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

Definitions

• the present invention relates to methods and compositions for sizing paper.
• the present invention relates to the use of a protein capable of binding to paper or a constituent of paper to size paper.
• the paper manufacturing process conventionally comprises the following steps: (l) 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 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, emulsions or suspensions which render the paper treated with the 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.
• the principal constituent of paper is cellulose.
• Cellulose may be in the form of wood fibre or annual crop 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.
• Xylanose 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, chitinases, ligninases, agarases, alginases, xylanases, mannases, amylases, cellulases and hemi-cellulases.
• cellulases and he i-cellulases cleave saccharide or polysaccharide molecules from cellulose and hemi-cellulose, respectively, and amylases cleave glucose from starch.
• binding domains of such proteins can be separated from the active-site domains by proteolysis.
• the isolated binding domains have been shown to retain binding capabilities (Van Tilbeurgh, et al . , FEBS Letters, 204(2) . 223-227, August 1986).
• Use of cellulose binding domains of cellulases has been proposed as a means of roughening the texture of the surface of cellulosic support, while use of cellulase active-site domains has been proposed as a means of smoothing the texture of such surfaces (International patent application WO93/05226) .
• binding domains have also been characterised at the genetic level (Ohmiya et al.,Microbial Utilisation of Renewal Resources, 8., 162-181, 1993) and have been subcloned to produce new fusion proteins (Kilburn et al .. Published International Patent Application WO90/00609; Ong et al . , Enzyme Microb. Technol, 1J3, 59-65, January 1991; Shoseyov et al.. Published International Patent Application W094/24158) . Some of these fusion proteins have then been used as anchor proteins for specific applications.
• Such proteins have been used as an aid to protein purification through adhesion of the fusion proteins to cellulosic support materials used in protein purification strategies (Kilburn et al . , United States Patent 5,137,819; Greenwood et al . , Biotechnology and Bioengineering, 44., 1295-1305, 1994) .
• the ability to immobilize fusion proteins onto cellulosic supports has also been suggested as a means of immobilization for enzyme bioreactors (Ong et al . , Bio/Technology, l_, 604-607, June 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 Application W093/21331) .
• proteins capable of binding to paper or a constituent of paper may be used to size paper.
• 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.
• 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.
• 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.
• the present invention provides a method of sizing paper.
• 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 proportions and optionally mixed with fine particles of inorganic materials such as oxides, carbonates and sulphates of metallic elements in various proportions.
• paper includes paperboard which refers to paper when the weight of the paper sheet or web is greater than 200g/m 2 .
• Vegetable sources of cellulose include wood, straws. Bagasse, Esparto, bamboo, Kanaf, Grass, Jute, Ramie, Hemp, Cotton, Flax.
• the crude vegetable derived cellulose is processed to form pulp, the material from which paper is made, either mechanically, chemically or both.
• Cellulose containing pulps may be described as mechanical, chemimechanical and chemithermomechanical, semi chemical, high yield chemical, full chemical (see “Pulp and Paper, Chemistry and Chemical Technology", Third Edition, Volume 1 pages 164, 165 edited by James P. Cassay ISBN 0-471-03175-5 (v.1)) 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 polysaccharides including hemi-celluloses such as xylanose, mannose and arabinose.
• proteins such as keratin, starch (including anionic, cationic or amphoteric starch) , pectin, guar, chitin, lignin, agar, alginate as well as other polysaccharides including hemi-celluloses such as xylanose, mannose and arabinose.
• the method of the present invention comprises contracting paper or a constituent of paper with a protein capable of binding to the paper or constituent 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.
• 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" 3 M.
• Kd dissociation constant of
• protein includes peptide, oligopeptide and polypeptide, as well as protein residues, protein-containing species, chains of amino acids and molecules containing a peptide linkage.
• a protein means a protein residue.
• the protein may comprise a naturally occurring protein, or fragment thereof or modified protein obtainable by chemical modification or synthesis or by expression of a genetically modified gene coding for the protein.
• modified protein includes chemical analogs of proteins capable of binding to paper or a constituent thereof.
• the protein comprises a naturally occurring enzyme or fragment thereof which is capable of binding to paper or a constituent of paper.
• 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.
• amylases include ⁇ - amylases, for example from Aspergillus oryzae (available as a Type X-A crude preparation from Sigma Aldrich Co Ltd) , and a yloglucosidases, for example from Aspergillus niger (available from Sigma Aldrich Co Ltd) .
• the protein comprises a protein capable of binding 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 expression of a genetically modified gene coding 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.
• cellulases examples include 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 Sigma 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) .
• the protein may be produced by recombinant DNA techniques as disclosed in, for example, International Patent application W094/24158.
• Cellulases generally comprise a cellulase binding domain and a domain responsible for cellulase activity.
• the present invention may employ the cellulase as a whole or a fragment thereof capable of binding to cellulose.
• a cellulase binding domain may be obtained from whole cellulase by treatment with protease(s) , such as papain.
• the cellulase may comprise an exo-cellulase or an endo-cellulase.
• Exo-cellulases 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 Humicola isolens .
• Endo-cellulases also known as Beta-1,4-
• Endoglucanases Endo-l,4-D-glucanases; 1,4-Beta-D-glucan glucanohydrolases; EC 3.2.1.4) cleave internal beta-1,4- glycosidic bonds yielding a mixture of glucose, cellobiose and other soluble cello-oligosaccharides.
• endo cellulases 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 endoglucanses (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 treasuretrove for biotechnology", Trends in Biotechnology 12:379-386.
• the protein employed in the present invention comprises cellulase obtainable from Humicola isolens (available as Celluzyme® from Novo Nordisk A/S, Bagsvaerd, Denmark) or cellulase obtainable from Trichoderma reesei (available as Celluclast® from Novo Nordisk A/S, Bagsvaerd, Denmark) . More preferably, the protein comprises cellulase obtainable 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.
• 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.
• the protein may be added to the formed paper product, for example, by immersing the paper in a bath containing the protein or by any suitable spraying, spreading, brushing, coating or printing process.
• the invention further 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.
• 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 to 20% by weight, more preferably 1 to 10% by weight.
• 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, alkalis, detergents (such as Tween®) , water soluble organic substances (such as alphatic alcohols) and chaotropic ions (such as I", C10 4 ", SCN “ , Li + , Mg 2+ , Ca 2+ and Ba 2+ ) .
• sizing is achieved by heating the paper.
• the paper may be heated at a temperature of 50 ⁇ C to 200°C, more preferably 70°C to 170°C, more preferably 80°C to 110°C, more preferably 100°C to 110°C.
• 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 temperatures.
• the length of time of heating required depends upon the temperature at which the paper is heated, longer times being required at lower temperature.
• 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.
• Pulp Water-leaf paper pulp was prepared by adding 10 g of 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 suspension
• a cellulase selected from the following:
• Humicola insolens - Cellulase derived from Humicola insolens available as Celluzyme® from Novo Nordisk A/S, Bagsvaerd, Denmark (875 ⁇ l corresponding to 8.7% weight per weight cellulose binding protein to dry weight pulp) .
• Trichoderma reesei available as Celluclast® from Novo
• Trichoderma viride Cellulose derived from Trichoderma vivide available from BDH Ltd UK.
• Incubation 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 cm 2 ) produced using a laboratory-designed paper making apparatus operated in the following manner: a suspension of paper pulp (0.2% wv "1 ) was poured into a plastic filter holder which houses a fine nylon filter mesh. By applying a vacuum for a few seconds the pulp was formed into a paper 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/Heating Paper sheets were dried in one of the following ways (a) air drying;
• drum drier - typically operating at a constant temperature of 80°C to 108°C with a contact time of 40 to 250 s;
• a flat aluminum plate was used to press the paper test sheets (sandwiched between blotting paper) against the hot plate.
• Test protocols The dried sheets were assessed for sizing by one or more of the following tests:-
• HST Hercules Size Test
• Trichoderma reesei cellulase preparation 70 ⁇ l Celluclast*, corresponding to 4.4 % ww '1 cellulose binding protein based on dry weight of cellulose fibre
• Trichoderma reesei cellulase preparation 70 ⁇ l Celluclast*, corresponding to 4.4 % ww '1 cellulose binding protein based on dry weight of cellulose fibre
• 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.
• the dried sheets were then assessed for sizing by the Ink Drop Test (IDT) .
• IDT Ink Drop Test
• test papers prepared under different drying regimes were prepared under different drying regimes.
• Tris-HCl 50 mM, pH 7.5, 10 ml
• the Humicola insolens cellulase preparation 875 ⁇ l
• the pulp samples were vortex mixed and diluted to 100 ml.
• Test papers were prepared in the standard manner and dried by a single pass through a drum drier lOO°C/250 s.
• the degree of sizing achieved by the different methods was assessed using the IDT method.
• Standardized 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 Humicola insolens ; Table 3) was added and the mixture vortex mixed. The pulp was incubated with gentle shaking 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. Each 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.
• Table3 Sizing achievedusing either Trichoderma reesei (Celluclast , Novo NordiskA/S, Bagsvaerd,
• Hmico.a insolens (CeUuzvme , Novo Nordisk A/S, Bagsvaerd, Denaark) cellulase preparation
• Example 4 The effects of buffer omission and high (160°C) 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.
• Table 4 Effect of temperature on the sizing achieved by either Trichoderma reesei cellulase 5 (Celluclast*, Novo Nordisk A/S, Bagsvaerd, Denmark) or Hunicola insolens cellulase (Celluzyme , Novo Nordisk A/S, Bagsvaerd, Denmark)
• Celluclast 4 9.4 Tris-HCl 160°C/30 ++ S + 108°C/250 s
• Celluclast 9.4 dH 2 0 108°C/250 +++ s
• HST Hercules Sizing Test
• Trichoderma 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.
• 2 % (wv" 1 ) pulp stock was incubated with 5 % (ww -1 ) 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 following post manufacture heat treatments: naturally aged for 24 h; 80°C for 10 min and 105°C for 10 min.
• Example 6 A Cellulase preparation from Trichoderma 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 distilled water) . The cellulase addition level was adjusted such that an equivalent cellulose binding protein 5 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) .
• thermocellum (NCIMB 10682) was grown on 1.0 % (wv _1 ) pulp in growth medium, comprising: 1000ml Basal Medium ((gl-1) yeast extract, 10; KH 2 P0 4 , 1.5; K 2 HPO 4 .3H 2 0, 2.9; (NH 4 ) 2 S0 4 , 1.3; 5 and FeS0 4 .7H 2 0, (1.0% wv _1 ) 1.0ml "1 ) with a cellulose source (pulp @1% wv" 1 ) which is then autoclaved to sterilize.
• Basal Medium ((gl-1) yeast extract, 10; KH 2 P0 4 , 1.5; K 2 HPO 4 .3H 2 0, 2.9; (NH 4 ) 2 S0 4 , 1.3; 5 and FeS0 4 .7H 2 0, (1.0% wv _1 ) 1.0ml "1 ) with a cellulose source (pulp @1% wv" 1 ) which is then
• the culture fluid was tested as a sizing agent by making paper using the Cl . thermocellum cultures.
• Water-leaf pulp (10% (wv -1 ) ; 2.16 g) was weighed into five 250 ml flasks.
• 0 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 standard paper making 5 method.
• the paper was dried at 80°C for 250 s using the drum drier. Sizing was measured the following day using the standard IDT method.
• thermocellum growth medium was added to the water-leaf pulp instead of the Cl . thermocellum culture fluid.
• 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 0 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 5 distilled water as compared 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. 0
• Trichoderma reesei cellulase preparation was employed ("Celluclast 1.5L" supplied by Novo Nordisk Bioindustry S.A. 92017 Nanterre Cedex. France).
• 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:
• the cellulase solution was added to the thick (2% consistency) stock.
• Two litres of the thick stock (containing 40g of fibre) was contained in a metal jug and stirred at the lowest possible speed to achieve a slow movement of the stock. Vigorous agitation should be avoided otherwise denaturing of the enzyme may occur during the incubation period.
• the stock was at ambient temperatures (20-25 * C).
• the incubation time was fifteen minutes. During this incubation period the movement of the stock may appear to become easier/faster. If this is apparent then reduce the stirrer speed as much as possible.
• 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.
• the white water box was filled with DEMI water for handsheet formation.
• the handsheet 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.
• the wet mat and handsheet wire were removed from the mould to the press.
• the moisture content of the pressed sheet should be 70%.
• the pressed sheet was then dried on an electrically heated drum dryer.
• the surface temperature of the dryer was 105 * C and the speed of the dryer was such that the pressed sheet was in contact with the hot surface for 35 seconds.
• the final moisture content of the sheet should be between 4 and 7% (typically 5%) .
• 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 sheet. Steps should be taken to avoid this.
• the surface temperature of the drum dryer is below 70'C, it is necessary to extend the contact time further or increase the initial pressing on the wet mat to remove more water or to do both. It is possible to reduce the moisture content of the pressed sheet to less than 60%.
• “oven cured” refers to treatment at 80 ⁇ C for 30 minutes.
• HAT seconds of handsheets made with Trichoderma reesei
• 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 using a starch glucose-release assay.
• the binding efficiencies of each protein were determined against a range of starches with BSA controls included in the assessment.
• 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.
• 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 "1 protein as measured by the Coomassie Blue technique. 100 ⁇ l 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. l ml fractions were collected and tested for reducing sugars released from starch suspensions as above.
• the sample was centrifuged at 13,000 rpm for 5 min and 100 ⁇ l samples loaded onto the HPLC column.
• the binding of amyloglucosidase was also tested against cationic starch.
• BSA was also used in the same way as a control. The final concentration of the BSA used was 0.2% (wv -1 ) in 0.1 M PBS.

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