Background and Prior Art Paper products consist largely of cellulose fibers. The fibers are connected with each other by hydrogen bonds. Paper products such as paper for writing and printing are easily torn. In addition, the tensile strength of such paper is greatly reduced further when the paper comes into contact with water.

Because of the low cost of paper products and of their environmental compatibility, it is desirable to use paper products also where high tensile strength and wet strength are essential. For instance, it is desired to provide paper labels for bottles, paperboard packaging for agricultural products, or paper for filtering liquids. The material used for these purposes must possess high tensile strength, particularly high wet strength. Prior art processes have been developed for binding compounds and particles, for imparting enhanced properties such as wet strength onto cellulose, for improving the liquid absorption capacity of cellulose fibers, and the like.

The prior art processes for improving wet strength include application of a water-resistant cover layer onto the paper or impregnating the paper with hydrophobic material. Usually, wax or derivatives thereof are used for this purpose. However, the use of wax in paper production is undesirable because of environmental concerns.

Prior art processes for imparting water-resistance to paper are known, in which the cellulose fibers are cross-linked with chemical polymers to form a matrix. Chemicals used in this process comprise e. g., polyamides and urea-formaldehyde. This process suffers from the disadvantage that when it is desired to recycle the paper product, the cross-linking cannot be broken up without degrading the fiber constituents. However, for the process of recycling, it is necessary to dissolve the paper product in an aqueous medium. Thus, the prior art methods of imparting water resistance on paper products lead to disadvantages at the stage of recycling such paper products. In addition, these methods are not desirable from an environmental point of view, as the polymers used to obtain the cross-linked cellulose matrix are not biodegradable.

US 5,207,826, Westland et al. describes a bacterial cellulose binding agent for use in binding small fragments of paper and wood. The bacterial cellulose binding agent, is derived from Acetobacter and consists of microfibrillated cellulose obtained by treatment by alkali, ph >13, at 60°C for 2 hours.

EP 212,289 describes a cellulose fiber material with increased absorbent properties which is resistant to compression. The process for producing this material involves beating plant material in order to break cell walls into microfibers according to a process known from US 4,474,949, and cross-linking the resulting pulp according to another known method using a chemical cross-linker, e. g., as described in US 3,241,553. The cross-linking process involves treatment of cellulose fibers with cross-linking agents such as glyoxal, formaldehyde, or the sodium thiosulfate derivative of divinyl sulfone. The cross-linking process is applied to cotton fibers or regenerated cellulose, such as Rayon, and the resulting material is used as tampon or as surgical dressing.

A number of prior art publications describe the cross-linking of proteins to cellulose. For instance, EP 243,151 relates to the cross-linking of cellulose materials with an animal cell binding protein such as laminin, collagen, or fibronectin. The so-treated cellulose materials are useful as wound covers, as the animal cell binding protein improves the adherence of the cellulose material to the wound.

Cellulose binding proteins A number of proteins or domains thereof that are capable of binding cellulose has been described. Kilburn et al., US 5,202,247, relates to cellulose binding proteins derived from Cellulomonas fimi endoglucanase.

The C. fimi endoglucanase-derived cellulose binding region exhibits a relatively low affinity to cellulose. The protein is dissociated substantially from the cellulose under moderate salt concentrations (0.5 M). Moreover, binding of C. fimi endoglucanase to cellulose disrupts the morphology of the cellulose fibers.

A publication in Applied Environmental Microbiology 54,2521,1988, relates to cloning of Thermomonospora endoglucanase. This enzyme binds crystalline cellulose. Once bound, the enzyme cannot be released by water, but only with the aid of strong denaturants such as guanidine hydrochloride.

Shoseyov et al., US 5,837,814, describe cloning and production of a cellulose binding domain (CDB) derived from Clostridium cellulovorans.

The CBD binds cellulose with high affinity. Once bound, CBD cannot be dissociated from the cellulose by water, but only by high concentrations (6M) of guanidinium hydrochloride. CBD binding does not lead to changes in cellulose morphology (decrease in crystallinity), as has been observed for cellulose binding regions of other sources.

Cellulose binding regions or domains have been suggested to be useful in biochemical applications. For instance, the cellulose matrix could serve as an inexpensive solid phase, to which bioactive molecules like antibodies or other reactants may be bound specifically using a recombinant cellulose binding region of CBD (see e. g., the above Kilburn et al., Shoseyov et al., US 5,670,623).

It has now surprisingly been found, and that is an object of the invention, that cellulose binding domains may be used as cross-linking agents to impart enhanced properties, such as tensile and in particular wet strength upon paper. It has further been found, and that is another object of the invention, that paper products cross-linked using a CBD may be easily recycled. The recycling is based on re-pulping of the waste paper under very strong shear forces. To assist the separation of fiber-fiber or fiber-CBD bonds, heat and chemical means may be used such as caustic solutions or oxidizing agents. Further objects and advantages of the invention will be come clear as the description proceeds.

Summary of the Invention The invention is directed to a carbohydrate polymer containing product comprising a carbohydrate substrate cross-linked with a biological cross-linking agent (BCA). The carbohydrate substrate is a cellulose-based carbohydrate substrate. The carbohydrate substrate of the invention is characterized by improved wet strength. Preferably, the carbohydrate substrate is also characterized by improved dry tensile strength.

The BCA comprises one or more cellulose binding domains. The cellulose binding domain (CBD) is preferably derived from a bacterial source, more preferably from C. cellulovorans.

In a preferred embodiment of the invention, the CBD binds crystalline cellulose. More preferably, the CBD binds Cellulose I. Further preferably, the binding affinity of the CBD to the cellulose substrate is characterized by a kd of between about 0.8 to about 1.5 pLM. Also preferably, the CBD does not exhibit amorphogenic effects upon binding to the cellulose.

The BCA preferably comprises two CBDs. Alternatively, or in addition, the BCA comprises a chemical cross-linking moiety reactable with cellulose.

The chemical cross-linking moiety is preferably an aldehyde group, a glyoxal group, or a sulfone group.

In a preferred embodiment of the invention, the BCA comprises two CBDs coupled by a linker. The linker may be derived from a bifunctional cross-linking agent, such as disuccinimidyl tartrate (DSS), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), and the like. A partial list of commercially available cross-linkers may be found at p. 0-90 to 0-104 of the 1994 Life Sciences Product Catalog and Handbook of PIERCE, Rockford, IL 61105 USA.

In another embodiment of the invention, the linker connecting the two CBD regions is a peptide linker. Preferably, the peptide linker and the two CBD regions are recombinantly produced as a single chain protein.

The binding of the CBD to the cellulose is not disrupted by water.

Preferably, the binding of the CBD to cellulose is not disrupted by high salt concentration.

The CBD is preferably derived from a cbdA gene of a bacterium. More preferably, the CBD is derived from the cbdA gene of C. Cellulovorans. Most preferably, the CBD comprises 162 amino acids of the CBD as described in Shoseyov et al., US 5,837,814.

The cellulose substrate is preferably, but not limitatively, crystalline cellulose. More preferably, the cellulose is in the form of fibers. Most preferably, the cellulose is a paper, paperboard, or the like material. The cellulose may also be a cotton, rayon, or similar type material.

The invention also relates to a process for the manufacture of an improved wet strength substrate cross-linked with a biological cross-linking agent (BCA).

The process of the invention comprises the steps of a) providing a cellulose substrate suspended in an aqueous medium; b) providing a BCA comprising a CBD and optionally, a reactive group; c) contacting said substrate with said BCA under conditions and for a time sufficient to allow binding of said CBD to said substrate, and optionally, under conditions and for a time sufficient for said reactive group to attach to said substrate; d) draining the water from the cellulose-CBD product; e) pressing and drying the drained product of step d).

A cellulose material such as paper or paperboard consists of a three-dimensional structure of cellulose fibers. Smaller amounts of other chemicals and pigments are linked to and between the fibers. The production of a paper sheet is preferably carried out in the following stages, wherein the possibility of adding the CBD containing agent is pointed out and explained at each stage: I) Fiber source. The fibers may be derived from several sources as follows: a) Virgin fibers in water suspension are pumped directly from the pulp mill after manufacturing from wood by chemical or mechanical or combined process; b) The fibers may be used in dried form (for shipment). These fiber bales are suspended in water by immersion and applying high shearing forces; c) Fibers from a recycling process may be used. Different waste paper sources are used to produce different paper grades: Fibers that were prepared from recycled old corrugated boxes will be used to produce corrugating medium or other types of wrapping paper. Fibers derived from recycled labels will be used in production of tissue paper or printing paper. The waste paper is immersed in water and separated to individual fibers by high shear forces created by special device. To improve the process, the water is heated and/or chemicals are added, such as caustic or oxidizing materials. The suspended fibers are then screened in a few screening stages to remove contaminants. A BCA containing a CBD may be added here as this provides maximum reaction time.

II) Fiber preparation The Fibers from the different sources are"refined"prior to the paper sheet formation. In this process, the surface area of the fiber is increased, enabling more chemical bonds between fibers and between fibers and papermaking additives or CBD. The CBD-containing agent is preferably added in this step. This has the advantage that the surface of the fiber is more developed and the refining is done under high intensity conditions that may destroy CBD-fiber bonds that were developed prior to this stage.

III Sheet formation The fibers are diluted with water to a low consistency (0.2%-1.5% preferably 0.5%-1%) in one or more stages. While diluting, chemical additives are added to improve paper quality or to improve paper machine runability. Immediately after this step, the fiber suspension is distributed on a fine wire moving continuously. Most of the water is drained out under the wire to produce a wet sheet of paper. CBD-containing agents may be added in the dilution stage. In this step, there is excellent mixing and good control of the amounts added.

IV Pressing, drying and surface coating The water is further removed from the sheet formed in step III. In the middle of this drying process, the sheet surface may be coated (size-press).

Usually, starch is used, but other materials may also be utilized, such as latex, CMC, PVA, mixtures thereof optionally with added pigments. It is also possible to coat the paper on an off-machine coating device. CBD may be added in this stage. When CBD is added at this stage, there are no losses of CBD in the water circulation system. Some types of BCA will also create a starch-CBD fiber bond. This stage of addition is best for applications that require mainly improved surface strength.

In a preferred embodiment of the process of the invention, the substrate is cellulose, more preferably cellulose derived from plants, most preferably wood pulp. In another embodiment of the invention, the substrate is selected from paper, paperboard, and the like materials.

In a preferred embodiment thereof, the invention is directed to the preparation of a water-resistant label, according to the method of the invention.

In another preferred embodiment thereof, the invention is directed to the preparation of a water-resistant labels, towels, food wrapping and packages, packages for products to be stored under refrigerating conditions, technical materials such as filter paper, table cloths, maps, optionally corrugated paperboards, and the like, the said products being prepared in accordance with the process of the invention and having been imparted thereby with improved wet strength, optionally with improved dry strength.

Brief description of the Drawings Fig. 1A shows a scheme of CBD-crosslinked cellulose fibers, wherein the linear strands represent cellulose fibers, to which the CBD (square forms) are attached. Two CBD molecules are bound to each other via a linker (wavy line); Fig. 1B schematically shows the attachment of a functional group or molecule (a) by means of CBD (b) to a cellulose fiber (c); Fig. 2 shows a scheme of the expression vector used for CBD, wherein Ori denotes the bacterial origin of replication, bla denotes the antibiotic resistance marker, the empty box 3'to the cbdA gene denotes an optional terminator sequence, and T7 denotes the T7 polymerase promoter, such plasmids comprising the T7 promoter and their use are described in Studier, J. Mol. Biol. 219: 37-44,1991; and Fig. 3 shows a scheme of the expression vector for a bifunctional CBD, the horizontally lined segment between the two cbdA genes denotes a linker sequence, and the box 3'to the second cbdA gene denotes an optional terminator sequence.

The above figures are self explanatory, and will therefore not be further discussed below. However, reference should be made to them when reviewing the description to follow.

Detailed Description of the invention The following terms are meant to be understood herein as defined below: -carbohydrate polymer, a polymer of natural or synthetic origin containing cellulose. Examples of materials that contain carbohydrate polymers according to the invention include wood-derived materials such as wood pulp, plant-derived materials such as paper, paperboard, plant-derived pulp, recycled paper products, and the like; -carbohydrate substrate, a structured cellulose fiber material, such as microcrystalline cellulose, microfiber-containing cellulose, pulp, and the like; -biological cross-linking agent (BCA), an agent being at least bifunctional and comprising in at least one function a cellulose binding domain for cross-linking. The BCA may be a cross-linking agent, being able to link the CBD it comprises to another substance, such as a cellulose fiber or a substance that it is desired to add to the cellulose product. Alternatively, the BCA may comprise the substance that it is desired to add to the cellulose product, and may bind said substance to the cellulose fibers of said cellulose product via its CBD; mechanical properties, properties of a cellulose-derived material, such as paper and paperboard, resulting mainly in resistance to various mechanical stresses. These properties may be improved by the method of the invention and include various properties such as tensile strength, wet tensile strength, bursting strength, tear strength, ring crush strength, short span compressive strength, and wet rub; tensile strength, the maximum tensile force per unit width developed in a test specimen at rupture or break; wet tensile strength or wet strength, tensile strength measured in a material such as paper that was aged in high humidity; Bursting strength, the maximum hydrostatic pressure, applied through a rubber diaphragm, required to produce rupture of a material such as paper that is held rigidly against the diaphragm; Tear strength, the resistance of a sheet-like material. Such as a paper sheet, to tearing; Ring crush strength, compressive strength that causes a ring of a material such as paper held in a special jig to collapse; Short span compressive strength, the maximum force causing failure to a specimen such as a paper specimen that is compressed between two close clamps; -Wet rub, the ability of a material such as paper to withstand the lifting of fibers from the surface when rubbed by or on another surface.

Usually, the sample, e. g., then paper sample, is soaked in water before the test; -cellulose binding domain, a protein domain capable of binding cellulose with high affinity and of retaining bound cellulose under wet conditions, including retaining bound cellulose in high salt solutions.

The invention is directed to a carbohydrate polymer containing product comprising a carbohydrate substrate cross-linked with a biological cross-linking agent (BCA). The carbohydrate substrate is a cellulose-based carbohydrate substrate. The carbohydrate substrate of the invention is characterized by improved properties, such as improved wet strength.

Preferably, the carbohydrate substrate is also characterized by improved dry tensile strength.

The BCA comprises one or more cellulose binding domains from any suitable source. The cellulose binding domain (CBD) can be, for instance, derived from a bacterial source, such as C. cellulovorans.

In a preferred embodiment of the invention, the CBD binds crystalline cellulose. More preferably, the CBD binds Cellulose I. Further preferably, the binding affinity of the CBD to the cellulose substrate is characterized by a kd of between about 0.8 to about 1.5 pM. Also preferably, the CBD does not exhibit amorphogenic effects upon binding to the cellulose.

The BCA preferably comprises two CBDs. Alternatively, or in addition, In many products of the paper industry, it is desirable to impart wet strength upon the paper product. However the prior art methods of imparting wet strength are accompanied by undesirable side effects: for instance, covering of paper products with wax is environmentally undesirable. Moreover, any fault in the wax layer will result in weakening of the underlying cellulose paper under humid conditions.

The prior art methods of chemically cross-linking cellulose are equally undesirable, because the monomers used are not environment-friendly.

All of the prior art methods of imparting wet strength upon a cellulose product suffer from the fact that upon recycling, the cellulose fibers cannot easily be dissociated and suspended in water. When a polymer-forming cross-linking agent is used, this is due to the fact that the cross-linking cannot be reversed.

The present invention overcomes all of these problems associated with prior art water-resistant paper. In addition, the material used in the present invention for cross-linking the cellulose strand is derivable from bacteria, thus inexpensive and available in large quantities.

Moreover, the cross-linking material of the present invention is a biological molecule. Therefore, the cross-linked cellulose product and its manufacturing process are environment-friendly and the final product is biodegradable.

The essence of the invention lies in the discovery that CBD, a protein domain characterized by its ability to tightly bind to cellulose, may be used as a cross-linker for cellulose fibers, thereby imparting improved wet strength and other desirable characteristics upon the cellulose.

Preferably, the biological cross-linking agent (BCA) comprises two CBD regions. This may be achieved, in a preferred embodiment of the invention, by cross-linking CBD protein. Cross-linking proteins is well known in the art, see e. g., Chemistry of Protein Conjugation and Cross-linking, Shan S.

Wong, CRC Press, 1991. Proteins may be cross-linked by their functional groups. Usually, the SH or NH2 groups of proteins are used for that purpose. Chemical groups that react with SH groups include e. g., dithio groups, including pyridyldithio groups, haloacetamido groups, including iodoacetamido groups, maleimido groups, including alkylmaleimido groups, and the like groups known to the skilled person. Amino groups may be coupled using optionally sulfonated N-hydroxysuccinimide ester groups, imidoester groups, including methyl pimelimidate and methyl suberimidate groups, or carbodiimide groups. Also free carboxyl groups of a protein may be used for cross-linking, e. g. using an amino group such as an alkylamino group, and providing a dehydrating agent in the reaction.

The preferred CBD is the CBD derived from C. cellulovorans, disclosed in the above US 5,837,814, which is included herein in its entirety by reference. Most preferred is the 162 amino acid CBD peptide disclosed in said US patent under SEQ ID No. 2. This sequence comprises 10 Lysine residues and one Arginine residue. The free amino groups of these residues may be advantageously used in cross-linking the CBD molecule.

The CBD may be cross-linked to a second CBD. In that case, the cross-linker should be bifunctional. The cross-linker may be homobifunctional or heterobifunctional. In addition, the cross-linker may be cleavable or non-cleavable. The use of a cleavable cross-linker may be advantageous in the preparation of a cellulose material that is easily recyclable. Examples for homobifunctional cross-linkers include disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG) and dimethyl suberimidate (DMS). Examples for heterobifunctional cross-linkers include m-maleimideobenzoyl-N-hydroxysuccinimide ester (MBS) and N-gamma-maleimidobutyryloxy-succinimide ester (GMBS).

Examples for cleavable cross-linkers include disuccinimidyl tartrate (DST), dimethyl 3,3'-dithiobispropionimidate (DTBP), ethyleneglycolbis-[succinimidylsuccinate] (EGS), and 3,3' dithiobis [sulfosuccinimidylpropionate]. IN yet another embodiment of the invention, a cross-linker is capable of reacting unspecifically with proteins, for instance by photoactivation. Examples for photoreactive groups are e. g., the azidobenoyl, azido-nitrobenzoyl, azido-hydroxybenzoyl or azido-coumarin groups. Examples of photoreactive cross-linkers include p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP) and azidobenzoyl hydrazide.

The CBD domain may also be cross-linked directly to cellulose. In this embodiment of the invention, a carbohydrate-reactive cross-linker is used.

Carbohydrate reactive groups include e. g., the aldehyde group, the glyoxal group, or the sulfone group. Cross-linkers reactive with carbohydrates include e. g., the above azidobenzoyl hydrazide, 4- [m-maleimidomethyl]-cyclohexane-1-carboxylhydrazide (M2C2H), or 4- (4-N-maleimidophenyl)-butyric acid hydrazide (MPBH). Advantageously, as the preferred CBD domain of the invention does not comprise cystine residues, a photoreactive cross-linker or an amino-reactive cross-linker is used, such as the activated N-hydroxy succinimide derivative of the above M2C2H or MPBH, e. g., 4- (4- (succinimido-N-oxo)-phenyl)-butyric acid hydrazide. Alternatively, when it is desired to use the above carbohydrate and sulfhydryl-reactive cross-linkers, a second cross-linker may be used, which may be linked to the sulfhydryl-reactive moiety of the first cross-linker. The protein may then be coupled via the second functionality of the second cross-linker, which advantageously is a group reactive with amino groups, such as an activated N-hydroxy succinimide ester group.

The above note cross-linkers are commercially available, e. g., from the above-noted PIERCE, as listed at p. O-90 to 0-104 of the 1994 Life Sciences Product Catalog and Handbook of PIERCE, Rockford, IL 61105 USA, or from other suppliers in the field of organic chemistry, such as e. g., Sigma, St. Louis, USA.

The cross-linker should advantageously comprise a spacer. The spacer is preferably at least 5 Angstrom in length, more preferably at least 10 Angstrom.

In addition, a number of cross-linkers for specific purposes have been described in the prior art. US 5,002,883 discloses conjugation of proteins, to solid supports via NH2 groups. A set of cross-linkers is described for that purpose. The cross-linkers of US 5,002,883 may be used to cross-link the CBD domain of the present invention to a second CBD domain.

US 5,399,501 describes the conjugation of immunologically active proteins, e. g. antibodies, to a solid phase via a rather elaborate set of three distinct molecules: first, a cross-linker which binds to amino, carboxyl or thiol groups on the surface of the solid phase and provides a group capable of reacting with thiols (e. g. maleimide); second, a cross-linker that binds to NH2 groups of the protein to be conjugated and also provides a group capable of reacting with thiols (e. g. maleimide), and third, a dithiol reagent capable of joining the solid-phase bound thiol-reactive group with the protein-bound thiol-reactive group. This set of cross-linkers may also be used in the present invention, for the purpose of cross-linking CBD domains, as the CBD protein comprises amino groups.

In another embodiment of the invention, the CBD protein is cross-linked directly to cellulose. This may be accomplished e. g., by using a bifunctional cross-linker capable of reacting with carbohydrates on the one hand, and with proteins on the other. For instance, the cross-linker azidobenzoyl hydrazide (ABH) may be coupled to CBD, using the photoactivatable azide group of the cross-linker to nonspecifically bind to the CBD. In a second step, this"activated"CBD may then be reacted with cellulose fibers, whereby the carbohydrate-reactive hydrazide group of the cross-linker binds to glucose units in the cellulose fiber. The non-covalent CBD-cellulose bond may then be allowed to form, depending upon the reaction conditions, such as concentration of salt, presence of caustic solutions, reaction time, and the like, to form the desired product containing cellulose-CBD-ABH-cellulose cross-links.

The cross-linking of CBD proteins may be carried out using a peptide linker.

The peptide linker is a peptide of suitable amino acid sequence which is expected not to interfere with the secondary and tertiary structure of the CBD domains. The linker peptide may be connected to the CBD protein by a cross-linker, as described above for linking proteins. The necessary functional groups for cross-linking may be provided in the linker peptide by the choice of amino acids. For instance, Lysine or Arginine is chosen when it is desired to use amino groups for cross-linking. Cysteine residues are chosen when it is desired to use sulfhydryl groups for cross-linking.'Glutamic acid or Aspartic acid may be chosen when it is desired to use carboxylic acid groups for cross-linking. Groups that are not desired to be reacted may be protected by a suitable protection group as known in the art for amino, carboxyl, or sulfhydryl groups.

The linker is preferably between 10 and 50 amino acids in length. Further prepferably, the linker comprises small, uncharged amino acids, such as Glycine, Alanine, Valine, Serine, or Threonine. The linker contains preferably Glycine and Serine residues. The ratio between the Glycine and Serine residues is preferably about 3: 1 to 4: 1. An example for a 17 amino acid Glycine/Serine linker is GGGGSGGGGSGGGGSGG. Alternatively, two CBD peptides may be linked via a peptide, by using recombinant DNA technology. The coding sequence for CBD is fused with the coding sequence for the desired linker peptide, followed by a second copy of the coding sequence of the CBD. The entire cassette may be expressed using recombinant DNA technology, e. g., as described for the single CBD domain in the above US 5,837,814. Advantageously, bacteria are used for producing the CBD protein. These bacteria are advantageously devoid of proteases that may lead to decreased yield, such as the BL21 strain of E. coli.

Alternaitvely, the CBD protein may be expressed as a fusion protein with a protein-protein binding domain. Such domains comprise e. g., alpha-helica structures. A large number of protein-protein interaction domains have been described in recently, due to the technical advances represented by the yeast two-hybrid screening emthod, which allow the isolation of protein-protein interacting domains. Many of the domains found are located in transcription factor proteins, such as the jun and fos domains, the leucine zipper domain, and others known to the person of skill in the art.

Another option of cross-linking CBD domains is represented by the avidin-biotin system, as described in a large number of publications by Wilchek and Bayer. The avidin molecule is a peptide originally isolated from chicken egg protein. This peptide may be expressed as a fusion protein with the CBD domain. The fusion protein, or alternatively, unfused CBD protein, may then be cross-linked to biotin, which is a small vitamin molecule. Methods for cross-linking biotin to proteins are well known to the person of skill in the art, and have been widely published in many articles and text books.

The present invention is directed to a substrate cross-linked with a biological cross-linking agent (BCA). The substrate is a cellulose substrate.

The cellulose substrate is preferably crystalline cellulose, more preferably paper, paperboard, or a similar material. The substrate is characterized by improved wet strength.

Wet strength may be measured under various conditions, which are modeled according to the purpose for which the material is to be used.

In general, the tensile modulus of the material is determined according to methods known in the art. In order to obtain a reference value the material is placed under conditions of increased humidity, under standard conditions (50% relative humidity at 23°C), and the tensile modulus is measured. Finally, the material is soaked in water and the measurement is repeated. The above measurements allow testing of the material for suitability in various applications. For instance, bottle labels made from paper come into contact when they are applied on the bottles, as the glue used in that process contains water, and are exposed to condensated water on the bottle surface.

When paper materials are used as paperboard containers, e. g., for shipping food products, the humidity in the environment, particularly when the goods are shipped by sea or stored under refrigerating conditions, may exceed 85% relative humidity. Packaging materials made from paperboard may collapse under these conditions.

Further, when paper-based materials are used in shipping foodstuff that requires cooling, the packaging material is subjected to conditions of increased humidity and lowered temperature. The temperature conditions may change several times, depending upon the temporary storage of the packages. Therefore, it is desired to measure the tensile modulus of the materials of the invention under increased humidity and various temperatures, including cold conditions, such as 5-10 degrees Celsius.

The BCA comprises a cellulose binding domain. The cellulose binding domain (CBD) is preferably derived from a bacterial source, more preferably from C. cellulovorans. Most preferably, the CBD is the 162 amino acid fragment starting with the amino acid sequence AATSSMSVQFYNSNKS.... as listed in SEQ ID No. 2 of US 5,837,814.

In a preferred embodiment of the invention, the CBD binds crystalline cellulose. More preferably, the CBD binds Cellulose I. Further preferably, the binding affinity of the CBD to the cellulose substrate is characterized by a kd of between about 0.8 to about 1.5. Also preferably, the CBD does not exhibit amorphogenic effects upon binding to the cellulose.

CBD is preferably produced by molecular biology methods. Such methods, e. g., site-directed mutagenesis, PCR cloning, phage'library screening using oligonucleotide or cDNA probes, expression of cDNAs, analysis of the recombinant proteins, transformation of bacterial and yeast cells, and the like are well known to the skilled person and are described in many articles and textbooks, see e. g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory; ISBN: 0879693096,1989, Current Protocols in Molecular Biology, by F. M. Ausubel, ISBN: 047150338X, 1988, and Short Protocols in Molecular Biology, by F. M. Ausubel et al. (eds.) 3rd ed. John Wiley & Sons ISBN: 0471137812,1995. These publications are incorporated herein in their entirety by reference.

Briefly, the CBD gene is cloned into an expression vector, such as the pET vector. The promoter used is preferably a bacterial phage promoter, such as the T3 or T7 promoter. These promoters requires the presence in the bacterial cell of their cognate polymerase, i. e., the T3 or the T7 polymerase. As these polymerases, especially the T3 polymerase, are highly processive and selective, they will transcribe only genes that feature their cognate promoter. As the said phage promoters do not naturally occur in bacteria, this means that the phage promoter used for the expression of the CBD protein or CBD-linker-CBD faces no competition with bacterial promoters. Therefore, a very high amount of mRNA can be produced, using these conditions. Examples of CBD production in bacterial are detailed in the above US'5, 837,814.

CBD is purified from bacterial extracts by methods as known in the art.

Preferably, the protein readily is solubilized by addition of 6M guanidinium hydrochloride. The dissolved protein is then reatured by slow removal of the guanidinium hydrochloride by dialysis. Alternatively, the CBD protein may be used directly as crude bacterial extract, which may be advantageous as the purification steps may be saved. However, when CBD is used as crude bacterial extract, some of the protein will be denatured and unable to bind cellulose, as it is found, upon high-level expression, in inclusion bodies.

Therefore, an advantageous method of purification of CBD consists in preparation of a crude bacterial extract, solubilization of the protein, preferably in concentrated guanidinium hydrochloride, removal of the solubilization agent, at least to a point that allows the CBD to regain its ability to bind cellulose, and affinity-purification of the solubilized CBD on a cellulose matrix. The preparation of bacterial extracts, solubilization, renaturation, and affinity-purification of CBD is described in US 5,837,814.

The binding of CBD to cellulose is measured preferably by methods known in the art. Such methods are known to the skilled person. In particular, the above US 5,837,814 describes methods for'the measurement of CBD-cellulose binding constant and cellulose binding capacity of CBD. For instance, purified CBD is produced as described in the above US 5,837,814. The protein (about 1-50 ug) is added to a solution containing cellulose (1 mg) in phosphate buffer at neutral pH, e. g., PBS or 50 mM phosphate/12 mM citrate, pH 7. The phosphate buffer is supplemented with 1 mg/ml of a carrier protein, such as BSA. The assay tubes are rotated slowly (30 rpm) over a period of one hour at 37 degrees Celsius.

After termination of the incubation period, the cellulose is centrifuged, the pellet washed twice with phosphate buffer, and finally resuspended in the original volume (1 ml) of phosphate buffer. The amount of bound protein in the pellet may then be determined by a protein quantification method as known in the art. A residual amount of BSA will be detected in the pellet.

This amount of BSA is accounted for and subtracted by running a parallel assay with cellulose, but without CBD.

The binding of the CBD to the cellulose is not disrupted by water.

Preferably, the binding of the CBD to cellulose is not disrupted by high salt concentration.

The CBD is preferably derived from a cbdA gene of a bacterium. More preferably, the CBD is derived from the cbdA gene of C. Cellulovorans.

Most preferably, the CBD comprises 162 amino acids of the CBD as described in Shoseyov et al., US 5,837,814.

The cellulose substrate is preferably crystalline cellulose. More preferably, the cellulose is in the form of fibers. Most preferably, the cellulose is a paper, paperboard, or the like material. The cellulose may also be a cotton, rayon, or similar type material.

The invention also relates to a process for the manufacture of an improved wet strength substrate cross-linked with a biological cross-linking agent (BCA). The process of the invention comprises the steps of contacting a cellulose substrate with a BCA comprising a CBD under conditions and for a time sufficient to allow binding of said CBD to said substrate.

Thus, in one embodiment of the invention, a cellulose substrate, such as paper, or paperboard, is incubated with solution containing cross-linked CBD. Alternatively, the cellulose substrate is incubated with CBD activated by a cross-linker, and then a cross-linking reaction wherein the activated CBD is coupled to the cellulose, is carried out.

The incubation buffer is preferably a low-salt buffer adjusted to neutral pH, such as e. g., 50 mM/12 mM Citrate buffer at pH 7.0. The reaction is preferably carried out in the presence of carrier protein. Said carrier protein may be derived from the bacterial extract, if crude bacterial extract is used, or if partially purified CBD is used. Alternatively, the carrier protein may be added to the incubation solution. Preferred concentrations for the carrier protein range from about 0.2 to about 2.5 mg/ml, more preferably about lmg/ml. The carrier protein is preferably BSA.

The cellulose substrate may comprise long fibers of cellulose, or short fibers, such as microfibrils, obtained by beating the plant source material to a pulp, e. g., as described in EP 212,289, which is incorporated herein in its entirety by reference. Microfibrils may also be added to a cellulose substrate, such as a paper. The addition of microfibrils to a cellulose substrate, in the presence or prior to the addition of cross-linked CBD thereto, will serve as an additional cross-linking agent, forming long-range bonds between the cellulose fibers which consist of a molecule of cross-linked CBD bound to a strand of microfibril, which is by itself bound by another cross-linked CBD molecule.

In a preferred embodiment of the process of the invention, the substrate is cellulose, more preferably cellulose derived from plants, most preferably wood pulp. In another embodiment of the invention, the substrate is selected from paper, paperboard, and the like materials.

In a preferred embodiment thereof, the invention is directed to the preparation of a water-resistant label, according to the method of the invention.

In another preferred embodiment thereof, the invention is directed to the preparation of a water-resistant labels, towels, food wrapping and packages, packages for chilled products, technical materials such as filter paper, table cloths, maps, optionally corrugated paperboards, and the like, the said products being prepared in accordance with the process of the invention and having been imparted thereby with improved wet strength, optionally with improved dry strength.

The invention thus provides a cellulose substrate wherein cellulose strands including fibrils are cross-linked by CBD domains. The improved tensile strength of said substrate, particularly the improved tensile strength under conditions of high humidity, suggests the use of the cellulose substrate of the invention in a number of applications where such improved tensile strength is desired.

Thus, the invention provides a towel comprising cellulose substrate cross-linked with CBD according to the invention.' The invention further provides a paper, particularly a paper label for a bottle, comprising cellulose substrate cross-linked with CBD according to the invention.

The invention also provides a paper, paperboard, or corrugated paperboard material, comprising cellulose substrate cross-linked with CBD according to the invention. The corrugated paperboard preferably consists of outer layers comprising cellulose substrate cross-linked with CBD according to the invention, while the inner (corrugated) layer may be cross-linked by a different BCA which imparts higher humidity stiffness, or may be a conventional, non-crosslinked cellulose substrate. The said paper, paperboard, or corrugated paperboard is used preferably in the manufacture of packages. In a preferred embodiment of the invention, the packages are for transport of goods by sea. In another preferred embodiment of the invention, the packages are for use in transporting goods in cooled transportation equipment, such as cooling trucks. In yet another embodiment of the invention, the packages are for use as storage packages in cooling environments, such as refrigerators, and freezers. The freezers preferably provide a temperature of at least from-20 to-40 degrees Celsius, or lower.

Thus, in a more preferred embodiment of the invention, the packages are for use in the transportation of food, particularly agricultural products, such as fruits, vegetables, cheese, and meat. In a preferred embodiment of the invention, the packages are for the transportation and storage of foodstuff that requires chilling. In another preferred embodiment of the invention, the packages are for use in the transportation and storage of products that require chilling, such as medicaments, biochemical reagents, kits, and the like.

In yet another embodiment, the invention provides cellulose substrate cross-linked with CBD as substrate for the manufacture or technical materials, such as filter papers.

In a still further embodiment, the invention provides cellulose substrate cross-linked with CBD for the manufacture of table cloths and maps.

Example 1 In a first step, CBD protein is produced and purified, preferably as described in US 5,837,814. A heterobifunctional crosslinker containing a spacer is then attached to the purified CBD, via the succinimide group. As crosslinker, succinimidyl-4- (p-maleimido-phenyl) butyrate (SMPB) or sulfo-SMPB is used. Cross-linkers containing other spacers may also be suitable. An advantages of SMPB are its long spacer arm (14.5 Angstrom) and the presence of different functional groups in the crosslinker, which allow selective (directed) activation.

CBD protein in PBS (phosphate-buffered saline) or 50 mM phosphate, 12 mM citrate buffer at pH 7.0 is purified free of traces of detergents, denaturating agents, or conservating agents, by affinity chromatography on cellulose, and/or by ultrafiltration or dialysis.

The CBD solution is then adjusted to its original volume, and disuccinimidyl suberate (DSS) in DMSO is added (final DSS concentration 1 mM). For example, 5 pl of a 0. 1M stock solution of DSS in DMSO is added to 0.5 ml CBD solution. Advantageously, a freshly prepared solution of the cross-linker in double-distilled water or in PBS may be used. The mixture is incubated for 2 hrs at RT and tilted gently throughout the incubation period. In this step, DSS is covalently attached to the amino groups of the CBD protein, thereby cross-linking CBD proteins to each other.

Excess cross-linker is then removed by washing with phosphate buffer as above, or PBS, using ultrafiltration or dialysis. Optionally, the buffer conditions are adjusted to pH 7.0, and the cellulose substrate is added. Te reaction is incubated with gentle tilting either overnight at 4°C or for 1-2h at 37 degrees Celsius. The cellulose is then washed free of excess CBD and buffer constituents.

In case the cross-linker used contained maleimide or other labile groups, these groups are then quenched. Maleimide groups may be quenched by incubation with L-cysteine or D-cysteine, while residual free amino reactive groups may be quenched by incubation with amino acids such as glycine or with Tris. The quenching reagent is added at a concentration of 50 mM and incubated for 30 min. at room temperature. Quenching is followed by a washing step as above.

The cellulose substrate is then washed, optionally sonicated or treated mechanically to break aggregates that may have formed during the conjugation process, and stored, either in wet form or after drying.

Example 2 The coding sequence of the CBD protein, SEQ. No. 1 as described in US 5,837,814, is cloned into an expression vector, preferably the pET vector.

At the 3' (corresponding to the N-terminal) end of the CBD protein coding sequence, a sequence coding for an amino acid linker is then inserted. A preferred amino acid linker is the sequence GGGGSGGGGSGGGGSGG.

At the 3'end of the linker sequence, the CBD coding sequence is inserted a second time. Alternatively, a CBD coding sequence derived from a different cellulose binding protein may be used. The CBD-linker-CBD fusion protein is then expressed in a bacterial host, preferably a high-yield protease-deficient bacterial host, such as the E. coli strain BL-21.

The production and purification of CBD fusion protein is carried out essentially as described in US 5,837,814 for production and purification of the CBD domain. Briefly, the CBD fusion protein is then introduced into the bacterial host, and bacteria containing the pET-CBD fusion protein plasmid are identified by selection of colonies under selective pressure, preferably in the presence of ampicillin. A number of suitable colonies are then selected and expanded by growth in a small scale (e. g., 1.5 ml) Luria-Bertani or other suitable medium, preferably NZCYM medium, at 37 degrees Celsius with shaking. When a bacterial cell density of about 0.2 to about 0.6 (optical density at 600 nm) is reached, the expression of the CBD fusion protein is induced by the addition of IPTG to imM final concentration. After further incubating for about 4 hours, bacterial cells are centrifuged, resuspended in 50 mM phosphate, 12 mM citrate, pH 7 buffer, containing RNAse (10 microgram/ml) and DNAse I (1 microgram/ml). The cells are then lysed by repeated sonication on ice. The insoluble fraction containing the inclusion bodies is then centrifuged (30 min, 12000 rpm, 4 degrees Celsius). The pellet is resuspended in 6M guanidinium hydrochloride to bring the CBD fusion protein into solution.

After incubation for 30 min on ice, insoluble particles are removed by centrifugation as above. The guanidinium solution is then gradually diluted, over a period of two hours, 20-fold with 10 mM Tris, pH 7.0,0.1 mM EDTA, 0.1 mM DTT, 5% glycerol. The CBD fusion protein is then precipitated by addition of ammonium sulfate to 80%. After incubating a further four hours at 4 degrees Celsius, the precipitated protein is collected by centrifugation as above, resuspended in phosphate/citrate buffer as above, and dialyzed against phosphate/citrate buffer to renature the CBD fusion protein and remove traces of guanidinium salt.

The purified CBD fusion protein is then tested for its ability to bind to cellulose, as described further above. Bacterial strains producing high amounts of highly active CBD fusion protein are selected for large-scale production of the CBD fusion protein.

The bacterial are grown in a rich medium, such as Luria-Bertani, 2YT, or TB medium. The constituents of these media are well known in the art of microbiology and are described, e. g., in the above Sambrook et al. The bacterial culture is allowed to grow to a density that is most suitable for producing the protein. This density will generally be between 0.2 and 1. 0, more preferably between 0.4 and 0.6 optical density at 600 nm of the bacterial culture. The optimal density depends on the culture conditions, the media used (bacteria grown in 2YT or TB may be grown to higher densities compared to the poorer LB medium), and on the bacterial strain used.

After reaching the optimum density for protein production, the inducing agent, in this example, IPTG, is added at a concentration of 1 mM.

Incubation is then continued for about four hours, and the bacteria are harvested by centrifugation.

The bacterial cell wall may now be broken by mechanical force, e. g., using a French Press, by sonication, or the like method. This yields a crude extract, which contains large amount of the desired CBD fusion protein (about 70 mg/L). The protein partly contained within the extract insoluble form. Therefore, the extract may be used directly for incubation with the cellulose substrate which it is desired to cross-link. Alternatively, the extract may be treated with 6 M guanidinium salt, in order to dissolve fusion protein trapped in inclusion bodies. This treated extract may then be diluted, in order to allow renaturation of the fusion protein. When renaturation has completed, the treated and diluted extract may be used in the cross-linking. Further alternatively, the fusion protein may be purified from the treated extract, by purification methods as known in the art. A preferred method is the above-described purification, which takes advantage of the cellulose-binding capability of the fusion protein.

Example 3 This Example describes the preparation of a paper sheet according to the invention. The cellulose fibers used for the manufacture of the paper mat be derived from various sources. Virgin fibers derived from wood pulp, suspended in water, are obtained directly from the pulp mill according to processes known in the art, including chemical or mechanical processes, or a combination thereof (see G. A. Smook,"Handbook for Pulp & Paper Technologists", Chapter 4, Overview of Pulping Methodology, TAPPI and CPPA, 1982). Dried fiber bales are suspended in water by immersion and subjected to high shearing forces. Fibers from a recycling process may also be used. Fibers that were prepared from recycled old corrugated boxes are useful in the production of corrugating medium or other types of wrapping paper. Fibers derived from recycled labels are used in production of tissue paper or printing paper. The waste paper is immersed in water and separated to individual fibers by high shear forces (see G. A. Smook, ibid, Chapter 14, Secondary Fiber Utilization). The water is optionally heated and/or chemicals, such as caustic or oxidizing materials, are added. The suspended fibers are then screened to remove contaminants (see G. A.

Smook, ibid, Chapter 9, Paragraph 9.4, Screening).

The Fibers from different sources are"refined"in order to increase the surface area of the fiber, enabling more chemical bonds between fibers and between fibers and papermaking additives or CBD (see G. A. Smook, ibid, Chapter 13, Preparation of Stock for Papermaking, Paragraph 13.2, Beating and Refining).

The fibers are then diluted with water to a low consistency (0.2%-1.5% preferably 0.5%-1%) in one or more stages. While diluting, chemical additives are added to improve paper quality or to improve paper machine runability. Immediately after this step, the fiber suspension is distributed on a fine wire moving continuously. Most of the water is drained out under the wire to produce a wet sheet of paper.

The water is then further removed from the sheet. While the drying process is ongoing, the sheet surface may be coated (size-press) (see G. A.

Smook, ibid, Chapter 18, Surface Treatments, Paragraph 18.1, Surface Sizing, and 18.2, Coating). Usually, starch is used, but other materials may also be utilized, such as latex, CMC, PVA, mixtures thereof optionally with added pigments. It is also possible to coat the paper on an off-machine coating device.

The above-described process of manufacturing a paper sheet has four stages: (a) the obtention of the fibers, (b) the refinement thereof, (c) the dilution of the fiber suspension and draining of water to produce a sheet, and (d) the drying of the sheet, optionally accompanied by addition of surface treatment agents.

In stage (a), a BCA containing a CBD may be added, as this provides maximum reaction time. More preferably is the addition of a BCA in stage (b). This has the advantage that the surface of the fiber is more developed.

Moreover, the refining is done under high intensity conditions that may destroy CBD-fiber bonds that were developed in stage (a). In stage (c), the addition of BCA is advantageous because of the excellent mixing and good control of the amounts added during this stage. When CBD is added at stage (d), there are no losses of CBD in the water circulation system. Some types of BCA will also create a starch-CBD fiber bond. Addition of BCA in stage (d) is best for applications that require mainly improved surface strength.