Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ME~HODS AND CHEMICA~ COMPOUNDS FOR MODIFYING POLYMER~
1 Technical Field
The present invention relates to methods and chemical
compounds for modifying the physical properties of a
polymer. In particular, the present invention relates to
methods and chemical compounds for modifying the physical
properties of a polymer by binding to the polymer a chemical
compound, hereinafter referred to as an "effector moiety",
which confers on the polymer improved fluid, electrical or
strength properties.
2 Background
Polymers and materials containing polymers are a ubiquitous
feature of every day life. Naturally occurring polymers
include, for example, proteins (including keratin, which is
the principal component of wool), starch, pectin, guar,
chitin, lignin, agar, alginate, and polysaccharides such as
cellulose and hemi-celluloses (including xylan, mannose and
arabinose). Cellulose is encountered in the form of, for
example, wood fibre and annual crop fibre (for example,
hemp, straw, rice, flax, jute) based products such as paper,
and cotton, which may be in the form of fibres, yarns,
threads or a variety of woven and non-woven textile or
fabric products. Xylanose is the principal component of
xylan, otherwise known as hemi-cellUloSe which occurs in
grasses, cereal, straw, grain husks and wood. Starch occurs
in seeds, fruits, leaves, bulbs etc.
The physical properties of polymers and materials containing
polymers may be modified by a variety of chemical and
physical treatments. Such chemical and physical treatments
may be directed at modification of the polymer structure
~ itself or at modification of the bulk properties of the
material containing the polymer.
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-
The bulk properties of a material containing a polymer may,
for example, be modified by admixture to the material of
agents such as wet strength agents, dry strength agents or
other chemical compounds which modify the physical
properties of the material. Admixture of such chemical
compounds to the material typically does not bind the
compounds strongly to the polymer and problems may therefore
be experienced with wastage of the chemical compounds and
with the compounds leaching out of the material, resulting
in variations in the properties of the material. Leaching
out of the chemical compound may be reduced by a charge
balancing protocol in which the ionic charge of the chemical
compound is made equal and opposite to that of the polymer-
containing material. However, in practical systems, the
charge on both components varies widely requiring careful
and frequent control measures. The modifying effect of the
chemical compound may also rely on covalent binding to the
polymer in order to properly achieve a modifying effect. In
addition, promoters may be required to facilitate binding of
certain chemicals to the material.
Alternatively, the chemical compounds may be applied to the
surface of the material by, for example, immersion or
printing. Once again, however, the chemical compounds
typically do not bind to the surface of the material and
problems may be encountered with diffusion of the compounds
away from the intended site of application.
A variety of non-covalent binding interactions are known;
for example, the binding interaction between an antibody and
an antigen and the binding interaction between biotin and
avidin or streptavidin. Enzymes capable of modifying an
enzyme substrate also typically rely on a non-covalent
binding interaction with the enzyme substrate in order to
function.
one such class of enzymes comprise enzymes which degrade
polymers, for example proteinases, keratinases, chitinases,
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ligninases, agarases, alginases, xylanases, mannases,
amylases, cellulases 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: séguin~ Annu. Rev. Microbiol., 44,
219-248, 1990; cellulases and xylanases: Gilbert and
Hazelwood, Journal of General Microbiology, 139, 187-194,
1993). This group of enzymes include cellulases and hemi-
cellulases which comprise functionally distinct protein
domains. In particular, the domain responsible for
catalytic activity is structurally distinct from the
cellulose binding domain. These domains are evolutionarily
conserved sequences which are very similar in all such
proteins (Gilkes et al., Microbiological Reviews, 303-315,
June 1991).
The binding domains of such proteins can be separated from
the active-site domains by proteolysis. The isolated
binding domains have been shown to retain binding
capabilities (Van Tilbeurgh, et al ., FEBS Letters, 204(2),
223-227, August 1986). Use of cellulose binding domains of
cellulases has been proposed as a means of roughening the
texture of the surface of cellulosic support, while use of
cellulase active-site domains has been proposed as a means
of smoothing the texture of such surfaces (International
patent application W093/05226).
A number of binding domains have also been characterised at
the genetic level (Ohmiya et al . ,Microbial Utilisation of
Renewal Resources, 8, 162-181, 1993) and have been subcloned
to produce new fusion proteins (Kilburn et al., Published
International Patent Application W090/00609; Ong et al.,
Enzyme Microb. Technol, 13, 59-65, January 1991; Shoseyov et
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al., Published International Patent Application
WO94/24158). 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, 7, 604-607, June 1989; Le et al. Enzyme
Microb. Technol., 16, 496-500, June 1994), and as a means of
attaching a chemical "tag" to a cellulosic material
15 (International Patent Application W093/21331).
3 S~ 5~ry of the Invention
According to the present invention there is provided a
method of treating a polymer to achieve an improvement in at
least one property selected from fluid, electrical and
strength properties comprising binding an effector moiety to
said polymer via a protein linkage for the purpose of
achieving said improvement, said effector moiety being
different from said protein linkage and said protein linkage
being different from said polymer, said effector moiety and
said protein linkage being present in an amount effective to
achieve said improvement.
It will be appreciated that the polymer may comprise a
polymeric molecule or a polymeric material comprising
polymeric molecules. Furthermore, reference to an effector
moiety and a protein linkage refers to at least one effector
moiety and at least one protein linkage, respectively.
Accordingly, the present invention encompasses a method of
treating a polymer to achieve an improvement in at least one
property selected from fluid, electrical and strength
properties comprising binding at least one effector moiety
to at least one polymer via at least one protein linkage for
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the purpose of achieving said improvement, said at least one
effector moiety being different from said at least one
protein linkage and said at least one protein linkage being
different from said at least one polymer, said at least one
effector moiety and said at least one protein linkage being
present in an amount effective to achieve said improvement.
According to a further aspect of the invention, there is
provided a method of treating a polymer to achieve an
improvement in at least one property selected from fluid,
electrical and strength properties comprising contacting
~ said polymer with an effector moiety and a protein for the
purpose of achieving said improvement, said effector moiety
being different from said protein and also different from
said polymer, and said protein being different from said
polymer, and said effector moiety and said protein being
present in an amount effective to achieve said improvement.
The invention encompasses a method of treating a polymer to
achieve an improvement in at least one property selected
from fluid, electrical and strength properties comprising
contacting at least one polymer with at least one effector
moiety and at least one protein for the purpose of achieving
said improvement, said at least one effector moiety being
different from said at least one protein and also different
from said at least one polymer, and said at least one
protein being different from said at least one polymer, and
said at least one effector moiety and said at least one
protein being present in an amount effective to achieve said
improvement.
According to a further aspect of the present invention there
is provided a chemical composition comprising:
a) an effector moiety; and
b) a protein capable of binding said effector moiety to
a polymer;
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wherein said effector moiety is different from said protein
and wherein said composition is capable of achieving an
improvement in at least one property selected from fluid,
electrical and strength properties of said polymer.
The invention further provides composition of matter
comprising a polymer to which is bound an effector moiety
via a protein linkage, said effector moiety being different
from said protein linkage, wherein said effector moiety and
said protein linkage are present in an amount effective to
achieve an improvement in at least one property selected
from fluid, electrical and strength properties of said
polymer.
According to a further aspect of the invention there is
provided method of treating paper or the constituent fibres
of paper to achieve an improvement in at least one property
selected from fluid, electrical and strength properties
comprising binding at least one effector moiety to said
paper or constituent fibres of paper via at least one
protein linkage for the purpose of achieving said
improvement, said at least one effector moiety being
different from said at least one protein linkage and said at
least one protein linkage being different from said paper or
constituent fibres of paper, and said at least one effector
moiety and said at least one protein linkage being present
in an amount effective to achieve said improvement.
4 Detailed Description of the Invention
The present invention provides methods and chemical
compounds for modifying the fluid, electrical and/or
strength properties of a polymer or material containing a
polymer by binding to the polymer an effector moiety capable
of conferring the desired property.
The term polymer includes reference to materials containing
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a polymer. The polymer-containing material may consi~t
exclusively of polymer or may comprise polymer in
combination with other components.
~ 5 The polymer may comprise any polymer of any number of
monomeric units. Preferably, the polymer comprises a
naturally occurring polymer or a chemically modified
derivative thereof. The naturally occurring polymer may,
for example, comprise a protein such as keratin, or a
polysaccharide such as a starch, pectin, guar, chitin,
lignin, agar, alginate. Preferably, the polymer comprises
a polysaccharide. The polysaccharide may comprise any
polysaccharide, for example, mannose, xylanose, cellulose or
a hemi-cellulose, preferably cellulose. Materials
comprising cellulose may comprise wood-fibre or annual crop
fibre (for example, hemp, straw, rice, flax, jute) based
material, such as paper. Alternatively, the material may
comprise cotton in the form of fibre, thread or woven or
non-woven textile, fabric or cotton-based paper.
Preferably, the material comprises paper.
The present invention may be employed to modify any fluid,
electrical or strength property of the polymer. Properties
of the polymer that may be modified include wet strength and
dry strength, sizing, hydrophobicity, dye resistance and
stain resistance, fluid penetration, oil and water
repellency, electrical conductivity and resistance,
electrical capacitance, pH and biometallic properties.
The protein employed in the present invention may comprise
any protein capable of binding to the polymer. Preferably,
the protein is capable of binding the polymer with a
dissociation constant of (Kd) less than 1 x 10-3M. As used
herein, the term "protein" includes peptide, oligopeptide
and polypeptide, as well as protein residues, protein-
containing species, chains of amino acids and molecules
containing a peptide linkage. Where the context requires
(for example, when protein is bonded to another molecule),
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reference to a protein means a protein residue. The term
"protein linkage" refers to a protein or protein residue via
which an effector moiety is bound to a polymer. The protein
may comprise a naturally occurring protein, or fragment
thereof or modified protein obtainable by chemical
modification or synthesis or by expression of a genetically
modified gene coding for the protein. As used herein the
term "modified protein" includes chemical analogs of
proteins capable of binding to a polymer. Examples of
proteins capabie of binding polymers are well known and
include enzymes selected from the group comprising
cellulases, hemi-cellulases, mannases, xylanases,
proteinases, keratinases, chitinases, ligninases, agarases,
alginases and amylases. For example, a variety of
cellulases are known which are dependent upon binding to
cellulose for their activity. Examples of such cellulases
are those isolable from bacterial organisms such as
Cellulomonas fimi and fungal organisms such as Trichoderma
viride, Aspergillus niger, Penicillium funiculosum,
Trichoderma reesei and ~umicula insolens, available as
commercial preparations from Sigma Chemical Sigma-Aldrich
Company Ltd., Novo Nordisk A/S, BDH Ltd., or ICN Biomedicals
Ltd. Alternatively, the protein may be produced by
recombinant DNA techniques as disclosed in, for example,
International Patent application W094/24158. Cellulases
generally comprise a cellulase binding domain and a domain
responsible for cellulase activity. The present invention
may employ the cellulase as a whole or a fragment thereof
capable of binding to cellulose. A cellulase binding domain
may be obtained from whole cellulase by treatment with
protease(s)l such as papain. The present invention may
employ an exo-cellulase or an endo-cellulase.
Preferably, the protein comprises a naturally occurring
enzyme which is capable of binding to the polymer. More
preferably, in respect of paper, the catalytic activity is
deactivated. The catalytic activity of the enzyme may be
deactivated by, for example, attachment of the effector
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moiety or cross-linking of the enzyme. Cross-linking of ~he
enzyme may be achieved with any suitable protein cross-
linking agent such as a dialdehyde such as glutaraldehyde.
Preferably, the protein comprises a deactivated naturally
occurring cellulase.
The effector moiety may be attached to the protein capable
of binding to the polymer in any convenient manner. For
example, the effector moiety may be covalently bonded
directly to the protein, via suitable reactive functional
groups in the effector moiety and protein. Recognition of
suitable reactive functional groups and, if necessary, their
chemical modification to facilitate covalent bonding are
within the ability of a person of ordinary skill in the art.
Examples of covalent bond formation include formation of an
amide bond between a carboxyl group and an amine group, by
means of carbodiimide or dimethyl formamide activation of
the carboxyl group.
The effector moiety may be attached to any suitable part of
the polymer binding protein. The effector moiety may be
attached to the polymer binding protein at the N-terminal
end of the protein, for example via the N-terminal amino
group. Alternatively, it may be attached at the C-terminal
end of the protein, for example via the C-terminal carboxyl
group. Alternatively, the effector moiety may be attached
to the protein via an alternative functional group present,
for example, in the amino acid chain of the protein or in a
side chain thereof or introduced into the protein for the
purpose of attachment to the effector moiety. The effector
moiety may, for example, be attached via a thiol group
present in cysteine, a hydroxyl group present in serine or
threonine, an amino group present in lysine or arginine, an
amide group present in asparagine or glutamine, a carboxyl
~ 35 group present in aspartic acid or glutamic acid or an
aromatic or heteroaromatic group present in phenylalanine,
tyrosine, tryptophan or histidine, or derivatives thereof.
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The effector moiety may be attached to the protein via a
linker. The linker may, for example, comprise a
difunctional molecule capable of reacting with a reactive
site of the protein and a reactive site of the effector
moiety so as to link the protein and effector moiety. It
may be advantageous to include such a linker as a spacer
between the protein and effector moiety, so that the two
species are sufficiently spaced apart so as not to interfere
sterically with each other's activity. A linker may also be
advantageous in providing suitable functional group with
which to join the effector moiety and protein.
Alternatively, or as part of a linker, the effector moiety
may be attached to the protein via a non-covalent binding
pair of molecules. Examples of such non-covalent binding
pairs of molecules include biotin and avidin, streptavidin
or neutralite.
Accordingly, one possibility is that the effector moiety is
covalently attached to streptavidin whilst the polymer
binding protein is covalently attached to biotin. Combining
these components facilitates binding of the streptavidin and
biotin portions of each component and hence attachment of
the effector moiety to the polymer binding protein. It will
be appreciated that the effector-streptavidin component may
be mixed with the protein-biotin component either before or
after the protein component has been bound to the polymer.
It will be further appreciated that alternatively the
effector moiety may be covalently attached to biotin, whilst
the protein is covalently attached to avidin, streptavidin,
or neutralite.
It will be appreciated that more than one type of effector
moiety may be attached to the polymer. Two or more types of
effector moiety may be used in order to reinforce each
other's effect or to provide two or more effects
simultaneously.
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11
It will be appreciated that in general the effector moiety-
may be attached to the polymer binding protein either before
or after the polymer binding protein is bound to the
polymer. The method of the present invention may comprise
~ 5 contacting a conjugate of the effector moiety and the
protein with the polymer, or may comprise contacting the
effector moiety with a comjugate of the protein and polymer.
Alternatively, attachment of the effector moiety to the
protein and attachment of the protein to the polymer may be
accomplished in Situ in a one-step process.
The present invention is not limited as to the precise
nature of the manner in which the effector moiety is bound
to the protein linkage and the protein linkage is bound to
the polymer. Binding may be by means of a chemical bond
such as a covalent bond or by means of a non-covolent
physical interrelation, tie, association, attraction or
affinity.
The effector moiety may comprise any moiety capable of
conferring a desired physical property. The effector moiety
may comprise an atom, molecule or chemical compound or
residue thereof capable of conferring the desired physical
property. In one embodiment the effector moiety comprises
a chemical compound capable of conferring a desired physical
property. For example, the agent may comprise a wet
strength agent such as an aldehyde eg glutaraldehyde or
dialdehyde starch or its cationic derivative, polyamide
resin, polyacrylamide copolymer glyoxal, glyoxylated
polyacrylamide,polyethyleneimine,polyamineepichlorohydrin
polymers, polyamidoamine epichlorohydrin polymers, urea
formaldehyde and melamine formaldehyde polymers, synthetic
latexes, formaldehyde modified proteins or other polymers
used for the purpose of imparting wet strength to paper; a
dry strength agent such as starch, anionic or cationic
starch, polyacrylamide, amphoteric, cationic or anionic
polyacrylamide copolymers, anionic or cationic guar, locust
bean gum or anionic or cationic modifications thereof,
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polyvinyl alcohol, carboxymethyl cellulose; a sizing agent
such as rosin acids including abietic acid, adducted rosin
acids including saponified fumaric acid gum rosin adduct,
derivatives of rosin acids including tall oil, fatty acids
including myristic acid, palmitic acid or stearic aci~,
other hydrophobic agents including alkenyl succinic
anhydride (ASA) or 2-oxetanone compounds such as alkyl or
alkynyl ketene dimer or multimer (AKD) or derivatives of ASA
or AKD, gum, adducted gum, wood or tall oil rosin, saturated
or unsaturated carboxylic acids with linear or branched
chain lengths of from about 4 carbon atoms chain length to
30 carbon atoms chain length, alkyl ketene dimers made from
such carboxylic acids, alkyl succinic anhydride of chain
length from about 4 carbon atoms to about 30 carbon atoms,
fully or partially fluorinated carboxylic acids or alkyl
ketene dimer derived therefrom, fully or partially
fluorinated alkyl succinic anhydride; a dye resistance or
stain resistance agent; an oil or water repellant agent such
as fluorochemical including a fluorinated fatty acid or
fluorinated derivative of ASA or AKD; an agent capable of
conferring softness such as an agent capable of disrupting
cellulose hydrogen bonding including surfactants,
detergents, fatty amides or enzymatic agents such as
expansin (McQueen-Mason et al., Proc. Natl. Acad. Sci. USA,
91, 6574-6578 (July 1995)); an agent capable of conferring
electrical conductivity such as a metal; an agent capable of
conferring stiffness; an agent capable of conferring
absorbency; an agent capable of conferring hydrophilicity;
an agent capable of modifying density; a metallising agent;
an agent capable of modifying pH, such as a buffer (for
example, to impart resistance to acid degradation).
In another embodiment, the effector moiety may comprise a
cross-linking or matrix forming agent or residue thereof,
which may itself serve to modify the physical properties of
the polymer, or may serve to modify the properties of the
protein and hence the physical properties of the polymer, or
may serve to entrap a further agent capable of modifying the
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physical properties of the polymer. Preferred examples of
cross-linking matrix forming agents comprises dialdehydes,
such as glutaraldehyde. Dialdehydes such as glutaraldehyde
can for example form a matrix with a cellulase derived
protein. The cellulase/glutaraldehyde matrix confers
improved wet strength and dry strength on paper, sizes the
paper and/or may entrap further agents such as Tioi or
CaCo3 .
An extensive review of compounds useful in papermaking is
provided by Roberts et al. (Paper Chemistry, Chapman Hall
New York, 1991) the entire contents of which are
incorporated herein by reference. This reference
particularly reviews retention aids, wet strength additives,
dry strength additives, sizing agents and fillers.
As used herein, the term "paper" refers to any material in
the form of a coherent sheet or web, comprising an
interlaced network of cellulose containing fibres derived
from vegetable 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. 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
processed to form pulp, the material from which paper is
made, either mechanically, chemically or both. Cellulose
containing pulps may be described as mechanical,
chemimechanical and chemithermomechanical, semi chemical,
high yield chemical, full chemical (see "Pulp and Paper,
Chemistry and Chemical Technology", Third Edition, Volume 1
pages 164, 165 edited by James P. Cassay ISBN 0-471-03175-5
(v.l)) according to the method of pulp preparation and
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14
purification.
The effector moiety may be attached to the polymer at any
suitable stage in the manufacture and processing of the
polymer or material containing the polymer.
If the effector moiety is to be applied to paper, it may be
attached 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. Alternatively, the
effector moiety may be attached to the formed paper product
by immersing the paper in a bath containing the reagents for
attaching the effector moiety or by any suitable spraying,
spreading, brushing, coating or printing process.
If the effector moiety is to be attached to cotton, it may
again be attached at any stage in the processing of the
cotton fibre. It may be attached to cotton fibre, thread,
yarn or to woven or non-woven cotton fabric or textiles.
The effector moiety may be attached by immersing the
material in a bath containing the reagents for attaching the
effector moiety or by any suitable spraying, spreading,
brushing, coating or printing process.
By choosing the point in the manufacture of the polymer or
material containing the polymer at which the effector moiety
is attached, control may be exercised as to whether the
effector moiety is distributed throughout the polymer
material or is substantially restricted to the surface
levels of the material.
In cases where the effector moiety is directed at modifying
the bulk properties of the material, it may be advantageous
to ensure even distribution of the effector moiety uniformly
throughout the material. Accordingly, the effector moiety
should be attached at an early stage in the manufacture. For
example in the manufacture of paper where the effector
SUBSTITUTE S~E~T ~aJLE 26)
:
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moiety is directed at modifying the bulk properties of the
paper, the ef~ector moiety should be applied at the pulp
stage.
In cases where the effector moiety is directed at modifying
the surface properties of the material, it may be sufficient
to restrict the effector moiety to the surface levels of the
material, with an attendant advantage in reducing the
quantities of effector moiety required. Accordingly, the
effector moiety should preferably be supplied at a late
stage in the manufacture. For example, in the manufacture
of paper, where the effector moiety is directed at modifying
the surface properties of the paper the effector moiety
should be applied to the paper surface.
Depending on the application it may be desirable to apply
the effector moiety to one or both planar surfaces of the
paper. Treating both surfaces of the paper with for example
an effector moiety comprising a wet strength agent, whilst
leaving one or more of the edges untreated, facilitates
preparation of a sandwich structure, in which a layer of
paper having poor wet strength properties but good liquid
absorption properties is sandwiched between two layers of
paper having good wet strength properties. Such a structure
is capable of transporting liquids through its middle layer
by capillary action and is particularly useful in the
manufacture of dip-stick type diagnostic assays.
A particular feature of the present invention concerns the
ability to modify the physical properties of the polymer or
material containing the polymer in a reversible ~nn~r.
Conventional treatment of polymers to impart particular
physical properties are often non-reversible. Furthermore,
the conventional treatments often render the polymer
unsuitable for recycling. In connection with recycling
paper, the repulping of paper is made more difficult and may
be impossible if the paper is treated with conventional wet
strength agents. The present invention lends itself to the
~iEST~TUTE SHEET tRlJLE 26~
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provision of means to permit release of the effector moiety
to permit recycling of the material. The effector moiety
may, for example, be released from the polymer-containing
material by treatment with a protease which cleaves the
protein attaching the effector moiety to the polymer;
alternatively, the effector moiety may be attached to the
protein by means of a selectively cleavable linker; cross-
linking agents such as aldehyde-substituted starch may be
cleaved by amylase.
A further advantage of the present invention lies in the
fact that the desired physical property is imparted
essentially immediately to the material. In conventional
treatments to impart wet strength to paper, heat treatment
and curing over several weeks may be re~uired.
The invention will now be described with reference to the
following figures and examples. In the Figures:
Figure 1 shows the effect of cellulase concentration on
glutaraldehyde cross-linked cellulase imparted wet strength.
Figure 2 shows the effect of glutaraldehyde concentration on
glutaraldehyde cross-linked cellulase imparted wet strength;
Figure 3 shows the effect of pH on glutaraldehyde cross-
linked cellulase imparted wet strength;
Figure 4 shows the effect of temperature on glutaraldehyde
cross-linked cellulase imparted wet strength;
Figure 5 shows the effect of incubation time on
glutaraldehyde cross-linked cellulase imparted wet strength;
Figure 6 shows the effect of pre-incubation time on
glutaraldehyde cross-linked cellulase imparted wet strength;
Figure 7 shows the effect of glutaraldehyde cross-linked
.
~U~STITU~E S~E~T (RULE 26)
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cellulase on the wet strength of paper produced from
different wood pulps.
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.
EXPF~R TM~NTAL
Principles and'Applications of Effector Moiety Attachment
The protocols defined below represent the techniques used to
characterize the use of cellulase as a biobridging agent for
the attachment of effector moieties to cellulose.
For initial stock PreParation one-third strength Phosphate
Buffered Saline (1/3 PBS) was used. The formulation for 1/3
PBS was as follows:
200 litres of deionized or demineralized water
(DEMI water)
197g of anhydrous sodium dihydrogen phosphate
( NaH2P04 )
767g of anhydrous disodium hydrogen phosphate
(Na2HP~4)
389g of sodium chloride (NaCl)
Anhydrous materials are not essential but the above
mentioned weights should be recalculated to take into
account any "water of crystallization" in the hydrated
salts.
The cellulases that have been used were derived from fungal
sources and are available either as aqueous solutions or
- 35 freeze dried powders.
Penicillium funiculosum
Cellulase derived from Penicillium funiculosum (Sigma
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18
Aldrich Co. Ltd., Poole, Dorset, U.K.) is availablç as a tan
powder and should be stored at below O C.
When used as an additive for handsheets the cellulase was
5 first be prepared as a 20% total solids solution in 1/3 PBS.
Into a large shallow beaker was placed 200g of the dry
enzyme preparation. To this was then added slowly 800g of
ll3 PBS. The mixture was gently stirred with a glass rod.
Vigorous agitation of the solution should NOT be used to
10 disperse the powder as denaturing of the enzyme may occur.
Any clumps of enzyme preparation may be broken up gently
with the glass rod. If the cellulase solution is prepared
the day before use then it should be stored at 4 C.
15 Trichoderma Reesei
Cellulase derived from Trichoderma Reesei is available
either as freeze dried powder from Sigma Aldrich Co. Ltd.
Poole, Dorset, U.K. or as an aqueous solution from Novo
Nordisk A/S, Bagsvaerd, Denmark. When using the powder, the
20 procedure and handling practises for preparation of the
aqueous solution with Penicillium funiculosum apply here as
well.
The cellulase solution was added to the stock on the basis
25 of the total protein content of the enzyme solution (e.g. 10
parts of dry protein per 100 parts of dry fibre). The total
protein content of the prepared cellulase solution was
determined by the W absorbance ( = 62Onm) of the protein
stained with Coomassie Brilliant Blue G250 dye (Sedmak and
30 Grassberg (Analytical Biochemistry, 79, 544-552 (1977)).
1. To assay for the binding of the cellulase to cellulose
Samples (typically between 25 to 500 mg and normally 100 mg)
35 of cellulose, such as microcrystalline cellulose (Avicel,
SigmaCell) or water-leaf paper pulp, were weighed into a
series of tubes/flasks.
SUBST~TUTE SHEET [RULE 26~
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,, 19
Cellulase solutions (typically containing between 200-600 mg
protein ml~l in 3 ml buffer), were added to each tube. The
exact concentration of protein added initially was
experimentally determined at the start of the binding assay
using the assay developed by Sedmak and Grassberg
(Analytical Biochemistry, 79, 544-552 (1977)).
The tubes were shaken at the desired temperature (typically
between 4~C and 30~C but usually at room temperature), for
a period of time (typically 1 to 90 min, usually between 5
to 15 min). Samples (0.5 - lml) were then taken for assay.
The samples were centrifuged in a 1 ml Eppendorf tube using
a bench-top microfuge for 5 min and the supernatant retained
for determination of protein concentration remaining in the
supernatant (unbound cellulase).
The supernatant protein concentration was subtracted from
the initial protein concentration thereby defining the
amount of cellulase associated with the cellulose pellet.
Bovine serum albumin (BSA) was used in the assay as a
control.
The results were presented as either the amount of protein
bound to the cellulose as a percentage of the protein added,
or as the amount of protein bound to the cellulose as a
percentage of the protein/cellulose (%w/w).
2. Visualization of the effector moiety attachment using
chemiluminescence
Pre~aration of cellulase for the ECL detection sYstem
1. Biotinylation of cellulase
A solution of biotinamido N-hydrosuccinimide ester
(BcapNHS) in N,N-dimethylformamide (DMF) was prepared (1
SUBST~TUTE SH~ RULE 26~
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mg ml~l). A solution of cellulase was prepared in
distilled water (77 mg ml~1).
lml of the cellulase solution was added to lml of the
BCapNHS solution and the mixture incubated for 2.5 h at
room temperature with shaking. The reaction was then
exhaustively dialysed against 500 ml 1/3 PBS buffer
(PBS, pH 7.5: Na2HP04, 11.5g; NaH2P04, 2.96g; NaCl, 5.84
g diluted to lL with distilled water) for 1 h.
2. Binding of the Biotinylated cellulase to paper sheets
[Application of the cellulase to the surface of a paper
sheet]
A water-leaf paper sheet, usually 2 cmZ, was incubated
with biotinylated cellulase at a range of concentrations
between 0.05 to 100 ~g ml 1 protein in 1/3 PBS (10 ml)
for 45 min to 2 h at 4~C in a shallow Petri-dish with
shaking. Experiments using PBS containing Tween 20
(0.1% vv~1) were also performed.
3. Binding of the biotinylated cellulase to paper pulp and
subsequent production of a paper sheet [Application of
the cellulase to the paper matrix~
Paper pulp was incubated with the biotinylated cellulase
in 1/3 PBS containing Tween 20 (0.1% vv 1) for 45 min at
room temperature with shaking. A disc of paper was
formed from the paper pulp-biotinylated cellulase using
the paper making filter. The paper disc was removed
from the filter, rolled and allowed to dry overnight.
4. Binding of HRP-labelled streptavidin to the biotinylated
cellulase
Binding of HRP-labelled streptavidin and ECL detection
of the biotinylated cellulase was subsequently performed
according to the manufacturer's recommendations
SUBSTITUTE SHEE~ (RULE 2~
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(Amersham Ltd., Amersham, U.K.; Whitehead, T.P. et al.,
Clin. Chem. 26, 1531-1546, 1979).
The paper was incubated with milk powder (4% wv~1) in PBS
for 45 min at e ither 4~C or room temperature with
shaking to block non-specific binding of the HRP-
streptavidin conjugate. The paper was then washed, 3x
3 min, using 0.5% (wv 1) milk powder in 1/3 PBS
containing Tween 20 (0.1%vv l). The solution was
discarded and replaced after each wash.
The horseradish peroxidase (HRP) - streptavidin
conjugate was prepared as a 1:1000 part solution using
milk powder (0.5% wv~1) made up in 1/3 PBS containing
Tween 20 (0.1%vv~1). A suitable volume (2 to 10 ml) was
added to cover the paper sheet which was then incubated
for 45 min at room temperature with shaking.
The paper was then washed 3x 5 min, in 1/3 PBS
containing milk powder (0.5% wv~l) and Tween 20 (0.1% w-
~). The wash solution was discarded and replaced after
each wash. The paper was then washed 3x 5 min using 1/3
PBS and again the wash solution was discarded and
replaced after each wash.
The cellulose bound cellulase-biotin-HRP-streptavidin
conjugate was then visualised by the ECL method or
quantified using the OPD methodology.
3. Enhanced chemiluminescence (ECL) method
It is necessary to carry out this method in a photographic
darkroom.
Amersham ECL Detection Reagents 1 + 2 were mixed together in
equal volumes (required approximately 0.13 ml cm2 paper).
Excess buffer was then drained from the paper and the
SUBSTiTUTE ~EET (RUL~ 26)
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22
detection reagents added to completely cover the paper
surface.
The paper was incubated for exactly 1 min at room
temperature without agitation. The detection reagent was
drained off and the paper was blotted between two pieces of
tissue paper to remove excess reagent. The blotted paper
was then transferred to a piece of cling film and wrapped
securely to remove any air pockets.
The paper was placed in a film cassette minimising the delay
between incubating the paper and exposing it to the
Hyperfilm. The film was carefully placed on top of the
paper and the film exposed for 15 s ensuring that the film
did not move during exposure. This first sheet of film was
then removed and immediately replaced with a second film
which was then exposed for 1 min.
The films were then immediately developed to visualize the
results. If necessary further sheets of film can be exposed
with exposures of 1 to 60 min.
4. The (OPD) method for quantification of effector moiety
bound to cellulose
The substrate buffer was prepared by dissolving 1 OPD tablet
(60 mg; o-phenylenediamine dihydrochloride, Sigma Chemicals,
UK) in 150 ml 0.06 M phosphate-citrate buffer (0.2 M
Na2HPO4, 121.5 ml; 0.1 M citric acid 121.5 ml made up to 500
ml distilled water and the pH adjusted to 5.0) to give a
final OPD concentration of 0.4 mg ml_1. Note that this
reagent is light sensitive. 10 ~l of fresh 30% H202 per 25
ml of substrate buffer was added immediately prior to use.
.
The paper samples containing the biotinylated cellulase were
placed into a 50 ml Falcon tube. 25 ml of the complete
substrate buffer solution was added to the tube and shaken
at room temperature for 30 s to 20 min, and usually between
SU~S~TUTE SHEET ~ULE 26)
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23
5 and 15 min, then the reaction was stopped by adding 1 ml
of 3M H2SO4. The absorbence was then determined at 492 nm
and reference was made to a standard curve of OD492 vs
biotinylated cellulase concentration in order to calculate
the concentration of biotinylated cellulase present on or in
the paper.
.
5. The coupling of paper effector moieties to enzyme
peptides using carbodiimide
Carbodiimides react with carboxylate groups to form
activated carboxyls. Amino groups then attack these
activated carboxyls to form covalent peptide bonds. This
chemistry can be used to attach paper effector chemicals
which contain free carboxyl groups to the amino groups on
peptides.
The carbodiimide chemistry used in the linkage of a paper
effect chemical to the cellulase was based on conventional
methodology (Hoare et al ., J. Biol. Chem, 242(10), 2447-
2453, 1967).
In the method described below, abietic acid was coupled to
cellulase.
Cellulase (21 mg ml~l) was dissolved in distilled water, and
abietic acid (100 mg) was dissolved in 25 ml of 10% (w-1)
methanol. 0.5 ml 1-(3-dimethylamino propyl)-3-ethyl
carbodiimide-HCl (WS-CDI; 63 mg ml~1) was added to 1.0 ml
abietic acid solution and the pH adjusted to pH 4.5 + 0.5
using HCl (0.1 N). The mixture was then stirred at room
temperature (5 min). 2 ml cellulase solution was then added
and the mixture left at room temperature with stirring (16
h).
The reaction was then stopped by the addition of sodium
acetate (0.1 M; pH 5.0) and excess abietic acid and WS-CDI
was removed by exhaustive dialysis in phosphate buffer.
SUBSTITU~E SH~ET ~ULE 26~
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24
The coupled cellulase was then used to bridge the abietic
acid onto cellulose as described above.
6. Demonstration of wet tensile strength properties using
glutaraldehyde cross-linked cellulase.
The application of glutaraldehyde cross-linked cellulase to
water-leaf paper pulp has been performed and demonstrated to
impart wet-strength properties to the paper sheet.
Water-leaf paper pulp slurry was produced in the following
manner: 10 g water-leaf paper was cut into 1 cmZ squares and
macerated in a domestic herb mill (CH100, Kenwood Ltd. UK)
for 3 min with 100 ml distilled water.
2.15 g of a water-leaf paper pulp slurry (10% wv~l)
containing 0.2 g cellulose was taken and the following
additions were made:
l 10 ml of 1/3 strength phosphate buffered saline (PBS),
pH 7.0 as a negative control.
2 10 ml of 1/3 PBS containing T. reesei cellulase (2 mg
ml-l )
3 10 ml of 1/3 PBS containing glutaraldehyde (25 ~l ml~l)
4a 10 ml of 1/3 PBS containing T. reesei (2 mg ml 1) and
glutaraldehyde (25 ~l ml~1) incubated together for 1 h at
room temperature prior to addition to the pulp
4b 10 ml of 1/3 PBS containing ~. reesei cellulase (2 mg
ml~1) and glutaraldehyde (25 ~l ml~1) added directly to
the pulp
All the samples were then incubated for 1 h at room
temperature on an orbital shaker before production of the
paper sheets.
To produce the paper sheets, the volume was increased to 100
ml with distilled water and paper sheets (6 cm2) produced
SUBSTITUTE S~IEET (~ULE 26)
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using a laboratory-designed paper making apparatus operated
in the following manner: a suspension of paper pulp (0.2%
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 adsorbent paper towels. The paper sheet
was carefully removed from the paper-making mesh, flattened
by rolling and allowed to dry overnight.
Wet-strength was determined in the following ways;
A Paper stability in water
Samples from each test paper sheet (1. 5 cm2 ) were placed in
Universal bottles and 25 ml distilled water added to each
one. The tubes were shaken and periodically examined for
signs of loss of integrity of the paper samples.
The results are given in Table 1
~U~ST~TUTE SHEET (R~JEE 26)
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26
Table 1 determination of the stability of the paper samples
in water
5 8smple Replicate Condition Initial Total
Number Number after shaking Disruption Disruption
shaking in
H20
lO 1 1 Disintegrated - -
2 Disintegrated
2 l Disintegrated
2 Disintegrated
3 1 Disintegrated
2 Disintegrated - -
4a 1 Intact <18 h 36 h
2 Intact <18 h 36 h
4b 1 Intact >8 d >8 d
2 Intact >8 d >8 d
In Table 1, "-" means not applicable.
B Paper strength
Samples from each test paper sheet (4 cm x 1 cm) were taken and
25 ~l of distilled water was pipetted across the middle of the
paper ensuring an even distribution. The paper was suspended
between two bull-dog clips and a container was secured to the
bottom clip. Water was added to the container and the weight
of water necessary to cause the paper to tear was determined.
The results are given in Table 2 and illustrate that the
samples prepared using glutaraldehyde cross-linked cellulase
demonstrated the greatest wet tensile strength.
Table 2 determination of paper strength
8 mple Replicate Added Weight
Number Number (g)
SUBSTITUTE SHEET lR~LE 26)
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27
1 l 27.43
2 43.48
2 l ~22.00
2 <22.00
3 1 <22.00
2 34.63
4a 1 66. 34
2 49.96
4b 1 >77. 33
2 64.20
The wet-strength of the paper samples were retested to include
BSA controls to assess the specificity of action of the
bridging protein. The paper samples were prepared as follows
1 10 ml of 1/3 strength phosphate buffered saline (PBS), pH7.0
2 10 ml of 1/3 PBS containing T. reesei cellulase (2 mg ml~1)
3 10 ml of 1/3 PBS containing glutaraldehyde (25 ,lLl ml~l)
4 10 ml of 1/3 PBS containing BSA (2 mg ml~1)
25 5 10 ml of 1/3 PBS containing T. reesei cellulase (2 mg ml 1)
and glutaraldehyde (25 ,ul ml~l) added to the pulp and
incubated for 1 h at room temperature on an orbital shaker
before production of the paper sheets
6 10 ml of l/3 PBS containing BSA (2 mg ml 1) and
glutaraldehyde (25 ~l ml~l) added to the pulp and incubated
for 1 h at room temperature on an orbital shaker before
production of the paper sheets.
The paper samples were placed in 50 ml Universal bottles with
25 ml water and vortexed using a laboratory mixer until
complete disintegration of the paper samples occurred. The
results are given in Table 3.
SUBSTITUTE SEIF~T (~JLE 26)
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28
Table 3 Determination of the stability of the paper when vortex
mixed in water.
5 8ample Replicate Time required until
Number Number complete disintegration
(sec)
1 1 <5
2 <5
2 l <5
2 <5
15 3 1 <5
2 <5
4 1 <5
2 <5
1 >1020
2 >1080
6 1 10
2 10
Although samples prepared using cross-linked BSA (Sample 6)
showed an increased wet tensile strength compared to the
controls, this was 100 fold less than that of the
- glutaraldehyde cross-linked cellulase.
To determine optimum conditions for glutaraldehyde/cellulase
treatment of paper to improve wet-strength, the following
parameters were varied in turn. For the purposes of this work
the control parameters were set at: cellulase (2 mg ml~l);
glutaraldehyde (0.6% vv~l) added separately to the pulp; pulp
suspended in buffer (p~ 7.0) at 25~C. The mix was incubated
SUBSTITUTE SHE~ (RULE 26)
CA 02229~88 1998-02-16
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29
for 60 min before paper sheet formation. Each parameter was
varied in turn as follows: cellulase (0.5 to 8 mg ml~l);
glutaraldehyde (0.1 to 2. 5 vv~l); pH (5.0 to 10.0); temperature
25~, 37~ and 45~C; incubation time (5 to 120 min); and time of
pre-incubation of the cellulase and glutaraldehyde (15 to 60
min).
All the paper sheets were allowed to dry overnight at ambient
temperature prior to wet tensile strength testing. The results
are illustrated in Figure 1 to 6.
7. Demonstration of Glutaraldehyde Cross-linked Cellulase
(GCC)-Wet ~ensile Strength Properties with Different Pulp
Types.
The GCC wet strength composition was applied to paper produced
from different types of pulp: ground wood pulp (GWP), chemo-
thermo-mechanical pulp (CTMP), hard wood pulp (HWP), soft wood
pulp (SWP) and water-leaf pulp (W-LP; 70~ HW: 30% SW). The
pulps were prepared in the usual manner, however, GWP and CTMP
pulps were soaked in water overnight before blending to promote
dispersion of the fibres.
The pulps were treated with either PBS buffer (10 ml); PBS
buffer (10 ml) + cellulase (20 mg) + glutaraldehyde (0.6%
vv~l). Then paper sheets (6 cmZ) were prepared from the pulp
samples as described above before wet tensile strength testing.
The results are given in Table 4 and illustrated graphically
in Figure 7.
The results indicate that there was an improvement in the wet
tensile strength of all the pulps tested. The final strength
of the paper sheets produced using either GWP or CTMP was
greater than that of the HWP, SWP and W-LP. The GCC
composition did however induce a greater percentage increase
in tensile strength in the HWP and SWP samples.
SU~ST~TUTE SHEET (RU~ 26~
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Table 4: Wet Tensile Strength of Paper Sheets Produced from
Different Pulps.
Pulp Treatment V o r t e x Wet Ten~ile
m i x i n g 1 S tr en g t h
(seconds) (g)
Ground Buffer 135 105.4
wood Cellulase 120 45.0
Glutaraldehyde 315 94. 5
Cellulase + > 1200 249
Glutaraldehyde
CTMP Buffer 480 78.5
Cellulase 420 88
Glutaraldehyde 420 71. 5
Cellulase + > 1200 242.5
Glutaraldehyde
20 HWP Buffer < 5 < 15.3
Cellulase < 10 < 15.3
Glutaraldehyde < 10 < 15.3
Cellulase + > 300 119
Glutaraldehyde
SWP Buffer < 5 < 15. 3
Cellulase < 5 < 15.3
Glutaraldehyde < 5 < 15.3
Cellulase + > 300 207
Glutaraldehyde
W-LP Buffer ~ 5 < 15. 3
Cellulase ND ND
Glutaraldehyde ND ND
Cellulase + > 300 193
Glutaraldehyde
l Time needed to complete disruption
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31
8. Demonstration of the reversibility of glutar~ldehyde cross-
linked cellulase imparted wet tensile strength
To determine the reversibility of wet strength imparted by the
glutaraldehyde cross-linked cellulase the following protease
~ solutions were prepared using commercial protease preparation
supplied by Sigma Chemical Sigma-Aldrich Company Ltd., Fancy
Road, Poole, Dorset, BH17 7NH: ficin (4~1 ml 1 solution in PBS
buffer at pH 6.5); papain (5~1 ml 1 solution in PBS buffer at
pH 6.5); Protease K (2.8 mg ml~l solution in PBS buffer pH
8.0); ~-chymotrypsin (1.0 mg ml 1 solution in PBS buffer at pH
8.0).
Paper squares (1.5 x 1.5 cm) prepared from water-leaf paper
pulp strengthened with glutaraldehyde cross-linked cellulase
were taken and incubated with the following treatments outlined
in Table 5.
SUBSTITUTE S~EET (RUL~ 26~
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32
Table 5 Wet strength reversibility treatments
8ample No. Treatment
5 1 Ficin (10 ml) + PBS buffer (pH 6.5)
2 Papain (10 ml) + 10 ml PBS buffer (pH 6.5)
3 Protease K (1 ml) + 19 ml PBS buffer (pH 8.0)
4 ~-chymotrypsin (1 ml) + 19 ml PBS buffer (pH 8.0)
Ficin (10 ml) + Papain (10 ml)
15 6 Protease K (1 ml) + ~-chymortrypsin (1 ml) +
18 ml PBS buffer (pH 8.0)
7 Ficin (10 ml) + Papain (10 ml) + Protease K (1
ml) + ~-chymotrypsin (1 ml)
8 0. 2 M Phosphate buffer pH 6.5 (20 ml)
9 0.2 M Phosphate buffer pH 8.0 (20 ml)
The samples were incubated at 30~C on an orbital shaker at 70
rpm. The samples were examined after 4h and 20h and were
vortex mixed for lO sec after 20 h if the paper was still
intact. The determinations were performed in duplicate and the
results are given in Table 6.
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33
T~ble 6 determination of paper disruption in the pres~nce of
various treatments
Treatment Incubation Time (h) After further
4 20 10 sec vortex
Ficin X XX XXXX
X XX XXXX
lO Papain ~ XX XXXX --_
XX XXXX ------
Protease K XX XX XXXX
XX XX XXXX
~-chymotrypsin O XX XXXX
o XX XXXX
Papain + Ficin XX XXXX ---
XX XXXX ---
~-chym + Prot K X XX XXXX
X XX XXXX
25 All 4 proteases XX XXXX ---
XX XXX XXXX
C o n t r o l O O ~
Phosphate O ~ ~ .
30 buffer (pH 6.5)
O O O
C o n t r o l O O ~
Phosphate
buffer (pH 8.0)
The key to the qualitative observations is given as an
arbitrary scale of O to XXXX where O represents no visible
SU~STITUTE SHEET (~ULE 26~
CA 02229~88 1998-02-16
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34
disruption of the paper and XXXX represents total paper
disruption; ~ represents previous 105s of paper integrity,
9. To determine the effect of protease treatment on the
recyclability of paper wet strengthened by glutaraldehyde
cross-linked cellulase
Paper sheets prepared either from water-leaf paper pulp (0.2
g) wet strengthened with glutaraldehyde cross-linked cellulase
or with pulp (~0.2 g) prepared without any wet strength agent
were taken and subjected to a series of treatments.
Treatment 1 A paper sheet (0.2 g) made from pulp wet
strengthened with glutaraldehyde cross-linked cellulase was cut
into 1 cm x 1 cm squares which were placed in a petri-dish with
20 ml 0.2 m phosphate buffer (pH 8.0) containing Protease K (14
mg). The sample was incubated at 37~C for 2 h with shaking (60
rpm). The squares were then removed and dipped into phosphate
buffer (pH 8.0) and placed in a universal bottle containing 20
ml fresh phosphate buffer (pH 8.0). The sample was then vortex
mixed to mascerate the paper. Any fibres left in the petri-
dishes after the original incubation were harvested by
centrifugation at 6,000 rpm, washed with phosphate buffer (pH
8.0) and added to the macerated sample in the universal bottle.
2 ml T. reesei cellulase (10 mg ml~l) and 0.5 ml glutaraldehyde
solution (25~) were added and the sample was incubated at 25~C
for 60 min. The sample was then used to form a new sheet of
paper.
Tre~tment 2 A water-leaf paper sheet (0.2 g) made from
pulp prepared with PBS without any wet strength agent was
placed in a universal bottle with 14 ml 1/3 strength PBS. The
sample was vortex mixed to macerated the paper and the pulp was
made into a fresh piece of paper.
Treatment 3 squares of paper made from pulp wet
strengthened with glutaraldehyde cross-linked cellulase (0.4
g) were mascerated in a blender with 30 ml of 1/3 strength PBS.
SUBSTITUTE S}~EET ~RULE 26)
CA 02229588 1998-02-16
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The resultant pulp was removed and made into a fresh square of
paper.
Treatment 4 A glutaraldehyde cross-linked cellulase
strengthened paper sheet (0.2g) was cut into 1 cmZ pieces and
mascerated in a blender in 30 ml of 1/3 strength PBS. 20 mg
T. reesei cellulase (10 mg ml~l) and 0.5 ml glutaraldehyde
solution (25~ vv~l) were added. The sample was incubated on an
orbital shaker for 60 min at room temperature. The pulp was
used to prepare a new sheet of paper.
Each paper sheet was allowed to dry overnight at ambient
temperature before testing the integrity of 1 cm2 samples to
destruction in water using the vortex mixer. The results of
the paper determination are given in Table 7.
Table 7: Integrity of Recycled Paper
8trategy Vortex mix Description of
(T ime, s) paper
1 300 Paper broken into
3 pieces
25 2 5 Paper totally
disintegrated
3 50 Lots of small
fragments
4 345 Small hole in
middle of paper
These results indicate that GCC - containing pulp, when made
into a new paper sheet, retains some wet tensile strength
properties; that pulp produced by protease treatment, as
opposed to physical disruption, generates stronger paper when
SUBSTITUTE SHE~T (RULE 26]
CA 02229~88 1998-02-16
W O 97/07203 PCT/~5G/'~2~09
36
recycled and that the further addition of GCC imparts the best
wet tensile strength properties to the recycled sheets.
10. Demonstration wet-strength, dry-strength and sizing in
paper following treatment with glutaraldehyde and
cellulase
Experiments were performed to determine the effect of
cellulase (protein ligand) and glutaraldehyde (effector
moiety) on the wet strength, dry strength and sizing of
paper. In the experiments, the following materials and
general protocols were employed:-
Cel l ul ase
An aqueous Trl choderma reesei cellulase preparation
was employed ("Cellulast 1.5L" supplied by Novo
Nordisk Bioindustry S.A. 92017 Nanterre Cedex.
France)
Glutaraldehyde
The glutaraldehyde used in the following examples
was a 25% aqueous solution commercially available
from Merck Ltd. (Poole, Dorset, U.K.)
- Stock Preparation
~xcept 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 l/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 fibres were dispersed by vigorous agitation.
This stock was then transferred to the Hollander
and beaten until a freeness value of 25OSR was
attained (time taken was usually 30 to 35 minutes).
The stock was then adjusted to a final consistency
of 2% with further l/3 PBS as necessary.
SUBST~TUTE SHE~T (E~UL~ 26)
CA 02229~88 1998-02-16
W O 97107203 PCT/~,"~2C~9
37
Addition/Incubation of Additives
Both the cellulase solution and glutaraldehyde
solution were added to the thick (2~ consistency)
stock. Two litres of the thick stock (containing
40g of fibre) was contained in a metal jug and
stirred at the lowest possible speed to achieve a
slow movement of the stock. Vigorous agitation
should be avoided otherwise denaturing cf the
enzyme may occur during the incubation period. The
stock was at ambient temperatures (20-25 C). The
cellulase solution was added first to the stock
(avoiding any splashing or splattering of the
solution). When one minute had elapsed from the
addition of the enzyme, the aqueous glutaraldehyde
was added.
The incubation time of the additives was fifteen
minutes, starting from the end of enzyme addition.
During this incubation period the movement of the
stock may appear to become easier/faster. If this
is apparent then reduce the stirrer speed as much
as possible.
After the fifteen minute incubation period had
elapsed the thick stock was then added to the
proportioner.
Proportioner
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.
Handsheet Formation
The white water box was filled with DEMI water for
handsheet formation. With the handsheet forming
wire in place in the mould assembly, one litre of
stock from the proportioner was added to the Deckle
SUBSTITUTE SI~ET ~r..ULE 26)
_
CA 02229~88 1998-02-16
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38
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.
Dep~ending 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.
Handsheet Pressing and Dryinq
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
between 60'C and 105-C and the speed of the dryer
was such that the pressed sheet was in contact with
the hot surface for 35 to 180 seconds. The final
moisture content of the sheet should be between 4
and 7% (typically 5%).
If the moisture content of the sheet after pressing
is less than 70%, then the sheet may stick to the
surface of the drum dryer when the above conditions
are employed. This may occur because of nonuniform
press pressures being applied across the width of
the sheet. Steps should be taken to avoid this.
When the surface temperature of the drum dryer is
less than 105-C but is 70 C or higher, longer
SU8S 111 UTE S}~F~ (RULE 26)
-
CA 02229~88 1998-02-16
W O 97/07203 PCT/GB96/02009
39
contact times are required in order for the
handsheet to have a final moisture content of 5%.
If the surface temperature of the drum dryer is
below 70 C, it is necessary to extend the contact
time further or increase the initial pressing on
the wet mat to remove more water or to do both. It
is possible to reduce the moisture content of the
pressed sheet to less than 60%.
Tes tinq
Conditioning and testing 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
30348, USA, ISBN 0 - 89852 - 200 - 5 (vol 1 and 2). The wet
tensile breaking strength of paper and paper board is defined
by method T 456 om - 87; the tensile breaking properties of
paper and paper board is T494 om - 81; the HST (Hercules Sizing
Test) is defined as size test for paper by ink resistance T 530
pm - 83; and the Cobb test is defined by T 441 om - 90.
A series of experiments were performed in which the cellulase
concentration, glutaraldehyde concentration, drying time and
temperature, aging time and temperature were each varied. The
results are presented in the following tables in which:-
"naturally aged" refers to storage for the specified timeat 23~C + 1~C in relative humidity 50.0 + 2% as specified
in T4020m-83;
"oven cured" refers to treatment at 80~C for 30 minutes;
"standard drying conditions" refers to drying at 105~C for
35 seconds;
The wet and dry tensile strengths were determined by methods
T4560m-87 and T4s4om-81, respectively, and the ratio of wet to
dry tensile strength expressed as a percentage. These are the
data presented in the tables where the higher the value, the
better the wet strength. The sizing effect was measured by the
SUB~TITUTE SHEET (RULE 26~
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HST (~ercules size test) (TAPPI method T530pm-83) and the data
recorded in seconds. The higher the value, the better the
sizing. Preferably the HST value is greater than 20g, more
preferably greater than 120g, more preferably greater than
200g. Size effect was also measured by the Cobb test (TAPPI
method T441Om-90) and the data recorded in grams/m2. "Fully
saturated" means that the paper showed no sizing at all. The
lower the Cobb value, the better the sizing. Preferably, the
Cobb value is less than 30g/m2, more preferably less than
10 2lg/m2.
Wet Strength and Sizing Performance of Cellulase/Glutaraldehyde
System in Handsheets dried under standard conditions
"~ Wet Strenqth after 24h naturallY aqed"
Protein on fibre
0% 5% 10%
Glutaraldehyde 10% 0.62 1.63 2.64
20 on fibre 20%0.80 3.44 6.24
40%0.97 5.00 8.86
Control:- 0.25% Cymene S_X = 4.57~;
"% Wet Strenqth after 2 weeks naturallY aqed"
Protein on fibre
0% 5% 10%
Glutaraldehyde 10% 0.94 1.97 3.01
on fibre 20%0.99 3.57 6.06
40%1.05 5.56 10.30
Control:- 0.25% ~ymene LX = 9.0_%
~iUBSTITUTE SHEET lRUlE 26)
CA 02229588 1998-02-16
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41
"HST (seconds) after 24h naturallY a~ed"
Protein on fibre
0% 5% 10%
Glutaraldehyde 10% 1 51 81
on fibre 20% 1 89 128
40% 1 108 159
'ontrol:- 0.25% .Cymene LX = ls
"HST (seconds) after 2 weeks naturally aqed"
Protein on fibre
0% 5% 10%
Glutaraldehyde 10% 1 66 125
on fibre 20% 1 86 163
40% 1 120 132
~ontrol:- 0.2S% .Cymene LX = ls
"HST (seconds) after oven curing"
Protein on fibre
0% 5% 10%
Glutaraldehyde 10% 1 60 108
on fibre 20% 1 101 165
40% 1 108 149
~ontrol:- 0.25% .Cymene .LX = ls
"Cobb (qsm) after 24h naturally aqeinq"
Protein on fibre
0% 5% 10%
Glutaraldehyde 10% Fully saturated 39.0 25.3
on fibre 20% Fully saturated 24.7 22.3
40% Fully saturated 24.4 21.6
Control:- 0.25% .Cymene SLX = Fully satu~ated
SUBSTITU~E St~EET ~U~E 26~
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42
"Cobb (gsm) after 2 weeks naturallY aqeing"
Protein on fibre
0% 5% 10%
Glutaraldehyde 10% Fully saturated35.3 21.1
on fibre 20% Fully saturated24.1 20.4
40% FU11Y saturated23.1 20.3
~ontrol:- Kymene SLX = FU11Y saturated
"Cobb (qsm) after oven curinq"
Protein on fibre
0% 5% 10%
Glutaraldehyde 10% FU11Y saturated 35.0 22.8
on fibre 20% Fullysaturated 29.2 20.7
40% Fullysaturated 24.5 20.3
'ontrol:- 0. 25% .Cymene SLX = Fully satu~ated
Wet Strength and sizing Performance on Cellulase/Glutaraldehy~le
system in hanasheets dried under alternative conditions
"% Wet Strenqth after 24 h natural 1Y aqed"
Protein/Glutaraldehyde on
fibre
5%/20% 10%/40%
Drying 23%C/ 0. 65 4.13
conditions overnight
(surface 60~C/1205 2.00 5.65
/contact 70~C/1805 2.54 6.50
time)
105~C/355 2. 86 6.97
'ontrol:- 0.2 % Kymene SLX (1~5~C/355) = 6.03%
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43
"% Wet Strenath after oven curinq"
Protein/Glutaraldehyde on
fibre
5%/20% 10%/40%
Drying 23%C/ 2.14 5.66
5 conditions overnight
(surface 60~C/120s 3.04 6.11
/contact 70~C/180s 3.25 7.28
time)
~105~C/35s2.99 8.82
10 ~ontrol:- 0.2 % Kymene SLX (1~5~C/35s) = 11.38
"HST (seconds) after 24h naturally aqed"
Protein/Glutaraldehyde on
fibre
5%/ 20% 10%/4096
Drying 23%C/ 3 3
conditions overnight
20 (surface 60~C/120s 51 39
/contact 70~Cll80s 154 184
time)
105~C/35s 83 208
'ontrol:-- 0.2 96 Kymene SLX (1~5~C/35s) = ls
"HST (seconds) after 2 weeks naturally aqed"
Protein/Glutaraldehyde on
fibre
5%/20% 10%/40
30 Drying 23%C/ 3 2
conditionsovernight
temperature60~C/120s 55 56
/contact 70~C/180s 158 221
time)
105~C/35s 82 231
~'ontrol:- 0.2 % Kymene SLX (1~5~C/355) = ls
SUBSTITUTE SHEET (RULE 26~
CA 02229588 1998-02-16
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44
"HST (seconds) after oven curinq"
Protein/Glutaraldehyde on
fibre
5%/20% 10%/40%
Drying 23%C/ 2 2
5 condi.ions overnight
(surface 60~C/120s 56 42
/contact 70~C/180s 134 193
time)
~105~C/35s 96 157
10 ontrol:- 0.2 % Kymene SLX (1~5~C/35s) = ls
"Cobb (qsm) after 24h naturally aqed"
Protein/Glutaraldehyde on
fibre
5%/20~ 10%/40%
20 Drying 23%C/ Fully Fully
conditions overnight saturated saturated
(surface60~C/120s 26.8 26.9
/contact70~C/180s 21.9 19.6
25 time)
105~C/35s 28.0 21.1
Control:- 0.2 ~ Kymene SLX (1~5~C/35s) = Fully saturated
"Cobb (qsm) after 2 weeks naturallY aqed"
Protein/Glutaraldehyde on
fibre
5%/20% 10%/40%
Drying 23%C/ Fully Fully
conditions overnight saturated saturated
35 temperature60~C/120s25.6 25.8
/contact70~C/180s 20.4 20.3
time)
105~C/35s 22.7 19.3
ontrol:- 0.2 % Kymene SLX (1~5~C/35s) = Fully saturated
SU~STITUTE SHEET (-~ULE 26~
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W O 97/07203 PCT/G~g~'.2-_9
Cobb (qsm) after oven curing"
Protein/Glutaraldehyde on
fibre
5%/20% 10%/40%
Drying 23~C/ Fully Fully
5 conditions overnightsaturated saturated
(surface
temperature60~C/120s26.3 26.3
/contact 70~C/180s 21.5 20.6
time)
105~C/35s 22.9 20.0
~ontrol:- 0.2 % Kymene SLX (1~5~C/35s) = Fully saturated
Wet/Dry Strength and Sizing Performance of
Cellulase/Glutaraldehyde system dried under two stage
co~ditions
"% Wet Strenqth after 48h naturallY aqed"
Dry Conditions (Surface temp/contact time) %Wet Strength
Expt CodeDryer ADryer B
1 40~C/60s70~C/180s 4.66
a 55~C/60s70~C/180s 3.73
b 40~C/180s70~C/180s S.29
ab 55~C/180s70~C/180s 2.61
c 40~C/60s105~C/35s 5.57
ac 55~C/60s105~C/35s 5.58
bc 40~C/180s105~C/35s 6.21
abc 55~C/180s105~C/35s 5.01
'ontrol:- 0.25% Kymene SLX (10 ~C/35s) = 8.48~
"~ Wet Strenqth after oven curinq"
Dry Conditions (Surface temp/contact time) %Wet Strength
Expt CodeDryer A Dryer B
1 40~C/60s 70~C/180s 5.38
a 55~C/60s 70~C/180s 4.56
b 40~C/180s 70~C/180s 5.82
SUBSTITUTE S~EET (RUL~ 26~
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46
ab 55~C/180s 70~C/180s 2.84
c 40~C/60s 105~C/35s 5.17
ac 55~C/60s 105~C/35s 5.49
bc 40~C/180s 105~C/35s 6.03
5 abc 55~C/180s 105~C/35s 5.53
Conlrol:- 0.25% Kymene SLX (10 ~C/35s) = 12.5.%
"DrY strenqth after 48h naturallY aqed"
Dry Conditions (Surface temp/contact time) Dry Strength
/kNm~
Expt Code Dryer A Dryer B
1 40~C/60s 70~C/180s 5.40
a 55~C/60s 70~C/180s 5.12
15 b 40~C/180s 70~C/180s 5.43
ab 55~C/180s 70~C/180s 4.77
c 40~C/60s 105~C/35s 5.09
ac 55~C/60s 105~C/35s 5.50
bc 40~C/180s 105~C/35s 5.13
20abc 55~C/180s 105~C/35s 5.27
ontrols:- Blank (105~C/35s) = 4.28kNm l; C.25% Kymene SL~
(105~C/35s) = 4.41kNm~l
"Dr~ strenqth after oven curinq"
Dry Conditions (Surface temp/contact time) Dry Strength
/kNm~
Expt Code Dryer A Dryer B
1 40~C/60s 70~C/180s 5.24
a 55~C/60s 70~C/180s 4.65
b 40~C/180s 70~C/180s 5.11
ab 55~C/180s 70~C/180s 4.75
c 40~C/60s 105~C/35s 5.09
ac 55~C/60s 105~C/35s 5.18
bc 40~C/180s 105~C/35s 5.46
abc 55~C/180s 105~C/35s 4.78
Controls:- Blank (105~C/3ss) = 3.93 kNm 1; ~.25% Kymene SL~ --
(105~C/35s) = 4.64kNm
SIJBSTITUTE St~EET tRuLE 26)
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47
"HST after 48h naturallY aged"
Dry Conditions (Surface temp/contact time) HST / seconds
Expt Code Dryer A Dryer B
1 40~C/60s 70~C/180s 277
a 55~C/60s 70~C/180s 216
b 40~C/180s70~C/180s 243
ab 55~C/180s70~C/180s 169
c 40~C/60s 105~C/35s 258
ac 55~C/60s 105~C/35s 274
bc 40~C/180s105~C/35s 310
abc 55~C/180s105~C/35s 195
Control:- 0.25; Kymene SLX (1~5~C/35s) = ls
"HST after 2 weeks naturallY aqed"
Dry Conditions (Surface temp/contact time) HST / seconds
20Expt Code .Dryer A Dryer B
1 40~C/60s 70~C/180s 304
a 55~C/60s 70~C/180s 241
b 40~C/180s70~C/180s 190
ab 55~C/180s70~C/180s 178
c 40~C/60s 105~C/35s 239
ac 55~C/60s 105~C/35s 251
bc 40~C/180s105~C/35s 290
abc 55~C/180s105~C/35s 171
'ontrol:- 0.25~ Kymene SLX (1~5~C/35s) = ls
"HST after oven curinq"
Dry Conditions (Surface temp/contact time) HST / seconds
3 5Expt Code Dryer A Dryer B
1 40~C/60s 70~C/180s 314
a 55~C/60s 70~C/180s 205
b 40~C/180s70~C/180s 242
ab 55~C/180s70~C/1 80s 149
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48
c 40~C/60s 105~C/35s 212
ac 55~C/60s 105~C/35s 275
bc 40~C/180s 105~C/35S 308
abc 55~C/180s 105~C/35s 210
5'ontrol:- 0.25~iKymene SLX (1~)5~C/35s) = ls
"Cobb after 48 h naturally aqed"
Dry Conditions (Surface temp/contact time) Cobb / gsm
Expt Code Dryer A Dryer B
1 40~C/60s 70~C/180s 21.7
a 55~C/60s 70~C/180s 21.9
b 40~C/180s 70~C/180s 22.0
15ab 55~C/180s 70~C/180s 25.1
c 40~C/60s 105~C/35s 20.7
ac 55~C/60s 105~C/35s 20.1
bc 40~C/180s 105~C/35s 22.1
abc 55~C/180s 105~C/35s 25.9
20_ontrol:-- 0.25~Kymene SLX (1~5~C/35s) = Fully saturated
"Cobb after 2 weeks naturallY aqed"
25 Dry Conditions (Surface temp/contact time) Cobb / gsm
Expt Code Dryer A Dryer B
1 40~C/60s 70~C/180s 20.8
a 55~C/60s 70~C/180s 19.2
b 40~C/180s 70~C/180s 28.2
30ab 55~C/180s 70~C/180s 21.2
c 40~C/60s 105~C/35s 22.1
ac 55~C/60s 105~C/35s 20.6
bc 40~C/180s 105~C/35s 21.7
abc 55~C/180s 105~C/35s 21.9
35 ~ontrol:- 0.25~Kymene SLX (1.)5~C/35s) = Ful_y saturated -
SUBSTI~UTE CHEET Ir~ULE 26j
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WO 97/07203 PCT/GB96/02009
49
"Cobb after oven curinq"
Dry Conditions (Surface temp/contact time) Cobb / gsm
Expt CodeDryer A Dryer B
l 40~C/60s 70~C/180s 21.7
a 55~C/60s 70~C/180s 20.4
b 40~C/180s 70~C/180s 22.3
ab 55~C/180s 70~C/180s 22.3
c 40~C/60s 105~C/35s 20.5
ac 55~C/60s 105~C/35s 20.4
bc 40~C/180s 105~C/35s 20.9
abc 55~C/180s 105~C/35s 22.3
Control:- 0.25; Kymene SLX (105~C/35s) = Ful:y saturated
Comparison of the Effect of Different Drying Regimes on Wet
Strength and Sizing Performance of Cellulase/Glutaraldehyde
System
"Effect of Different Drying Regimes on % Wet Strength (oven
cured data)"
Additive Drying Conditions % Wet
(Surface Temp/Contact Time) Strength
Dryer A Dryer B
5% Protein/20% 105~C/35s - 2.99
Glutaraldehyde
5% Protein/20% 70~C/180s - 3.25
Glutaraldehyde
5% Protein/20% 40~C/60s 105~C/35s 5.17
Glutaraldehyde
5% Protein/20% 55~C/180s 70~C/180s 5.49
Glutaraldehyde
0.25% Kymene105~C/35s - 12.53
SLX
"Effect of Different DrYinq Regimes on HST roven cured data)"
SU~ITUTE SHE-T (P~ULE 26)
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W097/07203 PCT/G~6/~2~G~
AdditiveDrying Conditions HST / seconds
(Surface Temp/Contact Time)
Dryer A Dryer B
5~ Protein/20% 105~C/35s - 96
Glutaraldehyde
5~ Protein/20% 70~C/180s - 134
5 Glutaraldehyde
5% Protein/20% 40~C/60s 105~C/35s 212
Glutaraldehyde
5% Protein/20~ 55~C/180s 70~C/180s 149
Glutaraldehyde
0.25% Kymene 105~C/35s
SLX
"Effect of Different Dryinq Reqimes on Cobb (oven cured data)"
Additive Drying Conditions Cobb / gsm
(Surface Temp/Contact
Time)
Dryer A Dryer B
5% Protein/20% 105~C/35s - 22.9
Glutaraldehyde
5% Protein/20% 70~C/180s - 21.5
Glutaraldehyde
5% Protein/20% 40~C/60s 105~C/35s 20.5
Glutaraldehyde
5% Protein/20% 55~C/180s 70~C/180s 22.3
Glutaraldehyde
0.25% Kymene 105~C/35s - Fully saturated
SLX
In conclusion, the above data demonstrates that
cellulase/glutaraldehyde treatment of paper leads to
improvement in the wet strength, dry strength and sizing of the
paper.
11. Demonstration of Bio-metalization of Paper
The bio-metalization of water-leaf paper was demonstrated. The
technique was based on the affinity of streptavidin for biotin.
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51
The biotin label was linked to the cellulase which in turn was
linked to streptavidin labelled with gold particles.
Biotinylated cellulase was incubated with the paper pulp in 1/3
PBS buffer (pH 7.4), containing Tween 20 (0.1% vv 1) for 45 min
at room temperature with shaking. A paper square (6 cmZ) was
formed, rolled and allowed to dry overnight at ambient
temperature.
Samples of the paper (1. 5 Cm2) containing the biotinylated
cellulase and control samples lacking the biotinylated
cellulase, were incubated with 5% (WV 1~ BSA in 10 mM PBS pH
7.4, for 30 min at ambient temperature with shaking.
The Auroprobe BLplus labelled streptavidin conjugate and
enhancer (Amersham Ltd., Amersham, U.K.) was used according to
the manufacturer's recommendation to attach and visualize the
gold particles (Fostel et al., Chromosoma, 90, 254, (1984);
Hutchinson et al., J. Cell Biol., 95, 609, (1982)). The
enhancer solution coated the gold particles with silver to
create an orange/brown colour which was indicative of the
presence of the metals. Control sheets which did not contain
the biotinylated cellulase did not develop the orange/brown
colouration and hence were not coated with the metal.
12. Capacitance Measurement of Biometalized Paper
The capacitance of biometalized paper sheets was compared to
control sheets to determine if the presence of the gold-
labelled cellulase altered the capacitance characteristics ofpaper.
Paper sheets were produced from W-LP containing either
cellulase, gold labelled cellulase, enhanced gold labelled
- 35 cellulase and cellulase-free controls. The sheets were each
held between two metal plates connected to a capacitance meter.
The metal plates were held in position in a jig which ensured
that a constant and reproducible distance was maintained
SUBSTITIJTE SI~ET (RULE 26~
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52
between the plates.
The capacitance (C) was calculated using the following equation
C = ~o~rA
d
where d = distance between the two plates
A = area
~o = constant
~r = relative permeativity
The measurements obtained indicated an increase in the
capacitance of the paper sheets in the presence of gold
labelled cellulase. The results of the determination are given
in Table 7.
Table 7: Capacitance Determination
20 8ample Capacitance (pF)
Machine Calibration (control) 10.00
Paper without cellulase 10.97
Paper + cellulase 10.65
25 Paper + gold labelled cellulase 13.86
13. Demonstration of binding amylase enzymes to starch
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.
A solution of 32 mg ml~l (dry weight) of ~-amylase was made up
SUBSTITUTE SHEET ~RULE 26)
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53
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 starch
suspension using the standard 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 ~l phenol reagent (0.128M phenol in O.lM phosphate buffer
pH7.0)
10 ~l amino pyrine reagent (19.7mM 4-amino phenazone in O.lM
phosphate buffer pH7.0)
10 ~l peroxidase in O.lM phosphate buffer pH 7.0 (to give
800Eu/ml)
10 ~l glucose oxidase in O.lM phosphate buffer pH 7.0 (to give
250Eu/ml)
60 ~l O.lM phosphate buffer pH7.0
These reagent components were mixed and added to the wells of
a microtitre dish. Test samples 100 ~l were added followed by
an excess 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~1 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 suspensions as
above.
The ability of ~-amylase and amyloglucosidase to bind to normal
starch in suspension was assessed. Starch (0.2 g; Roquette)
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CA 02229~88 1998-02-16
W 097/07203 PCT/G~961'~00
54
was added to 9 ml 0.1 M PBS (pH 7.0) and l 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
control. The final concentration of the BSA used was 0.2% (wv~l)
in 0.1 M PBS.
The results of the binding experiments are shown in the
following Table.
Starch binding profiles
Enzyme Substrate % Bound
20~-amylase starch 32
amyloglucosidase starch 27
amyloglucosidasecationic starch 45
BSA starch 7
BSA cationic starch 6
These results indicate that both ~-amylases and
amyloglucosidases specifically bind to both starch and cationic
starch and are therefore suitable for use as protein linkages
for binding effector moieties to starches.
SU8STITUTE S~tE~ (RULE 2~i)
