CA2229358A1 - Methods and compositions for sizing paper - Google Patents
Methods and compositions for sizing paper Download PDFInfo
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- CA2229358A1 CA2229358A1 CA 2229358 CA2229358A CA2229358A1 CA 2229358 A1 CA2229358 A1 CA 2229358A1 CA 2229358 CA2229358 CA 2229358 CA 2229358 A CA2229358 A CA 2229358A CA 2229358 A1 CA2229358 A1 CA 2229358A1
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- Prior art keywords
- paper
- protein
- cellulase
- sizing
- pulp
- Prior art date
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Classifications
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H21/00—Non-fibrous material added to the pulp, characterised by its function, form or properties; Paper-impregnating or coating material, characterised by its function, form or properties
- D21H21/14—Non-fibrous material added to the pulp, characterised by its function, form or properties; Paper-impregnating or coating material, characterised by its function, form or properties characterised by function or properties in or on the paper
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
- D21H17/20—Macromolecular organic compounds
- D21H17/21—Macromolecular organic compounds of natural origin; Derivatives thereof
- D21H17/22—Proteins
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H21/00—Non-fibrous material added to the pulp, characterised by its function, form or properties; Paper-impregnating or coating material, characterised by its function, form or properties
- D21H21/14—Non-fibrous material added to the pulp, characterised by its function, form or properties; Paper-impregnating or coating material, characterised by its function, form or properties characterised by function or properties in or on the paper
- D21H21/16—Sizing or water-repelling agents
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
- D21H17/005—Microorganisms or enzymes
Abstract
The present invention relates to methods and compositions for sizing paper. In particular, the invention relates to a method of sizing paper comprising the steps of a) contacting said paper or a constituent of said paper with a protein capable of binding to said paper or said constituent of paper; and b) denaturing or heating said protein bound to said paper.
Description
CA 022293~8 1998-02-11 W O 97/07Z82 PCT/~L~ 1012 :L
h~v8 AND CONPOSIT:~ON8 FOR 8IZING PAPER
1 Technical Field The present invention relates to methods and compositions for sizing paper. In particular, the present invention relates to the use of a protein capable of bi n~ in~ to paper or a constituent of paper to size paper.
h~v8 AND CONPOSIT:~ON8 FOR 8IZING PAPER
1 Technical Field The present invention relates to methods and compositions for sizing paper. In particular, the present invention relates to the use of a protein capable of bi n~ in~ to paper or a constituent of paper to size paper.
2 Rac~ ~u~a While there are a myriad of details for manufacturing paper, the paper manufacturing process conventionally comprises the following steps: (1) forming an aqueous suspension of cellulosic fibers, commonly known as pulp; (2) adding various processing and paper enhancing materials, such as strengthening and/or sizing materials; (3) sheeting and drying the fibers to form a desired cellulosic web; and (4) post-treating the web to provide various desired ZO characteristics to the resulting paper, including surface application of sizing materials, and the like.
Sizing materials are typically in the form of aqueous solutions, dispersions, emu]sions or suspensions which render the paper treated with ~he sizing agent, namely sized paper, resistant to penetration or wetting by an aqueous liquid, including other treatment additives, printing inks, and the like.
A sizing agent may be applied to the surface of paper as a "surface" size or may be incorporated within the paper as an "internal" size. Many chemical sizing agents are known including rosin-based and ketene dimer-based sizing compositions. There remains, however, a need for improved sizing compositions and methods of sizing.
Typically, the principal constituent of paper is cellulose.
Cellulose may be in the form of wood fibre or ~nntl~l crop CA 022293~8 1998-02-11 fibre (for example, hemp, straw, rice, flax, jute or cotton). Other constituents of paper may include other polymeric materials, including naturally occurring polymers such as starch, pectin, guar, chitin, lignin, agar, alginate as well as other polysaccharides including hemi-celluloses such as xylanose, mannose and arabinose. Xyl~no~e is the principal component of xylan, otherwise known as hemi-cellulose which occurs in grasses, cereal, straw, grain husks and wood. Starch occurs in seeds, fruits, leaves, bulbs etc.
Enzymes which are capable of modifying an enzyme substrate 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, kerati~ases, chiti~ c~ liqn; ~e~, agarases, alginA~?~, xy~ c~c~ ~nn~rQc, amylases, cell~ e~ and hemi-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; celllll~ and xyl~naces: Gilbert and Hazelwood, Journal of General Microbiology, 139, 187-194, 1993). This group of enzymes include celllll~ and hemi-celllllA~e~ which comprise functionally distinct proteindomains. In particular, the domain responsible for catalytic activity is structurally distinct from the cellulose binding domain. These dc ~i n~ are evolutionarily ~o~rved sequences which are very similar in all such proteins (Gilkes et al., Microbiological Reviewfi, 303-315, June 1991~.
The binding domains of such proteins can be separated from _ the active-site domains by proteolysis. The isolated b~ nA i n~ domains have been shown to retain b; n~ i n~
capabilities (Van Tilbeurgh, et al ., FEBS Letters, 204(2), 223-227, August 1986). use of cellulose bin~ing domains of cellulases has been proposed as a means of roughening the texture of the surface of cellulosic 5U~pOl L, while use of cellulase active-site domains has been proposed as a means of smoothing the texture of such surfaces (International patent application W093/05226).
A number of b;n~in~ 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, 13, 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 ~u~o~L materials used in protein purification strategies (Kilburn et al ., United States Patent 5,137,819; Greenwood et al., Biotechnology and Bioengineering, 44, 1295-1305, 1994). The ability to immcbilize fusion proteins onto cellulosic supports has also been suggested as a means of immobilization for enzyme bioreactors (ong et al., Bio/Technology, 7, 604-607, Jlune 1989; Le et al. Enzyme Microb. Technol., 6, 496-500, June 1994), and as a means of attaching a chemical "tag" to a cellulosic material (International Patent Applica1:ion W093/21331).
It has now been found that proteins capable of binding to paper or a constituent of paper may be used to size paper.
Sizing materials are typically in the form of aqueous solutions, dispersions, emu]sions or suspensions which render the paper treated with ~he sizing agent, namely sized paper, resistant to penetration or wetting by an aqueous liquid, including other treatment additives, printing inks, and the like.
A sizing agent may be applied to the surface of paper as a "surface" size or may be incorporated within the paper as an "internal" size. Many chemical sizing agents are known including rosin-based and ketene dimer-based sizing compositions. There remains, however, a need for improved sizing compositions and methods of sizing.
Typically, the principal constituent of paper is cellulose.
Cellulose may be in the form of wood fibre or ~nntl~l crop CA 022293~8 1998-02-11 fibre (for example, hemp, straw, rice, flax, jute or cotton). Other constituents of paper may include other polymeric materials, including naturally occurring polymers such as starch, pectin, guar, chitin, lignin, agar, alginate as well as other polysaccharides including hemi-celluloses such as xylanose, mannose and arabinose. Xyl~no~e is the principal component of xylan, otherwise known as hemi-cellulose which occurs in grasses, cereal, straw, grain husks and wood. Starch occurs in seeds, fruits, leaves, bulbs etc.
Enzymes which are capable of modifying an enzyme substrate 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, kerati~ases, chiti~ c~ liqn; ~e~, agarases, alginA~?~, xy~ c~c~ ~nn~rQc, amylases, cell~ e~ and hemi-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; celllll~ and xyl~naces: Gilbert and Hazelwood, Journal of General Microbiology, 139, 187-194, 1993). This group of enzymes include celllll~ and hemi-celllllA~e~ which comprise functionally distinct proteindomains. In particular, the domain responsible for catalytic activity is structurally distinct from the cellulose binding domain. These dc ~i n~ are evolutionarily ~o~rved sequences which are very similar in all such proteins (Gilkes et al., Microbiological Reviewfi, 303-315, June 1991~.
The binding domains of such proteins can be separated from _ the active-site domains by proteolysis. The isolated b~ nA i n~ domains have been shown to retain b; n~ i n~
capabilities (Van Tilbeurgh, et al ., FEBS Letters, 204(2), 223-227, August 1986). use of cellulose bin~ing domains of cellulases has been proposed as a means of roughening the texture of the surface of cellulosic 5U~pOl L, while use of cellulase active-site domains has been proposed as a means of smoothing the texture of such surfaces (International patent application W093/05226).
A number of b;n~in~ 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, 13, 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 ~u~o~L materials used in protein purification strategies (Kilburn et al ., United States Patent 5,137,819; Greenwood et al., Biotechnology and Bioengineering, 44, 1295-1305, 1994). The ability to immcbilize fusion proteins onto cellulosic supports has also been suggested as a means of immobilization for enzyme bioreactors (ong et al., Bio/Technology, 7, 604-607, Jlune 1989; Le et al. Enzyme Microb. Technol., 6, 496-500, June 1994), and as a means of attaching a chemical "tag" to a cellulosic material (International Patent Applica1:ion W093/21331).
It has now been found that proteins capable of binding to paper or a constituent of paper may be used to size paper.
3 8ummary of the Invention According to the present invention there is provided a CA 022293~8 1998-02-11 W O 97/07282 PCT/~ 012 method of sizing paper comprising the steps of a) contacting said paper or a constituent of said paper with a protein capable of binding to said paper or said constituent of paper, and b) denaturing or heating said protein bound to said paper.
According to a further aspect of the present invention there i8 provided a method of sizing paper comprising a) contacting said paper or a constituent of said paper with a protein capable of binding to said paper or said constituent of paper, and b) heating said paper.
According to a further aspect of the present invention there i8 provided use of a protein capable of binding paper or a constituent of paper for the purpose of sizing paper. The invention further provides paper sized according to a method of the present invention.
~ Detailed Description of the Invention The present invention provides a method of sizing paper. 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 sources optionally mixed with fibres from vegetable, mineral, animal or synthetic sources in various ~ Lions 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 sources of cellulose include wood, straws, Bagasse, Esparto, Bamboo, Kanaf, Grass, Jute, Ramie, Hemp, Cotton, Flax. The crude vegetable derived cellulose is proceC~ to form pulp, the material from which paper is made, either mechanically, chemically or both. Cellulose ContA; n~ ~g pulps may be described as mechanical, CA 022293~8 1998-02-11 WO 97t07282 PCT/GB96/02012 ~h~ hAn;cal and chemithermomechA~iCal~ 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.
Paper may also comprise other naturally occurring polymers such as proteins such as keratin, starch (including anionic, cationic or amphoteric starch), pectin, guar, chitin, lignin, agar, alginate as well as other poly~Acch~rides including hemi-celluloses such as xylanose, mannose and arabinose.
The method of the present invention comprises co~ acting paper or a constituent of paper with a protein capable of b~ n~ j n~ to the paper or const:ituent of paper followed by denaturing or heating the protein.
The protein employed in the present invention may comprise any protein capable of binding to the paper or constituent of paper. The protein may for example comprise a protein capable of binding cellulose or any other polymeric substance present as a constituent of the paper.
Preferably, the protein is capable of specific binding to cellulose or any other polymeric substance present as a constituent of paper. More preferably, the protein is capable of binding 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 (for example, when protein is hon~e~ to another molecule), reference to a protein means a protein residue. The protein may co~prise 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 CA 022293~8 1998-02-11 W O 97/07282 PCT/~Lr~~012 herein the term "modified protein" includes chemical analogs of proteins capable of binding to paper or a constituent thereof. Preferably/ the protein comprises a naturally occurring enzyme or fragment thereof which is capable of bin~ing to paper or a constituent of paper. Examples of proteins capable of binding paper or a constituent of paper are well known and include enzymes selected from the group comprisingcellulases, hemi-cellulases, mannases, xylanases, chitinases, ligninases, agarases, alginases and amylases.
The protein may for example comprise an amylase or fragment thereof capable of binding to starch (such as anionic, cationic or amphoteric starch) when present as a constituent of paper or paper pulp. Examples of amylases include ~-amylases, for example from Aspergillus oryzae (available asa Type X-A crude preparation from Sigma Aldrich Co Ltd), and amyloglucosidases, for example from Aspergillus niger (available from Sigma Aldrich Co Ltd).
Preferably, the protein comprises a protein capable of b~n~;nq to cellulose. More preferably, the protein comprises a cellulase or fragment thereof. The cellulase may comprise a naturally occurring cellulase, or fragment thereof, or modified cellulase obtainable by chemical modification of a naturally occurring cellulase or synthesis or by e~LessiGn of a genetically modified gene co~ for a cellulase. The cellulase may, for example, be modified to remove or deactivate the active-site domain. A variety of cellulases are known which bind to cellulose. Examples of such cell~ are those isolable from bacterial organisms such as Cellulomonas fimi and fungal organisms such as Trichoderma viride, Aspergillus niger, Fusarium oxysporum, Penicillium funiculosum, Trichoderma reesei and Humicola insolens, available as commercial preparations from Sig~a Chemical Sigma-Aldrich Company Ltd., Novo Nordisk A/S, BDH
Ltd., or ICN Biomedicals Ltd. (Fusarium oxysporum is available for example under deposit No. DSM 2672).
Alternatively, the protein may be produced by recombinant CA 022293~8 lsss-02-ll wos7/07zsz PCT/GB96/02012 DNA techn;~ues as disclosed in, for example, International Patent application WO94/24158. Cell~l A~ 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 obt~;ne~ from whole cellulase by treatment with protease(s), such as papain. The cellulase may comprise an exo-cellulase or an endo-cellulase. Exo-celll~l AS~ (also known as cellobiohydrolases, CBH; exoglucanaes; 1,4-beta-D-glucan cellobiohydrolases; EC 3.2.1.91) act on the non-reducing end of a cellulose molecule. Exo-cellulases may release terminal cellobiose units (a disaccharide) or release terminal glucose units (monosaccharide). Examples of exo-cellulases include cellulase obtainable from Numicol ai~olens. Endo-cellulases (also known as Beta-1,4-Endoglucanases; Endo-1,4-D-glucanases; 1,4-Beta-D-glucan gll~cAnohydrolases; EC 3.2.1.4) cleave internal beta-1,4-glycosidic bonds yielding a mixture of glucose, cellobiose and other soluble cello-oligosaccharides. Examples of endo celllll~ce~ include cellulase obtainable from Trichoderma reesei .
The protein may also comprise cellulosomes. Cellulosomes comprise a cellulase system comprising discrete, multifunctional, multienzyme complexes. They typically contain at least 14 distinct polypeptides including numerous endoglucA~e~ (endocellulases) and xylanases and at least one beta-glucanase. These are associated with scaffolding proteins. Cellulosomes are described in detail in Bayer E.A., Morag E., Lamed R. (1994), "The cellulosome - a trea~LeLLo~e for biotechnology", Trends in Biotechnology 12:379-386.
Preferably, the protein employed in the present invention comprises cellulase obtainable from ~umicol a isol ens (available as Celluzyme~ from Novo Nordisk A/S, Bagsvaerd, Denmark) or cellulase obtainable from Trichoderma reesei W O 97107282 PCT/~r~012 (available a Celluclast0 from Novo Nordisk A/S, Bagsvaerd, Denmark). More preferably, the protein comprises cellulase obtAinAhle from Humicola isolens .
The protein may be added to the paper at any suitable stage in the manufacture and processing of the paper. It may be added at the pulp stage or at any stage during the formation of the wet pulp matrix or during the pressing and rolling of the matrix to form paper. Thus, according to the present invention there is provided a method of manufacturing sized paper comprising the steps of a) preparing a paper pulp, b) adding a protein capable of binding to a constituent of said pulp, c) forming paper from said pulp, and d) heating said paper.
Alternatively, the protein may be added to the formed paper product, for example, by immersing the paper in a bath cont~;n;ng the protein or by any suitable spraying, spr~ gt brushing, coating or printing process. Thus, the invention furthcr provides a method of manufacturing sized paper comprising the steps of a) applying to paper a protein capable of binding said paper and b) heating said paper.
By choosing the point in the manufacture of the paper at which the protein is added, control may be exercised as to whether the protein is distributed throughout the paper or is substantially restricted to the surface levels of the paper.
The protein should be incubated with the paper or paper pulp for sufficient time to allow binding of the protein to the paper or paper pulp. Typically, 15 minutes has been found adequate, although shorter incubation times may be suitable.
The protein may be added in an amount suitable to achieve the desired level of sizing. The protein may be added in an amount of 0.01-40% by weight of the dry weight of the paper pulp. Preferably the protein is added in an amount of 0.1 _ CA 022293~8 1998-02-11 W 097107282 PCT/~Lr~ 012 to 20% by weight, more preferably 1 to 10% by weight.
Following incorporation of t:he protein in the paper or application of the protein to the surface of the paper, sizing of the paper is achieved by denaturing or heating the protein. The protein may be denatured by the application of a chemical protein denaturant to the paper. Chemical protein denaturants include urea, guanidine, acids, ~k~]i~, detergents (such as Tween~), water soluble organic subst~n~ (such as alphatic alcohols) and chaotropic ions (such as I-, Cl04-, SCN-, Li+, Mg2+, Ca2+ and Ba2+).
Preferably, sizing is achieved by heating the paper.
Preferably, the paper may be heated at a temperature of 50~C
to 200~~, more preferably 70~C to 170~C, more preferably 80~C to 110~C, more preferably 100~C to 110~C. Typically, the paper may be heated to approximately 105~C on steam heated rollers. The paper may be subjected to one or more heat treatments at different 1_emperatures.
The length of time of heating required depends upon the temperature at which the paper is heated, longer times being required at lower temperature. Typically, the paper may be heated for between 15 and 500 seconds, preferably between 25 and 300 seconds. The paper may also be subjected to post manufacture heat treatments to age or cure the paper.
The invention will now be described with reference to the following examples.
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.
W O 97/07282 PCT/~r5,'~012 EXP~ r~iL
Materials and Protocols Excep~ as indicated below, the following materials and protocols were used in the Examples to characterise the use of proteins as biosizing agents.
~le: Water-leaf paper pulp was prepared by A~ i ng 10 g of 10 water-leaf paper (70:30 Hardwood (birch) : softwood (pine)) to 100 ml distilled water. After 5 min the paper was blended to an homogenous pulp. Samples of pulp Cll~p~cion (e~e~ponding to 0.2 g dry paper) were weighed into Universal bottles.
TncubatiQn To each bottle 10.0 ml of one of the following 1~Cllh~tion buffer solutions was added:
(a) Tris-HCl, 50 mM, pH 7.5 (b) Phosphate buffered saline (PBS) 500 mM, pH 7.5 ~ ulases: To the incubation suspension was added a cellulase selected from the following:
1. ~umicola insolens - Cellulase derived from Humicola ~nsolens available as Celluzyme~ from Novo Nordisk A/S, Bagsvaerd, Denmark (875~1 corresponding to 8.7%
weight per weight cellulose binding protein to dry weight pulp).
2. Trichoderma reesei - Cellulase derived from Trichoderma reesei available as Celluclast~ from Novo Nordisk A/S, Bagsvaerd, Denmark (70~1 corresponding to 0.28~mols protein corresponding to 4.4% weight per weight cellulose binding protein to dry weight pulp).
CA 02229358 l998-02-ll W O 91~07282 PCTW L~J~012 3. Trichoderma viride - Cellulose derived from Trichoderma vivide available from BDH Ltd UK.
Control experiments in the absence of cellulase were also performed.
Tncubation: The mixtures were incubated for 15 min at room temperature with gentle agitation.
Test Sheet Preparation: To produce the paper test sheets, the volume was 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 (O.2~ wv-l) 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 sheet supported by the mesh. The filter mesh was removed from the apparatus and the paper sheet sandwiched between a second nylon mesh and blotted between blotting paper. The paper sheet was carefully removed from the paper-making mesh, flattened by rolling and then dried.
Test Sheet Drying/Heatinq: Paper sheets were dried in one of the following ways (a) air drying;
(b) drum drier - typically operating at a constant temperature of 80~C to 10~~C with a contact time of 40 to 250 ~;
(c) hot plate press - typically producing a maximum surface temperature of 160~C with a 30 s contact time.
A flat aluminum plate was used to press the paper test sheets (sandwiched between blotting paper) against the hot plate.
Test ~rotocols: The dried sheets were ~cs~-cced for sizing by one or more of the following t:ests:-(a) Ink drop test (IDT) - i~ which a 15 ~1 drop of Parker Super Quink Ink (Permanent Blue-Black) was dropped CA 022293~8 1998-02-11 W O 97/07282 PCT/G~9~ 2012 onto the test piece of paper and the time taken for complete absorption measured and recorded. Results were recorded in seconds or on an empirical scale of 0 to ~+++ in which:-0 means less than 100 s for complete absorption.
means 100-500 s for complete absorption.
++ means 500-1000 s for complete absorption.
+++ means 1000-2000 s for complete absorption.
++++ means greater than 2000 s for complete absorption.
(b) Hercules Size Test (HST) - defined in "Tappi Test Methods" published by TAPPI, Technology Park, Atlanta, P0 Box 105113, GA 30348, USA, ISBN 0-89852-200-5 (vol 1 and 2), (1987). The HST is defined as size test for paper by ink resistance T530pm-83. The data are recorded in seconds; the higher the value, the better the sizing. Pre~erably an HST value greater than 20 secon~, more preferably greater than 120 E~conA~, more preferably greater than 200 ~econ~c~ is obtained.
(c) Cobb Size Test - defined in "Tappi Test Methods"
(Ibid) by T441Om-84. Data are 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 a Cobb value less than 30g/m2, more preferably less than 21g/m2, is obtA;n~d.
Ex~mpl~ 1 An investigation into the effect of temperature on the sizing imparted by a Trichoderma reesei cellulase preparation (Celluclast~,_Novo Nordisk A/S, Bagsvaerd, Denmark;) was carried out. For these studies a drum drier was used in conjunction with a variable temperature hot plate. The drum drier gave a constant temperature of 80~C
with a contact time of 250 s and the hot plate gave a maximum surface temperature of 160~C with a 30 s contact time. A flat aluminium plate was used to press the paper sheets (sandwiched between blotting paper) against the hot W O 97/07282 . PCT/~,~'~1012 plate.
Trichoderma reesei cellulase preparation (70 ~1 Celluclast~, corresponding to 4.4 % ww~1 cellulose binding protein based on dry weight of cellulose fibre) was used and the mixLuLe ~cl~h~ted for 15 min at room temperature with gentle agitation. Control experiments without cellulase were also performed.
Paper test sheets were prepared as described above. Test sheets were initially pressed between blotting paper then dried in one of the three following ways.
(a) air drying;
(b) drum-drier: 80~C/250 s; OL-(c) hot plate press: 160~C/30 s followed by drum drier 80~C/250 8.
The dried sheets were then assessed for sizing by the Ink Drop Test (IDT).
The following sizing results were obtained from test papers prepared under different drying regimes.
T~ble1: Effect of pper dr~ e on the cellul~ ' ~ sizinll ~t Drying regime Additions to pulp Air dried80~C/250 s160~C/30 8 80~C/250 s Cel_u~last~+ Tris-HCl 0 +++ ++++
3~ Cel~u~,last~ + PBS - - ++
Tric-ICl - 0 PBS
H20 _ 0 - = Not tested.
No sizing was observed when papers were made without enzyme, ruling out the possibility that the buffer salts were interacting with the cellulose in such a way as to affect sizing. Sizing was observed with Tris-~. reesei cellulase WO 97/07282 PCr/~jL5r~ 012 (Celluclast~) inC~lhAtions and a greater degree of sizing was observed when papers were dried initially at the higher temperature followed by drum drying at the lower temperature. Cellulase/PBS paper also gave sizing at the higher temperature but the degree of sizing was lower than that obtained in the presence of Tris-HCl.
E~mpl~ 2 The effect of incubation time on the levels of sizing imparted using a Humicol a insol ens cellulase preparation (Celluzyme-, Novo Nordisk A/S, Bagsvaerd, Denmark) was investigated.
Tris-HCl (50 mM, pH 7.5, 10 ml) was added to 0.2g pulp in dist~lle~ water and the Humicol a insol ens cellulase preparation (875 ~1) was added and the mixtures incubated for either 15 min or 90 min at room temperature. At the end o~ the incubation period, the pulp samples were vortex mixed and diluted to 100 ml. Test papers were prepared in the ~t~n~rd -nne~ and dried by a single pass through a drum drier 100~C/250 s.
The degree of sizing achieved by the different methods was A~e-S~ using the IDT method.
T ble2:
Enzyme wt dry IDT Incubati test sizing on paper period (g) (min) Celluzyme' (875 0.207 +++ 15 3c ~1) Celluzyme' (875 0.157 +++ 90 ~Ll) Control 0.218 0 90 3S Increasing the incubation time from 15 min to 90 min did not significantly increase the level of sizing.
WO 97/07282 PCT/~9~,~2012 1!5 ~x~mple 3 To establish a quantitative relationship between sizing achieved in test papers and the quantity of Trich~rma reesei or ~umicola insolens cellulase preparations added, a further set of experiments were undertaken.
St~AA~dized paper making conditions were employed as follows: to a sample of pulp in distilled water (equivalent to 0.2 g dry paper) 10.0 ml buffer was added (50 mM Tris HCl, pH 7.5). Various amounts of cellulase (Trichoderma reesei or Humicol a insol ens ; Table 3) was added and the mixture vortex mixed. The pulp was ;ncllh~ted with gentle chAki~g at room temperature for 15 min, after which time the mixture was diluted to 150 ml with distilled water and the test paper sheets produced. Eac:h test sheet was removed from the mesh and pressed between a folded sheet of dry 3 MM
blotting paper using a hand-held roller. The sheet (still folded in the blotting paper) was passed once through a drum drier at 100-104~C with a 250 s contact time. The results are given in Table 3.
T ble 3: Sizir~ ~chieved usin~l either TriC ' ~ reesei ~Cellucl#t , ~lovo ~lordidcA~S, ~ _ J, Den or~) or Nu~icoln insolens (Celluey~e , Uovo Uordisk ~5, r _ d, Dononr~) cellul Le ~ . ~ io..
CelluzymeCelluzyme IDTCelluclastCellucla~t IDT
(/11) a~~Bindin~ protein~izing (lll) aQ ~Bindin9 ~L~c~ng receivedKlded ~brsed on rating received protein ~dded rating weight of fibre) ~b~red on X ww 1 weight of fibre) X ww 1 O O O O O O
0.5 + 25 1.6 +
0.7 + 50 3.1 +
100 1.0 + 70 4.4 +
150 1.5 +~ 100* 6.3 +
200 2.0 +++ 150 9.4 +
W O 97/07282 PcT/~b~ 2 250 2.5 +++ 200 12.5 +
300 3.0 +++ 300 18.8 ++
625* 6.2 +++ 400 25.1 ++
875 8.7 +++ 600 37.6 ++
*Represent equivalent addition levels.
The results show that whilst both cellulases impart sizing, the N. insolens cellulase preparation imparted greater levels of sizing than the T. reesei cellulase under the conditions used.
Bxample 4 The effects of buffer omission and high (160~C) o temperature drying on levels of sizing achieved with either a Trichoderma reesei cellulase preparation (Celluclast~, Novo Nordisk A/S, Bagsvaerd, Denmark) or a Humicola insolens cellulase preparation (Celluzyme~, Novo Nordisk A/S, Bagsvaerd, Denmark) were investigated.
Paper sheets were prepared as described previously, with the various conditions as described in Table 4. Sizing was measured the following day by the standard IDT method.
The results show that Celluclast~ benefits from higher drying temperatures (160~C) than Celluzyme2 to confer good sizing.
Teble ~: Effect of I . ~ ~ on the sizing ~chieved by either Tri~ ' ree~ei cellul-se 2 5 (Cellucl-st , ~ovo ~ordis~ ~JS, _ ~- n~, Den~0rk) or Humicoln in601ens cellul-~e ~Celluzyme , Hovo Hordis~ ~JS, ~ _ rd, Den~cr~) EnzymeB~nding protein Incubation Drying IDT
( 1 5 0 ,(L~ eight of ~bre) buffer regime rating 3c Celluclast' 9 4 dH20160~C/30 ++~+
108~C/250 Celluclast' 9.4 Tris-HCl160~C/30 ++
1 0 8 ~ C / 2 5 0 CA 02229358 l998-02-ll W 0 97/07282 PCT/~ al2 Celluclast9.4 dH20 108~C/250 +++
Celluclast~ 9.4 Tris-HCl 108~C/250 +
No enzyme 0 Tris-HCl 160~C/30 0 s +
c 108~C/250 No enzyme ~ dH20 160~C/30 o s +
108~C/2S0 ~ Celluzyme~ 1.5 dH20160~C/30 l~
108~C/250 Celluzyme~ 1.5 Tris-HCl 160~C/30 ++
108~C/250 Celluzyme~ 1.5 dH20 108~C/250++++
Celluzyme' 1.5 Tris-~Cl 108~C/250 +
s Examplc 5 The Hercules Sizing Test (HST) was used to determine the degree of sizing imparted by either Trichode~ma reesei cellulase (Celluclast~, Novo Nordisk A/S, Bagsvaerd, Denmark) or Humicola insolens cellulase (Celluzyme~, Novo Nordisk A/S, Bagsvaerd, Denmark) with and without the addition of Tris. To prepare the sheets, 2 %
(w-1) pulp stock was incubated with 5 % (ww~l) cellulase protein (based on dry weight of fibre) for 5 min at 25~C
before forming the paper sheets. The sheets were dried on a drum dryer at 105~C for 40, 80, 160 or 240s and were subjected to one of the fol].owing post manufacture heat treatments: naturally aged for 24 h; 80~C for 10 min and 105~C for 10 min.
The results show that Humicola insolens and Trichoderma reesei cellulases confer moderate sizing as measured by the HST after drum drying at 105~C.
_ W O 97/07282 PCTiGB9~2012 T bl~! 5 HST ~ . of peper ~;heets ~ized ~lith either T~ ei cellul#-~Cellucl-st, llovo llordid~ A/S, E _ J, Der~arlc) or ,llu-icol- insolens cellul~Ihn~ ~lordi~lc ~JS, ~ _ 1, Da_~rk).
HST (s) I -dH20 ¦ 25 mmoles Tris Drul Cellulase Drier N/A 80~C 105~C N/A 800C 105~C
Contac t Time (S) l~Celluclast' 40 10 13 18 10 11 15 Celluzyme~ 40 121 118 127 131 121 93 ~:s~mple 6 A Cellulase preparation from Tricho~ern2a viride (BDH Ltd.) was tested as a biosizing agent. Samples were added to aliquots of pulp stock (0.2 g dry weight fibre in 15 ml di~tilled water). The cellulase addition level was ad~usted such that an equivalent cellulose binding protein concentrations (corresponding to 8.7% ww-1 based on fibre weight) were added to enable direct comparison with Humicola insolens cellulase (Celluzyme~, Novo Nordisk A/S, Bagsvaerd, Denmark).
The pulp and cellulase samples were inc~hAted at room temperature for 15 min prior to preparation of the hand sheets. The sheets were dried by a single pass through a drum drier at 105~C and left at room temperature overnight before testing for sizing using the IDT. The results are shown in Table 6.
The results indicate that when using an equivalent protein concentrations and compared with ~umicol a insol ens cellulase, the Trichoderma viride cellulase imparted a moderate sizing effect.
W O 97/07282 PCT/~r~'~2012 Tab1Q 6 8izing achieved l~y ~ffer~nt det _ ; ~9~ by the C~-IDT ~88ay .
Cellulase Source Supplier IDT
sizing rating Trichoderma viride BDH Ltd. +
Humicola insolens Novo Nordisk A/S +++
C~-LLI 11 o o Example 7 To test the application of Clostridium thermocellum cellulosomes as a sizing agent Cl. thermocellum (NCIMB 10682) was grown on 1.0 % (wv~l) pulp in growth medium, comprising: lOOOml Basal Medium ((gl-l) yeast extract, 10; KH2P04, 1.5; K2HP04.3H20, 2.9; (NH4)2S04, 1 3;
and FeS04.7H20, (1.0% wv~1) l.Oml~1) with a cellulose s'ource (pulp Q1% wv~1) which is then autoclaved to sterilize. 25 ml Salts Solution (MgC12, 2%wv~1; CaC12 0.2% wv~1, autoclaved to sterilize). 15 ml Cysteine Solution (50 gl~1, filter sterilized). The growth medium (11 in a Duran bottle) was purged with nitrogen for 5 min and heated to 60~C prior to inoclllAtion. The culture was then i~c~lhAted for 240h. The culture was harvested and the cells and pulp debris separated from the culture fluid by centrifugation. Sodium azide (0.02 % (wv~1)) was added to both the culture fluid 2s and to the pulp debris to prevent further microbial growth.
The culture fluid was tested as a sizing agent by ~king ;~= paper using the Cl. thermocellum cultures. Water-leaf pulp (10% (wv~l); 2.16 g) was weighed into five 250 ml flasks.
Cl. thermocellum culture fluid (200; 100; 50; 25; and 0 ml) was then added to each flask and the volume adjusted to 200 ml with distilled water. The mixture was then stirred at room temperature for 15 min and a paper sheet was made from the contents of each flask using the stAn~Ard paper making method. The paper was dried at 80~C for 250 s using the drum drier. Sizing was measured the following day using the st~n~Ard IDT method.
CGl.L ol sheets of paper were also prepared and tested as W O 97/07282 PCT/GL,.'~2012 above wherein non-inoculated Cl. thel -~e7 lum growth medium was added to the water-leaf pulp instead of the Cl.
thermocellum culture fluid.
It was also decided to test for sizing using the pelleted pulp debris and cells. The pulp debris and cells (2.16 g;
percentage pulp now unknown) were weighed into two 250 ml flasks. To one flask 200 ml of Cl. thermocellum growth medium was added to resuspend the pulp debris and to the lo other flask 200 ml of distilled water waC added. Both flasks were stirred at room temperature for 15 min and a paper sheet made from the contents of both flasks using the st~n~Ard paper making method. The paper was dried at 90~C
for 250 s using the drum drier. Sizing was measured the following day using the IDT method.
The Cl. thermocellum culture fluid did not impart sizing to the paper it is believed because of the very low levels of cellulosomes free in the culture fluid. Paper sheets made from the pulp debris showed sizing (Table 4) and a significant lowering in the degree of sizing was noted when distilled water was used compared to the Cl. thermocellum growth medium. It is believed that the use of distilled water causes a lowering of the salt/ionic strength of the 2s distilled water as comr~red to use of the growth medium, resulting in elution of cellulosomes from the pulp surface thereby reducing the degree of sizing. The results confirm that the presence of the Cl. thermocellum cellulosome preparation imparts sizing to the paper sheet.
T ble 7: 5izir~ test: pulp h)~drolysed by Cl. 1~
Diluent (ml) Ink-drop test (s) Pulp debris + Cl. thermocellum140/175*
3r cells growth medium (2.16 g) (200 ml) Pulp debris + dH20 (200 ml) 37/35*
cellF
(2.16 g) * Duplicate tests.
-W O 97/07282 PCT/G~9~ 012 Exunple 8 A further series of experiments were performed to determine the effect of cellulase on the sizing of paper. In the experiments, the following materials and general protocols were employed:-Ce 7 lulase An aqueous ~richoderma reesei cellulase preparation was employed ("Celluclast 1.5L" supplied by Novo Nordisk Bioindustry S.A. 92017 Nanterre Cedex. France).
In addition Cellulase derived from Penici ~ 7 il~m funiculosum available as a tan powder from Sigma Aldrich Co. Ltd. Poole, Dorset, U.K. was used.
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 was prepared with 1/3 PBS and no fillers were added. The procedure was as follows:
280g of bleached hardwood pulp and 120g of bleached softwood pulp were added to 18 litres of 1/3 PBS. The 2S fibres were dispersed by vigorous agitation. This stock was then transferred to the Hollander and beaten until a freeness value of 250SR was att~i~e~ (time taken was usually 30 to 35 minutes). The stock was then adjusted to a final consistency of 2% with further 1/3 PBS as necessary.
Addition/Incubation of Additives The cellulase solution was added to the thick (2~
consistency) stock. Two litres of the thick stock (cont~;n;ng 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 stock was at ambient -CA 022293~8 1998-02-11 W O 97/07282 PCT/~ 12 temperatures (20-25 C).
The ;ncl~h~tion time was fifteen minutes. During this incllh~tion period the movement of the stock may Arr~
to become easier/faster. If this is apparent then reduce the stirrer speed as much as possible.
After the fifteen minute incubation period had el~p~
the thick stock was then added to the p~poLLioner.
Prop~rt~;oner The thick stock in the proportioner was then diluted to a consistency of 0.25~ using DEMI water only. Normal agitation speeds in the proportioner were employed to mix the stock.
~n~heet Form~tion The white water box was filled with DEMI water for h~Che~t formation. With the ~n~cheet forming wire in place in the mould assembly, 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 times). After the fifth stroke the agitator was rested 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 persist after the initial wet mat is 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.
W O 97/07282 PCT/~L5r~012 ~3 n~.eheet Pressin~ and Dryi ng The wet mat and h~n~heet wire were l~ -ved from the mould to the press. The moisture content of the presséd sheet should be 70%. The pressed sheet was s then dried on an electrically heated drum dryer. The surface temperature of the dryer was 105-C and the speed of the dryer was such that the pressed sheet was in contact with the hot surface for 35 ~e~on~. The final moi~ture content of the sheet should be between lo 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 press pressures being applied across the width of the ~heet.
Steps should be taken to avoid this.
When the surface tempera~ure of the drum dryer is less than 105-C but is 70-C or higher, longer contact times are re~uired in order for the h~n~cheet 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 1o do both. It is possible to reduce the moisture content of the pressed sheet to less than 60%.
Tes~ i ng Conditioning and testinq of the paper is done according to procedures laid out in the "Tappi Test Methods" published by TAPPI, Technology Park Atlanta, P0 Box 105113, Atlanta GA
35 30348, USA, ISBN 0 - 89852 - 200 - 5 (vol 1 and 2). The HST
(Hercules Sizing Test) is defined as size test for paper by ink resistance T 530 pm - 83; and the Cobb test is defined by T 441 om - 84.
-WO 97/07282 PC r/~L~ GI2 A series of experiments were performed in which the cellulase concentration, aging time and t~ ~ature were each varied. The results are presented in the following tables in which:-"naturally aged" refers to storage for the specifiedtime at 23~C + 1~C in relative humidity 50.0 + 2% as ~pecified in T402Om-83;
"oven cured" refers to treatment at -80~C for ~0 ominutes.
8~zing Perform~nce of Handsheets made with Cellula~e and ~r~e~ under ~t~n~rd conditions "~8T (secQ~ of hanasheets ma~e with Penici 77in~
~uniculosum Protein added Ageing Condition 24h naturally 2 weeks oven aged naturally cured aged 20 bl.nk 1 % 1 7 6 1 % 3 11 18 "~ t~sCon~Q) of handsheets made with Tr ~h~rma reeseia Protein added Ageing Cold.. tion 24h naturally , ~eek~ oven aged naturally cured aged blank 5% 13 27 28 10% 34 53 61 "Cobb (gs~) of hanasheets made with Tri~hoA~rma , Protein added Ageing Condition 24h naturally 2 weeks oven aged naturally cured aged blank Fully Fully Fully saturated saturated saturated 5% 78.6 67.2 74.2 10% 68.5 51.2 50.7 W O 97/07Z82 PCT/Cn9'r~012 ~5 ~mpl~ g In a further series of experiments conducted under the protocols described in Example 8 above, the degree to which the added protein tCelluclast (Trichoderma reesei, Novo Nordisk) and Celluzyme (Humicola insolens, Novo Nordisk)] is retained by the paper was investigated.
In separate experiments Celluclast and Celluzyme were added to paper pulp and test sheets prepared described above (24 h naturally aged). The amount of protein retA i n~ in the paper (as opposed to that remaining in the pulp supernatant when forming the paper web) was estimated on the basis of the nitrogen content of the paper, assuming that the nitrogen content of both proteins is 16% w/w. The nitrogen content was measured by Antec micropyrolysis.
The results are presented in the following table:
20 Additive Addition ~ ~ HST Cobb (g l~vel Nitrogen Protein (secon~) water/m2 w/w db in dry retained fibre paper w/w in pap~r w/w db fibre Blank - 0.007 - 1 fully ~aturated Cellucla~t 20 0.356 2.225 273 15.5 Celluzyme 5 0.374 2.3375 263 28.6 The results show that addition levels of Celluzyme one quarter those of Celluclast gave rise to similar protein retention levels. At similar protein retention levels, both celluloses gave similar sizing effects.
Ex~mpl~ 10 ~ In the following example, the binding amylase enzymes to starch is demonstrated. Two amylase enzymes were characterized using HPLC: an ~-amylase (Type X-A crude preparation) from Aspergillus oryzae and amyloglucosidase from A. niger (available from Sigma Aldrich Co. Ltd., Poole, Dorset, United Kingdom). The main catalytic peaks of each preparation were determined U5 ing a starch glucose-release assay. The binding efficiencies of each protein were determined against a range of starches with BSA CG~L~ 018 included in the assessment.
A solution of 32 mg ml~1 (dry weight) of ~-amylase was made up in 0.1 M PBS (pH7.0). 100~1 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~l. Fractions (1 ml) were collected and tested for reducing sugars released from a lo starch suspension using the st~n~rd microtitre assay (for glucose).
The following qualitative assay 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 ~1 phenol reagent (0.128M phenol in O.lM phosphate buffer pH7.0) 10 ~1 amino pyrine reagent (19.7mM 4-amino phenazone in O.lM
phosphate buffer pH7.0) 10 ~1 peroxidase in O.lM phosphate buffer pH 7.0 (to give 800Eu/ml) 10 ,Ul glucose oxidase in 0.lM phosphate buffer pH 7.0 (to give 250Eu/ml) 60 ~1 O.lM phosphate buffer pH7.0 These reagent components were mixed and added to the wells of a microtitre dish. Test samples 100 ~1 were added followed by an e~C~cfi of substrate (starch). The appearance of a red colour was indicative of the presence of amylase.
.
The same methods were also used to produce an HPLC profile for the amyloglucosidase. The amyloglucosidase was a liquid preparation containing approximately 262 mg ml~l protein as measured by the Coomassie Blue technique. 100 ~1 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 sùspensions as above.
The ability of cr-amylase and amyloglucosidase to bind to normal starch in suspension was assessed. Starch (0.2 g;
Roquette) was added to 9 ml 0.1 M PBS (pH 7.0) and 1 ml ~-amylase solution (9.5 mg ml~1 by Coomassie Blue assay) was added. This was incubated on a shaker for 20 min.
The sample was centrifuged at 13,000 rpm for 5 min and 100 ~l samples loaded onto the HPLC column. The peak profile of the 20 min bound ~-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 ~u~-LLol. The final concentration of the BSA used was O.2% (wv 1) in 0.1 M PBS.
The results of the binding experiments are shown in the following Table.
8t~rch b~ n~ profiles Enzyme Substrate % Bound 25~-amylase starch 32 amyloglucosidase st,arch 27 amyloglucosidasecationic starch 45 BSA starch 7 BSA cationic starch 6 30 These results in~icate that both c~-amylases and amyloglucosidases specifically bind to both starch and cationic starch.
-
According to a further aspect of the present invention there i8 provided a method of sizing paper comprising a) contacting said paper or a constituent of said paper with a protein capable of binding to said paper or said constituent of paper, and b) heating said paper.
According to a further aspect of the present invention there i8 provided use of a protein capable of binding paper or a constituent of paper for the purpose of sizing paper. The invention further provides paper sized according to a method of the present invention.
~ Detailed Description of the Invention The present invention provides a method of sizing paper. 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 sources optionally mixed with fibres from vegetable, mineral, animal or synthetic sources in various ~ Lions 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 sources of cellulose include wood, straws, Bagasse, Esparto, Bamboo, Kanaf, Grass, Jute, Ramie, Hemp, Cotton, Flax. The crude vegetable derived cellulose is proceC~ to form pulp, the material from which paper is made, either mechanically, chemically or both. Cellulose ContA; n~ ~g pulps may be described as mechanical, CA 022293~8 1998-02-11 WO 97t07282 PCT/GB96/02012 ~h~ hAn;cal and chemithermomechA~iCal~ 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.
Paper may also comprise other naturally occurring polymers such as proteins such as keratin, starch (including anionic, cationic or amphoteric starch), pectin, guar, chitin, lignin, agar, alginate as well as other poly~Acch~rides including hemi-celluloses such as xylanose, mannose and arabinose.
The method of the present invention comprises co~ acting paper or a constituent of paper with a protein capable of b~ n~ j n~ to the paper or const:ituent of paper followed by denaturing or heating the protein.
The protein employed in the present invention may comprise any protein capable of binding to the paper or constituent of paper. The protein may for example comprise a protein capable of binding cellulose or any other polymeric substance present as a constituent of the paper.
Preferably, the protein is capable of specific binding to cellulose or any other polymeric substance present as a constituent of paper. More preferably, the protein is capable of binding 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 (for example, when protein is hon~e~ to another molecule), reference to a protein means a protein residue. The protein may co~prise 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 CA 022293~8 1998-02-11 W O 97/07282 PCT/~Lr~~012 herein the term "modified protein" includes chemical analogs of proteins capable of binding to paper or a constituent thereof. Preferably/ the protein comprises a naturally occurring enzyme or fragment thereof which is capable of bin~ing to paper or a constituent of paper. Examples of proteins capable of binding paper or a constituent of paper are well known and include enzymes selected from the group comprisingcellulases, hemi-cellulases, mannases, xylanases, chitinases, ligninases, agarases, alginases and amylases.
The protein may for example comprise an amylase or fragment thereof capable of binding to starch (such as anionic, cationic or amphoteric starch) when present as a constituent of paper or paper pulp. Examples of amylases include ~-amylases, for example from Aspergillus oryzae (available asa Type X-A crude preparation from Sigma Aldrich Co Ltd), and amyloglucosidases, for example from Aspergillus niger (available from Sigma Aldrich Co Ltd).
Preferably, the protein comprises a protein capable of b~n~;nq to cellulose. More preferably, the protein comprises a cellulase or fragment thereof. The cellulase may comprise a naturally occurring cellulase, or fragment thereof, or modified cellulase obtainable by chemical modification of a naturally occurring cellulase or synthesis or by e~LessiGn of a genetically modified gene co~ for a cellulase. The cellulase may, for example, be modified to remove or deactivate the active-site domain. A variety of cellulases are known which bind to cellulose. Examples of such cell~ are those isolable from bacterial organisms such as Cellulomonas fimi and fungal organisms such as Trichoderma viride, Aspergillus niger, Fusarium oxysporum, Penicillium funiculosum, Trichoderma reesei and Humicola insolens, available as commercial preparations from Sig~a Chemical Sigma-Aldrich Company Ltd., Novo Nordisk A/S, BDH
Ltd., or ICN Biomedicals Ltd. (Fusarium oxysporum is available for example under deposit No. DSM 2672).
Alternatively, the protein may be produced by recombinant CA 022293~8 lsss-02-ll wos7/07zsz PCT/GB96/02012 DNA techn;~ues as disclosed in, for example, International Patent application WO94/24158. Cell~l A~ 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 obt~;ne~ from whole cellulase by treatment with protease(s), such as papain. The cellulase may comprise an exo-cellulase or an endo-cellulase. Exo-celll~l AS~ (also known as cellobiohydrolases, CBH; exoglucanaes; 1,4-beta-D-glucan cellobiohydrolases; EC 3.2.1.91) act on the non-reducing end of a cellulose molecule. Exo-cellulases may release terminal cellobiose units (a disaccharide) or release terminal glucose units (monosaccharide). Examples of exo-cellulases include cellulase obtainable from Numicol ai~olens. Endo-cellulases (also known as Beta-1,4-Endoglucanases; Endo-1,4-D-glucanases; 1,4-Beta-D-glucan gll~cAnohydrolases; EC 3.2.1.4) cleave internal beta-1,4-glycosidic bonds yielding a mixture of glucose, cellobiose and other soluble cello-oligosaccharides. Examples of endo celllll~ce~ include cellulase obtainable from Trichoderma reesei .
The protein may also comprise cellulosomes. Cellulosomes comprise a cellulase system comprising discrete, multifunctional, multienzyme complexes. They typically contain at least 14 distinct polypeptides including numerous endoglucA~e~ (endocellulases) and xylanases and at least one beta-glucanase. These are associated with scaffolding proteins. Cellulosomes are described in detail in Bayer E.A., Morag E., Lamed R. (1994), "The cellulosome - a trea~LeLLo~e for biotechnology", Trends in Biotechnology 12:379-386.
Preferably, the protein employed in the present invention comprises cellulase obtainable from ~umicol a isol ens (available as Celluzyme~ from Novo Nordisk A/S, Bagsvaerd, Denmark) or cellulase obtainable from Trichoderma reesei W O 97107282 PCT/~r~012 (available a Celluclast0 from Novo Nordisk A/S, Bagsvaerd, Denmark). More preferably, the protein comprises cellulase obtAinAhle from Humicola isolens .
The protein may be added to the paper at any suitable stage in the manufacture and processing of the paper. It may be added at the pulp stage or at any stage during the formation of the wet pulp matrix or during the pressing and rolling of the matrix to form paper. Thus, according to the present invention there is provided a method of manufacturing sized paper comprising the steps of a) preparing a paper pulp, b) adding a protein capable of binding to a constituent of said pulp, c) forming paper from said pulp, and d) heating said paper.
Alternatively, the protein may be added to the formed paper product, for example, by immersing the paper in a bath cont~;n;ng the protein or by any suitable spraying, spr~ gt brushing, coating or printing process. Thus, the invention furthcr provides a method of manufacturing sized paper comprising the steps of a) applying to paper a protein capable of binding said paper and b) heating said paper.
By choosing the point in the manufacture of the paper at which the protein is added, control may be exercised as to whether the protein is distributed throughout the paper or is substantially restricted to the surface levels of the paper.
The protein should be incubated with the paper or paper pulp for sufficient time to allow binding of the protein to the paper or paper pulp. Typically, 15 minutes has been found adequate, although shorter incubation times may be suitable.
The protein may be added in an amount suitable to achieve the desired level of sizing. The protein may be added in an amount of 0.01-40% by weight of the dry weight of the paper pulp. Preferably the protein is added in an amount of 0.1 _ CA 022293~8 1998-02-11 W 097107282 PCT/~Lr~ 012 to 20% by weight, more preferably 1 to 10% by weight.
Following incorporation of t:he protein in the paper or application of the protein to the surface of the paper, sizing of the paper is achieved by denaturing or heating the protein. The protein may be denatured by the application of a chemical protein denaturant to the paper. Chemical protein denaturants include urea, guanidine, acids, ~k~]i~, detergents (such as Tween~), water soluble organic subst~n~ (such as alphatic alcohols) and chaotropic ions (such as I-, Cl04-, SCN-, Li+, Mg2+, Ca2+ and Ba2+).
Preferably, sizing is achieved by heating the paper.
Preferably, the paper may be heated at a temperature of 50~C
to 200~~, more preferably 70~C to 170~C, more preferably 80~C to 110~C, more preferably 100~C to 110~C. Typically, the paper may be heated to approximately 105~C on steam heated rollers. The paper may be subjected to one or more heat treatments at different 1_emperatures.
The length of time of heating required depends upon the temperature at which the paper is heated, longer times being required at lower temperature. Typically, the paper may be heated for between 15 and 500 seconds, preferably between 25 and 300 seconds. The paper may also be subjected to post manufacture heat treatments to age or cure the paper.
The invention will now be described with reference to the following examples.
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.
W O 97/07282 PCT/~r5,'~012 EXP~ r~iL
Materials and Protocols Excep~ as indicated below, the following materials and protocols were used in the Examples to characterise the use of proteins as biosizing agents.
~le: Water-leaf paper pulp was prepared by A~ i ng 10 g of 10 water-leaf paper (70:30 Hardwood (birch) : softwood (pine)) to 100 ml distilled water. After 5 min the paper was blended to an homogenous pulp. Samples of pulp Cll~p~cion (e~e~ponding to 0.2 g dry paper) were weighed into Universal bottles.
TncubatiQn To each bottle 10.0 ml of one of the following 1~Cllh~tion buffer solutions was added:
(a) Tris-HCl, 50 mM, pH 7.5 (b) Phosphate buffered saline (PBS) 500 mM, pH 7.5 ~ ulases: To the incubation suspension was added a cellulase selected from the following:
1. ~umicola insolens - Cellulase derived from Humicola ~nsolens available as Celluzyme~ from Novo Nordisk A/S, Bagsvaerd, Denmark (875~1 corresponding to 8.7%
weight per weight cellulose binding protein to dry weight pulp).
2. Trichoderma reesei - Cellulase derived from Trichoderma reesei available as Celluclast~ from Novo Nordisk A/S, Bagsvaerd, Denmark (70~1 corresponding to 0.28~mols protein corresponding to 4.4% weight per weight cellulose binding protein to dry weight pulp).
CA 02229358 l998-02-ll W O 91~07282 PCTW L~J~012 3. Trichoderma viride - Cellulose derived from Trichoderma vivide available from BDH Ltd UK.
Control experiments in the absence of cellulase were also performed.
Tncubation: The mixtures were incubated for 15 min at room temperature with gentle agitation.
Test Sheet Preparation: To produce the paper test sheets, the volume was 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 (O.2~ wv-l) 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 sheet supported by the mesh. The filter mesh was removed from the apparatus and the paper sheet sandwiched between a second nylon mesh and blotted between blotting paper. The paper sheet was carefully removed from the paper-making mesh, flattened by rolling and then dried.
Test Sheet Drying/Heatinq: Paper sheets were dried in one of the following ways (a) air drying;
(b) drum drier - typically operating at a constant temperature of 80~C to 10~~C with a contact time of 40 to 250 ~;
(c) hot plate press - typically producing a maximum surface temperature of 160~C with a 30 s contact time.
A flat aluminum plate was used to press the paper test sheets (sandwiched between blotting paper) against the hot plate.
Test ~rotocols: The dried sheets were ~cs~-cced for sizing by one or more of the following t:ests:-(a) Ink drop test (IDT) - i~ which a 15 ~1 drop of Parker Super Quink Ink (Permanent Blue-Black) was dropped CA 022293~8 1998-02-11 W O 97/07282 PCT/G~9~ 2012 onto the test piece of paper and the time taken for complete absorption measured and recorded. Results were recorded in seconds or on an empirical scale of 0 to ~+++ in which:-0 means less than 100 s for complete absorption.
means 100-500 s for complete absorption.
++ means 500-1000 s for complete absorption.
+++ means 1000-2000 s for complete absorption.
++++ means greater than 2000 s for complete absorption.
(b) Hercules Size Test (HST) - defined in "Tappi Test Methods" published by TAPPI, Technology Park, Atlanta, P0 Box 105113, GA 30348, USA, ISBN 0-89852-200-5 (vol 1 and 2), (1987). The HST is defined as size test for paper by ink resistance T530pm-83. The data are recorded in seconds; the higher the value, the better the sizing. Pre~erably an HST value greater than 20 secon~, more preferably greater than 120 E~conA~, more preferably greater than 200 ~econ~c~ is obtained.
(c) Cobb Size Test - defined in "Tappi Test Methods"
(Ibid) by T441Om-84. Data are 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 a Cobb value less than 30g/m2, more preferably less than 21g/m2, is obtA;n~d.
Ex~mpl~ 1 An investigation into the effect of temperature on the sizing imparted by a Trichoderma reesei cellulase preparation (Celluclast~,_Novo Nordisk A/S, Bagsvaerd, Denmark;) was carried out. For these studies a drum drier was used in conjunction with a variable temperature hot plate. The drum drier gave a constant temperature of 80~C
with a contact time of 250 s and the hot plate gave a maximum surface temperature of 160~C with a 30 s contact time. A flat aluminium plate was used to press the paper sheets (sandwiched between blotting paper) against the hot W O 97/07282 . PCT/~,~'~1012 plate.
Trichoderma reesei cellulase preparation (70 ~1 Celluclast~, corresponding to 4.4 % ww~1 cellulose binding protein based on dry weight of cellulose fibre) was used and the mixLuLe ~cl~h~ted for 15 min at room temperature with gentle agitation. Control experiments without cellulase were also performed.
Paper test sheets were prepared as described above. Test sheets were initially pressed between blotting paper then dried in one of the three following ways.
(a) air drying;
(b) drum-drier: 80~C/250 s; OL-(c) hot plate press: 160~C/30 s followed by drum drier 80~C/250 8.
The dried sheets were then assessed for sizing by the Ink Drop Test (IDT).
The following sizing results were obtained from test papers prepared under different drying regimes.
T~ble1: Effect of pper dr~ e on the cellul~ ' ~ sizinll ~t Drying regime Additions to pulp Air dried80~C/250 s160~C/30 8 80~C/250 s Cel_u~last~+ Tris-HCl 0 +++ ++++
3~ Cel~u~,last~ + PBS - - ++
Tric-ICl - 0 PBS
H20 _ 0 - = Not tested.
No sizing was observed when papers were made without enzyme, ruling out the possibility that the buffer salts were interacting with the cellulose in such a way as to affect sizing. Sizing was observed with Tris-~. reesei cellulase WO 97/07282 PCr/~jL5r~ 012 (Celluclast~) inC~lhAtions and a greater degree of sizing was observed when papers were dried initially at the higher temperature followed by drum drying at the lower temperature. Cellulase/PBS paper also gave sizing at the higher temperature but the degree of sizing was lower than that obtained in the presence of Tris-HCl.
E~mpl~ 2 The effect of incubation time on the levels of sizing imparted using a Humicol a insol ens cellulase preparation (Celluzyme-, Novo Nordisk A/S, Bagsvaerd, Denmark) was investigated.
Tris-HCl (50 mM, pH 7.5, 10 ml) was added to 0.2g pulp in dist~lle~ water and the Humicol a insol ens cellulase preparation (875 ~1) was added and the mixtures incubated for either 15 min or 90 min at room temperature. At the end o~ the incubation period, the pulp samples were vortex mixed and diluted to 100 ml. Test papers were prepared in the ~t~n~rd -nne~ and dried by a single pass through a drum drier 100~C/250 s.
The degree of sizing achieved by the different methods was A~e-S~ using the IDT method.
T ble2:
Enzyme wt dry IDT Incubati test sizing on paper period (g) (min) Celluzyme' (875 0.207 +++ 15 3c ~1) Celluzyme' (875 0.157 +++ 90 ~Ll) Control 0.218 0 90 3S Increasing the incubation time from 15 min to 90 min did not significantly increase the level of sizing.
WO 97/07282 PCT/~9~,~2012 1!5 ~x~mple 3 To establish a quantitative relationship between sizing achieved in test papers and the quantity of Trich~rma reesei or ~umicola insolens cellulase preparations added, a further set of experiments were undertaken.
St~AA~dized paper making conditions were employed as follows: to a sample of pulp in distilled water (equivalent to 0.2 g dry paper) 10.0 ml buffer was added (50 mM Tris HCl, pH 7.5). Various amounts of cellulase (Trichoderma reesei or Humicol a insol ens ; Table 3) was added and the mixture vortex mixed. The pulp was ;ncllh~ted with gentle chAki~g at room temperature for 15 min, after which time the mixture was diluted to 150 ml with distilled water and the test paper sheets produced. Eac:h test sheet was removed from the mesh and pressed between a folded sheet of dry 3 MM
blotting paper using a hand-held roller. The sheet (still folded in the blotting paper) was passed once through a drum drier at 100-104~C with a 250 s contact time. The results are given in Table 3.
T ble 3: Sizir~ ~chieved usin~l either TriC ' ~ reesei ~Cellucl#t , ~lovo ~lordidcA~S, ~ _ J, Den or~) or Nu~icoln insolens (Celluey~e , Uovo Uordisk ~5, r _ d, Dononr~) cellul Le ~ . ~ io..
CelluzymeCelluzyme IDTCelluclastCellucla~t IDT
(/11) a~~Bindin~ protein~izing (lll) aQ ~Bindin9 ~L~c~ng receivedKlded ~brsed on rating received protein ~dded rating weight of fibre) ~b~red on X ww 1 weight of fibre) X ww 1 O O O O O O
0.5 + 25 1.6 +
0.7 + 50 3.1 +
100 1.0 + 70 4.4 +
150 1.5 +~ 100* 6.3 +
200 2.0 +++ 150 9.4 +
W O 97/07282 PcT/~b~ 2 250 2.5 +++ 200 12.5 +
300 3.0 +++ 300 18.8 ++
625* 6.2 +++ 400 25.1 ++
875 8.7 +++ 600 37.6 ++
*Represent equivalent addition levels.
The results show that whilst both cellulases impart sizing, the N. insolens cellulase preparation imparted greater levels of sizing than the T. reesei cellulase under the conditions used.
Bxample 4 The effects of buffer omission and high (160~C) o temperature drying on levels of sizing achieved with either a Trichoderma reesei cellulase preparation (Celluclast~, Novo Nordisk A/S, Bagsvaerd, Denmark) or a Humicola insolens cellulase preparation (Celluzyme~, Novo Nordisk A/S, Bagsvaerd, Denmark) were investigated.
Paper sheets were prepared as described previously, with the various conditions as described in Table 4. Sizing was measured the following day by the standard IDT method.
The results show that Celluclast~ benefits from higher drying temperatures (160~C) than Celluzyme2 to confer good sizing.
Teble ~: Effect of I . ~ ~ on the sizing ~chieved by either Tri~ ' ree~ei cellul-se 2 5 (Cellucl-st , ~ovo ~ordis~ ~JS, _ ~- n~, Den~0rk) or Humicoln in601ens cellul-~e ~Celluzyme , Hovo Hordis~ ~JS, ~ _ rd, Den~cr~) EnzymeB~nding protein Incubation Drying IDT
( 1 5 0 ,(L~ eight of ~bre) buffer regime rating 3c Celluclast' 9 4 dH20160~C/30 ++~+
108~C/250 Celluclast' 9.4 Tris-HCl160~C/30 ++
1 0 8 ~ C / 2 5 0 CA 02229358 l998-02-ll W 0 97/07282 PCT/~ al2 Celluclast9.4 dH20 108~C/250 +++
Celluclast~ 9.4 Tris-HCl 108~C/250 +
No enzyme 0 Tris-HCl 160~C/30 0 s +
c 108~C/250 No enzyme ~ dH20 160~C/30 o s +
108~C/2S0 ~ Celluzyme~ 1.5 dH20160~C/30 l~
108~C/250 Celluzyme~ 1.5 Tris-HCl 160~C/30 ++
108~C/250 Celluzyme~ 1.5 dH20 108~C/250++++
Celluzyme' 1.5 Tris-~Cl 108~C/250 +
s Examplc 5 The Hercules Sizing Test (HST) was used to determine the degree of sizing imparted by either Trichode~ma reesei cellulase (Celluclast~, Novo Nordisk A/S, Bagsvaerd, Denmark) or Humicola insolens cellulase (Celluzyme~, Novo Nordisk A/S, Bagsvaerd, Denmark) with and without the addition of Tris. To prepare the sheets, 2 %
(w-1) pulp stock was incubated with 5 % (ww~l) cellulase protein (based on dry weight of fibre) for 5 min at 25~C
before forming the paper sheets. The sheets were dried on a drum dryer at 105~C for 40, 80, 160 or 240s and were subjected to one of the fol].owing post manufacture heat treatments: naturally aged for 24 h; 80~C for 10 min and 105~C for 10 min.
The results show that Humicola insolens and Trichoderma reesei cellulases confer moderate sizing as measured by the HST after drum drying at 105~C.
_ W O 97/07282 PCTiGB9~2012 T bl~! 5 HST ~ . of peper ~;heets ~ized ~lith either T~ ei cellul#-~Cellucl-st, llovo llordid~ A/S, E _ J, Der~arlc) or ,llu-icol- insolens cellul~Ihn~ ~lordi~lc ~JS, ~ _ 1, Da_~rk).
HST (s) I -dH20 ¦ 25 mmoles Tris Drul Cellulase Drier N/A 80~C 105~C N/A 800C 105~C
Contac t Time (S) l~Celluclast' 40 10 13 18 10 11 15 Celluzyme~ 40 121 118 127 131 121 93 ~:s~mple 6 A Cellulase preparation from Tricho~ern2a viride (BDH Ltd.) was tested as a biosizing agent. Samples were added to aliquots of pulp stock (0.2 g dry weight fibre in 15 ml di~tilled water). The cellulase addition level was ad~usted such that an equivalent cellulose binding protein concentrations (corresponding to 8.7% ww-1 based on fibre weight) were added to enable direct comparison with Humicola insolens cellulase (Celluzyme~, Novo Nordisk A/S, Bagsvaerd, Denmark).
The pulp and cellulase samples were inc~hAted at room temperature for 15 min prior to preparation of the hand sheets. The sheets were dried by a single pass through a drum drier at 105~C and left at room temperature overnight before testing for sizing using the IDT. The results are shown in Table 6.
The results indicate that when using an equivalent protein concentrations and compared with ~umicol a insol ens cellulase, the Trichoderma viride cellulase imparted a moderate sizing effect.
W O 97/07282 PCT/~r~'~2012 Tab1Q 6 8izing achieved l~y ~ffer~nt det _ ; ~9~ by the C~-IDT ~88ay .
Cellulase Source Supplier IDT
sizing rating Trichoderma viride BDH Ltd. +
Humicola insolens Novo Nordisk A/S +++
C~-LLI 11 o o Example 7 To test the application of Clostridium thermocellum cellulosomes as a sizing agent Cl. thermocellum (NCIMB 10682) was grown on 1.0 % (wv~l) pulp in growth medium, comprising: lOOOml Basal Medium ((gl-l) yeast extract, 10; KH2P04, 1.5; K2HP04.3H20, 2.9; (NH4)2S04, 1 3;
and FeS04.7H20, (1.0% wv~1) l.Oml~1) with a cellulose s'ource (pulp Q1% wv~1) which is then autoclaved to sterilize. 25 ml Salts Solution (MgC12, 2%wv~1; CaC12 0.2% wv~1, autoclaved to sterilize). 15 ml Cysteine Solution (50 gl~1, filter sterilized). The growth medium (11 in a Duran bottle) was purged with nitrogen for 5 min and heated to 60~C prior to inoclllAtion. The culture was then i~c~lhAted for 240h. The culture was harvested and the cells and pulp debris separated from the culture fluid by centrifugation. Sodium azide (0.02 % (wv~1)) was added to both the culture fluid 2s and to the pulp debris to prevent further microbial growth.
The culture fluid was tested as a sizing agent by ~king ;~= paper using the Cl. thermocellum cultures. Water-leaf pulp (10% (wv~l); 2.16 g) was weighed into five 250 ml flasks.
Cl. thermocellum culture fluid (200; 100; 50; 25; and 0 ml) was then added to each flask and the volume adjusted to 200 ml with distilled water. The mixture was then stirred at room temperature for 15 min and a paper sheet was made from the contents of each flask using the stAn~Ard paper making method. The paper was dried at 80~C for 250 s using the drum drier. Sizing was measured the following day using the st~n~Ard IDT method.
CGl.L ol sheets of paper were also prepared and tested as W O 97/07282 PCT/GL,.'~2012 above wherein non-inoculated Cl. thel -~e7 lum growth medium was added to the water-leaf pulp instead of the Cl.
thermocellum culture fluid.
It was also decided to test for sizing using the pelleted pulp debris and cells. The pulp debris and cells (2.16 g;
percentage pulp now unknown) were weighed into two 250 ml flasks. To one flask 200 ml of Cl. thermocellum growth medium was added to resuspend the pulp debris and to the lo other flask 200 ml of distilled water waC added. Both flasks were stirred at room temperature for 15 min and a paper sheet made from the contents of both flasks using the st~n~Ard paper making method. The paper was dried at 90~C
for 250 s using the drum drier. Sizing was measured the following day using the IDT method.
The Cl. thermocellum culture fluid did not impart sizing to the paper it is believed because of the very low levels of cellulosomes free in the culture fluid. Paper sheets made from the pulp debris showed sizing (Table 4) and a significant lowering in the degree of sizing was noted when distilled water was used compared to the Cl. thermocellum growth medium. It is believed that the use of distilled water causes a lowering of the salt/ionic strength of the 2s distilled water as comr~red to use of the growth medium, resulting in elution of cellulosomes from the pulp surface thereby reducing the degree of sizing. The results confirm that the presence of the Cl. thermocellum cellulosome preparation imparts sizing to the paper sheet.
T ble 7: 5izir~ test: pulp h)~drolysed by Cl. 1~
Diluent (ml) Ink-drop test (s) Pulp debris + Cl. thermocellum140/175*
3r cells growth medium (2.16 g) (200 ml) Pulp debris + dH20 (200 ml) 37/35*
cellF
(2.16 g) * Duplicate tests.
-W O 97/07282 PCT/G~9~ 012 Exunple 8 A further series of experiments were performed to determine the effect of cellulase on the sizing of paper. In the experiments, the following materials and general protocols were employed:-Ce 7 lulase An aqueous ~richoderma reesei cellulase preparation was employed ("Celluclast 1.5L" supplied by Novo Nordisk Bioindustry S.A. 92017 Nanterre Cedex. France).
In addition Cellulase derived from Penici ~ 7 il~m funiculosum available as a tan powder from Sigma Aldrich Co. Ltd. Poole, Dorset, U.K. was used.
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 was prepared with 1/3 PBS and no fillers were added. The procedure was as follows:
280g of bleached hardwood pulp and 120g of bleached softwood pulp were added to 18 litres of 1/3 PBS. The 2S fibres were dispersed by vigorous agitation. This stock was then transferred to the Hollander and beaten until a freeness value of 250SR was att~i~e~ (time taken was usually 30 to 35 minutes). The stock was then adjusted to a final consistency of 2% with further 1/3 PBS as necessary.
Addition/Incubation of Additives The cellulase solution was added to the thick (2~
consistency) stock. Two litres of the thick stock (cont~;n;ng 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 stock was at ambient -CA 022293~8 1998-02-11 W O 97/07282 PCT/~ 12 temperatures (20-25 C).
The ;ncl~h~tion time was fifteen minutes. During this incllh~tion period the movement of the stock may Arr~
to become easier/faster. If this is apparent then reduce the stirrer speed as much as possible.
After the fifteen minute incubation period had el~p~
the thick stock was then added to the p~poLLioner.
Prop~rt~;oner The thick stock in the proportioner was then diluted to a consistency of 0.25~ using DEMI water only. Normal agitation speeds in the proportioner were employed to mix the stock.
~n~heet Form~tion The white water box was filled with DEMI water for h~Che~t formation. With the ~n~cheet forming wire in place in the mould assembly, 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 times). After the fifth stroke the agitator was rested 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 persist after the initial wet mat is 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.
W O 97/07282 PCT/~L5r~012 ~3 n~.eheet Pressin~ and Dryi ng The wet mat and h~n~heet wire were l~ -ved from the mould to the press. The moisture content of the presséd sheet should be 70%. The pressed sheet was s then dried on an electrically heated drum dryer. The surface temperature of the dryer was 105-C and the speed of the dryer was such that the pressed sheet was in contact with the hot surface for 35 ~e~on~. The final moi~ture content of the sheet should be between lo 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 press pressures being applied across the width of the ~heet.
Steps should be taken to avoid this.
When the surface tempera~ure of the drum dryer is less than 105-C but is 70-C or higher, longer contact times are re~uired in order for the h~n~cheet 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 1o do both. It is possible to reduce the moisture content of the pressed sheet to less than 60%.
Tes~ i ng Conditioning and testinq of the paper is done according to procedures laid out in the "Tappi Test Methods" published by TAPPI, Technology Park Atlanta, P0 Box 105113, Atlanta GA
35 30348, USA, ISBN 0 - 89852 - 200 - 5 (vol 1 and 2). The HST
(Hercules Sizing Test) is defined as size test for paper by ink resistance T 530 pm - 83; and the Cobb test is defined by T 441 om - 84.
-WO 97/07282 PC r/~L~ GI2 A series of experiments were performed in which the cellulase concentration, aging time and t~ ~ature were each varied. The results are presented in the following tables in which:-"naturally aged" refers to storage for the specifiedtime at 23~C + 1~C in relative humidity 50.0 + 2% as ~pecified in T402Om-83;
"oven cured" refers to treatment at -80~C for ~0 ominutes.
8~zing Perform~nce of Handsheets made with Cellula~e and ~r~e~ under ~t~n~rd conditions "~8T (secQ~ of hanasheets ma~e with Penici 77in~
~uniculosum Protein added Ageing Condition 24h naturally 2 weeks oven aged naturally cured aged 20 bl.nk 1 % 1 7 6 1 % 3 11 18 "~ t~sCon~Q) of handsheets made with Tr ~h~rma reeseia Protein added Ageing Cold.. tion 24h naturally , ~eek~ oven aged naturally cured aged blank 5% 13 27 28 10% 34 53 61 "Cobb (gs~) of hanasheets made with Tri~hoA~rma , Protein added Ageing Condition 24h naturally 2 weeks oven aged naturally cured aged blank Fully Fully Fully saturated saturated saturated 5% 78.6 67.2 74.2 10% 68.5 51.2 50.7 W O 97/07Z82 PCT/Cn9'r~012 ~5 ~mpl~ g In a further series of experiments conducted under the protocols described in Example 8 above, the degree to which the added protein tCelluclast (Trichoderma reesei, Novo Nordisk) and Celluzyme (Humicola insolens, Novo Nordisk)] is retained by the paper was investigated.
In separate experiments Celluclast and Celluzyme were added to paper pulp and test sheets prepared described above (24 h naturally aged). The amount of protein retA i n~ in the paper (as opposed to that remaining in the pulp supernatant when forming the paper web) was estimated on the basis of the nitrogen content of the paper, assuming that the nitrogen content of both proteins is 16% w/w. The nitrogen content was measured by Antec micropyrolysis.
The results are presented in the following table:
20 Additive Addition ~ ~ HST Cobb (g l~vel Nitrogen Protein (secon~) water/m2 w/w db in dry retained fibre paper w/w in pap~r w/w db fibre Blank - 0.007 - 1 fully ~aturated Cellucla~t 20 0.356 2.225 273 15.5 Celluzyme 5 0.374 2.3375 263 28.6 The results show that addition levels of Celluzyme one quarter those of Celluclast gave rise to similar protein retention levels. At similar protein retention levels, both celluloses gave similar sizing effects.
Ex~mpl~ 10 ~ In the following example, the binding amylase enzymes to starch is demonstrated. Two amylase enzymes were characterized using HPLC: an ~-amylase (Type X-A crude preparation) from Aspergillus oryzae and amyloglucosidase from A. niger (available from Sigma Aldrich Co. Ltd., Poole, Dorset, United Kingdom). The main catalytic peaks of each preparation were determined U5 ing a starch glucose-release assay. The binding efficiencies of each protein were determined against a range of starches with BSA CG~L~ 018 included in the assessment.
A solution of 32 mg ml~1 (dry weight) of ~-amylase was made up in 0.1 M PBS (pH7.0). 100~1 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~l. Fractions (1 ml) were collected and tested for reducing sugars released from a lo starch suspension using the st~n~rd microtitre assay (for glucose).
The following qualitative assay 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 ~1 phenol reagent (0.128M phenol in O.lM phosphate buffer pH7.0) 10 ~1 amino pyrine reagent (19.7mM 4-amino phenazone in O.lM
phosphate buffer pH7.0) 10 ~1 peroxidase in O.lM phosphate buffer pH 7.0 (to give 800Eu/ml) 10 ,Ul glucose oxidase in 0.lM phosphate buffer pH 7.0 (to give 250Eu/ml) 60 ~1 O.lM phosphate buffer pH7.0 These reagent components were mixed and added to the wells of a microtitre dish. Test samples 100 ~1 were added followed by an e~C~cfi of substrate (starch). The appearance of a red colour was indicative of the presence of amylase.
.
The same methods were also used to produce an HPLC profile for the amyloglucosidase. The amyloglucosidase was a liquid preparation containing approximately 262 mg ml~l protein as measured by the Coomassie Blue technique. 100 ~1 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 sùspensions as above.
The ability of cr-amylase and amyloglucosidase to bind to normal starch in suspension was assessed. Starch (0.2 g;
Roquette) was added to 9 ml 0.1 M PBS (pH 7.0) and 1 ml ~-amylase solution (9.5 mg ml~1 by Coomassie Blue assay) was added. This was incubated on a shaker for 20 min.
The sample was centrifuged at 13,000 rpm for 5 min and 100 ~l samples loaded onto the HPLC column. The peak profile of the 20 min bound ~-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 ~u~-LLol. The final concentration of the BSA used was O.2% (wv 1) in 0.1 M PBS.
The results of the binding experiments are shown in the following Table.
8t~rch b~ n~ profiles Enzyme Substrate % Bound 25~-amylase starch 32 amyloglucosidase st,arch 27 amyloglucosidasecationic starch 45 BSA starch 7 BSA cationic starch 6 30 These results in~icate that both c~-amylases and amyloglucosidases specifically bind to both starch and cationic starch.
-
Claims (19)
1. A method of sizing paper comprising the steps of a) contacting said paper or a constituent of said paper with a protein capable of specific binding to said paper or said constituent of paper; and b) denaturing said protein bound to said paper.
2. A method according to claim 1 wherein the protein is denatured by means of heat.
3. A method according to claim 2 wherein the paper is heated to a temperature of 70°C to 170°C.
4. A method according to claim 3 wherein the paper is heated to a temperature of 80°C to 110°C.
5. A method according to claim 1 wherein the protein is denatured by means of treatment with a chemical denaturant.
6. A method according to any preceding claim wherein the protein is capable of binding a polysaccharide.
7. A method according to any preceding claim wherein the protein is capable of binding cellulose.
8. A method according to any preceding claim wherein the protein is a cellulase or fragment thereof.
9. A method according to claim 8 wherein the protein is a cellulase selected from the group comprising Cellumonas fimi, Trichoderma viride, Trichoderma reesei, Aspergillus niger, Fusarum Oxysporum, Penicillium funiculosum and Humicola insolens.
10. A method according to claim 8 wherein the protein is an exo cellulase or fragment thereof.
11. A method according to claim 10 wherein the protein is cellulase derived from Humicola isolens..
12. A method according to claim 8 wherein the protein is an endo cellulase or fragment thereof.
13. A method according to claim 12 wherein the protein is cellulase derived from Trichoderma reesei.
14. A method according to any one of claims 1 to 5 wherein the protein is capable of specific binding to starch.
15. A method according to claim 14 wherein the protein is an amylase or fragment thereof.
16. A method of manufacturing sized paper comprising the steps of a) preparing a paper pulp, b) adding a protein capable of specific binding to a constituent of said pulp, c) forming paper from said pulp, and d) heating the paper to denature the protein.
17. A method of manufacturing sized paper comprising the steps of a) applying to paper a protein capable of binding said paper and b) heating said paper to denature the protein.
18. Paper sized according to a method of any one of claims 1 to 17.
19. Use of a protein capable of specific binding to paper or a constituent of paper for the purpose of sizing paper.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9516766.4 | 1995-08-16 | ||
| GBGB9516766.4A GB9516766D0 (en) | 1995-08-16 | 1995-08-16 | Method and chemical compounds for modifying polymers |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2229358A1 true CA2229358A1 (en) | 1997-02-27 |
Family
ID=10779311
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2229358 Abandoned CA2229358A1 (en) | 1995-08-16 | 1996-08-16 | Methods and compositions for sizing paper |
| CA 2229588 Abandoned CA2229588A1 (en) | 1995-08-16 | 1996-08-16 | Methods and chemical compounds for modifying polymers |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2229588 Abandoned CA2229588A1 (en) | 1995-08-16 | 1996-08-16 | Methods and chemical compounds for modifying polymers |
Country Status (11)
| Country | Link |
|---|---|
| EP (2) | EP0845060B1 (en) |
| JP (2) | JPH11510701A (en) |
| CN (2) | CN1199439A (en) |
| AU (2) | AU6824896A (en) |
| BR (2) | BR9610327A (en) |
| CA (2) | CA2229358A1 (en) |
| DE (1) | DE69610841T2 (en) |
| GB (1) | GB9516766D0 (en) |
| PT (1) | PT845060E (en) |
| TW (1) | TW353092B (en) |
| WO (2) | WO1997007282A1 (en) |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9711984D0 (en) * | 1997-06-11 | 1997-08-06 | Vincent Julian F V | Biodegradable waterproofing of paper & paper products |
| US6146497A (en) * | 1998-01-16 | 2000-11-14 | Hercules Incorporated | Adhesives and resins, and processes for their production |
| AU7275498A (en) * | 1998-05-01 | 1999-11-23 | Procter & Gamble Company, The | Laundry detergent and/or fabric care compositions comprising a modified enzyme |
| US6468955B1 (en) | 1998-05-01 | 2002-10-22 | The Proctor & Gamble Company | Laundry detergent and/or fabric care compositions comprising a modified enzyme |
| WO1999064678A1 (en) * | 1998-06-08 | 1999-12-16 | ALBUPRO Ltd | Water resistant fibrous material |
| IL133134A0 (en) * | 1999-11-25 | 2001-03-19 | American Israeli Paper Mills | Improved paper products |
| US7364890B2 (en) | 2001-07-28 | 2008-04-29 | Midwest Research Institute | Thermal tolerant avicelase from Acidothermus cellulolyticus |
| JP4058412B2 (en) * | 2001-10-16 | 2008-03-12 | スウェツリー・テクノロジーズ・アクチボラゲット | Method for modifying polymeric carbohydrate materials |
| AT412733B (en) * | 2003-09-04 | 2005-06-27 | Fine Foods Handels Und Beteili | Method for coating paper, cardboard or similar materials |
| US20070131368A1 (en) * | 2005-12-14 | 2007-06-14 | Sonoco Development, Inc. | Paperboard with discrete densified regions, process for making same, and laminate incorporating same |
| US7842362B2 (en) | 2006-02-17 | 2010-11-30 | Sonoco Development, Inc. | Water-resistant wound paperboard tube |
| GB0609477D0 (en) * | 2006-05-12 | 2006-06-21 | Ciba Sc Holding Ag | Process for making paper and paperboard |
| WO2010105357A1 (en) * | 2009-03-20 | 2010-09-23 | Fpinnovations | Cellulose materials with novel properties |
| CN102086611B (en) * | 2010-11-30 | 2012-11-14 | 王祥槐 | Composition for changing and improving property of fiber surface and paper making method |
| US9284687B2 (en) * | 2011-09-09 | 2016-03-15 | Novozymes A/S | Properties of paper materials |
| WO2014058557A1 (en) * | 2012-10-10 | 2014-04-17 | Buckman Laboratories International, Inc. | Methods for enhancing paper strength |
| CN108755216B (en) * | 2018-05-07 | 2021-04-13 | 希杰尤特尔(山东)生物科技有限公司 | Method for improving strength of broad leaf pulp fibers by using complex enzyme |
| CN109082936B (en) * | 2018-08-16 | 2020-11-24 | 内江师范学院 | Paper surface sizing agent and preparation method thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1187912B (en) * | 1961-10-31 | 1965-02-25 | Basf Ag | Use of polyvinyl sulfonic acid or its salts in paper sizing |
| US3809605A (en) * | 1972-10-30 | 1974-05-07 | American Cyanamid Co | Fibrous mats and sheets containing immobilized enzymes entrapped in their interstices |
| FI82734C (en) * | 1987-12-07 | 1991-04-10 | Enso Gutzeit Oy | Process for making a paper or board product and a product produced by the process |
| US5340731A (en) * | 1988-07-08 | 1994-08-23 | University Of British Columbia | Method of preparing a B-1,4 glycan matrix containing a bound fusion protein |
| WO1993005226A1 (en) * | 1991-08-29 | 1993-03-18 | University Of British Columbia | Method for modification of polysaccharide fibres |
-
1995
- 1995-08-16 GB GBGB9516766.4A patent/GB9516766D0/en active Pending
-
1996
- 1996-08-16 CN CN 96197614 patent/CN1199439A/en active Pending
- 1996-08-16 BR BR9610327-2A patent/BR9610327A/en not_active Application Discontinuation
- 1996-08-16 PT PT96928510T patent/PT845060E/en unknown
- 1996-08-16 AU AU68248/96A patent/AU6824896A/en not_active Abandoned
- 1996-08-16 WO PCT/GB1996/002012 patent/WO1997007282A1/en active IP Right Grant
- 1996-08-16 EP EP96928510A patent/EP0845060B1/en not_active Expired - Lifetime
- 1996-08-16 CA CA 2229358 patent/CA2229358A1/en not_active Abandoned
- 1996-08-16 EP EP96927804A patent/EP0845031A1/en not_active Withdrawn
- 1996-08-16 BR BR9610219A patent/BR9610219A/en not_active Application Discontinuation
- 1996-08-16 CN CN 96197613 patent/CN1199421A/en active Pending
- 1996-08-16 JP JP9509064A patent/JPH11510701A/en active Pending
- 1996-08-16 WO PCT/GB1996/002009 patent/WO1997007203A1/en not_active Application Discontinuation
- 1996-08-16 CA CA 2229588 patent/CA2229588A1/en not_active Abandoned
- 1996-08-16 JP JP9509067A patent/JPH11510861A/en active Pending
- 1996-08-16 AU AU67502/96A patent/AU6750296A/en not_active Abandoned
- 1996-08-16 DE DE69610841T patent/DE69610841T2/en not_active Expired - Fee Related
- 1996-10-19 TW TW085112821A patent/TW353092B/en active
Also Published As
| Publication number | Publication date |
|---|---|
| EP0845060B1 (en) | 2000-11-02 |
| WO1997007203A1 (en) | 1997-02-27 |
| DE69610841T2 (en) | 2001-03-01 |
| EP0845031A1 (en) | 1998-06-03 |
| CN1199439A (en) | 1998-11-18 |
| TW353092B (en) | 1999-02-21 |
| CA2229588A1 (en) | 1997-02-27 |
| DE69610841D1 (en) | 2000-12-07 |
| BR9610219A (en) | 1999-06-15 |
| GB9516766D0 (en) | 1995-10-18 |
| AU6750296A (en) | 1997-03-12 |
| JPH11510861A (en) | 1999-09-21 |
| WO1997007282A1 (en) | 1997-02-27 |
| AU6824896A (en) | 1997-03-12 |
| BR9610327A (en) | 2005-09-06 |
| JPH11510701A (en) | 1999-09-21 |
| PT845060E (en) | 2001-03-30 |
| EP0845060A1 (en) | 1998-06-03 |
| CN1199421A (en) | 1998-11-18 |
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