both. It is possible to reduce the moisture content of the presεed sheet to less than 60%.

Testing

Conditioning and testing of the paper is done according to procedures laid out in the "Tappi Test Methods" published by TAPPI, Technology Park Atlanta, PO Box 105113, Atlanta GA 30348, USA, ISBN 0 - 89852 - 200 - 5 (vol 1 and 2). The wet tensile breaking strength of paper and paper board is defined by method T 456 om - 87; the tensile breaking properties of paper and paper board is T494 om - 81; the HST (Hercules Sizing Test) iε defined as size test for paper by ink resiεtance T 530 pm - 83; and the Cobb test is defined by T 441 om - 90.

A series of experiments were performed in which the cellulase concentration, glutaraldehyde concentration, drying time and temperature, aging time and temperature were each varied. The results are presented in the following tables in which:-

"naturally aged" refers to storage for the specified time at 23°C ± l°C in relative humidity 50.0 ± 2% as specified in T402om-83; "oven cured" refers to treatment at 80°C for 30 minutes;

"standard drying conditions" refers to drying at 105°C for 35 seconds;

The wet and dry tensile strengths were determined by methods T456om-87 and T494om-81, respectively, and the ratio of wet to dry tensile strength expressed as a percentage. These are the data presented in the tables where the higher the value, the better the wet strength. The sizing effect was measured by the HST (Hercules size test) (TAPPI method T530pm-83) and the data recorded in seconds. The higher the value, the better the sizing. Preferably the HST value is greater than 20g, more preferably greater than 12Og, more preferably greater than 200g. Size effect was also measured by the Cobb test (TAPPI method T441om-90) and the data recorded in grams/m². "Fully saturated" means that the paper showed no sizing at all. The lower the Cobb value, the better the sizing. Preferably, the Cobb value is lesε than 30g/m², more preferably leεs than 2lg/m'^!

Wet Strength and Sizing Performance of Cellulase/Glutaraldehyde Svstem in Handsheets dried under standard conditions

Wet Strength after 24h naturally aged"

Protein on fibre

0% 5% 10%

Glutaraldehyde 10% 0.62 1.63 2.64 on fibre

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 naturally aged"

Protein on fibre

0% 5% 10%

Glutaraldehyde 10% 0.94 1.97 3.01 on fibre

20% 0.99 3.57 6.06

40% 1.05 5.56 10.30 Control:- 0.25% Kymene SLX = 9.01% "HST (seconds) after 24h naturally aged"

Protein on fi .bre

0% 5% 10%

Glutaraldehyde 10% 1 51 81 on fibre

20% 1 89 128

40% 1 108 159

Control:- 0.25% Kymene SLX = ls

"HST (seconds) after 2 weeks naturally aged"

Protein on fibre

0% 5% 10%

Glutaraldehyde 10% 1 66 125 on fibre

20% 1 86 163

40% 1 120 132 Control:- 0.25% Kymene SLX = ls

"HST (seconds) after oven curing"

Protein on fibre

0% 5% 10%

Glutaraldehyde 10% 1 60 108 on fibre

20% 1 101 165

40% 1 108 149

Control:- 0.25% Kymene SLX = ls

"Cobb (gsm) after 24h naturally ageing"

Protein on fibre

0% 5% 10%

Glutaraldehyde 10% Fully saturated 39.0 25.3 on fibre

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 naturally ageing"

Protein on fibre

0% 5% 10%

Glutaraldehyde 10% Fully saturated 35.3 21.1 on fibre

20% Fully saturated 24.1 20.4

40% Fully saturated 23.1 20.3

Control:- Kymene SLX = Fully saturated

"Cobb (gsm) after oven curing"

Protein on fibre

0% 5% 10%

Glutaraldehyde 10% Fully saturated 35.0 22.8 on fibre

20% Fully saturated 29.2 20.7

40% Fully saturated 24.5 20.3 Control:- 0.25% Kymene SLX = Fully saturated

Wet Strength and Sizing Performance on Cellulase/Glutaraldehyde svstem in handsheets dried under alternative conditions

"% Wet Strength after 24h naturally aged"

Protein/Glutaraldehyde on fibre

5%/20% 10%/40%

Drying 23%C/ 0.65 4.13 conditions overnight

(surface temperature 60°C/120s 2.00 5.65

/contact 70°C/180s 2.54 6.50 time)

105°C/35s 2.86 6.97

Control:- 0.2 5% Kymene SLX (IC )5°C/35s) = 6.03% "% Wet Strength after oven curing"

"HST (seconds) after 24h naturally aged"

"HST (seconds) after 2 weeks naturally aged"

"HST (seconds) after oven curing"

"Cobb (gεm) after 24h naturally aged"

"Cobb (gsm) after 2 weeks naturally aged"

"Cobb (gsm) after oven curing"

Wet/Dry Strength and Sizing Performance of Cellulase/Glutaraldehyde system dried under two stage conditions

"% Wet Strength after 48h naturally aged"

Dry Conditions (Surface temp/contact time) %Wet Strength

Expt Code Dryer A Dryer B

1 40°C/60s 70°C/180s 4.66 a 55°C/60ε 70°C/180s 3.73 b 40°C/180ε 70°C/180s 5.29 ab 55°C/180ε 70°C/180s 2.61 c 40°C/60ε 105°C/35s 5.57 ac 55°C/60s 105°C/35s 5.58 be 40°C/180s 105°C/35S 6.21 abc 55°C/180s 105°C/35ε 5.01 Control:- 0.25% Kymene SLX (105°C/35s) = 8.48%

"% Wet Strength after oven curing"

Dry Conditions (Surface temp/contact time) %Wet Strength

Expt Code Dryer A Dryer B

1 40°C/60s 70°C/180s 5.38 a 55°C/60s 70°C/180s 4.56 b 40°C/180s 70°C/180s 5.82 ab 55°C/180s 70°C/180ε 2.84 c 40°C/60ε 105°C/35s 5.17 ac 55°C/60ε 105°C/35s 5.49 be 40°C/180ε 105°C/35s 6.03 abc 55°C/180ε 105°C/35s 5.53

Control:- 0.25% Kymene SLX (105°C/35ε) = 12.53 3

"Drv strength after 48h naturally aged"

Dry Conditions (Surface temp/contact time) Dry Strength /kNm^"1

Expt Code Dryer A Dryer B

1 40°C/60s 70°C/180s 5.40 a 55°C/60ε 70°C/180s 5.12 b 40°C/180ε 70°C/180s 5.43 ab 55°C/180ε 70°C/180ε 4.77 c 40°C/60s 105°C/35ε 5.09 ac 55°C/60s 105°C/35s 5.50 be 40°C/180ε 105°C/35s 5.13 abc 55°C/180s 105°C/35s 5.27

Controls:- Blank (105°C/35s) = 4.28kNm^_i; 0.25% Kymene SLX (105°C/35s) = 4.41kNm^_1

"Drv strength after oven curing"

Dry Conditions (Surface temp/contact time) Dry Strength /kNm^-1

Expt Code Dryer A Dryer B

1 40°C/60s 70°C/180s 5.24 a 55°C/60ε 70°C/180ε 4.65 b 40°C/180s 70°C/180ε 5.11 ab 55°C/180s 70°C/180ε 4.75 c 40°C/60ε 105°C/35s 5.09 ac- 55°C/60ε 105°C/35s 5.18 be 40°C/180s 105°C/35s 5.46 abc 55°C/180s 105°C/35s 4.78

( 105 ° C/ 35s ) = 4 . 64kNm -1 "HST after 48h naturally aged"

Dry Conditions (Surface temp/contact time) HST / seconds

Expt Code Dryer A Dryer B

1 40°C/60s 70°C/180s 277 a 55°C/60s 70°C/180s 216 b 40°C/180s 70°C/180s 243 ab 55°C/180s 70°C/180S 169 c 40°C/60s 105°C/35s 258 ac 55°C/60ε 105°C/35s 274 be 40°C/180ε 105°C/35s 310 abc 55°C/180ε 105°C/35s 195

Control:- 0.25% Kymene SLX (105°C/35s) = lε

"HST after 2 weeks naturally aged"

Dry Conditions (Surface temp/contact time) HST / seconds

Expt Code Dryer A Dryer B

1 40°C/60s 70°C/180s 304 a 55°C/60s 70°C/180s 241 b 40°C/180s 70°C/180s 190 ab 55°C/180s 70°C/180s 178 c 40°C/60s 105°C/35s 239 ac 55°C/60s 105°C/35ε 251 be 40°C/180ε 105°C/35ε 290 abc 55°C/180ε 105°C/35s 171

Control:- 0.25% Kymene SLX (105°C/35s) = ls

"HST after oven curing"

Dry Conditions (Surface temp/contact time) HST / seconds

Expt Code Dryer A Dryer B

1 40°C/60s 70°C/180ε 314 a 55°C/60s 70°C/180s 205 b 40°C/180s 70°C/180s 242 ab 55°C/180S 70°C/180s 149

"Cobb after 48h naturally aσed"

"Cobb after 2 weeks naturally aged"

Dry Conditions (Surface temp/contact time) Cobb / gsm

Expt Code Dryer A Dryer B

1 40°C/60s 70°C/180s 20.8 a 55°C/60s 70°C/180s 19.2 b 40°C/180s 70°C/180s 28.2 ab 55°C/180ε 70°C/180s 21.2 c 40°C/60s 105°C/35s 22.1 ac 55°C/60s 105°C/35s 20.6 be 40°C/180s 105°C/35s 21.7 abc 55°C/180s 105°C/35s 21.9 Control:- 0.25% Kymene SLX (105°C/35s) = Fully saturated "Cobb after oven curing"

Dry Conditions (Surface temp/contact time) Cobb / gsm

Expt Code Dryer A Dryer B

1 40°C/60s 70°C/180ε 21.7 a 55°C/60ε 70°C/180s 20.4 b 40°C/180ε 70°C/180ε 22.3 ab 55°C/180ε 70°C/180ε 22.3 c 40°C/60s 105°C/35s 20.5 ac 55°C/60s 105°C/35s 20.4 be 40°C/180s 105°C/35s 20.9 abc 55°C/180s 105°C/35s 22.3

Control:- 0.25% Kymene SLX (105°C/35ε) = Fully saturated

Comparison of the Effect of Different Drying Regimes on Wet Strength and Sizing Performance of Cellulase/Glutaraldehyde Svstem

"Effect of Different Drying Regimes on % Wet Strength (oven cured data) "

Additive Drying Conditions % Wet

(Surface Temp/Contact Time) Strength

Dryer A Dryer B

5% Protein/20% 105°C/35s - 2.99 Glutaraldehyde

5% Protein/20% 70°C/180s - 3.25 Glutaraldehyde

5% Protein/20% 40°C/60ε 105°C/35ε 5.17 Glutaraldehyde

5% Protein/20% 55°C/180S 70°C/180ε 5.49 Glutaraldehyde

0.25% Kymene 105°C/35s - 12.53 SLX

"Effect of Different Drying Regimes on HST (oven cured data)" Additive Drying Conditions HST / seconds

(Surface Temp/Contact Time)

Dryer A Dryer B

5% Protein/20% 105°C/35s - 96 Glutaraldehyde

5% Protein/20% 70°C/180s - 134 Glutaraldehyde

5% Protein/20% 40°C/60s 105°C/35s 212 Glutaraldehyde

5% Protein/20% 55°C/180s 70°C/180s 149 Glutaraldehyde

0.25% Kymene 105°C/35s - 1 SLX

"Effect of Different Drying Regimes on Cobb (oven cured data)"

Additive Drying Conditions Cobb / gεm (Surface Temp/Contact Time)

Dryer A Dryer B

5% Protein/20% 105°C/35s - 22.9 Glutaraldehyde

5% Protein/20% 70°C/180s - 21.5 Glutaraldehyde

5% Protein/20% 40°C/60s 105°C/35S 20.5 Glutaraldehyde

5% Protein/20% 55°C/180s 70°C/180s 22.3 Glutaraldehyde

0.25% Kymene 105°C/35s - Fully εaturated SLX

In conclusion, the above data demonstrates that cellulase/glutaraldehyde treatment of paper leads to improvement in the wet strength, dry strength and sizing of the paper.

11. Demonstration of Bio-metalization of Paper

The bio-metalization of water-leaf paper was demonstrated. The technique was based on the affinity of streptavidin for biotin. The biotin label was linked to the cellulase which in turn was linked to streptavidin labelled with gold particles.

Biotinylated cellulase was incubated with the paper pulp in 1/3 PBS buffer (pH 7.4), containing Tween 20 (0.1% vv^"1) for 45 min at room temperature with shaking. A paper square (6 cm²) was formed, rolled and allowed to dry overnight at ambient temperature.

Samples of the' paper (1.5 cm²) containing the biotinylated cellulaεe and control samples lacking the biotinylated cellulase, were incubated with 5% (wv^-1) BSA in 10 mM PBS pH 7.4, for 30 min at ambient temperature with shaking.

The Auroprobe BLplus labelled streptavidin conjugate and enhancer (Amersham Ltd., Amersham, U.K.) was used according to the manufacturer's recommendation to attach and visualize the gold particleε (Fostel et al . , Chromosoma, £0, 254, (1984); Hutchinson et al . , J. Cell Biol., 95, 609, (1982)). The enhancer solution coated the gold particles with silver to create an orange/brown colour which was indicative of the presence of the metalε. Control εheets which did not contain the biotinylated cellulase did not develop the orange/brown colouration and hence were not coated with the metal.

12. Capacitance Measurement of Eiometalized Paper

The capacitance of bio etalized paper sheets was compared to control sheets to determine if the presence of the gold- labelled cellulase altered the capacitance characteristics of paper.

Paper sheets were produced from W-LP containing either cellulase, gold labelled cellulase, enhanced gold labelled cellulase and cellulase-free controlε. The sheetε were each held between two metal plates connected to a capacitance meter. The metal plates were held in position in a jig which ensured that a constant and reproducible distance was maintained between the plates.

The capacitance (C) was calculated using the following equation

C = eoεrA d

where d = distance between the two plates A = area eo = constant er = relative permeativity

The measurements obtained indicated an increase in the capacitance of the paper sheets in the presence of gold labelled cellulase. The results of the determination are given in Table 7.

Table 7: Capacitance Determination

Sample Capacitance (pF)

Machine Calibration (control) 10.00

Paper without cellulase 10.97

Paper + cellulaεe 10.65 Paper + gold labelled cellulaεe 13.86

13. Demonstration of binding amylase enzymes to starch

Two amylase enzymes were characterized using HPLC: an α-amylase (Type X-A crude preparation) from Aspergillus oryzae and amyloglucosidaεe from A . niger (available from Sigma Aldrich Co. Ltd., Poole, Dorεet, United Kingdom). The main catalytic peakε of each preparation were determined using a starch glucose-releaεe assay. The binding efficiencies of each protein were determined against a range of starches with BSA controls included in the assessment.

A solution of 32 mg ml^"1 (dry weight) of α-amylase was made up in 0.1 M PBS (pH7.0) . lOOμl of this was loaded onto an HPLC using a Bio-Sil SEC gel permeation column running 0.1 M phosphate buffer at 1 ml min^"1. Fractions (l ml) were collected and tested for reducing sugars released from a starch suspenεion uεing the standard microtitre asεay (for glucose) .

The following qualitative aεsay was used to detect glucose and cellobiose in test samples. The assay was carried out in a micro titre dish at room temperature.

Reagent Components:

10 μl phenol reagent (0.128M phenol in 0.1M phosphate buffer

PH7.0) 10 μl amino pyrine reagent (l9.7mM 4-amino phenazone in 0.1M phosphate buffer pH7.0)

10 μl peroxidase in 0.1M phosphate buffer pH 7.0 (to give

800Eu/ml)

10 μl glucose oxidase in 0.1M phosphate buffer pH 7.0 (to give 250Eu/ml)

60 μl 0.1M phosphate buffer pH7.0

These reagent components were mixed and added to the wells of a microtitre dish. Test samples 100 μl were added followed by an excess of subεtrate (εtarch) . The appearance of a red colour was indicative of the presence of amylase.

The same methodε were alεo uεed to produce an HPLC profile for the amyloglucosidase. The am loglucosidaεe waε a liquid preparation containing approximately 262 mg ml^"1 protein aε measured by the Coomassie Blue technique. 100 μl of a 0.007 dilution in 0.1 M PBS (pH 7.0) was loaded onto the HPLC and monitored at 230 nm 0.1 AUS. 1 ml fractions were collected and tested for reducing sugars released from starch suspensions as above.

The ability of α-amylase and amyloglucosidase to bind to normal starch in suspension was asεessed. Starch (0.2 g; Roquette) was added to 9 ml 0.1 M PBS (pH 7.0) and l ml α-amylase solution (9.5 mg ml^"1 by Coomassie Blue asεay) waε added. This was incubated on a shaker for 20 min.

The sample was centrifuged at 13,000 rpm for 5 min and 100 μl samples loaded onto the HPLC column. The peak profile of the 20 min bound α-amylase was compared with a T = 0 sample. From this data the percentage binding of the enzyme was calculated. The binding of amyloglucosidase was also tested against cationic starch. BSA was also used in the same way as a control. The final concentration of the BSA used was 0.2% (wv^-1) in 0.1 M PBS.

The resultε of the binding experiments are shown in the following Table.

Starch binding profiles

Enzyme Substrate % Bound α-amylase starch 32 amyloglucosidaεe starch 27 amyloglucosidase cationic starch 45

BSA starch 7

BSA cationic starch 6

These resultε indicate that both α-amylases and amyloglucosidases specifically bind to both starch and cationic starch and are therefore suitable for use as protein linkages for binding effector moieties to starches.

Claims

1. A method of treating a polymer to achieve an improvement in at least one property selected from fluid, electrical and strength properties comprising binding an effector moiety to said polymer via a protein linkage for the purpose of achieving said improvement, said effector moiety being different from said protein linkage and said protein linkage being different from said polymer, said effector moiety and said protein linkage being present in an amount effective to achieve said improvement.

2. A method of treating a polymer according to claim 1 to achieve an improvement in the fluid penetration properties of said polymer.

3. A method of treating a polymer according to claim 1 to achieve an improvement in the sizing properties of said polymer.

4. A method of treating a polymer according to claim 1 to achieve an improvement in the electrical conductivity properties of εaid polymer.

5. A method of treating a polymer according to claim 1 to achieve an improvement in the metallic properties of said polymer.

6. A method of treating a polymer according to claim 1 to achieve an improvement in the wet strength properties of said polymer.

7. A method of treating a polymer according to claim 1 to achieve an improvement in the dry strength properties of said polymer.

8. A method according to any preceding claim wherein said polymer is a polysaccharide.

9. A method according to claim 8 wherein said polymer is cellulose.

10. A method according to any preceding claim wherein said polymer is paper or the constituent fibres of paper.

11. A method according to any preceding claim wherein said protein linkage comprises a naturally occurring enzyme or fragment thereof.

12. A method according to any claim 1 wherein said protein linkage comprises an enzyme selected from the group comprising cellulases, hemi-cellulases, mannases, xylanases, proteinases, keratinases, chitinases, ligninases, agarases, alginases and amylases or fragment thereof.

13. A method according to any preceding claim wherein said protein linkage comprises a polysaccharidase or fragment thereof.

14. A method according to claim 13 wherein said protein linkage comprises a cellulase or fragment thereof.

15. A method according to claim 14 wherein said protein linkage comprises a celluloεe binding domain of a cellulase.

16. A method according to any preceding claim wherein said effector moiety is attached to said protein linkage via a linker.

17. A method according to claim 16 wherein said linker comprises a non-covalent binding pair.

18. A method according to any one of claims 1 to 16 wherein said effector moiety is covalently bonded to said protein linkage.

19. A method according to any preceding claim wherein said effector moiety is selectively cleavable from said polymer.

20. A chemical composition comprising:

a) an effector moiety; and

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

wherein said effector moiety is different from said protein and wherein said composition iε capable of achieving an improvement in at least one property selected from fluid, electrical and strength propertieε of εaid polymer.

21. A compoεition of matter compriεing a polymer to which is bound an effector moiety via a protein linkage, said effector moiety being different from said protein linkage, wherein said effector moiety and said protein linkage are present in an amount effective to achieve an improvement in at least one property selected from fluid, electrical and strength properties of said polymer.

22. A method^* of treating a polymer to achieve an improvement in at least one property selected from fluid, electrical and strength properties comprising contacting said polymer with an effector moiety and a protein for the purpose of achieving said improvement, said effector moiety being different from said protein and also different from said polymer, and said protein being different from said polymer, and said effector moiety and said protein being present in an amount effective to achieve said improvement.

23. A method according to claim 22 comprising the step of contacting a conjugate of said effector moiety and said protein with said polymer.

24. A method according to claim 22 comprising the step of contacting said effector moiety with a conjugate of said protein and said polymer.

25. A method of treating a polymer to achieve an improvement in at least one property selected from fluid, electrical and strength properties compriεing binding at least one effector moiety to at least one polymer via at least one protein linkage for the purpose of achieving said improvement, said at least one effector moiety being different from said at least one protein linkage and said at least one protein linkage being different from said at least one polymer, said at leaεt one effector moiety and εaid at least one protein linkage being preεent in an amount effective to achieve εaid improvement.

26. A method of treating a polymer to achieve an improvement in at least one property selected from fluid, electrical and strength propertieε comprising contacting at least one polymer with at least one effector moiety and at least one protein for the purpose of achieving said improvement, said at least one effector moiety being different from said at least one protein and also different from said at least one polymer, and said at least one protein being different from said at least one polymer, and said at least one effector moiety and said at least one protein being preεent in an amount effective to achieve εaid improvement.

27. A method of treating paper or the conεtituent fibres of paper to achieve an improvement in at least one property selected from fluid, electrical and εtrength properties compriεing binding at leaεt one effector moiety to said paper or constituent fibres of paper via at least one protein linkage for the purpose of achieving said improvement, said at least one effector moiety being different from said at least one protein linkage and said at least one protein linkage being different from said paper or constituent fibres of paper, and εaid at least one effector moiety and said at least one protein linkage being present in an amount effective to achieve said improvement.

28. A method of treating paper or the constituent fibres of paper to achieve an improvement in at least one property selected from fluid, electrical and εtrength properties comprising binding an effector moiety to said paper or constituent fibreε of paper via a protein linkage for the purpoεe of achieving εaid improvement, said effector moiety being different from said protein linkage and said protein linkage being different from said paper or constituent fibreε of paper, and effector moiety and said protein linkage being present in an amount effective to achieve said improvement.

29. A method according to claim 28 wherein said effector moiety is capable of conferring improved wet strength on said paper.

30. A method according to claim 28 wherein said effector moiety is capable of conferring improved dry strength on said paper.

31. A method according to claim 28 wherein said effector moiety is capable of conferring improved sizing on said paper.

32. A method according to claim 28 wherein said effector moiety is a crosε-linking agent.

33. A method according to claim 32 wherein εaid effector moiety iε a dialdehyde cross-linking agent.

34. A method according to claim 33 wherein said effector moiety is glutaraldehyde.

35. A method according to any one of claims 28 to 34 wherein said protein linkage iε a cellulaεe.

36. Use of an effector moiety and a protein in a method of treating a polymer to achieve an improvement in at least one property selected from fluid, electrical and strength properties of said polymer.