Note: Descriptions are shown in the official language in which they were submitted.
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:L
h~v8 AND CONPOSIT:~ON8 FOR 8IZING PAPER
1 Technical Field
The present invention relates to methods and compositions
for sizing paper. In particular, the present invention
relates to the use of a protein capable of bi n~ in~ to paper
or a constituent of paper to size paper.
2 Rac~ ~u~a
While there are a myriad of details for manufacturing paper,
the paper manufacturing process conventionally comprises the
following steps: (1) forming an aqueous suspension of
cellulosic fibers, commonly known as pulp; (2) adding
various processing and paper enhancing materials, such as
strengthening and/or sizing materials; (3) sheeting and
drying the fibers to form a desired cellulosic web; and (4)
post-treating the web to provide various desired
ZO characteristics to the resulting paper, including surface
application of sizing materials, and the like.
Sizing materials are typically in the form of aqueous
solutions, dispersions, emu]sions or suspensions which
render the paper treated with ~he sizing agent, namely sized
paper, resistant to penetration or wetting by an aqueous
liquid, including other treatment additives, printing inks,
and the like.
A sizing agent may be applied to the surface of paper as a
"surface" size or may be incorporated within the paper as an
"internal" size. Many chemical sizing agents are known
including rosin-based and ketene dimer-based sizing
compositions. There remains, however, a need for improved
sizing compositions and methods of sizing.
Typically, the principal constituent of paper is cellulose.
Cellulose may be in the form of wood fibre or ~nntl~l crop
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fibre (for example, hemp, straw, rice, flax, jute or
cotton). Other constituents of paper may include other
polymeric materials, including naturally occurring polymers
such as starch, pectin, guar, chitin, lignin, agar, alginate
as well as other polysaccharides including hemi-celluloses
such as xylanose, mannose and arabinose. Xyl~no~e is the
principal component of xylan, otherwise known as hemi-
cellulose which occurs in grasses, cereal, straw, grain
husks and wood. Starch occurs in seeds, fruits, leaves,
bulbs etc.
Enzymes which are capable of modifying an enzyme substrate
typically rely on a non-covalent binding interaction with
the enzyme substrate in order to function. One such class
of enzymes comprise enzymes which degrade polymers, for
example proteinases, kerati~ases, chiti~ c~ liqn; ~e~,
agarases, alginA~?~, xy~ c~c~ ~nn~rQc, amylases,
cell~ e~ and hemi-cellulases. For example, cellulases and
hemi-cellulases cleave saccharide or polysaccharide
molecules from cellulose and hemi-cellulose, respectively,
and amylases cleave glucose from starch.
The interactions between cellulose and cellulase proteins,
in particular those that bind to the cellulose fibres as a
prerequisite to catalytic activity have been described and
reviewed (cellulase: Beguin, Annu. Rev. Microbiol., 44,
219-248, 1990; celllll~ and xyl~naces: Gilbert and
Hazelwood, Journal of General Microbiology, 139, 187-194,
1993). This group of enzymes include celllll~ and hemi-
celllllA~e~ which comprise functionally distinct proteindomains. In particular, the domain responsible for
catalytic activity is structurally distinct from the
cellulose binding domain. These dc ~i n~ are evolutionarily
~o~rved sequences which are very similar in all such
proteins (Gilkes et al., Microbiological Reviewfi, 303-315,
June 1991~.
The binding domains of such proteins can be separated from
_
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the active-site domains by proteolysis. The isolated
b~ nA i n~ domains have been shown to retain b; n~ i n~
capabilities (Van Tilbeurgh, et al ., FEBS Letters, 204(2),
223-227, August 1986). use of cellulose bin~ing domains of
cellulases has been proposed as a means of roughening the
texture of the surface of cellulosic 5U~pOl L, while use of
cellulase active-site domains has been proposed as a means
of smoothing the texture of such surfaces (International
patent application W093/05226).
A number of b;n~in~ domains have also been characterised at
the genetic level (Ohmiya et al . ,Microbial Utilisation of
Renewal Resources, 8, 162-181, 1993) and have been subcloned
to produce new fusion proteins (Kilburn et al., Published
International Patent Application Wo90/00609; Ong et al.,
Enzyme Microb. Technol, 13, 59-65, January 1991; Shoseyov et
al., Published International Patent Application
W094/24158). Some of these fusion proteins have then been
used as anchor proteins for specific applications. Such
proteins have been used as an aid to protein purification
through adhesion of the fusion proteins to cellulosic
~u~o~L materials used in protein purification strategies
(Kilburn et al ., United States Patent 5,137,819; Greenwood
et al., Biotechnology and Bioengineering, 44, 1295-1305,
1994). The ability to immcbilize fusion proteins onto
cellulosic supports has also been suggested as a means of
immobilization for enzyme bioreactors (ong et al.,
Bio/Technology, 7, 604-607, Jlune 1989; Le et al. Enzyme
Microb. Technol., 6, 496-500, June 1994), and as a means of
attaching a chemical "tag" to a cellulosic material
(International Patent Applica1:ion W093/21331).
It has now been found that proteins capable of binding to
paper or a constituent of paper may be used to size paper.
3 8ummary of the Invention
According to the present invention there is provided a
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method of sizing paper comprising the steps of a) contacting
said paper or a constituent of said paper with a protein
capable of binding to said paper or said constituent of
paper, and b) denaturing or heating said protein bound to
said paper.
According to a further aspect of the present invention there
i8 provided a method of sizing paper comprising a)
contacting said paper or a constituent of said paper with a
protein capable of binding to said paper or said constituent
of paper, and b) heating said paper.
According to a further aspect of the present invention there
i8 provided use of a protein capable of binding paper or a
constituent of paper for the purpose of sizing paper. The
invention further provides paper sized according to a method
of the present invention.
~ Detailed Description of the Invention
The present invention provides a method of sizing paper. As
used herein, the term "paper" refers to any material in the
form of a coherent sheet or web, comprising an interlaced
network of cellulose containing fibres derived from
vegetable sources optionally mixed with fibres from
vegetable, mineral, animal or synthetic sources in various
~ Lions and optionally mixed with fine particles of
inorganic materials such as oxides, carbonates and sulphates
of metallic elements in various proportions. The term
"paper" includes paperboard which refers to paper when the
weight of the paper sheet or web is greater than 200g/m2.
Vegetable sources of cellulose include wood, straws,
Bagasse, Esparto, Bamboo, Kanaf, Grass, Jute, Ramie, Hemp,
Cotton, Flax. The crude vegetable derived cellulose is
proceC~ to form pulp, the material from which paper is
made, either mechanically, chemically or both. Cellulose
ContA; n~ ~g pulps may be described as mechanical,
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~h~ hAn;cal and chemithermomechA~iCal~ semi chemical,
high yield chemical, full chemical (see "Pulp and Paper,
Chemistry and Chemical Technology", Third Edition, Volume 1
pages 164, 165 edited by James P. Cassay ISBN 0-471-03175-5
(v.l)) according to the method of pulp preparation and
purification.
Paper may also comprise other naturally occurring polymers
such as proteins such as keratin, starch (including anionic,
cationic or amphoteric starch), pectin, guar, chitin,
lignin, agar, alginate as well as other poly~Acch~rides
including hemi-celluloses such as xylanose, mannose and
arabinose.
The method of the present invention comprises co~ acting
paper or a constituent of paper with a protein capable of
b~ n~ j n~ to the paper or const:ituent of paper followed by
denaturing or heating the protein.
The protein employed in the present invention may comprise
any protein capable of binding to the paper or constituent
of paper. The protein may for example comprise a protein
capable of binding cellulose or any other polymeric
substance present as a constituent of the paper.
Preferably, the protein is capable of specific binding to
cellulose or any other polymeric substance present as a
constituent of paper. More preferably, the protein is
capable of binding with a dissociation constant of (Kd) less
than 1 x 10-3M. As used herein, the term "protein" includes
peptide, oligopeptide and polypeptide, as well as protein
residues, protein-containing species, chains of amino acids
and molecules containing a peptide linkage. Where the
context requires (for example, when protein is hon~e~ to
another molecule), reference to a protein means a protein
residue. The protein may co~prise a naturally occurring
protein, or fragment thereof or modified protein obtainable
by chemical modification or synthesis or by expression of a
genetically modified gene coding for the protein. As used
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herein the term "modified protein" includes chemical analogs
of proteins capable of binding to paper or a constituent
thereof. Preferably/ the protein comprises a naturally
occurring enzyme or fragment thereof which is capable of
bin~ing to paper or a constituent of paper. Examples of
proteins capable of binding paper or a constituent of paper
are well known and include enzymes selected from the group
comprisingcellulases, hemi-cellulases, mannases, xylanases,
chitinases, ligninases, agarases, alginases and amylases.
The protein may for example comprise an amylase or fragment
thereof capable of binding to starch (such as anionic,
cationic or amphoteric starch) when present as a constituent
of paper or paper pulp. Examples of amylases include ~-
amylases, for example from Aspergillus oryzae (available asa Type X-A crude preparation from Sigma Aldrich Co Ltd), and
amyloglucosidases, for example from Aspergillus niger
(available from Sigma Aldrich Co Ltd).
Preferably, the protein comprises a protein capable of
b~n~;nq to cellulose. More preferably, the protein
comprises a cellulase or fragment thereof. The cellulase
may comprise a naturally occurring cellulase, or fragment
thereof, or modified cellulase obtainable by chemical
modification of a naturally occurring cellulase or synthesis
or by e~LessiGn of a genetically modified gene co~ for
a cellulase. The cellulase may, for example, be modified to
remove or deactivate the active-site domain. A variety of
cellulases are known which bind to cellulose. Examples of
such cell~ are those isolable from bacterial organisms
such as Cellulomonas fimi and fungal organisms such as
Trichoderma viride, Aspergillus niger, Fusarium oxysporum,
Penicillium funiculosum, Trichoderma reesei and Humicola
insolens, available as commercial preparations from Sig~a
Chemical Sigma-Aldrich Company Ltd., Novo Nordisk A/S, BDH
Ltd., or ICN Biomedicals Ltd. (Fusarium oxysporum is
available for example under deposit No. DSM 2672).
Alternatively, the protein may be produced by recombinant
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DNA techn;~ues as disclosed in, for example, International
Patent application WO94/24158. Cell~l A~ generally
comprise a cellulase binding domain and a domain responsible
for cellulase activity. The present invention may employ
the cellulase as a whole or a fragment thereof capable of
binding to cellulose. A cellulase binding domain may be
obt~;ne~ from whole cellulase by treatment with protease(s),
such as papain. The cellulase may comprise an exo-cellulase
or an endo-cellulase. Exo-celll~l AS~ (also known as
cellobiohydrolases, CBH; exoglucanaes; 1,4-beta-D-glucan
cellobiohydrolases; EC 3.2.1.91) act on the non-reducing end
of a cellulose molecule. Exo-cellulases may release
terminal cellobiose units (a disaccharide) or release
terminal glucose units (monosaccharide). Examples of exo-
cellulases include cellulase obtainable from Numicol ai~olens. Endo-cellulases (also known as Beta-1,4-
Endoglucanases; Endo-1,4-D-glucanases; 1,4-Beta-D-glucan
gll~cAnohydrolases; EC 3.2.1.4) cleave internal beta-1,4-
glycosidic bonds yielding a mixture of glucose, cellobiose
and other soluble cello-oligosaccharides. Examples of endo
celllll~ce~ include cellulase obtainable from Trichoderma
reesei .
The protein may also comprise cellulosomes. Cellulosomes
comprise a cellulase system comprising discrete,
multifunctional, multienzyme complexes. They typically
contain at least 14 distinct polypeptides including numerous
endoglucA~e~ (endocellulases) and xylanases and at least
one beta-glucanase. These are associated with scaffolding
proteins. Cellulosomes are described in detail in Bayer
E.A., Morag E., Lamed R. (1994), "The cellulosome - a
trea~LeLLo~e for biotechnology", Trends in Biotechnology
12:379-386.
Preferably, the protein employed in the present invention
comprises cellulase obtainable from ~umicol a isol ens
(available as Celluzyme~ from Novo Nordisk A/S, Bagsvaerd,
Denmark) or cellulase obtainable from Trichoderma reesei
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(available a Celluclast0 from Novo Nordisk A/S, Bagsvaerd,
Denmark). More preferably, the protein comprises cellulase
obtAinAhle from Humicola isolens .
The protein may be added to the paper at any suitable stage
in the manufacture and processing of the paper. It may be
added at the pulp stage or at any stage during the formation
of the wet pulp matrix or during the pressing and rolling of
the matrix to form paper. Thus, according to the present
invention there is provided a method of manufacturing sized
paper comprising the steps of a) preparing a paper pulp, b)
adding a protein capable of binding to a constituent of said
pulp, c) forming paper from said pulp, and d) heating said
paper.
Alternatively, the protein may be added to the formed paper
product, for example, by immersing the paper in a bath
cont~;n;ng the protein or by any suitable spraying,
spr~ gt brushing, coating or printing process. Thus, the
invention furthcr provides a method of manufacturing sized
paper comprising the steps of a) applying to paper a protein
capable of binding said paper and b) heating said paper.
By choosing the point in the manufacture of the paper at
which the protein is added, control may be exercised as to
whether the protein is distributed throughout the paper or
is substantially restricted to the surface levels of the
paper.
The protein should be incubated with the paper or paper pulp
for sufficient time to allow binding of the protein to the
paper or paper pulp. Typically, 15 minutes has been found
adequate, although shorter incubation times may be suitable.
The protein may be added in an amount suitable to achieve
the desired level of sizing. The protein may be added in an
amount of 0.01-40% by weight of the dry weight of the paper
pulp. Preferably the protein is added in an amount of 0.1
_
CA 022293~8 1998-02-11
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to 20% by weight, more preferably 1 to 10% by weight.
Following incorporation of t:he protein in the paper or
application of the protein to the surface of the paper,
sizing of the paper is achieved by denaturing or heating the
protein. The protein may be denatured by the application of
a chemical protein denaturant to the paper. Chemical
protein denaturants include urea, guanidine, acids, ~k~]i~,
detergents (such as Tween~), water soluble organic
subst~n~ (such as alphatic alcohols) and chaotropic ions
(such as I-, Cl04-, SCN-, Li+, Mg2+, Ca2+ and Ba2+).
Preferably, sizing is achieved by heating the paper.
Preferably, the paper may be heated at a temperature of 50~C
to 200~~, more preferably 70~C to 170~C, more preferably
80~C to 110~C, more preferably 100~C to 110~C. Typically,
the paper may be heated to approximately 105~C on steam
heated rollers. The paper may be subjected to one or more
heat treatments at different 1_emperatures.
The length of time of heating required depends upon the
temperature at which the paper is heated, longer times being
required at lower temperature. Typically, the paper may be
heated for between 15 and 500 seconds, preferably between 25
and 300 seconds. The paper may also be subjected to post
manufacture heat treatments to age or cure the paper.
The invention will now be described with reference to the
following examples.
It will be appreciated that the following is by way of
example only and modification of detail may be made within
the scope of the invention.
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EXP~ r~iL
Materials and Protocols
Excep~ as indicated below, the following materials and
protocols were used in the Examples to characterise the use
of proteins as biosizing agents.
~le: Water-leaf paper pulp was prepared by A~ i ng 10 g of
10 water-leaf paper (70:30 Hardwood (birch) : softwood (pine))
to 100 ml distilled water. After 5 min the paper was blended
to an homogenous pulp. Samples of pulp Cll~p~cion
(e~e~ponding to 0.2 g dry paper) were weighed into
Universal bottles.
TncubatiQn To each bottle 10.0 ml of one of the following
1~Cllh~tion buffer solutions was added:
(a) Tris-HCl, 50 mM, pH 7.5
(b) Phosphate buffered saline (PBS) 500 mM, pH 7.5
~ ulases: To the incubation suspension was added a
cellulase selected from the following:
1. ~umicola insolens - Cellulase derived from Humicola
~nsolens available as Celluzyme~ from Novo Nordisk
A/S, Bagsvaerd, Denmark (875~1 corresponding to 8.7%
weight per weight cellulose binding protein to dry
weight pulp).
2. Trichoderma reesei - Cellulase derived from
Trichoderma reesei available as Celluclast~ from Novo
Nordisk A/S, Bagsvaerd, Denmark (70~1 corresponding
to 0.28~mols protein corresponding to 4.4% weight per
weight cellulose binding protein to dry weight pulp).
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3. Trichoderma viride - Cellulose derived from
Trichoderma vivide available from BDH Ltd UK.
Control experiments in the absence of cellulase were also
performed.
Tncubation: The mixtures were incubated for 15 min at room
temperature with gentle agitation.
Test Sheet Preparation: To produce the paper test sheets,
the volume was increased to 100 ml with distilled water and
paper sheets (6 cm2) produced using a laboratory-designed
paper making apparatus operated in the following manner: a
suspension of paper pulp (O.2~ wv-l) was poured into a
plastic filter holder which houses a fine nylon filter mesh.
By applying a vacuum for a few seconds the pulp was formed
into a paper sheet supported by the mesh. The filter mesh
was removed from the apparatus and the paper sheet
sandwiched between a second nylon mesh and blotted between
blotting paper. The paper sheet was carefully removed from
the paper-making mesh, flattened by rolling and then dried.
Test Sheet Drying/Heatinq: Paper sheets were dried in one of
the following ways
(a) air drying;
(b) drum drier - typically operating at a constant
temperature of 80~C to 10~~C with a contact time of 40
to 250 ~;
(c) hot plate press - typically producing a maximum
surface temperature of 160~C with a 30 s contact time.
A flat aluminum plate was used to press the paper test
sheets (sandwiched between blotting paper) against the
hot plate.
Test ~rotocols: The dried sheets were ~cs~-cced for sizing by
one or more of the following t:ests:-
(a) Ink drop test (IDT) - i~ which a 15 ~1 drop of Parker
Super Quink Ink (Permanent Blue-Black) was dropped
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onto the test piece of paper and the time taken for
complete absorption measured and recorded. Results
were recorded in seconds or on an empirical scale of
0 to ~+++ in which:-
0 means less than 100 s for complete absorption.
means 100-500 s for complete absorption.
++ means 500-1000 s for complete absorption.
+++ means 1000-2000 s for complete absorption.
++++ means greater than 2000 s for complete
absorption.
(b) Hercules Size Test (HST) - defined in "Tappi Test
Methods" published by TAPPI, Technology Park,
Atlanta, P0 Box 105113, GA 30348, USA, ISBN 0-89852-
200-5 (vol 1 and 2), (1987). The HST is defined as
size test for paper by ink resistance T530pm-83. The
data are recorded in seconds; the higher the value,
the better the sizing. Pre~erably an HST value
greater than 20 secon~, more preferably greater than
120 E~conA~, more preferably greater than 200
~econ~c~ is obtained.
(c) Cobb Size Test - defined in "Tappi Test Methods"
(Ibid) by T441Om-84. Data are recorded in grams/m2.
"Fully saturated" means that the paper showed no
sizing at all. The lower the Cobb value, the better
the sizing. Preferably a Cobb value less than 30g/m2,
more preferably less than 21g/m2, is obtA;n~d.
Ex~mpl~ 1
An investigation into the effect of temperature on the
sizing imparted by a Trichoderma reesei cellulase
preparation (Celluclast~,_Novo Nordisk A/S, Bagsvaerd,
Denmark;) was carried out. For these studies a drum drier
was used in conjunction with a variable temperature hot
plate. The drum drier gave a constant temperature of 80~C
with a contact time of 250 s and the hot plate gave a
maximum surface temperature of 160~C with a 30 s contact
time. A flat aluminium plate was used to press the paper
sheets (sandwiched between blotting paper) against the hot
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plate.
Trichoderma reesei cellulase preparation (70 ~1 Celluclast~,
corresponding to 4.4 % ww~1 cellulose binding protein based
on dry weight of cellulose fibre) was used and the mixLuLe
~cl~h~ted for 15 min at room temperature with gentle
agitation. Control experiments without cellulase were also
performed.
Paper test sheets were prepared as described above. Test
sheets were initially pressed between blotting paper then
dried in one of the three following ways.
(a) air drying;
(b) drum-drier: 80~C/250 s; OL-
(c) hot plate press: 160~C/30 s followed by drum drier
80~C/250 8.
The dried sheets were then assessed for sizing by the Ink
Drop Test (IDT).
The following sizing results were obtained from test papers
prepared under different drying regimes.
T~ble1: Effect of pper dr~ e on the cellul~ ' ~ sizinll ~t
Drying regime
Additions to pulp Air dried80~C/250 s160~C/30 8
80~C/250 s
Cel_u~last~+ Tris-HCl 0 +++ ++++
3~ Cel~u~,last~ + PBS - - ++
Tric-ICl - 0
PBS
H20 _ 0
- = Not tested.
No sizing was observed when papers were made without enzyme,
ruling out the possibility that the buffer salts were
interacting with the cellulose in such a way as to affect
sizing. Sizing was observed with Tris-~. reesei cellulase
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(Celluclast~) inC~lhAtions and a greater degree of sizing was
observed when papers were dried initially at the higher
temperature followed by drum drying at the lower
temperature. Cellulase/PBS paper also gave sizing at the
higher temperature but the degree of sizing was lower than
that obtained in the presence of Tris-HCl.
E~mpl~ 2
The effect of incubation time on the levels of sizing
imparted using a Humicol a insol ens cellulase preparation
(Celluzyme-, Novo Nordisk A/S, Bagsvaerd, Denmark) was
investigated.
Tris-HCl (50 mM, pH 7.5, 10 ml) was added to 0.2g pulp in
dist~lle~ water and the Humicol a insol ens cellulase
preparation (875 ~1) was added and the mixtures incubated
for either 15 min or 90 min at room temperature. At the end
o~ the incubation period, the pulp samples were vortex mixed
and diluted to 100 ml. Test papers were prepared in the
~t~n~rd -nne~ and dried by a single pass through a drum
drier 100~C/250 s.
The degree of sizing achieved by the different methods was
A~e-S~ using the IDT method.
T ble2:
Enzyme wt dry IDT Incubati
test sizing on
paper period
(g) (min)
Celluzyme' (875 0.207 +++ 15
3c ~1)
Celluzyme' (875 0.157 +++ 90
~Ll)
Control 0.218 0 90
3S Increasing the incubation time from 15 min to 90 min did not
significantly increase the level of sizing.
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1!5
~x~mple 3
To establish a quantitative relationship between sizing
achieved in test papers and the quantity of Trich~rma
reesei or ~umicola insolens cellulase preparations added, a
further set of experiments were undertaken.
St~AA~dized paper making conditions were employed as
follows: to a sample of pulp in distilled water (equivalent
to 0.2 g dry paper) 10.0 ml buffer was added (50 mM Tris
HCl, pH 7.5). Various amounts of cellulase (Trichoderma
reesei or Humicol a insol ens ; Table 3) was added and the
mixture vortex mixed. The pulp was ;ncllh~ted with gentle
chAki~g at room temperature for 15 min, after which time the
mixture was diluted to 150 ml with distilled water and the
test paper sheets produced. Eac:h test sheet was removed from
the mesh and pressed between a folded sheet of dry 3 MM
blotting paper using a hand-held roller. The sheet (still
folded in the blotting paper) was passed once through a drum
drier at 100-104~C with a 250 s contact time. The results
are given in Table 3.
T ble 3: Sizir~ ~chieved usin~l either TriC ' ~ reesei ~Cellucl#t , ~lovo ~lordidcA~S, ~ _ J,
Den or~) or Nu~icoln insolens (Celluey~e , Uovo Uordisk ~5, r _ d, Dononr~) cellul Le
~ . ~ io..
CelluzymeCelluzyme IDTCelluclastCellucla~t IDT
(/11) a~~Bindin~ protein~izing (lll) aQ ~Bindin9 ~L~c~ng
receivedKlded ~brsed on rating received protein ~dded rating
weight of fibre) ~b~red on
X ww 1 weight of
fibre) X ww 1
O O O O O O
0.5 + 25 1.6 +
0.7 + 50 3.1 +
100 1.0 + 70 4.4 +
150 1.5 +~ 100* 6.3 +
200 2.0 +++ 150 9.4 +
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250 2.5 +++ 200 12.5 +
300 3.0 +++ 300 18.8 ++
625* 6.2 +++ 400 25.1 ++
875 8.7 +++ 600 37.6 ++
*Represent equivalent addition levels.
The results show that whilst both cellulases impart sizing,
the N. insolens cellulase preparation imparted greater
levels of sizing than the T. reesei cellulase under the
conditions used.
Bxample 4 The effects of buffer omission and high (160~C)
o temperature drying on levels of sizing achieved with either
a Trichoderma reesei cellulase preparation (Celluclast~,
Novo Nordisk A/S, Bagsvaerd, Denmark) or a Humicola insolens
cellulase preparation (Celluzyme~, Novo Nordisk A/S,
Bagsvaerd, Denmark) were investigated.
Paper sheets were prepared as described previously, with the
various conditions as described in Table 4. Sizing was
measured the following day by the standard IDT method.
The results show that Celluclast~ benefits from higher
drying temperatures (160~C) than Celluzyme2 to confer good
sizing.
Teble ~: Effect of I . ~ ~ on the sizing ~chieved by either Tri~ ' ree~ei cellul-se
2 5 (Cellucl-st , ~ovo ~ordis~ ~JS, _ ~- n~, Den~0rk) or Humicoln in601ens cellul-~e ~Celluzyme ,
Hovo Hordis~ ~JS, ~ _ rd, Den~cr~)
EnzymeB~nding protein Incubation Drying IDT
( 1 5 0 ,(L~ eight of ~bre) buffer regime rating
3c Celluclast' 9 4 dH20160~C/30 ++~+
108~C/250
Celluclast' 9.4 Tris-HCl160~C/30 ++
1 0 8 ~ C / 2 5 0
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Celluclast9.4 dH20 108~C/250 +++
Celluclast~ 9.4 Tris-HCl 108~C/250 +
No enzyme 0 Tris-HCl 160~C/30 0
s +
c 108~C/250
No enzyme ~ dH20 160~C/30 o
s +
108~C/2S0
~ Celluzyme~ 1.5 dH20160~C/30 l~
108~C/250
Celluzyme~ 1.5 Tris-HCl 160~C/30 ++
108~C/250
Celluzyme~ 1.5 dH20 108~C/250++++
Celluzyme' 1.5 Tris-~Cl 108~C/250 +
s
Examplc 5 The Hercules Sizing Test (HST) was used to
determine the degree of sizing imparted by either
Trichode~ma reesei cellulase (Celluclast~, Novo Nordisk A/S,
Bagsvaerd, Denmark) or Humicola insolens cellulase
(Celluzyme~, Novo Nordisk A/S, Bagsvaerd, Denmark) with and
without the addition of Tris. To prepare the sheets, 2 %
(w-1) pulp stock was incubated with 5 % (ww~l) cellulase
protein (based on dry weight of fibre) for 5 min at 25~C
before forming the paper sheets. The sheets were dried on a
drum dryer at 105~C for 40, 80, 160 or 240s and were
subjected to one of the fol].owing post manufacture heat
treatments: naturally aged for 24 h; 80~C for 10 min and
105~C for 10 min.
The results show that Humicola insolens and Trichoderma
reesei cellulases confer moderate sizing as measured by the
HST after drum drying at 105~C.
_
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18
T bl~! 5 HST ~ . of peper ~;heets ~ized ~lith either T~ ei cellul#-
~Cellucl-st, llovo llordid~ A/S, E _ J, Der~arlc) or ,llu-icol- insolens cellul~Ihn~ ~lordi~lc ~JS, ~ _ 1, Da_~rk).
HST (s) I -
dH20 ¦ 25 mmoles Tris
Drul
Cellulase Drier N/A 80~C 105~C N/A 800C 105~C
Contac
t Time
(S)
l~Celluclast' 40 10 13 18 10 11 15
13 15 22 10 14 16
160 19 18 23 16 16 20
240 25 23 50 19 17 22
Celluzyme~ 40 121 118 127 131 121 93
1 80 137 141 149 134 128 104
160 134 135 138 155 103 126
240 111 106 117 112 98 113
~:s~mple 6 A Cellulase preparation from Tricho~ern2a viride
(BDH Ltd.) was tested as a biosizing agent. Samples were
added to aliquots of pulp stock (0.2 g dry weight fibre in
15 ml di~tilled water). The cellulase addition level was
ad~usted such that an equivalent cellulose binding protein
concentrations (corresponding to 8.7% ww-1 based on fibre
weight) were added to enable direct comparison with Humicola
insolens cellulase (Celluzyme~, Novo Nordisk A/S, Bagsvaerd,
Denmark).
The pulp and cellulase samples were inc~hAted at room
temperature for 15 min prior to preparation of the hand
sheets. The sheets were dried by a single pass through a
drum drier at 105~C and left at room temperature overnight
before testing for sizing using the IDT. The results are
shown in Table 6.
The results indicate that when using an equivalent protein
concentrations and compared with ~umicol a insol ens
cellulase, the Trichoderma viride cellulase imparted a
moderate sizing effect.
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19
Tab1Q 6 8izing achieved l~y ~ffer~nt
det _ ; ~9~ by the C~-IDT ~88ay .
Cellulase Source Supplier IDT
sizing
rating
Trichoderma viride BDH Ltd. +
Humicola insolens Novo Nordisk A/S +++
C~-LLI 11 o
o Example 7 To test the application of Clostridium
thermocellum cellulosomes as a sizing agent Cl. thermocellum
(NCIMB 10682) was grown on 1.0 % (wv~l) pulp in growth
medium, comprising: lOOOml Basal Medium ((gl-l) yeast
extract, 10; KH2P04, 1.5; K2HP04.3H20, 2.9; (NH4)2S04, 1 3;
and FeS04.7H20, (1.0% wv~1) l.Oml~1) with a cellulose s'ource
(pulp Q1% wv~1) which is then autoclaved to sterilize. 25
ml Salts Solution (MgC12, 2%wv~1; CaC12 0.2% wv~1, autoclaved
to sterilize). 15 ml Cysteine Solution (50 gl~1, filter
sterilized). The growth medium (11 in a Duran bottle) was
purged with nitrogen for 5 min and heated to 60~C prior to
inoclllAtion. The culture was then i~c~lhAted for 240h. The
culture was harvested and the cells and pulp debris
separated from the culture fluid by centrifugation. Sodium
azide (0.02 % (wv~1)) was added to both the culture fluid
2s and to the pulp debris to prevent further microbial growth.
The culture fluid was tested as a sizing agent by ~king
;~= paper using the Cl. thermocellum cultures. Water-leaf pulp
(10% (wv~l); 2.16 g) was weighed into five 250 ml flasks.
Cl. thermocellum culture fluid (200; 100; 50; 25; and 0 ml)
was then added to each flask and the volume adjusted to 200
ml with distilled water. The mixture was then stirred at
room temperature for 15 min and a paper sheet was made from
the contents of each flask using the stAn~Ard paper making
method. The paper was dried at 80~C for 250 s using the drum
drier. Sizing was measured the following day using the
st~n~Ard IDT method.
CGl.L ol sheets of paper were also prepared and tested as
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above wherein non-inoculated Cl. thel -~e7 lum growth medium
was added to the water-leaf pulp instead of the Cl.
thermocellum culture fluid.
It was also decided to test for sizing using the pelleted
pulp debris and cells. The pulp debris and cells (2.16 g;
percentage pulp now unknown) were weighed into two 250 ml
flasks. To one flask 200 ml of Cl. thermocellum growth
medium was added to resuspend the pulp debris and to the
lo other flask 200 ml of distilled water waC added. Both flasks
were stirred at room temperature for 15 min and a paper
sheet made from the contents of both flasks using the
st~n~Ard paper making method. The paper was dried at 90~C
for 250 s using the drum drier. Sizing was measured the
following day using the IDT method.
The Cl. thermocellum culture fluid did not impart sizing to
the paper it is believed because of the very low levels of
cellulosomes free in the culture fluid. Paper sheets made
from the pulp debris showed sizing (Table 4) and a
significant lowering in the degree of sizing was noted when
distilled water was used compared to the Cl. thermocellum
growth medium. It is believed that the use of distilled
water causes a lowering of the salt/ionic strength of the
2s distilled water as comr~red to use of the growth medium,
resulting in elution of cellulosomes from the pulp surface
thereby reducing the degree of sizing. The results confirm
that the presence of the Cl. thermocellum cellulosome
preparation imparts sizing to the paper sheet.
T ble 7: 5izir~ test: pulp h)~drolysed by Cl. 1~
Diluent (ml) Ink-drop test (s)
Pulp debris + Cl. thermocellum140/175*
3r cells growth medium
(2.16 g) (200 ml)
Pulp debris + dH20 (200 ml) 37/35*
cellF
(2.16 g)
* Duplicate tests.
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21
Exunple 8
A further series of experiments were performed to determine
the effect of cellulase on the sizing of paper. In the
experiments, the following materials and general protocols
were employed:-
Ce 7 lulase
An aqueous ~richoderma reesei cellulase preparation was
employed ("Celluclast 1.5L" supplied by Novo Nordisk
Bioindustry S.A. 92017 Nanterre Cedex. France).
In addition Cellulase derived from Penici ~ 7 il~m
funiculosum available as a tan powder from Sigma
Aldrich Co. Ltd. Poole, Dorset, U.K. was used.
Stock Preparation
Except where otherwise indicated, the furnish used was
a blend of ECF bleached hardwood and softwood pulps
(ratio of 70:30 HW/SW). The stock was prepared with
1/3 PBS and no fillers were added. The procedure was
as follows:
280g of bleached hardwood pulp and 120g of bleached
softwood pulp were added to 18 litres of 1/3 PBS. The
2S fibres were dispersed by vigorous agitation. This
stock was then transferred to the Hollander and beaten
until a freeness value of 250SR was att~i~e~ (time
taken was usually 30 to 35 minutes). The stock was
then adjusted to a final consistency of 2% with further
1/3 PBS as necessary.
Addition/Incubation of Additives
The cellulase solution was added to the thick (2~
consistency) stock. Two litres of the thick stock
(cont~;n;ng 40g of fibre) was contained in a metal jug
and stirred at the lowest possible speed to achieve a
slow movement of the stock. Vigorous agitation should
be avoided otherwise denaturing of the enzyme may occur
during the incubation period. The stock was at ambient
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temperatures (20-25 C).
The ;ncl~h~tion time was fifteen minutes. During this
incllh~tion period the movement of the stock may Arr~
to become easier/faster. If this is apparent then
reduce the stirrer speed as much as possible.
After the fifteen minute incubation period had el~p~
the thick stock was then added to the p~poLLioner.
Prop~rt~;oner
The thick stock in the proportioner was then diluted to
a consistency of 0.25~ using DEMI water only. Normal
agitation speeds in the proportioner were employed to
mix the stock.
~n~heet Form~tion
The white water box was filled with DEMI water for
h~Che~t formation. With the ~n~cheet forming wire
in place in the mould assembly, one litre of stock from
the proportioner was added to the Deckle Box, together
with water from the white water box. The contents of
the Deckle Box were agitated with the perforated
agitator (moved up and down five times). After the
fifth stroke the agitator was rested on the surface of
the water to help dampen the motion of the water in the
Deckle Box. The water was then pumped back to the
white water box and the initial wet mat was formed.
Depending on how vigorous the agitation has been some
foaming may occur in the Deckle Box. This foam may
still persist after the initial wet mat is formed and
can be quite substantial. Some of this foam can be
dispersed if the pump is kept on for a few seconds
after the water has been removed so that air can be
drawn through the mat.
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~3
n~.eheet Pressin~ and Dryi ng
The wet mat and h~n~heet wire were l~ -ved from the
mould to the press. The moisture content of the
presséd sheet should be 70%. The pressed sheet was
s then dried on an electrically heated drum dryer. The
surface temperature of the dryer was 105-C and the
speed of the dryer was such that the pressed sheet was
in contact with the hot surface for 35 ~e~on~. The
final moi~ture content of the sheet should be between
lo 4 and 7~ (typically 5%).
If the moisture content of the sheet after pressing is
less than 70~, then the sheet may stick to the surface
of the drum dryer when the above conditions are
employed. This may occur because of nonuniform press
pressures being applied across the width of the ~heet.
Steps should be taken to avoid this.
When the surface tempera~ure of the drum dryer is less
than 105-C but is 70-C or higher, longer contact times
are re~uired in order for the h~n~cheet to have a final
moisture content of 5%.
If the surface temperature of the drum dryer is below
70 C, it is necessary to extend the contact time
further or increase the initial pressing on the wet mat
to remove more water or 1o do both. It is possible to
reduce the moisture content of the pressed sheet to
less than 60%.
Tes~ i ng
Conditioning and testinq of the paper is done according to
procedures laid out in the "Tappi Test Methods" published by
TAPPI, Technology Park Atlanta, P0 Box 105113, Atlanta GA
35 30348, USA, ISBN 0 - 89852 - 200 - 5 (vol 1 and 2). The HST
(Hercules Sizing Test) is defined as size test for paper by
ink resistance T 530 pm - 83; and the Cobb test is defined
by T 441 om - 84.
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24
A series of experiments were performed in which the
cellulase concentration, aging time and t~ ~ature were
each varied. The results are presented in the following
tables in which:-
"naturally aged" refers to storage for the specifiedtime at 23~C + 1~C in relative humidity 50.0 + 2% as
~pecified in T402Om-83;
"oven cured" refers to treatment at -80~C for ~0
ominutes.
8~zing Perform~nce of Handsheets made with Cellula~e and
~r~e~ under ~t~n~rd conditions
"~8T (secQ~ of hanasheets ma~e with Penici 77in~
~uniculosum
Protein added Ageing Condition
24h naturally 2 weeks oven
aged naturally cured
aged
20 bl.nk
1 % 1 7 6
1 % 3 11 18
"~ t~sCon~Q) of handsheets made with Tr ~h~rma reeseia
Protein added Ageing Cold.. tion
24h naturally , ~eek~ oven
aged naturally cured
aged
blank
5% 13 27 28
10% 34 53 61
"Cobb (gs~) of hanasheets made with Tri~hoA~rma ,
Protein added Ageing Condition
24h naturally 2 weeks oven
aged naturally cured
aged
blank Fully Fully Fully
saturated saturated saturated
5% 78.6 67.2 74.2
10% 68.5 51.2 50.7
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~5
~mpl~ g
In a further series of experiments conducted under the
protocols described in Example 8 above, the degree to which
the added protein tCelluclast (Trichoderma reesei, Novo
Nordisk) and Celluzyme (Humicola insolens, Novo Nordisk)] is
retained by the paper was investigated.
In separate experiments Celluclast and Celluzyme were added
to paper pulp and test sheets prepared described above (24
h naturally aged). The amount of protein retA i n~ in the
paper (as opposed to that remaining in the pulp supernatant
when forming the paper web) was estimated on the basis of
the nitrogen content of the paper, assuming that the
nitrogen content of both proteins is 16% w/w. The nitrogen
content was measured by Antec micropyrolysis.
The results are presented in the following table:
20 Additive Addition ~ ~ HST Cobb (g
l~vel Nitrogen Protein (secon~) water/m2
w/w db in dry retained
fibre paper w/w in pap~r
w/w db
fibre
Blank - 0.007 - 1 fully
~aturated
Cellucla~t 20 0.356 2.225 273 15.5
Celluzyme 5 0.374 2.3375 263 28.6
The results show that addition levels of Celluzyme one
quarter those of Celluclast gave rise to similar protein
retention levels. At similar protein retention levels, both
celluloses gave similar sizing effects.
Ex~mpl~ 10
~ In the following example, the binding amylase enzymes to
starch is demonstrated. Two amylase enzymes were
characterized using HPLC: an ~-amylase (Type X-A crude
preparation) from Aspergillus oryzae and amyloglucosidase
from A. niger (available from Sigma Aldrich Co. Ltd., Poole,
Dorset, United Kingdom). The main catalytic peaks of each
preparation were determined U5 ing a starch glucose-release
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26
assay. The binding efficiencies of each protein were
determined against a range of starches with BSA CG~L~ 018
included in the assessment.
A solution of 32 mg ml~1 (dry weight) of ~-amylase was made
up in 0.1 M PBS (pH7.0). 100~1 of this was loaded onto an
HPLC using a Bio-Sil SEC gel permeation column running 0.1
M phosphate buffer at 1 ml min~l. Fractions (1 ml) were
collected and tested for reducing sugars released from a
lo starch suspension using the st~n~rd microtitre assay (for
glucose).
The following qualitative assay was used to detect glucose
and cellobiose in test samples. The assay was carried out
in a micro titre dish at room temperature.
Reagent Components:
10 ~1 phenol reagent (0.128M phenol in O.lM phosphate buffer
pH7.0)
10 ~1 amino pyrine reagent (19.7mM 4-amino phenazone in O.lM
phosphate buffer pH7.0)
10 ~1 peroxidase in O.lM phosphate buffer pH 7.0 (to give
800Eu/ml)
10 ,Ul glucose oxidase in 0.lM phosphate buffer pH 7.0 (to
give 250Eu/ml)
60 ~1 O.lM phosphate buffer pH7.0
These reagent components were mixed and added to the wells
of a microtitre dish. Test samples 100 ~1 were added
followed by an e~C~cfi of substrate (starch). The appearance
of a red colour was indicative of the presence of amylase.
.
The same methods were also used to produce an HPLC profile
for the amyloglucosidase. The amyloglucosidase was a liquid
preparation containing approximately 262 mg ml~l protein as
measured by the Coomassie Blue technique. 100 ~1 of a 0.007
dilution in 0.1 M PBS (pH 7.0) was loaded onto the HPLC and
-
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27
monitored at 230 nm 0.1 AUS. 1 ml fractions were collected
and tested for reducing sugars released from starch
sùspensions as above.
The ability of cr-amylase and amyloglucosidase to bind to
normal starch in suspension was assessed. Starch (0.2 g;
Roquette) was added to 9 ml 0.1 M PBS (pH 7.0) and 1 ml ~-
amylase solution (9.5 mg ml~1 by Coomassie Blue assay) was
added. This was incubated on a shaker for 20 min.
The sample was centrifuged at 13,000 rpm for 5 min and 100
~l samples loaded onto the HPLC column. The peak profile of
the 20 min bound ~-amylase was compared with a T = 0 sample.
From this data the percentage binding of the enzyme was
calculated. The binding of amyloglucosidase was also tested
against cationic starch. BSA was also used in the same way
as a ~u~-LLol. The final concentration of the BSA used was
O.2% (wv 1) in 0.1 M PBS.
The results of the binding experiments are shown in the
following Table.
8t~rch b~ n~ profiles
Enzyme Substrate % Bound
25~-amylase starch 32
amyloglucosidase st,arch 27
amyloglucosidasecationic starch 45
BSA starch 7
BSA cationic starch 6
30 These results in~icate that both c~-amylases and
amyloglucosidases specifically bind to both starch and
cationic starch.
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