Note: Descriptions are shown in the official language in which they were submitted.
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FUSION PROTEINS AND DETERGENT COMPOSITIONS COMPRISING THEM
TECHNICAL FIEhD
The present invention generally relates fusion proteins and
detergent compositions comprising them. More in particular,
it relates to detergent compositions comprising fusion
proteins which are useful for delivering a benefit agent to
a fabric.
BACKGROUND AND PRIOR ART
In a laundry cleaning process it is sometimes desirable to
deliver benefit agents onto the fabric. Such a benefit agent
may for example be a fabric softening agent, a perfume, a
polymeric lubricant, a photoprotective agent, a latex, a
resin, a dye fixative agent, an encapsulated material, an
antioxidant, an insecticide, a soil repelling agent or a
soil release agent.
From WO-A-98/00500 (Unilever) it is known to deliver a
benefit agent onto fabric by means of peptide or protein
Deposition Aid having a high affinity for fabric. The
benefit agent is attached or adsorbed to a peptide or
protein Deposition Aid having a high affinity for fabric.
Preferably, the deposition aid is the cellulose binding
domain of a cellulase enzyme. The compositions are said to
effectively deposit the benefit agent onto the fabric during
the wash cycle.
In order to overcome certain drawbacks and limitations of
WO-A-98/00500, it has been further proposed in WO-A-01/46357
(Unilever) to use fusion proteins to deposit the benefit
agents onto the fabric. The fusion proteins comprise a
cellulose binding domain for binding to the fabric and a
domain having a high binding affinity for the benefit agent.
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However, the stability of detergent compositions disclosed
in WO-A-01/46357, may under certain circumstances, leave to
be desired. It was found that many of the available
technologies to encapsulat a the payload of benefit agent may
not give sufficiently stable encapsulates when they are
formulated into a detergent composition. Subsequently, the
benefit agent may leak out of the capsule during processing
of the formulation or upon storage.
It is an object of the pre sent invention to provide an
alternative or improved detergent composition, which is
capable of delivering a benefit agent to a fabric during a
washing or rinsing proces s.
Importantly, the use of chemically defined capsules is key
to maintain the integrity of capsules during the formulation
and storage stages of a detergent formulation. However,
because these chemical species are very simple in structure,
the ability to obtain a protein-binding molecule to
specifically recognise the se cross-linked repeating haptens
cannot be readily envisage d.
Surprisingly, we have now found that these and other objects
of the invention may be achieved by the fusion protein of
the present invention, which is characterised in that it
comprises a Carbohydrate Blinding Domain, preferably a
Cellulose Binding domain, and a domain having a high binding
affinity for a melamine-type polymer. We have found that, by
means of such fusion prote ins, it is possible to enhance the
delivery and retention of perfume encapsulated in melamine /
formaldehyde based polymer capsules to fabric in a main wash
formulation.
This fusion molecule is bi -functional in its binding
ability, whereby the Carbohydrate Binding Domain region
binds to carbohydrate such as ce11u1osic fabric materials
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and the second domain binds to a melamine-type polymer. In
this way, melamine capsules containing benefit,agents may be
targeted to the fabric to deliver the benefit agent onto the
fabric.
Furthermore, the same cap sine form can be used to deliver a
whole range of benefit agents, therefore the same protein
binding molecules may be used to delivery a whole range of
benefit agents utilising a generic capsule approach.
DEFINITION OF THE INVENTZ ON
According to a first aspect of the invention, there is
provided a fusion protein comprising a Carbohydrate Binding
Domain and a domain havin g a high binding affinity for a
melamine-type polymer.
According to a second asp ect, there is provided a detergent
composition comprising such fusion protein and micro-
particles containing a benefit agent.
According to a third aspect, there is provided a process for
delivering a benefit agent to a fabric by treating said
fabric with a composition comprising said fusion protein and
a micro-particles contain ing a benefit agent.
DETAINED DESCRIPTION OF THE INVENTION
1.1 The Carbohydrate Binding Domain
In its first aspect, the invention relates to a fusion
protein comprising a Carbohydrate Binding Domain (CBD) and a
domain having a high binding affinity for a melamine-type
polymer. A Carbohydrate Binding Domain is a polypeptide that
has a high affinity for or binds to water-soluble or water-
insoluble forms of carbohydrate polymers such as cellulose
and chitin, including thei r crystalline forms. CBDs are
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sometimes als o referred to as polysaccharide binding domains
(PBDs).
Preferred examples of Carbohydrate Binding Domains are
Cellulose Binding Domains. The term Carbohydrate Binding
Domain is broader and also includes protein sequences such
as starch binding domains, mannose binding domains, xylan
binding domains and chitin binding domains. Henrissat B. and
Davies G.J. (Plant Physiology (2000) 124(4):1515-1519)
describe many examples of such Carbohydrate Binding Domains.
The term Carbohydrate Binding Domain (or CBD) will be
collectively used hereinafter for any of such binding
domains which can be used as part of the fusion protein
according to the present invention, unless indicated
otherwise.
A preferred Carbohydrate Binding Domain is the Cellulose
Binding Domain, which will be used hereinafter as a typical
example of the Carbohydrate Binding Domain part of the
~0 fusion protein according to the present invention. Thus, the
abbreviation CBD is used hereinafter to indicate
Carbohydrate Binding Domains in general, whereas a Cellulose
Binding Domain is a typical and preferred example of the
broader defin.i tion of Carbohydrate Binding Domains.
Carbohydrate Binding Domains are found as integral parts of
large protein complexes consisting of two or more different
polypeptides, for example in hydrolytic enzymes (hydrolases)
which typicall y are composed of a catalytic domain
containing the active site for substrate hydrolysis, and a
Carbohydrate Blinding Domain such as a Cellulose Binding
Domain for binding to the insoluble matrix. Such enzymes may
comprise more than one catalytic domain and one, two or
three CBDs and optionally one or more polypeptide regions
linking the CB D(s) with the catalytic domain(s), the latter
regions usually being denoted a "linker". Examples of
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hydrolytic enzymes comprising a CBD are cellulases,
xy1 anases, mannanases, arabinofuranosidases, acetyl
est erases and chitinases. CBDs have also been found in
algae, e.g. the red alga Porphyra purpurea as a non-
5 hydrolytic polysaccharide binding protein, see Peter Tomme
et al., "Cellulose Binding Domains: Classification and
Properties" in "Enzymatic Degradation o.f Insoluble
Carbohydrates", John N. Saddler and Michael H. Penner
(Eds.), ACS Symposium Series, No. 618, 1996. However, most
of the known CBDs are from cellulases and xylanases.
In this context, the term "Cellulose Binding Domain" is
intended to be understood as defined by Tomme et al., op.
cit_ This definition classifies more than 120 Cellulose
Binding Domains into 10 families (I-X) which may have
different functions or roles in connection with the
meth anism of substrate binding. However, it is anticipated
that new family representatives and additional CBD families
will appear in the future.
A Carbohydrate Binding Domain may be exemplified by a
binding domain recognising cellulose. Such a Cellulose
Bindd.ng Domain is a part of many cellulolytic enzymes and
can be obtained therefrom. Cellulose Binding Domains are
also obtainable from xylanase and other hemicellulase
degrading enzymes. Preferably, the Cellulose Binding Domain
is obtainable from a fungal enzyme origin such as Humicola,
Trick oderma, Thermomonospora, Phanerochaete, Aspergillus,
Meripilus or from a bacterial enzyme origin such as
Baci1 lus, Clostridium, Streptomyces, Ce11u1omonas and
Pseudomonas,
In th a fusion protein according to the invention, the
Carbohydrate Binding Domain is fused, using conventional
rDNA techniques, to a second domain having a high binding
affinity for a melamine-type polymer. Preferably, the
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Carbohydrate Binding Domain is connected to the domain
having a high binding affinity for the melamine-type polymer
by means of a linker consisting of about 0-20, preferably
about 2-15, more preferably of 2-5 amino acid residues.
The second domain having a high binding affinity for the
melamine-type polymer may, for instance, be an antibody or
an antibody fragment. Especially preferred are heavy chain
antibodies such as found in Camelidae.
Llama antibodies have been shown to be able to bind to both
proteins and dye haptens. Published work on their structure
and functionality states that raising single chain
antibodies against haptens turned out to be difficult if not
impossible, especially with hydrophobic ones (Journal of
Molecular Biology 2001 311, 123-129). It was shown that it
was possible to generate binders to a variety of dye
h aptens. These characteristically contained a number of
benzene rings and sulphonated side chains.
The present inventors have now is~lated 3 antibody binding
domains from a library size of 1011 that are capable of
blinding to melamine particles. It was surprising that
binders to melamine were found, considering that melamine
represents a repeating unit of C3N6H6 [1,3,5 triazine 2,4,6
triamine] .
The fusion protein according to the invention may comprise
more than two recognition domains. It is for example
possible to produce a CBD fusion protein with more than one
antibody domain, in which the antibody domains may bind to
the same or bind to different antigens. Conversely, it is
a1 so possible in the CBD antibody fusion format to produce a
molecule with one antibody domain with more than one CBD,
whereby the CBD's incorporated may be identical sequences or
from more than one source, or modified varieties thereof.
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Generally speaking, the degree of binding of a molecule A to
another molecule B can be generally expressed by the
chemical equilibrium constant Kd resulting from the
following reaction:
(AJ+ (BJ t~ (A = BJ
The chemical equilibrium constant Kd is then given by:
Kd= (AJx(BJ
(A--_- BJ
Whether the binding of a molecule to the fabric / ligand is
specific or not can be judged from the difference between
the binding (Kd value) of the molecule to one type of fabric
/ ligand, versus the binding to another type of fabric /
ligand material. For applications in laundry, said ligand
material will form part of or be associated with a benefit
agent. In this aspect of the invention, the CBD region of
the fusion protein binds to the fabric and the high affinity
domain region binds to the melamine-type polymer agent.
Alternatively, this approach can be reversed whereby the
fabric material, or a ligand bound to the fabric is targeted
by the high affinity domain of the fusion protein and the
cellulose binding domain region binds to a cellulosic based
or cellulosic containing benefit agent.
However, it will usually be more convenient to measure Kd
values and differences in Kd values on other materials such
as a polystyrene microtitre plate or a specialised surface
in an analytical biosensor. The difference between the two
binding constants should be minimally 10, preferably more
than 100, and more preferably, more that 1000. Typically,
the reagent should bind to the ligand / fabric, with a Kd
lower than 10-4 M, preferably lower than 10-6 M and could be
10-1° M or even less. Higher binding affinities (Kd of less
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than 10-5 M) and/or a larger difference between the one type
of ligand / fabric and another type of ligand / fabric (or
background) binding would increase the deposition of the
melamine-type polymer in the micro-particles containing the
benefit agent. Also, the weight efficiency of the reagent in
the total rinse composition would be increased and smaller
amounts of the reagent would be required.
Several classes of reagent or molecules can be envisaged
which deliver the capability of specific binding to fabrics
/ ligands, to which one would like to deliver the benefit
agent. In the following we will give a number of examples of
molecules having such capabilities, without pretending to be
exhaustive.
1.2.1. Antibodies.
Antibodies are specific binding proteins. Their function in
nature is to protect against disease by recognising (and
binding) foreign bodies, such as viruses or bacteria, but
not self-cells. Furthermore, methods are well-known in the
art to generate antibodies that are specific for almost any
protein, organic molecule, or cell surface, that is likely
to be encountered. This binding specificity has been
exploited in the Biotechnology industry, principally for
medical diagnostics. For example, many home-based pregnancy
test kits comprise an antibody that specifically binds to
the pregnancy marker hormone, human chorionic gonadotropin
(hCG), but not to other hormones present in urine.
More recently, the use of antibodies in laundry products has
been described. In particular, Unilever has described the
use of stain-specific antibodies to target bleaching enzymes
exclusively to stains but not to dyes - thus achieving
efficient stain removal without damaging surrounding fabric
(WO-A-98/56885).
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Antibodies are well known examples of molecules that are
capable of binding specifically to compounds against which
they were raised. Antibodies can be derived from several
sources. From mice, monoclonal antibodies can be obtained
which possess very high binding affinities. From such
antibodies, Fab, Fv or scFv fragments, can be prepared which
have retained their binding properties. Such antibodies or
fragments can be produced through recombinant DNA technology
by microbial fermentation. Well known production hosts for
antibodies and their fragments are yeast, moulds or
bacteria.
A class of antibodies of particular interest is formed by
the Heavy Chain antibodies as found in Camelidae, like the
camel or the llama. The binding domains of these antibodies
consist of a single polypeptide fragment, namely the
variable region of the heavy chain polypeptide (HC-V). In
contrast, in the classic antibodies (murine, human, etc.),
the binding domain consists of two polypeptide chains (the
variable regions of the heavy chain (Vh~ and the light chain
(V1)). Procedures to obtain heavy chain immunoglobulins from
Camelidae, or (functionalized) fragments thereof, have been
described in WO-A-94/04678 (Casterman and Hamers) and WO-A-
94/25591 (Unilever and Free University of Brussels).
Alternatively, binding domains can be obtained from the Vh
fragments of classical antibodies by a procedure termed
"camelization". Hereby the classical Vh fragment is
transformed, by substitution of a number of amino acids,
into a HC-V-like fragment, whereby its binding properties
are retained. This procedure has been described by Riechmann
et al. in a number of publications (J. Mol. Biol. (1996)
259, 957-969 Protein. Eng. (1996) 9, 531-537,
Bio/Technology (1995) 13, 475-479). Also HC-V fragments can
be produced through recombinant DNA technology in a number
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of microbial hosts (bacterial, yeast, mould), as described
in WO-A-94/29457 (Unilever).
Met hods for producing fusion proteins that comprise an
5 enzyme and an antibody or that comprise an enzyme and an
ant ibody fragment are already known in the art. One approach
is described by Neuberger and Rabbits (EP-A-194 276). A
method for producing a fusion protein comprising an enzyme
and an antibody fragment that was derived from an antibody
10 on ginating in Camelidae is described in WO-A-94/25591. A
met hod for producing bispecific antibody fragments is
de scribed by Holliger et al. (1993) PNAS 90, 6444-6448.
A particularly attractive feature of antibody binding
behaviour is their reported ability to bind to a "family" of
structurally related molecules. For example, in Gani et al.
(J_ Steroid Biochem. Molec. Biol. 48, 277-282) an antibody
is described that was raised against progesterone but also
binds to the structurally-related steroids, pregnanedione,
pregnanolone and 6-hydroxy-progesterone. Therefore, using
the same approach, antibodies could be isolated that bind to
a whole "family" of stain chromophores (such as the
po lyphenols, porphyrins, or caretenoids as described below).
A broad action antibody such as this could be used to treat
several different stains when coupled to a bleaching enzyme.
1. 2 . 2 . Peptides .
Peptides usually have lower binding affinities to the
sub stances of interest than antibodies. Nevertheless, the
binding properties of carefully selected or designed
peptides can be sufficient to deliver the desired
se 1 ectivity in an oxidation process. A peptide which is
capable of binding selectively to a fabric / ligand to which
on a would like to deliver a benefit agent, can for instance
be obtained from a protein which is known to bind to that
specific fabric / ligand. An example of such a peptide would
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be a binding region extracted from an antibody raised
against that fabric / ligand. A suitable peptide could be
analogous to the active centre of a protein analogous to a
non-catalytic binding domain of a protein, e.g. a receptor.
Alternatively, peptides that bind to such substance can be
obtained by the use of peptide combinatorial libraries. Such
a library may contain up to 101° peptides, from which the
peptide with the desired binding properties can be isolated.
(R. A. Houghten, Trends in Genetics, Vol 9, no &, 235-239).
Several embodiments have been described for this procedure
(J. Scott et al., Science (1990) 249, 386-390; Fodor et al.,
Science (1991) 251, 767-773; K. Lam et al., Nature (1991)
354, 82-84~ R.A. Houghten et al., Nature (1991) 354, 84-86).
Suitable peptides can be produced by organic synthesis,
using for example the Merrifield procedure (Merrifield
(1963) J.Am.Chem.Soc. 85, 2149-2154). Alternatively, the
peptides can be produced by recombinant DNA technology in
microbial hosts (yeast, moulds, bacteria)(K.N. Faber et al.
(1996) Appl. Microbiol. Biotechnol. 45, 72-79).
1.2.3. Peptidomimics.
In order to improve the stability and/or binding properties
of a peptide, the molecule can be modified by the
incorporation of non-natural amino acids and/or non-natural
chemical linkages between the amino acids. Such molecules
are called peptidomimics (H. U. Saragovi et al. (1991)
Bio/Technology 10, 773-778; S. Chen et al. (1992)
Proc.Natl.Acad. Sci. USA 89, 5872-5876). The production of
such compounds is restricted to chemical synthesis.
1.2.4. Other organic molecules.
It can be readily envisaged that other molecular structures,
which need not be related to proteins, peptides or
derivatives thereof, can be found which bind selectively to
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fabrics / ligands to which one would like to deliver a
benefit agent. For example, certain polymeric RNA molecules
which have been shown to bind small synthetic dye molecules
(A. Ellington et al. (1990) Nature 346, 818-822). Such
binding compounds can be obtained by the combinatorial
approach, as described for peptides (L.B. McGown et al.
(1995), Analytical Chemistry, 663A-668A).
This approach can also be applied for purely organic
compounds which are not polymeric. Combinatorial procedures
for synthesis and selection for the desired binding
properties have been described for such compounds (Weber et
al. (1995) Angew.Chem.Int.Ed.Engl. 34, 2280-2282; G. Lowe
(1995), Chemical Society Reviews 24, 309-317; L.A. Thompson
et al. (1996) Chem. Rev. 96, 550-600). Once suitable binding
compounds have been identified, they can be produced on a
larger scale by means of organic synthesis.
A further embodiment of the present invention would be for
the domain with a high binding affinity to the melamine-type
polymer to be a bi-specific reagent. Such a reagent could
fulfil the requirement of accumulating the benefit agent on
the fabric either by supplying said reagent together with
the benefit agent as a pre-formed non-covalent complex or by
supplying the two separately and allowing them to self-
assemble either in the wash liquor or on the fabric.
2. The melamine-type polymer
The fusion proteins according to the invention comprises a
part having a high binding affinity for melamine-based
polymers, such that they can bind to micro-capsules made of
or containing such chemicals and polymers. Utilising and
producing melamine capsules is well known in the art, see
for instance WO-A-01/51197, WO-A-01/49817, US-A-6 248 703.
They contain and are characterised by the repeating unit of
C3N6H6 [1,3,5 triazine 2,4,6 triamine~.
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These melamine polymers are advantageously used in the
manufacture of micro-capsules, preferably having a particle
size o f between 0.1 and 100 micrometer, more preferably of
between 50 And 10 micrometer. Such micro-capsules are well
known and have been described in US-A-2003078043, JP-A-
101398 17, WO-A-03/035245, US-A-6 080 418. These melamine-
polymer containing micro-capsules contain the benefit agents
which is to be deposited onto the fabric. Many processes for
micro encapsulation are known. These include methods for
capsul a formation such as described in US-A-2 730 456, US-A-
2 800 457 and US-A-2 800 458. Other useful methods for
micro capsule manufacture are described in: US-A-4 001 140,
US-A- 4 081 376 and US-A-4 089 802 describing a reaction
betwe en urea and formaldehyde; US-A-4 100 103 describing
react ion between melamine and formaldehyde; GB-A-2 062 570
descr~.bing a process for producing microcapsules having
walls produced by polymerization of melamine and
forma ldehyde in the presence of a styrenesulfonic acid.
Micro -encapsulation is also taught in US-A-2 730 457 and US-
A-4 1 97 346. Processes for forming microcapsules from urea-
forma ldehyde resin and/or melamine formaldehyde resin are
disclosed in US-A-4 001 140, US-A-4 081 376, US-A-4 089 802,
US-A- 4 100 103, US-A-4 105 823, US-A-4 444 699. Alkyl
acryl ate/acrylic acid copolymer capsules are taught in US-A-
4 552 811.
3. The detergent composition
In th a detergent composition according to the invention, the
fusio n protein can be used to deposit a benefit agent onto
the fabric by means of the Carbohydrate Binding Domain.
In a further embodiment, the detergent compositions of the
invention comprise micro-particles comprising melamine-type
polymers, sensitised with CBD-antibody fusions, and
confi guyed such that the micro-particles are loaded with the
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benefit agent and the antibody domain of the fusion protein
has a high affinity or specificity for a substance (or
" marker molecule") typically found on some regions of
fabrics but not on others. Examples of such marker molecules
include bleach-damaged dyes and microbes known to be
associated with malodour. The antibody domain targets the
benefit agent to its intended site of action and binds it
there. For example, Microbe-specific antibody based fusions
may target fragrance-containing particles to the regions of
malodour. Thus, a more efficient use of expensive
ingredients is achieved. Alternatively, antibody based
fusions specific for bleach-damaged dyes can target dyed
particles to faded regions, thus replenishing the colour
lost in the main wash cycle.
The CBD antibody fusion protein binds to the fabric via the
CBD region, thereby allowing the antibody domain to bind to
corresponding antigens / ligands that comprise or form part
of the benefit agent. The fusion protein can be dosed in
conjunction with the benefit agent or can be added to the
fabric prior to the said fabric coming into contact with the
benefit agent.
A further aspect of the invention is a process for
delivering a benefit agent to a fabric by treating said
fabric with a composition comprising a fusion protein
according to the invention and micro-capsules comprising a
benefit agent selected from the group consisting of
softening agents, finishing agents/ protective agents,
fragrances (perfumes) and bleaches.
Examples of softening agents are clays, cationic surfactants
or silicon compounds. Examples of finishing agents/
protective agents are polymeric lubricants, soil repelling
agents, soil release agents, photo-protective agents
(sunscreens), anti-static agents, dye-fixing agents, anti-
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bacteri al agents and anti-fungal agents. The fragrances or
perfume s inside the micro-capsules may be further
encapsulated, e.g. in latex micro-capsules or the may be
gelatin a based coacervates.
5
SuitabL a examples of bleaches are photobleaches. Examples of
photobL caches are given in EP-A-379 312 (British Petroleum),
which discloses a water-insoluble photobleach derived from
anionically substituted porphine, and in EP-A-035 470 (Ciba
10 Geigy), which discloses a textile treatment composition
comprising a photobleaching component.
Another advantage of the present invention is that it is
possibl a to target some benefit molecules to particular
15 regions of fabric. For example, dyes can be targeted to
colour-bleached regions to replenish dye lost in the main
wash or fragrance can be targeted to regions where it is
most needed, in particular to those regions where microbes
associated with malodour are present, such as the "underarm"
regions .
4. The fabrics
For laundry detergent applications, several classes of
natural or man-made fabrics can be envisaged, in particular
cotton_ In the embodiment of the invention whereby the
antibody region of the fusion protein targets the fabric,
such macromolecular compounds have the advantage that they
can have a more immunogenic nature, i.e. that it is easier
to rail a antibodies against them. Furthermore, they are more
accessible at the surface of the fabric than for instance
colour ed substances in stains, which generally have a low
molecu tar weight.
An imp ortant embodiment of the invention is to use binding
domain s (as described above) that bind to several different
types of fabrics. This would have the advantage of enabling
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a benefit agent to be deposited to several different types
of fabric using the CBD-antibody fusion molecule.
5. The Detergent Composition.
The fusion proteins of the invention can be used in a
detergent composition that is specifically suited for the
purpose, and this constitutes a second aspect of the
invention. When formulating a detergent composition, it is
important to ensure that the other ingredients of the
product are compatible with the activity of the fusion
protein. WO-A-98/07820 (P&G) discloses inter alia rinse
treatment compositions containing antibodies directed at
cellulase and standard softener actives such as DEQA. The
detergent product according to the present invention
preferably contains no softener or low levels of softener
active (e. g. HEQ).
To that extent, the detergent composition comprises one or
more benefit agents and optionally other conventional
detergent ingredients. The invention in its second aspect
provides a detergent composition which comprises from 0.1 -
50 o by weight, based on the total composition, of one or
more surfactants. This surfactant system may in turn
comprise 0 - 95 o by weight of one or more anionic
surfactants and 5 - 100 o by weight of one or more nonionic
surfactants. The surfactant system may additionally contain
amphoteric or zwitterionic detergent compounds, but this in
not normally desired owing to their relatively high cost. It
may be advantageous to also include cationic surfactants
into the composition. Examples of suitable cationic
surfactants are given in WO-A-97/03160 and WO-A-98/17767
(Procter&Gamble).
In general, the nonionic and anionic surfactants of the
surfactant system may be chosen from the surfactants
described "Surface Active Agents" Vol. 1, by Schwartz &
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Perry, Interscience 1949, Vol. 2 by Schwartz, Perry & Berch,
I nterscience 1958, in the current edition of "McCutcheon's
Emulsifiers and Detergents" published by Manufacturing
Confectioners Company or in "Tenside-Taschenbuch", H.
Stache, 2nd Edn., Carl Hauser Verlag, 1981.
Suitable nonionic detergent compounds which may be used
include, in particular, the reaction products of compounds
having a hydrophobic group and a reactive hydrogen atom, for
example, aliphatic alcohols, acids, amides or alkyl phenols
with alkylene oxides, especially ethylene oxide either alone
or with propylene oxide. Specific nonionic detergent
compounds are C6-C22 alkyl phenol-ethylene oxide condensates,
generally 5 to 25 E0, i.e. 5 to 25 units of ethylene oxide
per molecule, and the condensation products of aliphatic C$-
C1$ primary or secondary linear or branched alcohols with
ethylene oxide, generally 5 to 40 EO.
Suitable anionic detergent compounds which may be used are
usually water-soluble alkali metal salts of organic
sulphates and sulphonates having alkyl radicals containing
from about 8 to about 22 carbon atoms, the term alkyl being
used to include the alkyl portion of higher acyl radicals.
Examples of suitable synthetic anionic detergent compounds
are sodium and potassium alkyl sulphates, especially those
obtained by sulphating higher Ca-C18 alcohols, produced for
example from tallow or coconut oil, sodium and potassium
alkyl C9-C2o benzene sulphonates, particularly sodium linear
secondary alkyl Clo-C15 benzene sulphonates; and sodium alkyl
glyceryl ether sulphates, especially those ethers of the
higher alcohols derived from tallow or coconut oil and
synthetic alcohols derived from petroleum. The preferred
anionic detergent compounds are sodium C11-C15 alkyl benzene
sulphonates and sodium C1~-Ci$ alkyl sulphates. Also
applicable are surfactants such as those described in EP-A-
328 177 (Unilever), which show resistance to salting-out,
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18
the alkyl polyglycoside surfactants described in EP-A-070
074, and alkyl monoglycosides.
Preferred surfactant systems are mixtures of anionic with
nonionic detergent active materials, in particular the
groups and examples of anionic and nonionic surfactants
pointed out in EP-A-346 995 (Unilever). Especially preferred
is surfactant system which is a mixture of an alkali metal
salt of a C16-C1$ primary alcohol sulphate together with a
C12-Cis primary alcohol 3-7 EO ethoxylate .
The nonionic detergent is preferably present in amounts
greater than 100, e.g. 25-90o by weight of the surfactant
system. Anionic surfactants can be present for example in
amounts in the range from about 5o to about 40o by weight of
the surfactant system.
The detergent composition may take any suitable physical
form, such as a powder, a tablet, an aqueous or non aqueous
liquid, a paste or a gel. The fusion protein according to
the invention will generally be used as a dilution in water
of about 0.05 to 20.
The detergent composition in accordance with the invention
comprising the fusion protein can have any suitable form,
i.e. the form of a granular composition, a liquid or a
slurry of the enzyme, or with carrier material (e.g. as in
EP-A-258 068 and the Savinase (TM) and Zipolase (TM)
products of Novo Nordisk). A good way of adding the fusion
protein to a liquid detergent product is in the form of a
slurry containing from 0.005 to 50 o by weight of the
complex in an ethoxylated alcohol nonionic surfactant.
The detergent compositions of the invention comprise about
0.001 to 50 mg, preferably from 0.01 to 10 mg of fusion
protein per liter of the rinse liquor in use. A concentrated
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19
detergent composition before use will comprise about 1 to
1000 mg/l, preferably from 10 mg to 100 mg per liter of the
detergent product.
The invention will now be further illustrated in the
following, non-limiting Examples. In the accompanying
drawings:
Figure 1 illustrates the ability of two isolated clones to
bind to melamine and a variety of other antigens,
Figure ~ illustrates the dilution factors used to
demonstrate the antibody / antigen binding event of four
chosen antibody proteins,
Figures 3 and 4 illustrate the ability of the fusion protein
to enhance the delivery of melamine based micro-capsules
(containing fragrance notes) to cotton fabric both in a
water rinse formulation (Figure 3) or in a formulated OMO
washing powder rinse (Figure 4),
Figure 5 gives the gene sequences of three melamine-binding
proteins VhhM-1E7, VhhM-1C8, VhhM-16711 which were isolated
out of the antibody library.
Example 1
Identification of melamine capsule binders
Figure 1 illustrates the ability of isolated clones to bind
to melamine and a variety of other antigens. With such a
small repeating epitope in the melamine particle structure,
the very fact that we have isolated binders is surprising.
Interestingly the binders also cross-react with gelatin
cross-linked microspheres, suggesting that the binding
epitope may also be present in these particles in the form
of an amine side chain or a common cross-linked motif
whereby formaldehyde or urea cross-linking is common with
amine containing micro-particles. Figure 2 illustrates the
dilution factors used to demonstrate the antibody / antigen
binding event of the chosen antibody proteins. Figure 5
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gives the gene sequences of the 3 melamine binding proteins
isolated out of the antibody library.
Example 2
5 Binding perfumed melamine particles to cotton.
Figures 3 and 4 illustrate the ability of the fusion protein
to enhance the delivery of melamine based microcapsules
(containing fragrance notes) to cotton fabric both in a
water rinse formulation (figure 3) or in a formulated OMO
10 washing powder rinse (figure 4). OMO is a commercially
available laundry detergent powder.
