U.S. patent application number 15/562180 was filed with the patent office on 2018-04-19 for detergent composition.
This patent application is currently assigned to NOVOZYMES A/S. The applicant listed for this patent is NOVOZYMES A/S. Invention is credited to Klaus Gori, Rune Lyngklip Jensen, Henrik Lund.
Application Number | 20180105772 15/562180 |
Document ID | / |
Family ID | 52814928 |
Filed Date | 2018-04-19 |
United States Patent
Application |
20180105772 |
Kind Code |
A1 |
Gori; Klaus ; et
al. |
April 19, 2018 |
DETERGENT COMPOSITION
Abstract
The present invention concerns a detergent comprising a
deoxyribonuclease (DNase). The present invention further relates to
methods and uses of the detergent comprising a deoxyribonuclease
(DNase) for laundering.
Inventors: |
Gori; Klaus; (Dyssegaard,
DK) ; Jensen; Rune Lyngklip; (Bagsvaerd, DK) ;
Lund; Henrik; (Kobenhavn N, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVOZYMES A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
NOVOZYMES A/S
Bagsvaerd
DK
|
Family ID: |
52814928 |
Appl. No.: |
15/562180 |
Filed: |
April 11, 2016 |
PCT Filed: |
April 11, 2016 |
PCT NO: |
PCT/EP2016/057912 |
371 Date: |
September 27, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C11D 11/0017 20130101;
C12N 9/22 20130101; C11D 1/72 20130101; C11D 17/0017 20130101; C12Y
301/21001 20130101; C11D 3/38636 20130101; C11D 1/22 20130101; C11D
1/29 20130101 |
International
Class: |
C11D 3/386 20060101
C11D003/386; C11D 1/22 20060101 C11D001/22; C11D 1/29 20060101
C11D001/29; C11D 17/00 20060101 C11D017/00; C12N 9/22 20060101
C12N009/22 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 10, 2015 |
EP |
15163230.4 |
Claims
1. A detergent composition comprising; (i) a polypeptide having
deoxyribonuclease activity, and (ii) at least one surfactant,
wherein the total amount of surfactant(s) in said composition is in
the range of 3.6 w/w % to 28.5 w/w %.
2. The detergent composition of claim 1, wherein said at least one
surfactant is selected from the group consisting of anionic
surfactant, cationic surfactant and non-ionic surfactant.
3. The detergent composition of claim 2, wherein said composition
comprises at least one anionic surfactant and the amount of said
anionic surfactant(s) in said composition is in the range of 2.5
w/w % to 19.6 w/w %.
4. The detergent composition according to claim 1, wherein said
composition comprises linear alkylbenzene sulfonate (LAS).
5. The detergent composition according to claim 4, wherein the
amount of linear alkylbenzene sulfonate (LAS) in said composition
is in the range of 1.2 w/w % to 9.6 w/w %.
6. The detergent composition according to claim 1, wherein said
composition comprises at least one alkyl ethoxysulfate (AEOS).
7. The detergent composition according to claim 6, wherein the
amount of said at least one alkyl ethoxysulfate (AEOS) in said
composition is in the range of 0.7 w/w % to 5.6 w/w %.
8. The detergent composition according to claim 1, wherein said
composition comprises at least one non-ionic surfactant and the
amount of said non-ionic surfactant(s) in said composition is in
the range of 1.1 w/w % to 8.8 w/w %.
9. The detergent composition according to claim 8, wherein said at
least one non-ionic surfactant is AEO Biosoft N25-7.
10. The detergent composition according to claim 1 further
comprising a builder.
11. The detergent composition according to claim 1, wherein said
composition is in a solid form.
12. The detergent composition according to claim 1, wherein said
composition is in a liquid form.
13. The detergent composition according to claim 4, wherein said
detergent composition comprises a solvent.
14. The detergent composition according to claim 1, wherein said
polypeptide having deoxyribonuclease activity is selected from the
group consisting of a polypeptide having at least 85% sequence
identity to the mature polypeptide of SEQ ID NO: 1, a polypeptide
having at least 85% sequence identity to the amino acid sequence
set forth in SEQ ID NO: 2, a polypeptide having at least 85%
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 3, a polypeptide having at least 85% sequence identity to the
mature polypeptide of SEQ ID NO: 4, a polypeptide having at least
85% sequence identity to the mature polypeptide of SEQ ID NO: 5 and
a polypeptide having at least 85% sequence identity to the mature
polypeptide of SEQ ID NO: 6.
15. The detergent composition according to claim 1, wherein said
polypeptide having deoxyribonuclease activity is selected from the
group consisting of a polypeptide comprising or consisting of the
mature polypeptide of SEQ ID NO: 1, a polypeptide comprising or
consisting of the amino acid sequence set forth in SEQ ID NO: 2, a
polypeptide comprising or consisting of the amino acid sequence set
forth in SEQ ID NO: 3, a polypeptide comprising or consisting of
the mature polypeptide of SEQ ID NO: 4, a polypeptide comprising or
consisting of the mature polypeptide of SEQ ID NO: 5 and a
polypeptide comprising or consisting of the mature polypeptide of
SEQ ID NO: 6.
16. The detergent composition according to claim 1, wherein the
concentration of said polypeptide having deoxyribonuclease activity
in a wash dose of said detergent composition is within the range of
0.001 ppm to 100 ppm.
17. A method for preventing, reducing or removing biofilm from a
textile or fabric comprising; (i) contacting said textile or fabric
at least partly coated with a biofilm with a wash liquor comprising
the detergent composition according to claim 1, (ii) subjecting
said textile to at least one washing cycle, and (iii) optionally
rinsing said textile.
18. The method according to claim 17, wherein the temperature of
said wash liquor is in the range of 5.degree. C. to 95.degree.
C.
19. The method according to claim 17, wherein the concentration of
said polypeptide having deoxyribonuclease activity in the wash
liquor is within the range of 0.001 ppm to 100 ppm.
20. (canceled)
Description
REFERENCE TO A SEQUENCE LISTING
[0001] This application contains a Sequence Listing in computer
readable form. The computer readable form is incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention concerns a detergent comprising a
deoxyribonuclease (DNase). The present invention further relates to
methods and uses of the detergent comprising a deoxyribonuclease
(DNase) for laundering.
BACKGROUND OF THE INVENTION
[0003] Microorganisms generally live attached to surfaces in many
natural, industrial, and medical environments, encapsulated by
extracellular substances including biopolymers and macromolecules.
The resulting layer of slime encapsulated microorganism is termed a
biofilm. Biofilms are the predominant mode of growth of bacteria in
the natural environment, and bacteria growing in biofilms exhibit
distinct physiological properties. Compared to their planktonically
grown counterparts, the bacteria in a biofilm are more resistant to
antibiotics, UV irradiation, detergents and the host immune
response.
[0004] It has for many years been a known problem that laundry
items like shirts and blouses become more and more grey as time
goes by. For normal day clothing as well as sportswear sweat and
the resulting odor is a challenge. These stains usually consist of
a lot of different components adhering to the textile of the
clothing and can be difficult to dissolve and remove. When laundry
items like T-shirts or sportswear are used, they are contacted to
sweat and bacteria from the body of the user and for other kinds of
dirt from the rest of the environment in which they are used. Some
of these bacteria are capable of adhering to the laundry item and
form a biofilm on the item. The presence of bacteria implies that
the laundry items become sticky and therefore soil adheres to the
sticky areas. This soil has shown difficult to remove by
commercially available detergent compositions. Further, when very
dirty laundry items are washed together with less dirty laundry
items the dirt present in the wash liquor tend to stick to the
biofilm. As a result hereof the laundry item is more "soiled" and
more grey after wash than before wash.
[0005] Sportswear is a good example, because there is often soil,
clay and traffic dirt on the clothes washed together with very
sweaty shirts. From wash to wash the clothes become more and more
grey and they eventually appear as developed spots. This kind of
dirt is one reason why people discard their clothes. Although the
problem is well known to most garment the problem is very
pronounced for mixed fabrics. There is a European political desire
to conserve resources for laundry which has led to their adoption
of a labeling law for washing machines in the EU to exclude
machines with high demand. This means that cold water washing is
much more prevalent in the EU and thus come to resemble the rest of
the world wash circumstances better. However, the saving of energy
by washing at lower temperature may lead to consumers discarding
clothes and buying new because the sweat stains are not properly
removed.
[0006] International patent application WO 2011/098579 concerns
bacterial deoxyribonuclease compounds and methods for biofilm
disruption and prevention. International patent application WO
2014/087011 concerns detergent compositions comprising one or more
anionic surfactants, a bacterial deoxyribonuclease and a further
enzyme selected from the group consisting of a protease, a lipase,
a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a
mannanase, an arabinase, a galactanase, a xylanase, and an
oxidase.
SUMMARY OF THE INVENTION
[0007] In a first aspect, the invention provides a detergent
composition comprising:
[0008] (i) a polypeptide having deoxyribonuclease activity,
[0009] (ii) at least one surfactant,
[0010] wherein the total amount of surfactant(s) in said
composition is in the range of 3.6 w/w % to 28.5 w/w %.
[0011] A second aspect concerns a method for preventing, reducing
or removing biofilm from a textile or fabric comprising: [0012] (i)
contacting the a textile or fabric at least partly coated with a
biofilm with a wash liquor comprising the detergent composition of
the present invention, [0013] (ii) subjecting said textile to at
least one washing cycle, [0014] (iii) optionally rinsing said
textile.
[0015] A third aspect concerns the use of the detergent composition
of the present invention for preventing, reducing or removing
biofilm from a textile or a fabric.
Definitions
[0016] Allelic variant: The term "allelic variant" means any of two
or more alternative forms of a gene occupying the same chromosomal
locus. Allelic variation arises naturally through mutation, and may
result in polymorphism within populations. Gene mutations can be
silent (no change in the encoded polypeptide) or may encode
polypeptides having altered amino acid sequences. An allelic
variant of a polypeptide is a polypeptide encoded by an allelic
variant of a gene.
[0017] Biofilm: A biofilm is any group of microorganisms in which
cells stick to each other on a surface, such as a textile, dishware
or hard surface. These adherent cells are frequently embedded
within a self-produced matrix of extracellular polymeric substance
(EPS). Biofilm EPS is a polymeric conglomeration generally composed
of extracellular DNA, proteins, and polysaccharides. Biofilms may
form on living or non-living surfaces. The microbial cells growing
in a biofilm are physiologically distinct from planktonic cells of
the same organism, which, by contrast, are single-cells that may
float or swim in a liquid medium.
[0018] Bacteria living in a biofilm usually have significantly
different properties from free-floating bacteria of the same
species, as the dense and protected environment of the film allows
them to cooperate and interact in various ways. One benefit of this
environment is increased resistance to detergents and antibiotics,
as the dense extracellular matrix and the outer layer of cells
protect the interior of the community.
[0019] On laundry biofilm producing bacteria can be found among the
following species: Adnetobacter sp., Aeromicrobium sp.,
Brevundimonas sp., Microbacterium sp., Micrococcus luteus,
Pseudomonas sp., Staphylococcus epidermidis, and Stenotrophomonas
sp.
[0020] Color difference (L value): A Lab color space is a
color-opponent space with dimension L for lightness. L value, L*
represents the darkest black at L*=0, and the brightest white at
L*=100. In the context of the present invention L value is also
referred to as color difference.
[0021] Detergent adjunct ingredient: The detergent adjunct
ingredient is different to the DNase of this invention. The precise
nature of these additional adjunct components, and levels of
incorporation thereof, will depend on the physical form of the
composition and the nature of the operation for which it is to be
used. Suitable adjunct materials include, but are not limited to
the components described below such as surfactants, builders,
flocculating aid, chelating agents, dye transfer inhibitors,
enzymes, enzyme stabilizers, enzyme inhibitors, catalytic
materials, bleach activators, hydrogen peroxide, sources of
hydrogen peroxide, preformed peracids, polymeric dispersing agents,
clay soil removal/anti-redeposition agents, brighteners, suds
suppressors, dyes, perfumes, structure elasticizing agents, fabric
softeners, carriers, hydrotropes, builders and co-builders, fabric
huing agents, anti-foaming agents, dispersants, processing aids,
and/or pigments.
[0022] Detergent Composition: The term "detergent composition"
refers to compositions that find use in the removal of undesired
compounds from items to be cleaned, such as textiles. The detergent
composition may be used to e.g. clean textiles for both household
cleaning and industrial cleaning. The terms encompass any
materials/compounds selected for the particular type of cleaning
composition desired and the form of the product (e.g., liquid, gel,
powder, granulate, paste, or spray compositions) and includes, but
is not limited to, detergent compositions (e.g., liquid and/or
solid laundry detergents and fine fabric detergents; fabric
fresheners; fabric softeners; and textile and laundry
pre-spotters/pretreatment). In addition to containing the enzyme of
the invention, the detergent formulation may contain one or more
additional enzymes (such as proteases, amylases, lipases,
cutinases, cellulases, endoglucanases, xyloglucanases, pectinases,
pectin lyases, xanthanases, peroxidases, haloperoxygenases,
catalases and mannanases, or any mixture thereof), and/or detergent
adjunct ingredients such as surfactants, builders, chelators or
chelating agents, bleach system or bleach components, polymers,
fabric conditioners, foam boosters, suds suppressors, dyes,
perfume, tannish inhibitors, optical brighteners, bactericides,
fungicides, soil suspending agents, anti-corrosion agents, enzyme
inhibitors or stabilizers, enzyme activators, transferase(s),
hydrolytic enzymes, oxido reductases, bluing agents and fluorescent
dyes, antioxidants, and solubilizers.
[0023] DNase (deoxyribonuclease): The term "DNase" means a
polypeptide with DNase activity that catalyzes the hydrolytic
cleavage of phosphodiester linkages in the DNA backbone, thus
degrading DNA. For purposes of the present invention, DNase
activity is determined according to the procedure described in the
Assay I. In one embodiment of the present invention, the DNase
activity of polypeptide having is at least 105%, e.g., at least
110%, at least 120%, at least 130%, at least 140%, at least 160%,
at least 170%, at least 180%, or at least 200% with reference to
the DNase activity of the mature polypeptide of SEQ ID NO: 1, a
polypeptide comprising or consisting of the sequence set forth in
SEQ ID NO: 2, a polypeptide comprising or consisting of the
sequence set forth in SEQ ID NO: 3, a polypeptide comprising or
consisting of the mature polypeptide of SEQ ID NO: 4, a polypeptide
comprising or consisting of the mature polypeptide of SEQ ID NO: 5
or a polypeptide comprising or consisting of the mature polypeptide
of SEQ ID NO: 6.
[0024] Enzyme Detergency benefit: The term "enzyme detergency
benefit" is defined herein as the advantageous effect an enzyme may
add to a detergent compared to the same detergent without the
enzyme. Important detergency benefits which can be provided by
enzymes are stain removal with no or very little visible soils
after washing and/or cleaning, prevention or reduction of
redeposition of soils released in the washing process (an effect
that also is termed anti-redeposition), restoring fully or partly
the whiteness of textiles which originally were white but after
repeated use and wash have obtained a greyish or yellowish
appearance (an effect that also is termed whitening). Textile care
benefits, which are not directly related to catalytic stain removal
or prevention of redeposition of soils, are also important for
enzyme detergency benefits. Examples of such textile care benefits
are prevention or reduction of dye transfer from one fabric to
another fabric or another part of the same fabric (an effect that
is also termed dye transfer inhibition or anti-backstaining),
removal of protruding or broken fibers from a fabric surface to
decrease pilling tendencies or remove already existing pills or
fuzz (an effect that also is termed anti-pilling), improvement of
the fabric-softness, colour clarification of the fabric and removal
of particulate soils which are trapped in the fibers of the fabric
or garment. Enzymatic bleaching is a further enzyme detergency
benefit where the catalytic activity generally is used to catalyze
the formation of bleaching components such as hydrogen peroxide or
other peroxides.
[0025] Fragment: The term "fragment" means a polypeptide having one
or more (e.g., several) amino acids absent from the amino and/or
carboxyl terminus of a mature polypeptide or domain; wherein the
fragment has DNase activity. In one embodiment, a fragment contains
at least 206 amino acid residues (e.g., amino acids 38 to 243 of
SEQ ID NO: 1), at least 205 amino acid residues (e.g., amino acids
39 to 243 of SEQ ID NO: 1), or at least 204 amino acid residues
(e.g., amino acids 40 to 243 of SEQ ID NO: 1).
[0026] Bacterial: In the context of the present invention, the term
"bacterial" in relation to polypeptide (such as an enzyme, e.g. a
DNase) refers to a polypeptide encoded by and thus directly
derivable from the genome of a bacteria, where such bacteria has
not been genetically modified to encode said polypeptide, e.g. by
introducing the encoding sequence in the genome by recombinant DNA
technology. In the context of the present invention, the term
"bacterial DNase" or "polypeptide having DNase activity obtained
from a "bacterial source" thus refers to a DNase encoded by and
thus directly derivable from the genome of a bacterial species,
where the bacterial species has not been subjected to a genetic
modification introducing recombinant DNA encoding said DNase. Thus,
the nucleotide sequence encoding the bacterial polypeptide having
DNase activity is a sequence naturally in the genetic background of
a bacterial species.
[0027] The bacterial polypeptide having DNase activity encoding by
such sequence may also be referred to a bacterial wildtype DNase
(or bacterial parent DNase). In a further embodiment, the
polypeptides having DNase activity are substantially homologous to
a bacterial DNase. In the context of the present invention, the
term "substantially homologous" denotes a polypeptide having DNase
activity which is at least 80%, preferably at least 85%, more
preferably at least 90%, more preferably at least 95%, even more
preferably at least 96%, 97%, 98%, and most preferably at least 99%
identical to the amino acid sequence of a selected bacterial
DNase.
[0028] Fungal: In the context of the present invention, the term
"fungal" in relation to polypeptide (such as an enzyme, e.g. a
DNase) refers to a polypeptide encoded by and thus directly
derivable from the genome of a fungus, where such fungus has not
been genetically modified to encode said polypeptide, e.g. by
introducing the encoding sequence in the genome by recombinant DNA
technology. In the context of the present invention, the term
"fungal DNase" or "polypeptide having DNase activity obtained from
a fungal source" thus refers to a DNase encoded by and thus
directly derivable from the genome of a fungal species, where the
fungal species has not been subjected to a genetic modification
introducing recombinant DNA encoding said DNase. Thus, the
nucleotide sequence encoding the fungal polypeptide having DNase
activity is a sequence naturally in the genetic background of a
fungal species. The fungal polypeptide having DNase activity
encoding by such sequence may also be referred to a fungal wildtype
DNase (or fungal parent DNase). In a further embodiment, the
polypeptides having DNase activity are substantially homologous to
another DNase. In the context of the present invention, the term
"substantially homologous" denotes a polypeptide having DNase
activity which is at least 80%, preferably at least 85%, more
preferably at least 90%, more preferably at least 95%, even more
preferably at least 96%, 97%, 98%, and most preferably at least 99%
identical to the amino acid sequence of any of the mature
polypeptides of SEQ ID NO 1, 4, 5 or 6.
[0029] Improved wash performance: The term "improved wash
performance" is defined herein as an enzyme displaying an increased
wash performance in a detergent composition relative to the wash
performance of same detergent composition without the enzyme e.g.
by increased stain removal or less redeposition. The term "improved
wash performance" includes wash performance in laundry.
[0030] Laundering: The term "laundering" relates to both household
laundering and industrial laundering and means the process of
treating textiles with a solution containing a cleaning or
detergent composition of the present invention. The laundering
process can for example be carried out using e.g. a household or an
industrial washing machine or can be carried out by hand.
[0031] By the term "malodor" is meant an odor which is not desired
on clean items. The cleaned item should smell fresh and clean
without malodors adhered to the item. One example of malodor is
compounds with an unpleasant smell, which may be produced by
microorganisms. Another example is unpleasant smells can be sweat
or body odor adhered to an item which has been in contact with
human or animal. Another example of malodor can be the odor from
spices, which sticks to items for example curry or other exotic
spices which smells strongly. One way of measuring the ability of
an item to adhere malodor is by using Assay II disclosed
herein.
[0032] Mature polypeptide: The term "mature polypeptide" means a
polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. In one
embodiment, the mature polypeptide is amino acids 38 to 243 of SEQ
ID NO: 1 and amino acids 1 to 22 of SEQ ID NO: 1 are a signal
peptide and amino acids 23 to 37 of SEQ ID NO: 1 are a propeptide.
It is known in the art that a host cell may produce a mixture of
two of more different mature polypeptides (i.e., with a different
C-terminal and/or N-terminal amino acid) expressed by the same
polynucleotide. It is also known in the art that different host
cells process polypeptides differently, and thus, one host cell
expressing a polynucleotide may produce a different mature
polypeptide (e.g., having a different C-terminal and/or N-terminal
amino acid) as compared to another host cell expressing the same
polynucleotide. In one embodiment, a mature polypeptides contains
up to 206 (such as 204) consecutive amino acid residues of the
sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., amino
acids 38 to 243 of SEQ ID NO: 1 or amino acids 1 to 206 of SEQ ID
NO: 2 or amino acids 1 to 204 of SEQ ID NO: 3), or up to 204 amino
acid residues (e.g., amino acids 40 to 243 of SEQ ID NO: 1). In
another embodiment, the mature polypeptide consists of the of the
amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. In
yet another embodiment, the mature polypeptide comprises or
consists of the consecutive amino acid residues 18 to 205 of SEQ ID
NO: 4. In one embodiment, the mature polypeptide comprises or
consists of the consecutive amino acid residues 34 to 142 of SEQ ID
NO: 5. In one embodiment, the mature polypeptide comprises or
consists of the consecutive amino acid residues 27 to 136 of SEQ ID
NO: 6.
[0033] Sequence identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity". For purposes of the present
invention, the sequence identity between two amino acid sequences
is determined using the Needleman-Wunsch algorithm (Needleman and
Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the
Needle program of the EMBOSS package (EMBOSS: The European
Molecular Biology Open Software Suite, Rice et al., 2000, Trends
Genet. 16: 276-277), preferably version 5.0.0 or later. The
parameters used are gap open penalty of 10, gap extension penalty
of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution
matrix. The output of Needle labeled "longest identity" (obtained
using the -nobrief option) is used as the percent identity and is
calculated as follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment)
[0034] For purposes of the present invention, the sequence identity
between two deoxyribonucleotide sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as
implemented in the Needle program of the EMBOSS package (EMBOSS:
The European Molecular Biology Open Software Suite, Rice et al.,
2000, supra), preferably version 5.0.0 or later. The parameters
used are gap open penalty of 10, gap extension penalty of 0.5, and
the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
The output of Needle labeled "longest identity" (obtained using the
-nobrief option) is used as the percent identity and is calculated
as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment).
[0035] Textile: The term "textile" means any textile material
including yarns, yarn intermediates, fibers, non-woven materials,
natural materials, synthetic materials, and any other textile
material, fabrics made of these materials and products made from
fabrics (e.g., garments and other articles). The textile or fabric
may be in the form of knits, wovens, denims, non-wovens, felts,
yarns, and toweling. The textile may be cellulose based such as
natural cellulosics, including cotton, flax/linen, jute, ramie,
sisal or coir or manmade cellulosics (e.g. originating from wood
pulp) including viscose/rayon, cellulose acetate fibers (tricell),
lyocell or blends thereof. The textile or fabric may also be
non-cellulose based such as natural polyamides including wool,
camel, cashmere, mohair, rabbit and silk or synthetic polymers such
as nylon, aramid, polyester, acrylic, polypropylene and
spandex/elastane, or blends thereof as well as blends of cellulose
based and non-cellulose based fibers. Examples of blends are blends
of cotton and/or rayon/viscose with one or more companion material
such as wool, synthetic fiber (e.g. polyamide fiber, acrylic fiber,
polyester fiber, polyvinyl chloride fiber, polyurethane fiber,
polyurea fiber, aramid fiber), and/or cellulose-containing fiber
(e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate
fiber, lyocell). Fabric may be conventional washable laundry, for
example stained household laundry. When the term fabric or garment
is used it is intended to include the broader term textiles as
well. In the context of the present invention, the term "textile"
also covers fabrics.
[0036] Variant: The term "variant" means a polypeptide having same
activity as the parent enzyme comprising an alteration, i.e., a
substitution, insertion, and/or deletion, at one or more (e.g.,
several) positions. A substitution means replacement of the amino
acid occupying a position with a different amino acid; a deletion
means removal of the amino acid occupying a position; and an
insertion means adding an amino acid adjacent to and immediately
following the amino acid occupying a position. In the context of
the present invention, a variant of an identified DNase has the
enzymatic activity of the parent, i.e. the capacity of catalyzing
the hydrolytic cleavage of phosphodiester linkages in the DNA
backbone (deoxyribonuclease activity). In one embodiment, the
deoxyribonuclease activity of the variant is increased with
reference to the parent DNase, e.g. the mature polypeptide of a
polypeptide having deoxyribonuclease activity is selected from the
group consisting of a polypeptide comprising or consisting of the
mature polypeptide of SEQ ID NO: 1, a polypeptide comprising or
consisting of the sequence set forth in SEQ ID NO: 2, a polypeptide
comprising or consisting of the sequence set forth in SEQ ID NO: 3,
a polypeptide comprising or consisting of the mature polypeptide of
SEQ ID NO: 4, a polypeptide comprising or consisting of the mature
polypeptide of SEQ ID NO: 5 or a polypeptide comprising or
consisting of the mature polypeptide of SEQ ID NO: 6.
[0037] Wash cycle: The term "wash cycle" is defined herein as a
washing operation wherein textiles are immersed in the wash liquor,
mechanical action of some kind is applied to the textile in order
to release stains and to facilitate flow of wash liquor in and out
of the textile and finally the superfluous wash liquor is removed.
After one or more wash cycles, the textile is generally rinsed and
dried.
[0038] Wash liquor: The term "wash liquor" is defined herein as the
solution or mixture of water and detergent components optionally
including the enzyme of the invention.
[0039] Wash time: The term "wash time" is defined herein as the
time it takes for the entire washing process; i.e. the time for the
wash cycle(s) and rinse cycle(s) together.
[0040] Whiteness: The term "Whiteness" is defined herein as a broad
term with different meanings in different regions and for different
consumers. Loss of whiteness can e.g. be due to greying, yellowing,
or removal of optical brighteners/hueing agents. Greying and
yellowing can be due to soil redeposition, body soils, colouring
from e.g. iron and copper ions or dye transfer. Whiteness might
include one or several issues from the list below: colourant or dye
effects; incomplete stain removal (e.g. body soils, sebum etc.);
redeposition (greying, yellowing or other discolourations of the
object) (removed soils reassociate with other parts of textile,
soiled or unsoiled); chemical changes in textile during
application; and clarification or brightening of colours.
DETAILED DESCRIPTION OF THE INVENTION
[0041] Biofilm can develop on textile when microorganisms are
present on an item and stick together on the item. Some
microorganisms tend to adhere to the surface of items such as
textiles. Some microorganisms adhere to such surfaces and form a
biofilm on the surface. The biofilm may be sticky and the adhered
microorganisms and/or the biofilm may be difficult to remove.
Furthermore, the biofilm adhere soil due to the sticky nature of
the biofilm. Commercial laundry detergent compositions available on
the marked do not remove such adhered microorganisms or biofilm.
The inventors have found that polypeptides having deoxyribonuclease
(DNase) activity can be used for disrupting/eroding biofilm on
textiles and/or fabrics and that the effect of DNase on the
disruption/erosion of the biofilm is independent on the amount of
surfactant applied. This is surprising as many enzymes are not
working optimally if surfactant concentration is lowered from the
original concentration. The inventors have found that DNase,
however, does not loose performance if surfactant concentration is
reduced by e.g. 30%. Accordingly, detergent composition comprising
DNase may be formulated with reduced amount of surfactants without
affecting the beneficial effect of the DNase. (e.g. when the total
amount of surfactant(s) including soap in the detergent composition
is in the range of about 3.6 w/w % to about 28.5 w/w %, about 3.0
w/w % to about 35 w/w %, about 5.0 w/w % to about 25 w/w %, about
5.0 w/w % to about 20 w/w %, about 10.0 w/w % to about 20 w/w % or
about 10.0 w/w % to about 15 w/w %). The inventors have found that
the DNases perform maintain or e.g. improve its effects on biofilm
swatches when the amount of surfactant(s) including soap in the
detergents is below 20. This has been shown in example 1, table 2
of the application where the performance of DNases, such as DNase
from A. oryzae in detergent with different levels of surfactants
are compared, e.g. when comparing performance of the DNases, such
as A. oryzae, in model detergent A with surfactant level (total
amount of surfactants including soap) of about 30 (33.5) with the
performance of the DNases, A. oryzae, in model detergent B with
surfactant level (total amount of surfactant with soap) of about 20
(22.1) the performance on biofilm swatches is similar in the two
detergents. Thus in some aspects of the invention the detergent
composition of the invention comprises; i) a polypeptide having
deoxyribonuclease activity and (ii) at least one surfactant,
wherein the total amount of surfactant(s) in said detergent
composition is reduced by at least 5%, such as at least 10%, such
as at least 20% such as at least 30% such as at least 40% such as
at least 50% such as at least 60% such as at least 70% such as at
least 80% such as at least 90% compared to the original detergent.
Reducing the amount of surfactants in detergents is beneficial for
the environment and reduces the cost of the detergent
compositions.
Detergent Composition of the Invention
[0042] A first aspect of the present invention provides a detergent
composition comprising:
[0043] (i) a polypeptide having deoxyribonuclease activity,
[0044] (ii) at least one surfactant,
[0045] wherein the total amount of surfactant(s) in said
composition is in the range of 3.6 w/w % to 28.5 w/w %, optionally
in the range of 3 w/w % to 30 w/w %, optionally in the range of 5
w/w % to 20 w/w %, optionally in the range of 10 w/w % to 20 w/w %
or optionally in the range of 15 w/w % to 20 w/w %. If nothing else
is mentioned w/w % has its common meaning in the field of laundry
detergents and includes the amount of surfactant in the detergent
solution e.g. wherein w/w % means the percent by weight of
surfactant in the total weight of detergent solution. If nothing
else is mentioned the total amount of surfactant includes soap if
one or more soap(s) is present in the detergent. If nothing else is
mentioned the term soap has its common meaning in the field of
laundry detergents and includes the meaning that soap is a salt of
a fatty acid.
[0046] In some aspects of the invention the detergent composition
of the invention comprises;
[0047] i) a polypeptide having DNase activity, such as fungal
DNase, and/or such as a DNase having a polypeptide sequence which
has at least 60% sequence identity to the mature polypeptide of SEQ
ID NO 1, wherein the activity of the DNase polypeptide is at least
100%, e.g., at least 110%, at least 110%, at least 120%, at least
130%, at least 140%, at least 160%, at least 170%, at least 180%,
or at least 200% compared to the to the DNase activity of the
mature polypeptide of SEQ ID NO: 1, and ii) at least one
surfactant, wherein the total amount of surfactant(s) in the
composition is about 25 w/w %, such as about 24 w/w %, such as
about 23 w/w %, such as about 22 w/w %, such as about 21 w/w %,
such as about 20 w/w %, such as about 19 w/w %, such as about 18
w/w %, such as about 17 w/w %, such as about 16 w/w %, such as
about 15 w/w %, such as about 14 w/w %, such as about 13 w/w %,
such as about 12 w/w %, such as about 11 w/w %, such as about 10
w/w %, such as about 9 w/w %, such as about 8 w/w %, such as about
7 w/w %, such as about 6 w/w %, such as about 5 w/w %, such as
about 4 w/w %, such as about 3 w/w %, such as about 2 w/w %, such
as about 1 w/w % or wherein the total amount of surfactant(s) is in
the range of about 3.6 w/w % to about 28.5 w/w %, about 3.0 w/w %
to about 35 w/w %, about 5.0 w/w % to about 25 w/w %, about 5.0 w/w
% to about 20 w/w %, about 10.0 w/w % to about 20 w/w % or about
10.0 w/w % to about 15 w/w %, where total amount of surfactant(s)
e.g. one or more surfactant, is the total amount of surfactant(s)
(e.g. one or more surfactants) including soap if soap is present in
the detergent.
[0048] In some aspects of the invention, the detergent composition
of the invention comprises;
[0049] i) a polypeptide having DNase activity, such as fungal
DNase, and/or such as a DNase having a polypeptide sequence which
has at least 60% sequence identity to the mature polypeptide of SEQ
ID NO 4, wherein the activity of the DNase polypeptide is at least
100%, e.g., at least 110%, at least 110%, at least 120%, at least
130%, at least 140%, at least 160%, at least 170%, at least 180%,
or at least 200% compared to the to the DNase activity of the
mature polypeptide of SEQ ID NO: 4, and ii) at least one
surfactant, wherein the total amount of surfactant(s) in the
composition is about 25 w/w %, such as about 24 w/w %, such as
about 23 w/w %, such as about 22 w/w %, such as about 21 w/w %,
such as about 20 w/w %, such as about 19 w/w %, such as about 18
w/w %, such as about 17 w/w %, such as about 16 w/w %, such as
about 15 w/w %, such as about 14 w/w %, such as about 13 w/w %,
such as about 12 w/w %, such as about 11 w/w %, such as about 10
w/w %, such as about 9 w/w %, such as about 8 w/w %, such as about
7 w/w %, such as about 6 w/w %, such as about 5 w/w %, such as
about 4 w/w %, such as about 3 w/w %, such as about 2 w/w %, such
as about 1 w/w % or wherein the total amount of surfactant(s) is in
the range of about 3.6 w/w % to about 28.5 w/w %, about 3.0 w/w %
to about 35 w/w %, about 5.0 w/w % to about 25 w/w %, about 5.0 w/w
% to about 20 w/w %, about 10.0 w/w % to about 20 w/w % or about
10.0 w/w % to about 15 w/w %, where total amount of surfactant(s)
e.g. one or more surfactant, is the total amount of surfactant(s)
(e.g. one or more surfactants) including soap if soap is present in
the detergent.
[0050] In some aspects of the invention, the detergent composition
of the invention comprises; i) a polypeptide having DNase activity,
such as bacillus DNase, and/or such as a DNase having a polypeptide
sequence which has at least 60% sequence identity to the mature
polypeptide of SEQ ID NO 5, wherein the activity of the DNase
polypeptide is at least 100%, e.g., at least 110%, at least 110%,
at least 120%, at least 130%, at least 140%, at least 160%, at
least 170%, at least 180%, or at least 200% compared to the to the
DNase activity of the mature polypeptide of SEQ ID NO: 5, and ii)
at least one surfactant, wherein the total amount of surfactant(s)
in the composition is about 25 w/w %, such as about 24 w/w %, such
as about 23 w/w %, such as about 22 w/w %, such as about 21 w/w %,
such as about 20 w/w %, such as about 19 w/w %, such as about 18
w/w %, such as about 17 w/w %, such as about 16 w/w %, such as
about 15 w/w %, such as about 14 w/w %, such as about 13 w/w %,
such as about 12 w/w %, such as about 11 w/w %, such as about 10
w/w %, such as about 9 w/w %, such as about 8 w/w %, such as about
7 w/w %, such as about 6 w/w %, such as about 5 w/w %, such as
about 4 w/w %, such as about 3 w/w %, such as about 2 w/w %, such
as about 1 w/w % or wherein the total amount of surfactant(s) is in
the range of about 3.6 w/w % to about 28.5 w/w %, about 3.0 w/w %
to about 35 w/w %, about 5.0 w/w % to about 25 w/w %, about 5.0 w/w
% to about 20 w/w %, about 10.0 w/w % to about 20 w/w % or about
10.0 w/w % to about 15 w/w %, where total amount of surfactant(s)
e.g. one or more surfactant, is the total amount of surfactant(s)
(e.g. one or more surfactants) including soap if soap is present in
the detergent.
[0051] In some aspects of the invention, the detergent composition
of the invention comprises; i) a polypeptide having DNase activity,
such as bacillus DNase, and/or such as a DNase having a polypeptide
sequence which has at least 60% sequence identity to the mature
polypeptide of SEQ ID NO 6, wherein the activity of the DNase
polypeptide is at least 100%, e.g., at least 110%, at least 110%,
at least 120%, at least 130%, at least 140%, at least 160%, at
least 170%, at least 180%, or at least 200% compared to the to the
DNase activity of the mature polypeptide of SEQ ID NO: 6, and ii)
at least one surfactant, wherein the total amount of surfactant(s)
in the composition is about 25 w/w %, such as about 24 w/w %, such
as about 23 w/w %, such as about 22 w/w %, such as about 21 w/w %,
such as about 20 w/w %, such as about 19 w/w %, such as about 18
w/w %, such as about 17 w/w %, such as about 16 w/w %, such as
about 15 w/w %, such as about 14 w/w %, such as about 13 w/w %,
such as about 12 w/w %, such as about 11 w/w %, such as about 10
w/w %, such as about 9 w/w %, such as about 8 w/w %, such as about
7 w/w %, such as about 6 w/w %, such as about 5 w/w %, such as
about 4 w/w %, such as about 3 w/w %, such as about 2 w/w %, such
as about 1 w/w % or wherein the total amount of surfactant(s) is in
the range of about 3.6 w/w % to about 28.5 w/w %, about 3.0 w/w %
to about 35 w/w %, about 5.0 w/w % to about 25 w/w %, about 5.0 w/w
% to about 20 w/w %, about 10.0 w/w % to about 20 w/w % or about
10.0 w/w % to about 15 w/w %, where total amount of surfactant(s)
e.g. one or more surfactant, is the total amount of surfactant(s)
(e.g. one or more surfactants) including soap if soap is present in
the detergent.
[0052] In some aspects of the invention, at least one surfactant is
selected from the group consisting of anionic surfactant, cationic
surfactant and non-ionic surfactant. In one embodiment the ratio of
non-ionic surfactant versus anionic and/or cationic surfactant is
1:1, 1:2, 1:3 or 1:4.
[0053] In a further embodiment, the detergent composition comprises
at least one synthetic surfactant, i.e. an artificial surfactant
which is not available from a natural source such as crops, animal
fats. In yet a further embodiment, the at least one synthetic
surfactant is selected from the list consisting of a synthetic
anionic surfactant, synthetic cationic surfactant and synthetic
non-ionic surfactant. In a preferred embodiment, said composition
comprises at least one anionic surfactant and the amount of said
anionic surfactant(s) in said composition is in the range of 2.5
w/w % to 19.6 w/w %, such as in the range of about 2 w/w % to about
30 w/w %, about 2 w/w % to about 20 w/w %, such as in the range of
about 5 w/w % to about 20 w/w %, such as about 10 w/w % to about 20
w/w % or such as about 15 w/w % to about 20 w/w %, wherein w/w %
means the percent by weight of surfactant in the total weight of
detergent solution. If nothing else is mentioned the total amount
of surfactant(s) includes soap if one or more soap(s) is present in
the detergent. In some aspects the amount of surfactant(s) in the
composition of the present invention is in the range of 2 w/w % to
30 w/w % e.g. in the range of 2 w/w % to 29 w/w %, 2 w/w % to 28
w/w %, 2 w/w % to 27 w/w %, 2 w/w % to 26 w/w %, 2 w/w % to 25 w/w
%, 2 w/w % to 24 w/w %, 2 w/w % to 23 w/w %, 2 w/w % to 22 w/w %, 2
w/w % to 21 w/w %, 2 w/w % to 20 w/w %, 2 w/w % to 19 w/w %, 2 w/w
% to 18 w/w %, 2 w/w % to 17 w/w %, 2 w/w % to 16 w/w %, 2 w/w % to
15 w/w %, 2 w/w % to 14 w/w %, 2 w/w % to 13 w/w %, 2 w/w % to 12
w/w %, 2 w/w % to 11 w/w %, 2 w/w % to 10 w/w %, 2 w/w % to 9 w/w
%, 2 w/w % to 8 w/w %, 2 w/w % to 7 w/w %, 2 w/w % to 6 w/w %, 2
w/w % to 5 w/w %, 2 w/w % to 4 w/w % or 2 w/w % to 3 w/w %, where
total amount of surfactant(s) e.g. one or more surfactant, is the
total amount of surfactant(s) (e.g. one or more surfactant(s) in
the detergent solution.
Surfactants
[0054] The detergent composition of the present invention comprises
one or more surfactants, which may be anionic and/or cationic
and/or non-ionic and/or semi-polar and/or zwitterionic, or a
mixture thereof. In a particular embodiment, the detergent
composition includes a mixture of one or more nonionic surfactants
and one or more anionic surfactants. The total amount of
surfactant(s) is in the range of 3.6 w/w % to 28.5 w/w %, such as 4
w/w % to 25 w/w %, such as, such as 5% to 25%, or 10% to about 20%,
or 15% to about 20%, where total amount of surfactant(s) e.g. one
or more surfactant, is the total amount of surfactant(s) (e.g. one
or more surfactants) in the detergent solution. The surfactant(s)
is chosen based on the desired cleaning application, which is
laundry.
Anionic Surfactants
[0055] In a preferred embodiment of the present invention, the
detergent composition comprises at least one anionic surfactant.
When included therein the detergent composition, the amount of the
anionic surfactant(s) in said composition is in the range of about
2.5 to 20% by weight (w/w %), such as 2.5 w/w % to 19.6 w/w %, for
example from about 5 w/w % to about 15 w/w %, such as 10 w/w % to
about 15 w/w %. Non-limiting examples of anionic surfactants
include sulfates and sulfonates, in particular, linear
alkylbenzenesulfonates (LAS), isomers of LAS, branched
alkylbenzenesulfonates (BABS), phenylalkanesulfonates,
alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates,
alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and
disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate
(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates
(PAS), alcohol ethersulfates (AES or AEOS or FES, also known as
alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary
alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,
sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid
methyl esters (alpha-SFMe or SES) including methyl ester sulfonate
(MES), alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl
succinic acid (DTSA), fatty acid derivatives of amino acids,
diesters and monoesters of sulfo-succinic acid or salt of fatty
acids (soap), and combinations thereof.
[0056] In a preferred embodiment, the composition comprises linear
alkylbenzene sulfonate (LAS). In one embodiment, the amount of
linear alkylbenzene sulfonate (LAS) in the composition is in the
range of 1.2 w/w % to 9.6 w/w %, such as 2 w/w % to 9 w/w %, for
example 3 w/w % to 7 w/w %, such as 4 w/w % to 6 w/w %, for example
5 w/w % to 6 w/w %.
In some aspects of the present invention provides a detergent
composition comprising:
[0057] (i) a polypeptide having deoxyribonuclease activity,
[0058] (ii) at least one surfactant,
[0059] wherein the total amount of surfactant(s) in said
composition is in the range of 3.6 w/w % to 28.5 w/w %, optionally
in the range of 3 w/w % to 30 w/w %, optionally in the range of 5
w/w % to 20 w/w %, optionally in the range of 10 w/w % to 20 w/w %
or optionally in the range of 15 w/w % to 20 w/w %, wherein the
composition comprises from about 1.2 w/w % to about 9.6 w/w %, or
optionally from about 2 w/w % to about 9 w/w %, or optionally from
about 3 w/w % to about 7 w/w %, or optionally from about 4 w/w % to
about 6 w/w % or optionally from about 5 w/w % to about 6 w/w %
linear alkylbenzene sulfonate (LAS).
[0060] In another preferred embodiment, the composition comprises
at least one alkyl ethoxysulfate (AEOS). In one embodiment, the
amount of said least one alkyl ethoxysulfate (AEOS) in said
composition is in the range of 0.7 w/w % to 5.6 w/w %.
Nonionic Surfactants
[0061] In one embodiment, the composition comprises at least one
nonionic surfactant. When included therein the detergent
composition, the at least one nonionic surfactant(s) is in the
range of about 1 to 10% by weight (w/w %), such as 1.1 w/w % to 8.8
w/w %, for example from about 1 w/w % to about 9 w/w %, such as 2
w/w % to about 5 w/w %. Non-limiting examples of nonionic
surfactants include alcohol ethoxylates (AE or AEO), alcohol
propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty
acid alkyl esters, such as ethoxylated and/or propoxylated fatty
acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol
ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines,
fatty acid monoethanolamides (FAM), fatty acid diethanolamides
(FADA), ethoxylated fatty acid monoethanolamides (EFAM),
propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl
fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine
(glucamides, GA, or fatty acid glucamides, FAGA), as well as
products available under the trade names SPAN and TWEEN, and
combinations thereof. In a preferred embodiment, the least one
non-ionic surfactant is AEO Biosoft N25-7.
Cationic Surfactants
[0062] The detergent composition of the present invention may also
include one or more cationic surfactants. Non-limiting examples of
cationic surfactants include alkyldimethylethanolamine quat
(ADMEAQ), cetyltrimethylammonium bromide (CTAB),
dimethyldistearylammonium chloride (DSDMAC), and
alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds,
alkoxylated quaternary ammonium (AQA) compounds, ester quats, and
combinations thereof. When included therein the detergent
composition, the at least one cationic surfactant(s) is in the
range of about 1 to 10% by weight (w/w %), such as 1.1 w/w % to 8.8
w/w %, for example from about 1 w/w % to about 9 w/w %, such as 2
w/w % to about 5 w/w %.
Semipolar Surfactants
[0063] The detergent composition of the present invention may also
include one or more semipolar surfactants. Non-limiting examples of
semipolar surfactants include amine oxides (AO) such as
alkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide and
N-(tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, and
combinations thereof. Non-limiting examples of zwitterionic
surfactants include betaines such as alkyldimethylbetaines,
sulfobetaines, and combinations thereof. When included therein the
detergent composition, the at least one semipolar surfactant(s) is
in the range of about 1 to 10% by weight (w/w %), such as 1.1 w/w %
to 8.8 w/w %, for example from about 1 w/w % to about 9 w/w %, such
as 2 w/w % to about 5 w/w %.
Hydrotropes
[0064] A hydrotrope is a compound that solubilises hydrophobic
compounds in aqueous solutions (or oppositely, polar substances in
a non-polar environment). Typically, hydrotropes have both
hydrophilic and a hydrophobic character (so-called amphiphilic
properties as known from surfactants); however the molecular
structure of hydrotropes generally do not favor spontaneous
self-aggregation, see e.g. review by Hodgdon and Kaler (2007),
Current Opinion in Colloid & Interface Science 12: 121-128.
Hydrotropes do not display a critical concentration above which
self-aggregation occurs as found for surfactants and lipids forming
miceller, lamellar or other well defined meso-phases. Instead, many
hydrotropes show a continuous-type aggregation process where the
sizes of aggregates grow as concentration increases. However, many
hydrotropes alter the phase behavior, stability, and colloidal
properties of systems containing substances of polar and non-polar
character, including mixtures of water, oil, surfactants, and
polymers. Hydrotropes are classically used across industries from
pharma, personal care, food, to technical applications. Use of
hydrotropes in detergent compositions allow for example more
concentrated formulations of surfactants (as in the process of
compacting liquid detergents by removing water) without inducing
undesired phenomena such as phase separation or high viscosity.
[0065] The detergent may contain 0-10% by weight, for example 0-5%
by weight, such as about 0.5 to about 5%, or about 3% to about 5%,
of a hydrotrope. Any hydrotrope known in the art for use in
detergents may be utilized. Non-limiting examples of hydrotropes
include sodium benzenesulfonate, sodium p-toluene sulfonate (STS),
sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS),
sodium cymene sulfonate, amine oxides, alcohols and
polyglycolethers, sodium hydroxynaphthoate, sodium
hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and
combinations thereof.
Builders and Co-Builders
[0066] In one embodiment of the present invention, the detergent
composition further comprises a builder.
[0067] The detergent composition may contain about 0-65% by weight,
such as about 5% to about 50% of a detergent builder or co-builder,
or a mixture thereof. In a dish wash detergent, the level of
builder is typically 40-65%, particularly 50-65%. The builder
and/or co-builder may particularly be a chelating agent that forms
water-soluble complexes with Ca and Mg. Any builder and/or
co-builder known in the art for use in laundry detergents may be
utilized. Non-limiting examples of builders include zeolites,
diphosphates (pyrophosphates), triphosphates such as sodium
triphosphate (STP or STPP), carbonates such as sodium carbonate,
soluble silicates such as sodium metasilicate, layered silicates
(e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol
(MEA), diethanolamine (DEA, also known as 2,2'-iminodiethan-1-ol),
triethanolamine (TEA, also known as 2,2',2''-nitrilotriethan-1-ol),
and (carboxymethyl)inulin (CMI), and combinations thereof.
[0068] The detergent composition may also contain 0-50% by weight,
such as about 5% to about 30%, of a detergent co-builder. The
detergent composition may include include a co-builder alone, or in
combination with a builder, for example a zeolite builder.
Non-limiting examples of co-builders include homopolymers of
polyacrylates or copolymers thereof, such as poly(acrylic acid)
(PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA). Further
non-limiting examples include citrate, chelators such as
aminocarboxylates, aminopolycarboxylates and phosphonates, and
alkyl- or alkenylsuccinic acid. Additional specific examples
include 2,2',2''-nitrilotriacetic acid (NTA),
ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid
(IDS), ethylenediamine-N,N'-disuccinic acid (EDDS),
methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid
(GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP),
ethylenediaminetetra(methylenephosphonic acid) (EDTMPA),
diethylenetriaminepentakis(methylenephosphonic acid) (DTMPA or
DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic
acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid
(ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic
acid (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS),
N-(2-sulfoethyl)-aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic
acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL),
N-methyliminodiacetic acid (MIDA), .alpha.-alanine-N,N-diacetic
acid (.alpha.-ALDA), serine-N,N-diacetic acid (SEDA),
isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid
(PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic
acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and
sulfomethyl-N,N-diacetic acid (SMDA),
N-(2-hydroxyethyl)ethylenediamine-N,N,N''-triacetic acid (HEDTA),
diethanolglycine (DEG), diethylenetriamine
penta(methylenephosphonic acid) (DTPMP),
aminotris(methylenephosphonic acid) (ATMP), and combinations and
salts thereof. Further exemplary builders and/or co-builders are
described in, e.g., WO 09/102854, U.S. Pat. No. 5,977,053.
Bleaching Systems
[0069] The detergent may contain 0-30% by weight, such as about 1%
to about 20%, of a bleaching system. Any bleaching system known in
the art for use in detergents may be utilized. Suitable bleaching
system components include bleaching catalysts, photobleaches,
bleach activators, sources of hydrogen peroxide such as sodium
percarbonate, sodium perborates and hydrogen peroxide-urea (1:1),
preformed peracids and mixtures thereof. Suitable preformed
peracids include, but are not limited to, peroxycarboxylic acids
and salts, diperoxydicarboxylic acids, perimidic acids and salts,
peroxymonosulfuric acids and salts, for example, Oxone (R), and
mixtures thereof. Non-limiting examples of bleaching systems
include peroxide-based bleaching systems, which may comprise, for
example, an inorganic salt, including alkali metal salts such as
sodium salts of perborate (usually mono- or tetra-hydrate),
percarbonate, persulfate, perphosphate, persilicate salts, in
combination with a peracid-forming bleach activator. The term
bleach activator is meant herein as a compound which reacts with
hydrogen peroxide to form a peracid via perhydrolysis. The peracid
thus formed constitutes the activated bleach. Suitable bleach
activators to be used herein include those belonging to the class
of esters, amides, imides or anhydrides. Suitable examples are
tetraacetylethylenediamine (TAED), sodium
4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS),
4-(dodecanoyloxy)benzene-1-sulfonate (LOBS),
4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS
or DOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and/or those
disclosed in WO98/17767. A particular family of bleach activators
of interest was disclosed in EP624154 and particulary preferred in
that family is acetyl triethyl citrate (ATC). ATC or a short chain
triglyceride like triacetin has the advantage that it is
environmentally friendly Furthermore acetyl triethyl citrate and
triacetin have good hydrolytical stability in the product upon
storage and are efficient bleach activators. Finally ATC is
multifunctional, as the citrate released in the perhydrolysis
reaction may function as a builder. Alternatively, the bleaching
system may comprise peroxyacids of, for example, the amide, imide,
or sulfone type. The bleaching system may also comprise peracids
such as 6-(phthalimido)peroxyhexanoic acid (PAP). The bleaching
system may also include a bleach catalyst or booster. Some
non-limiting examples of bleach catalysts that may be used in the
compositions of the present invention include manganese oxalate,
manganese acetate, manganese-collagen, cobalt-amine catalysts and
manganese triazacyclononane (MnTACN) catalysts; particularly
preferred are complexes of manganese with
1,4,7-trimethyl-1,4,7-triazacyclononane (Me3-TACN) or
1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), in
particular Me3-TACN, such as the dinuclear manganese complex
[(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and
[2,2',2''-nitrilotris(ethane-1,2-diylazanylylidene-.kappa.N-methanylylide-
ne)triphenolato-.kappa.3O]manganese(III). The bleach catalysts may
also be other metal compounds, such as iron or cobalt
complexes.
[0070] In some embodiments, the bleach component may be an organic
catalyst selected from the group consisting of organic catalysts
having the following formulae:
##STR00001##
[0071] (iii) and mixtures thereof;
[0072] wherein each R.sup.1 is independently a branched alkyl group
containing from 9 to 24 carbons or linear alkyl group containing
from 11 to 24 carbons, preferably each R.sup.1 is independently a
branched alkyl group containing from 9 to 18 carbons or linear
alkyl group containing from 11 to 18 carbons, more preferably each
R.sup.1 is independently selected from the group consisting of
2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl,
tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl
and isopentadecyl. Other exemplary bleaching systems are described,
e.g. in WO2007/087258, WO2007/087244, WO2007/087259, EP1867708
(Vitamin K) and WO2007/087242. Suitable photobleaches may for
example be sulfonated zinc or aluminium phthalocyanines.
[0073] Preferably, the bleach component comprises a source of
peracid in addition to bleach catalyst, particularly organic bleach
catalyst. The source of peracid may be selected from (a) pre-formed
peracid; (b) percarbonate, perborate or persulfate salt (hydrogen
peroxide source) preferably in combination with a bleach activator;
and (c) perhydrolase enzyme and an ester for forming peracid in
situ in the presence of water in a textile treatment step.
Polymers
[0074] The detergent may contain 0-10% by weight, such as 0.5-5%,
2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymer known in the art
for use in detergents may be utilized. The polymer may function as
a co-builder as mentioned above, or may provide antiredeposition,
fiber protection, soil release, dye transfer inhibition, grease
cleaning and/or anti-foaming properties. Some polymers may have
more than one of the above-mentioned properties and/or more than
one of the below-mentioned motifs. Exemplary polymers include
(carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA),
poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethylene
oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin
(CMI), and polycarboxylates such as PAA, PAA/PMA, poly-aspartic
acid, and lauryl methacrylate/acrylic acid copolymers,
hydrophobically modified CMC (HM-CMC) and silicones, copolymers of
terephthalic acid and oligomeric glycols, copolymers of
poly(ethylene terephthalate) and poly(oxyethene terephthalate)
(PET-POET), PVP, poly(vinylimidazole) (PVI),
poly(vinylpyridine-N-oxide) (PVPO or PVPNO) and
polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary
polymers include sulfonated polycarboxylates, polyethylene oxide
and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate.
Other exemplary polymers are disclosed in, e.g., WO 2006/130575.
Salts of the above-mentioned polymers are also contemplated.
Fabric Hueing Agents
[0075] The detergent compositions of the present invention may also
include fabric hueing agents such as dyes or pigments, which when
formulated in detergent compositions can deposit onto a fabric when
said fabric is contacted with a wash liquor comprising said
detergent compositions and thus altering the tint of said fabric
through absorption/reflection of visible light. Fluorescent
whitening agents emit at least some visible light. In contrast,
fabric hueing agents alter the tint of a surface as they absorb at
least a portion of the visible light spectrum. Suitable fabric
hueing agents include dyes and dye-clay conjugates, and may also
include pigments. Suitable dyes include small molecule dyes and
polymeric dyes. Suitable small molecule dyes include small molecule
dyes selected from the group consisting of dyes falling into the
Colour Index (C.I.) classifications of Direct Blue, Direct Red,
Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic
Violet and Basic Red, or mixtures thereof, for example as described
in WO2005/03274, WO2005/03275, WO2005/03276 and EP1876226 (hereby
incorporated by reference). The detergent composition preferably
comprises from about 0.00003 wt % to about 0.2 wt %, from about
0.00008 wt % to about 0.05 wt %, or even from about 0.0001 wt % to
about 0.04 wt % fabric hueing agent. The composition may comprise
from 0.0001 wt % to 0.2 wt % fabric hueing agent, this may be
especially preferred when the composition is in the form of a unit
dose pouch. Suitable hueing agents are also disclosed in, e.g. WO
2007/087257 and WO2007/087243.
Enzymes
Deoxyribonuclease (DNases)
[0076] The detergent composition of the present invention comprises
a polypeptide having deoxyribonuclease activity (a
deoxyribonuclease). The polypeptide having deoxyribonuclease
activity is preferably a microbial deoxyribonuclease, such as a
bacterial or a fungal deoxyribonuclease.
[0077] In one embodiment, the polypeptides having DNase activity
are substantially homologous to a bacterial DNase. In the context
of the present invention, the term "substantially homologous"
denotes a polypeptide having DNase activity which is at least 80%,
preferably at least 85%, more preferably at least 90%, more
preferably at least 95%, even more preferably at least 96%, 97%,
98%, and most preferably at least 99% identical to the amino acid
sequence of a selected bacterial DNase.
[0078] In a further embodiment, the polypeptides having DNase
activity are substantially homologous to a fungal DNase. In the
context of the present invention, the term "substantially
homologous" denotes a polypeptide having DNase activity which is at
least 80%, preferably at least 85%, more preferably at least 90%,
more preferably at least 95%, even more preferably at least 96%,
97%, 98%, and most preferably at least 99% identical to the amino
acid sequence of a selected fungal DNase.
[0079] In one embodiment of the present invention, the detergent
composition comprises at least one polypeptide having
deoxyribonuclease activity, which polypeptide is selected from the
group consisting of a polypeptide comprising or consisting of the
mature polypeptide of SEQ ID NO: 1, a polypeptide comprising or
consisting of the amino acid sequence set forth in SEQ ID NO: 2, a
polypeptide comprising or consisting of the amino acid sequence set
forth in SEQ ID NO: 3, a polypeptide comprising or consisting of
the mature polypeptide of SEQ ID NO: 4, a polypeptide comprising or
consisting of the mature polypeptide of SEQ ID NO: 5 and a
polypeptide comprising or consisting of the mature polypeptide of
SEQ ID NO: 6. In a further embodiment, the detergent composition
comprises a polypeptide consisting of the amino acid sequence set
forth in SEQ ID NO: 2 and a polypeptide consisting of the amino
acid sequence set forth in SEQ ID NO: 3.
[0080] In one embodiment, the polypeptide contains up to 206 (such
as 204) consecutive amino acid residues of the sequence set forth
in SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., amino acids 38 to 243 of SEQ
ID NO: 1 or amino acids 1 to 206 of SEQ ID NO: 2 or amino acids 1
to 204 of SEQ ID NO: 3), or up to 204 amino acid residues (e.g.,
amino acids 40 to 243 of SEQ ID NO: 1). In another embodiment, the
polypeptide consists of the of the amino acid sequence set forth in
SEQ ID NO: 2 or SEQ ID NO: 3. In yet another embodiment, the
polypeptide comprises or consists of the consecutive amino acid
residues 18 to 205 of SEQ ID NO: 4. In one embodiment, the
polypeptide comprises or consists of the consecutive amino acid
residues 34 to 142 of SEQ ID NO: 5. In one embodiment, the
polypeptide comprises or consists of the consecutive amino acid
residues 27 to 136 of SEQ ID NO: 6.
[0081] In one embodiment of the present invention, the detergent
composition comprises at least one polypeptide having
deoxyribonuclease activity, wherein said polypeptide is a
polypeptide having at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to the mature polypeptide of SEQ ID NO: 1. In one
embodiment, one or more amino acids have been substituted, deleted
or inserted with the proviso that the deoxyribonuclease activity is
maintained, substantially maintained or increased. In a further
embodiment, up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10, have been substituted, deleted or inserted.
[0082] In one embodiment of the present invention, the detergent
composition comprises at least one polypeptide having
deoxyribonuclease activity, wherein said polypeptide is a
polypeptide having at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to the polypeptide having the amino acid sequence set
forth in SEQ ID NO: 2. In one embodiment, one or more amino acids
have been substituted, deleted or inserted with the proviso that
the deoxyribonuclease activity is maintained, substantially
maintained or increased. In a further embodiment, up to 10 amino
acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, have been
substituted, deleted or inserted.
[0083] In one embodiment of the present invention, the detergent
composition comprises at least one polypeptide having
deoxyribonuclease activity, wherein said polypeptide is a
polypeptide having at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to the polypeptide having the amino acid sequence set
forth in SEQ ID NO: 3. In one embodiment, one or more amino acids
have been substituted, deleted or inserted with the proviso that
the deoxyribonuclease activity is maintained, substantially
maintained or increased. In a further embodiment, up to 10 amino
acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, have been
substituted, deleted or inserted.
[0084] In one embodiment of the present invention, the detergent
composition comprises at least one polypeptide having
deoxyribonuclease activity, wherein said polypeptide is a
polypeptide having at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to the mature polypeptide of SEQ ID NO: 4. In one
embodiment, one or more amino acids have been substituted, deleted
or inserted with the proviso that the deoxyribonuclease activity is
maintained, substantially maintained or increased. In a further
embodiment, up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10, have been substituted, deleted or inserted.
[0085] In one embodiment of the present invention, the detergent
composition comprises at least one polypeptide having
deoxyribonuclease activity, wherein said polypeptide is a
polypeptide having at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to the mature polypeptide of SEQ ID NO: 5. In one
embodiment, one or more amino acids have been substituted, deleted
or inserted with the proviso that the deoxyribonuclease activity is
maintained, substantially maintained or increased. In a further
embodiment, up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10, have been substituted, deleted or inserted.
[0086] In one embodiment of the present invention, the detergent
composition comprises at least one polypeptide having
deoxyribonuclease activity, wherein said polypeptide is a
polypeptide having at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% sequence
identity to the mature polypeptide of SEQ ID NO: 6. In one
embodiment, one or more amino acids have been substituted, deleted
or inserted with the proviso that the deoxyribonuclease activity is
maintained, substantially maintained or increased. In a further
embodiment, up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10, have been substituted, deleted or inserted.
[0087] The amino acid changes may be of a minor nature, that is
conservative amino acid substitutions or insertions that do not
significantly affect the folding and/or activity of the protein;
small deletions, typically of 1-30 amino acids; small amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine
residue; a small linker peptide of up to 20-25 residues; or a small
extension that facilitates purification by changing net charge or
another function, such as a poly-histidine tract, an antigenic
epitope or a binding domain.
[0088] Examples of conservative substitutions are within the groups
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine). Amino acid substitutions that do
not generally alter specific activity are known in the art and are
described, for example, by H. Neurath and R. L. Hill, 1979, In, The
Proteins, Academic Press, New York. Common substitutions are
Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,
Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,
Leu/Val, Ala/Glu, and Asp/Gly.
[0089] Alternatively, the amino acid changes are of such a nature
that the physico-chemical properties of the polypeptides are
altered. For example, amino acid changes may improve the thermal
stability of the polypeptide, alter the substrate specificity,
change the pH optimum, and the like.
[0090] Essential amino acids in a polypeptide can be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,
1989, Science 244: 1081-1085). In the latter technique, single
alanine mutations are introduced at every residue in the molecule,
and the resultant mutant molecules are tested for DNase activity to
identify amino acid residues that are critical to the activity of
the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271:
4699-4708. The active site of the enzyme or other biological
interaction can also be determined by physical analysis of
structure, as determined by such techniques as nuclear magnetic
resonance, crystallography, electron diffraction, or photoaffinity
labeling, in conjunction with mutation of putative contact site
amino acids. See, for example, de Vos et al., 1992, Science 255:
306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver
et al., 1992, FEBS Lett. 309: 59-64. The identity of essential
amino acids can also be inferred from an alignment with a related
polypeptide.
[0091] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
[0092] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide.
[0093] The polypeptide having deoxyribonuclease activity may be a
hybrid polypeptide in which a region of one polypeptide is fused at
the N-terminus or the C-terminus of a region of another
polypeptide.
[0094] The polypeptide may be a fusion polypeptide or cleavable
fusion polypeptide in which another polypeptide is fused at the
N-terminus or the C-terminus of the polypeptide of the present
invention. A fusion polypeptide is produced by fusing a
polynucleotide encoding another polypeptide to a polynucleotide of
the present invention. Techniques for producing fusion polypeptides
are known in the art, and include ligating the coding sequences
encoding the polypeptides so that they are in frame and that
expression of the fusion polypeptide is under control of the same
promoter(s) and terminator. Fusion polypeptides may also be
constructed using intein technology in which fusion polypeptides
are created post-translationally (Cooper et al., 1993, EMBO J. 12:
2575-2583; Dawson et al., 1994, Science 266: 776-779).
[0095] A fusion polypeptide can further comprise a cleavage site
between the two polypeptides. Upon secretion of the fusion protein,
the site is cleaved releasing the two polypeptides. Examples of
cleavage sites include, but are not limited to, the sites disclosed
in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576;
Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson
et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al.,
1995, Biotechnology 13: 498-503; and Contreras et al., 1991,
Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25:
505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987;
Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:
240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
[0096] In one embodiment, the polypeptide is a fragment of any of
the deoxyribonucleases disclosed herein. The fragment may be
obtained by truncation of the amino and/or carboxyl terminus of the
deoxyribonuclease, for example truncation of the amino and/or
carboxyl terminus of a mature polypeptide or domain. The
deoxyribonuclease activity of the parent deoxyribonuclease is
maintained in the fragment. The deoxyribonuclease activity of the
fragment may be slightly lower than the parent deoxyribonuclease in
cases where truncation and optional further modification introduces
other advantageous properties.
[0097] In one embodiment, a fragment contains at least 206 amino
acid residues (e.g., amino acids 38 to 243 of SEQ ID NO: 1), at
least 205 amino acid residues (e.g., amino acids 39 to 243 of SEQ
ID NO: 1), or at least 204 amino acid residues (e.g., amino acids
40 to 243 of SEQ ID NO: 1).
[0098] The concentration of the DNase in the wash liquor is
typically in the range of 0.00004-100 ppm enzyme protein, such as
in the range of 0.00008-100 ppm enzyme protein, in the range of
0.0001-100 ppm enzyme protein, in the range of 0.0002-100 ppm
enzyme protein, in the range of 0.0004-100 ppm enzyme protein, in
the range of 0.0008-100 ppm enzyme protein, in the range of
0.001-100 ppm enzyme protein, 0.01-100 ppm enzyme protein,
preferably 0.05-50 ppm enzyme protein, more preferably 0.1-50 ppm
enzyme protein, more preferably 0.1-30 ppm enzyme protein, more
preferably 0.5-20 ppm enzyme protein, and most preferably 0.5-10
ppm enzyme protein. In one embodiment, the concentration of the
polypeptide having deoxyribonuclease activity in a wash dose of
said detergent composition is within the range of 0.001 ppm to 100
ppm.
[0099] The DNase of the present invention may be added to a
detergent composition in an amount corresponding to at least 0.002
mg of DNase protein, such as at least 0.004 mg of DNase protein, at
least 0.006 mg of DNase protein, at least 0.008 mg of DNase
protein, at least 0.01 mg of DNase protein, at least 0.02 mg of
DNase protein, at least 0.05 mg of DNase protein, at least 0.1 mg
of protein, at least 0.2 mg of DNase protein, preferably at least
0.01 mg of protein, preferably at least 0.02 mg of protein,
preferably at least 0.5 mg of protein, preferably at least 1 mg of
protein, more preferably at least 10 mg of protein, even more
preferably at least 15 mg of protein, most preferably at least 20
mg of protein, and even most preferably at least 25 mg of protein.
Thus, the detergent composition may comprise at least 0.00008%
DNase protein, preferably at least 0.002%, 0.003%, 0.004%, 0.005%,
0.006%, 0.008%, 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%,
0.6%, 0.7%, 0.8%, 0.9% or 1.0% of DNase protein.
[0100] In some aspects, the present invention provides a detergent
composition comprising:
[0101] (i) a polypeptide having deoxyribonuclease activity, wherein
the concentration of the DNase in said composition is at least
0.002 mg of active DNase protein per litre of detergent
composition, such as at least 0.004 mg of active DNase protein, at
least 0.006 mg of active DNase protein, at least 0.008 mg of active
DNase protein, or such as at least 0.01 mg of active DNase protein,
at least 0.02 mg of active DNase protein, at least 0.05 mg of
active DNase protein, at least 0.1 mg of active DNase protein, at
least 0.2 mg of active DNase protein or at least 0.5 mg of active
DNase protein e.g. the detergent composition may comprise at least
0.00008% DNase protein, preferably at least 0.002%, 0.003%, 0.004%,
0.005%, 0.006%, 0.008%, 0.01%, 0.02%, 0.03%, 0.05%, 0.1%, 0.2%,
0.3%, 0.4%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% of DNase protein,
and
[0102] (ii) at least one surfactant, wherein the total amount of
surfactant(s) in said composition is in the range of 3.6 w/w % to
28.5 w/w %, optionally in the range of 3 w/w % to 30 w/w %,
optionally in the range of 5 w/w % to 20 w/w %, optionally in the
range of 10 w/w % to 20 w/w % or optionally in the range of 15 w/w
% to 20 w/w %, optionally in the range of w/w % 10 to 15 w/w % or
optionally below 20 w/w %, such as below 15 w/w % but more than 0
w/w %.
[0103] In one aspect, at least one surfactant is LAS.
[0104] The DNase of the detergent composition of the invention may
be stabilized using conventional stabilizing agents, e.g. a polyol
such as propylene glycol or glycerol, a sugar or sugar alcohol,
lactic acid, boric acid, or a boric acid derivative, e.g. an
aromatic borate ester, or a phenyl boronic acid derivative such as
4-formylphenyl boronic acid, and the composition may be formulated
as described in, for example, WO92/19709 and WO92/19708.
[0105] A polypeptide of the present invention may also be
incorporated in the detergent formulations disclosed in WO97/07202,
which is hereby incorporated by reference.
[0106] The detergent composition of the present invention may
comprise one or more additional enzymes such as a protease, lipase,
cutinase, an amylase, carbohydrase, cellulase, pectinase,
mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a
laccase, and/or peroxidase.
[0107] In general, the properties of the selected enzyme(s) should
be compatible with the selected detergent, (i.e., pH-optimum,
compatibility with other enzymatic and non-enzymatic ingredients,
etc.), and the enzyme(s) should be present in effective amounts. In
a preferred embodiment, the detergent composition comprises a
protease.
Cellulases
[0108] Suitable cellulases include those of bacterial or fungal
origin. Chemically modified or protein engineered mutants are
included. Suitable cellulases include cellulases from the genera
Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium,
e.g., the fungal cellulases produced from Humicola insolens,
Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S.
Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No.
5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.
[0109] Especially suitable cellulases are the alkaline or neutral
cellulases having colour care benefits. Examples of such cellulases
are cellulases described in EP 0 495 257, EP 0 531 372, WO
96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase
variants such as those described in WO 94/07998, EP 0 531 315, U.S.
Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No.
5,763,254, WO 95/24471, WO 98/12307 and WO99/001544.
[0110] Other cellulases are endo-beta-1,4-glucanase enzyme having a
sequence of at least 97% identity to the amino acid sequence of
position 1 to position 773 of SEQ ID NO:2 of WO 2002/099091 or a
family 44 xyloglucanase, which a xyloglucanase enzyme having a
sequence of at least 60% identity to positions 40-559 of SEQ ID NO:
2 of WO 2001/062903.
[0111] Commercially available cellulases include Celluzyme.TM., and
Carezyme.TM. (Novozymes NS) Carezyme Premium.TM. (Novozymes A/S),
Celluclean.TM. (Novozymes A/S), Celluclean Classic.TM. (Novozymes
A/S), Cellusoft.TM. (Novozymes A/S), Whitezyme.TM. (Novozymes A/S),
Clazinase.TM., and Puradax HA.TM. (Genencor International Inc.),
and KAC-500(B).TM. (Kao Corporation).
Proteases
[0112] Suitable proteases include those of bacterial, fungal,
plant, viral or animal origin e.g. vegetable or microbial origin.
Microbial origin is preferred. Chemically modified or protein
engineered mutants are included. It may be an alkaline protease,
such as a serine protease or a metalloprotease. A serine protease
may for example be of the S1 family, such as trypsin, or the S8
family such as subtilisin. A metalloproteases protease may for
example be a thermolysin from e.g. family M4 or other
metalloprotease such as those from M5, M7 or M8 families.
[0113] The term "subtilases" refers to a sub-group of serine
protease according to Siezen et al., Protein Engng. 4 (1991)
719-737 and Siezen et al. Protein Science 6 (1997) 501-523. Serine
proteases are a subgroup of proteases characterized by having a
serine in the active site, which forms a covalent adduct with the
substrate. The subtilases may be divided into 6 sub-divisions, i.e.
the Subtilisin family, the Thermitase family, the Proteinase K
family, the Lantibiotic peptidase family, the Kexin family and the
Pyrolysin family.
[0114] Examples of subtilases are those obtained from Bacillus such
as Bacillus lentus, B. alkalophilus, B. subtilis, B.
amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described
in; U.S. Pat. No. 7,262,042 and WO09/021867, and subtilisin lentus,
subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis,
subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168
described in WO89/06279 and protease PD138 described in
(WO93/18140). Other useful proteases may be those described in
WO92/175177, WO01/016285, WO02/026024 and WO02/016547. Examples of
trypsin-like proteases are trypsin (e.g. of porcine or bovine
origin) and the Fusarium protease described in WO89/06270,
WO94/25583 and WO05/040372, and the chymotrypsin proteases obtained
from Cellumonas described in WO05/052161 and WO05/052146.
[0115] A further preferred protease is the alkaline protease from
Bacillus lentus DSM 5483, as described for example in WO95/23221,
and variants thereof which are described in WO92/21760, WO95/23221,
EP1921147 and EP1921148.
[0116] Examples of metalloproteases are the neutral metalloprotease
as described in WO07/044993 (Genencor Int.) such as those obtained
from Bacillus amyloliquefaciens. Examples of useful proteases are
the protease variants described in: WO92/19729, WO96/034946,
WO98/20115, WO98/20116, WO99/011768, WO01/44452, WO03/006602,
WO04/03186, WO04/041979, WO07/006305, WO11/036263, WO11/036264,
especially the protease variants with alterations in one or more of
the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95,
96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128,
129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222,
224, 232, 235, 236, 245, 248, 252 and 274 corresponding to the
positions in BPN' i.e. BPN' numbering. More preferred the protease
variants are variants of a subtilase variants which comprise one or
more of the mutations: S3T, V41, S9R, A15T, K27R, *36D, V68A, N76D,
N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V1041,Y,N,
S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A,
R170S, A194P, G195E, V199M, V2051, L217D, N218D, M222S, A232V,
K235L, Q236H, Q245R, N252K, T274A (using BPN' numbering). The
protease variants are preferably variants of the Bacillus Lentus
protease (Savinase.RTM.) shown in SEQ ID NO 1 of WO2016/001449 and
in SEQ ID NO 7, or of the Bacillus amylolichenifaciens protease
(BPN') shown in SEQ ID NO 2 of WO2016/001449. The protease variants
preferably have at least 80% sequence identity to SEQ ID NO 7 or
SEQ ID NO 1 or 2 of WO2016/001449. The term "BPN' numbering" has it
common meaning within the protease field and includes the numbering
according to the alignment of Savinase and BPN' as shown in
WO1991/000345. The amino acid preceding the position is the amino
acid present in sequence of the protease Savinase e.g. shown in SEQ
ID NO 1 of WO2016/001449 (or SEQ ID NO 7 of the present invention)
it is clear to the person skilled in the art that the amino acid to
be replaced or deleted can be any amino acid and depends on the
parent protease.
[0117] A protease variant comprising a substitution at one or more
positions corresponding to positions 171, 173, 175, 179, or 180 of
SEQ ID NO: 1 of WO2004/067737, wherein said protease variant has a
sequence identity of at least 75% but less than 100% to SEQ ID NO:
1 of WO2004/067737.
[0118] Suitable commercially available protease enzymes include
those sold under the trade names: Alcalase.RTM., Duralase.TM.,
Durazym.TM., Relase.RTM., Relase.RTM. Ultra, Savinase.RTM.,
Savinase.RTM. Ultra, Primase.RTM., Polarzyme.RTM., Kannase.RTM.,
Liquanase.RTM., Liquanase.RTM. Ultra, Ovozyme.RTM., Coronase.RTM.,
Coronase.RTM. Ultra, Blaze.RTM., Blaze Evity.RTM. 100T, Blaze
Evity.RTM. 125T, Blaze Evity.RTM. 150T, Neutrase.RTM.,
Everlase.RTM. and Esperase.RTM. (Novozymes A/S), those sold under
the tradename Maxatase.RTM., Maxacal.RTM., Maxapem.RTM., Purafect
Ox.RTM., Purafect OxP.RTM., Puramax.RTM., FN2.RTM., FN3.RTM.,
FN4.RTM., Excellase.RTM., Excellenz P1000.TM., Excellenz P1250.TM.,
Eraser.RTM., Preferenz P100.TM., Purafect Prime.RTM., Preferenz
P110.TM., Effectenz P1000.TM., Purafect.RTM..TM., Effectenz
P1050.TM., Purafect Ox.RTM..TM., Effectenz P2000.TM.,
Purafast.RTM., Properase.RTM., Opticlean.RTM. and Optimase.RTM.
(Danisco/DuPont), Axapem.TM. (Gist-Brocases N.V.), BLAP (sequence
shown in FIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof
(Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from
Kao.
[0119] In one embodiment of the invention, the inventive detergent
composition is formulated with a protease, which is of animal,
vegetable or microbial origin. The protease is chemically modified
or protein engineered. The protease can be a serine protease or a
metalloprotease, preferably an alkaline microbial protease or a
trypsin-like protease.
[0120] In one embodiment of the invention, the protease is selected
from the group consisting of Bacillus, e.g., subtilisin Novo,
subtilisin Carlsberg, subtilisin 309, subtilisin 147, subtilisin
168, trypsin of bovine origin, trypsin of porcine origin and
Fusarium protease. The protease can have at least 90%, such as at
least 95%, sequence identity to SEQ ID NO: 7. In one embodiment,
the protease has at least 90% identity to the amino acid sequence
of SEQ ID NO: 10 or a variant thereof with substitutions in one or
more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104,
120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274,
preferably the variant is an alkaline protease having at least 90%
identity to the amino acid sequence of SEQ ID NO: 7 with the
following substitution: M222S, or substitutions N76D+G195E.
Lipases and Cutinases
[0121] Suitable lipases and cutinases include those of bacterial or
fungal origin. Chemically modified or protein engineered mutant
enzymes are included. Examples include lipase from Thermomyces,
e.g. from T. lanuginosus (previously named Humicola lanuginosa) as
described in EP258068 and EP305216, cutinase from Humicola, e.g. H.
insolens (WO96/13580), lipase from strains of Pseudomonas (some of
these now renamed to Burkholderia), e.g. P. alcaligenes or P.
pseudoalcaligenes (EP218272), P. cepacia (EP331376), P. sp. strain
SD705 (WO95/06720 & WO96/27002), P. wisconsinensis
(WO96/12012), GDSL-type Streptomyces lipases (WO10/065455),
cutinase from Magnaporthe grisea (WO10/107560), cutinase from
Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipase from
Thermobifida fusca (WO11/084412), Geobacillus stearothermophilus
lipase (WO11/084417), lipase from Bacillus subtilis (WO11/084599),
and lipase from Streptomyces griseus (WO11/150157) and S.
pristinaespiralis (WO12/137147).
[0122] Other examples are lipase variants such as those described
in EP407225, WO92/05249, WO94/01541, WO94/25578, WO95/14783,
WO95/30744, WO95/35381, WO95/22615, WO96/00292, WO97/04079,
WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and
WO09/109500.
[0123] Preferred commercial lipase products include include
Lipolase.TM., Lipex.TM.; Lipolex.TM. and Lipoclean.TM. (Novozymes
A/S), Lumafast (originally from Genencor) and Lipomax (originally
from Gist-Brocades).
[0124] Still other examples are lipases sometimes referred to as
acyltransferases or perhydrolases, e.g. acyltransferases with
homology to Candida antarctica lipase A (WO10/111143),
acyltransferase from Mycobacterium smegmatis (WO05/56782),
perhydrolases from the CE 7 family (WO09/67279), and variants of
the M. smegmatis perhydrolase in particular the S54V variant used
in the commercial product Gentle Power Bleach from Huntsman Textile
Effects Pte Ltd (WO10/100028).
Amylases
[0125] Suitable amylases which can be used together with the enzyme
of the invention may be an alpha-amylase or a glucoamylase and may
be of bacterial or fungal origin. Chemically modified or protein
engineered mutants are included. Amylases include, for example,
alpha-amylases obtained from Bacillus, e.g., a special strain of
Bacillus licheniformis, described in more detail in GB
1,296,839.
[0126] Suitable amylases include amylases having SEQ ID NO: 2 in WO
95/10603 or variants having 90% sequence identity to SEQ ID NO: 3
thereof. Preferred variants are described in WO 94/02597, WO
94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, such as
variants with substitutions in one or more of the following
positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179,
181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304,
305, 391, 408, and 444.
[0127] Different suitable amylases include amylases having SEQ ID
NO: 6 in WO 02/010355 or variants thereof having 90% sequence
identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are
those having a deletion in positions 181 and 182 and a substitution
in position 193.
[0128] Other amylases which are suitable are hybrid alpha-amylase
comprising residues 1-33 of the alpha-amylase obtained from B.
amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and
residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ
ID NO: 4 of WO 2006/066594 or variants having 90% sequence identity
thereof. Preferred variants of this hybrid alpha-amylase are those
having a substitution, a deletion or an insertion in one of more of
the following positions: G48, T49, G107, H156, A181, N190, M197,
1201, A209 and Q264. Most preferred variants of the hybrid
alpha-amylase comprising residues 1-33 of the alpha-amylase
obtained from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO
2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having
the substitutions:
[0129] M197T;
[0130] H156Y+A181T+N190F+A209V+Q264S; or
[0131] G48A+T491+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S.
[0132] Further amylases which are suitable are amylases having SEQ
ID NO: 6 in WO 99/019467 or variants thereof having 90% sequence
identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are
those having a substitution, a deletion or an insertion in one or
more of the following positions: R181, G182, H183, G184, N195,
1206, E212, E216 and K269. Particularly preferred amylases are
those having deletion in positions R181 and G182, or positions H183
and G184.
[0133] Additional amylases which can be used are those having SEQ
ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO
96/023873 or variants thereof having 90% sequence identity to SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred
variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:
7 are those having a substitution, a deletion or an insertion in
one or more of the following positions: 140, 181, 182, 183, 184,
195, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO
96/023873 for numbering. More preferred variants are those having a
deletion in two positions selected from 181, 182, 183 and 184, such
as 181 and 182, 182 and 183, or positions 183 and 184. Most
preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID
NO: 7 are those having a deletion in positions 183 and 184 and a
substitution in one or more of positions 140, 195, 206, 243, 260,
304 and 476.
[0134] Other amylases which can be used are amylases having SEQ ID
NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or variants
thereof having 90% sequence identity to SEQ ID NO: 2 of WO
08/153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01/66712.
Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having
a substitution, a deletion or an insertion in one of more of the
following positions: 176, 177, 178, 179, 190, 201, 207, 211 and
264.
[0135] Further suitable amylases are amylases having SEQ ID NO: 2
of WO 09/061380 or variants having 90% sequence identity to SEQ ID
NO: 2 thereof. Preferred variants of SEQ ID NO: 2 are those having
a truncation of the C-terminus and/or a substitution, a deletion or
an insertion in one of more of the following positions: Q87, Q98,
S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202,
N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and
G475. More preferred variants of SEQ ID NO: 2 are those having the
substitution in one of more of the following positions: Q87E,R,
Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y,
N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E
and G475K and/or deletion in position R180 and/or S181 or of T182
and/or G183. Most preferred amylase variants of SEQ ID NO: 2 are
those having the substitutions:
[0136] N128C+K178L+T182G+Y305R+G475K;
[0137] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;
[0138] S125A+N128C+K178L+T182G+Y305R+G475K; or
[0139] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein the
variants are C-terminally truncated and optionally further
comprises a substitution at position 243 and/or a deletion at
position 180 and/or position 181.
[0140] Other suitable amylases are the alpha-amylase having SEQ ID
NO: 12 in WO01/66712 or a variant having at least 90% sequence
identity to SEQ ID NO: 12. Preferred amylase variants are those
having a substitution, a deletion or an insertion in one of more of
the following positions of SEQ ID NO: 12 in WO01/66712: R28, R118,
N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299,
K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439,
R444, N445, K446, Q449, R458, N471, N484. Particular preferred
amylases include variants having a deletion of D183 and G184 and
having the substitutions R118K, N195F, R320K and R458K, and a
variant additionally having substitutions in one or more position
selected from the group: M9, G149, G182, G186, M202, T257, Y295,
N299, M323, E345 and A339, most preferred a variant that
additionally has substitutions in all these positions.
[0141] Other examples are amylase variants such as those described
in WO2011/098531, WO2013/001078 and WO2013/001087.
[0142] Commercially available amylases are Duramyl.TM.,
Termamyl.TM., Fungamyl.TM., Stainzyme.TM., Stainzyme Plus.TM.,
Natalase.TM., Liquozyme X and BAN.TM. (from Novozymes A/S), and
Rapidase.TM., Purastar.TM./Effectenz.TM., Powerase and Preferenz
S100 (from Genencor International Inc./DuPont).
Peroxidases/Oxidases
[0143] A peroxidase according to the invention is a peroxidase
enzyme comprised by the enzyme classification EC 1.11.1.7, as set
out by the Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (IUBMB), or any fragment
obtained therefrom, exhibiting peroxidase activity.
[0144] Suitable peroxidases include those of plant, bacterial or
fungal origin. Chemically modified or protein engineered mutants
are included. Examples of useful peroxidases include peroxidases
from Coprinopsis, e.g., from C. cinerea (EP 179,486), and variants
thereof as those described in WO 93/24618, WO 95/10602, and WO
98/15257.
[0145] A peroxidase according to the invention also includes a
haloperoxidase enzyme, such as chloroperoxidase, bromoperoxidase
and compounds exhibiting chloroperoxidase or bromoperoxidase
activity. Haloperoxidases are classified according to their
specificity for halide ions. Chloroperoxidases (E.C. 1.11.1.10)
catalyze formation of hypochlorite from chloride ions.
[0146] In an embodiment, the haloperoxidase of the invention is a
chloroperoxidase. Preferably, the haloperoxidase is a vanadium
haloperoxidase, i.e., a vanadate-containing haloperoxidase. In a
preferred method of the present invention the vanadate-containing
haloperoxidase is combined with a source of chloride ion.
[0147] Haloperoxidases have been isolated from many different
fungi, in particular from the fungus group dematiaceous
hyphomycetes, such as Caldariomyces, e.g., C. fumago, Alternaria,
Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera,
Ulocladium and Botrytis.
[0148] Haloperoxidases have also been isolated from bacteria such
as Pseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S.
aureofaciens.
[0149] In a preferred embodiment, the haloperoxidase is derivable
from Curvularia sp., in particular Curvularia verruculosa or
Curvularia inaequalis, such as C. inaequalis CBS 102.42 as
described in WO 95/27046; or C. verruculosa CBS 147.63 or C.
verruculosa CBS 444.70 as described in WO 97/04102; or from
Drechslera hartlebii as described in WO 01/79459, Dendryphiella
salina as described in WO 01/79458, Phaeotrichoconis crotalarie as
described in WO 01/79461, or Geniculosporium sp. as described in WO
01/79460.
[0150] An oxidase according to the invention include, in
particular, any laccase enzyme comprised by the enzyme
classification EC 1.10.3.2, or any fragment obtained therefrom
exhibiting laccase activity, or a compound exhibiting a similar
activity, such as a catechol oxidase (EC 1.10.3.1), an
o-aminophenol oxidase (EC 1.10.3.4), or a bilirubin oxidase (EC
1.3.3.5).
[0151] Preferred laccase enzymes are enzymes of microbial origin.
The enzymes may be obtained from plants, bacteria or fungi
(including filamentous fungi and yeasts).
[0152] Suitable examples from fungi include a laccase derivable
from a strain of Aspergillus, Neurospora, e.g., N. crassa,
Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus,
Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R.
solani, Coprinopsis, e.g., C. cinerea, C. comatus, C. friesii, and
C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g.,
P. papilionaceus, Myceliophthora, e.g., M. thermophila,
Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P. pinsitus,
Phlebia, e.g., P. radiata (WO 92/01046), or Coriolus, e.g., C.
hirsutus (JP 2238885).
[0153] Suitable examples from bacteria include a laccase derivable
from a strain of Bacillus.
[0154] A laccase obtained from Coprinopsis or Myceliophthora is
preferred; in particular a laccase obtained from Coprinopsis
cinerea, as disclosed in WO 97/08325; or from Myceliophthora
thermophila, as disclosed in WO 95/33836.
[0155] The detergent enzyme(s) may be included in a detergent
composition by adding separate additives containing one or more
enzymes, or by adding a combined additive comprising all of these
enzymes. A detergent additive of the invention, i.e., a separate
additive or a combined additive, can be formulated, for example, as
a granulate, liquid, slurry, etc. Preferred detergent additive
formulations are granulates, in particular non-dusting granulates,
liquids, in particular stabilized liquids, or slurries.
[0156] Non-dusting granulates may be produced, e.g. as disclosed in
U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be coated
by methods known in the art. Examples of waxy coating materials are
poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean
molar weights of 1000 to 20000; ethoxylated nonylphenols having
from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in
which the alcohol contains from 12 to 20 carbon atoms and in which
there are 15 to 80 ethylene oxide units; fatty alcohols; fatty
acids; and mono- and di- and triglycerides of fatty acids. Examples
of film-forming coating materials suitable for application by fluid
bed techniques are given in GB 1483591. Liquid enzyme preparations
may, for instance, be stabilized by adding a polyol such as
propylene glycol, a sugar or sugar alcohol, lactic acid or boric
acid according to established methods. Protected enzymes may be
prepared according to the method disclosed in EP 238,216.
Other Materials
[0157] Any detergent components known in the art for use in
detergents may also be utilized. Other optional detergent
components include anti-corrosion agents, anti-shrink agents,
anti-soil redeposition agents, anti-wrinkling agents, bactericides,
binders, corrosion inhibitors, disintegrants/disintegration agents,
dyes, enzyme stabilizers (including boric acid, borates, CMC,
and/or polyols such as propylene glycol), fabric conditioners
including clays, fillers/processing aids, fluorescent whitening
agents/optical brighteners, foam boosters, foam (suds) regulators,
perfumes, soil-suspending agents, softeners, suds suppressors,
tarnish inhibitors, and wicking agents, either alone or in
combination. Any ingredient known in the art for use in detergents
may be utilized. The choice of such ingredients is well within the
skill of the artisan.
Dispersants
[0158] The detergent compositions of the present invention can also
contain dispersants. In particular powdered detergents may comprise
dispersants. Suitable water-soluble organic materials include the
homo- or co-polymeric acids or their salts, in which the
polycarboxylic acid comprises at least two carboxyl radicals
separated from each other by not more than two carbon atoms.
Suitable dispersants are for example described in Powdered
Detergents, Surfactant science series volume 71, Marcel Dekker,
Inc.
Dye Transfer Inhibiting Agents
[0159] The detergent compositions of the present invention may also
include one or more dye transfer inhibiting agents. Suitable
polymeric dye transfer inhibiting agents include, but are not
limited to, polyvinylpyrrolidone polymers, polyamine N-oxide
polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,
polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.
When present in a subject composition, the dye transfer inhibiting
agents may be present at levels from about 0.0001% to about 10%,
from about 0.01% to about 5% or even from about 0.1% to about 3% by
weight of the composition.
Fluorescent Whitening Agent
[0160] The detergent compositions of the present invention will
preferably also contain additional components that may tint
articles being cleaned, such as fluorescent whitening agent or
optical brighteners. Where present the brightener is preferably at
a level of about 0.01% to about 0.5%. Any fluorescent whitening
agent suitable for use in a laundry detergent composition may be
used in the composition of the present invention. The most commonly
used fluorescent whitening agents are those belonging to the
classes of diaminostilbene-sulfonic acid derivatives,
diarylpyrazoline derivatives and bisphenyl-distyryl derivatives.
Examples of the diaminostilbene-sulfonic acid derivative type of
fluorescent whitening agents include the sodium salts of:
4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)
stilbene-2,2'-disulfonate,
4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino)
stilbene-2.2'-disulfonate,
4,4'-bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-s-triazin-6-ylamin-
o) stilbene-2,2'-disulfonate,
4,4'-bis-(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2'-disulfonate and
sodium
5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benz-
enesulfonate. Preferred fluorescent whitening agents are Tinopal
DMS and Tinopal CBS available from Ciba-Geigy AG, Basel,
Switzerland. Tinopal DMS is the disodium salt of
4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)
stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of
2,2'-bis-(phenyl-styryl)-disulfonate. Tinopal CBS-X is a
4.4'-bis-(sulfostyryl)-biphenyl disodium salt also known as
Disodium Distyrylbiphenyl Disulfonate. Also preferred are
fluorescent whitening agents is the commercially available
Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai,
India. Other fluorescers suitable for use in the invention include
the 1-3-diaryl pyrazolines and the 7-alkylaminocoumarins.
[0161] Suitable fluorescent brightener levels include lower levels
of from about 0.01, from 0.05, from about 0.1 or even from about
0.2 wt % to upper levels of 0.5 or even 0.75 wt %.
Soil Release Polymers
[0162] The detergent compositions of the present invention may also
include one or more soil release polymers which aid the removal of
soils from fabrics such as cotton and polyester based fabrics, in
particular the removal of hydrophobic soils from polyester based
fabrics. The soil release polymers may for example be nonionic or
anionic terephthalte based polymers, polyvinyl caprolactam and
related copolymers, vinyl graft copolymers, polyester polyamides
see for example Chapter 7 in Powdered Detergents, Surfactant
science series volume 71, Marcel Dekker, Inc. Another type of soil
release polymers are amphiphilic alkoxylated grease cleaning
polymers comprising a core structure and a plurality of alkoxylate
groups attached to that core structure. The core structure may
comprise a polyalkylenimine structure or a polyalkanolamine
structure as described in detail in WO 2009/087523 (hereby
incorporated by reference). Furthermore random graft co-polymers
are suitable soil release polymers. Suitable graft co-polymers are
described in more detail in WO 2007/138054, WO 2006/108856 and WO
2006/113314 (hereby incorporated by reference). Other soil release
polymers are substituted polysaccharide structures especially
substituted cellulosic structures such as modified cellulose
derivatives such as those described in EP 1867808 or WO 2003/040279
(both are hereby incorporated by reference). Suitable cellulosic
polymers include cellulose, cellulose ethers, cellulose esters,
cellulose amides and mixtures thereof. Suitable cellulosic polymers
include anionically modified cellulose, nonionically modified
cellulose, cationically modified cellulose, zwitterionically
modified cellulose, and mixtures thereof. Suitable cellulosic
polymers include methyl cellulose, carboxy methyl cellulose, ethyl
cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl
cellulose, ester carboxy methyl cellulose, and mixtures
thereof.
Anti-Redeposition Agents
[0163] The detergent compositions of the present invention may also
include one or more anti-redeposition agents such as
carboxymethylcellulose (CMC), polyvinyl alcohol (PVA),
polyvinylpyrrolidone (PVP), polyoxyethylene and/or
polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers
of acrylic acid and maleic acid, and ethoxylated
polyethyleneimines. The cellulose based polymers described under
soil release polymers above may also function as anti-redeposition
agents.
Rheology Modifiers
[0164] The detergent compositions of the present invention may also
include one or more rheology modifiers, structurants or thickeners,
as distinct from viscosity reducing agents. The rheology modifiers
are selected from the group consisting of non-polymeric
crystalline, hydroxy-functional materials, polymeric rheology
modifiers which impart shear thinning characteristics to the
aqueous liquid matrix of a liquid detergent composition. The
rheology and viscosity of the detergent can be modified and
adjusted by methods known in the art, for example as shown in EP
2169040.
[0165] Other suitable adjunct materials include, but are not
limited to, anti-shrink agents, anti-wrinkling agents,
bactericides, binders, carriers, dyes, enzyme stabilizers, fabric
softeners, fillers, foam regulators, hydrotropes, perfumes,
pigments, sod suppressors, solvents, and structurants for liquid
detergents and/or structure elasticizing agents.
Formulation of Detergent Products
[0166] The detergent composition of the invention may be in any
convenient form, e.g., a bar, a homogenous tablet, a tablet having
two or more layers, a pouch having one or more compartments, a
regular or compact powder, a granule, a paste, a gel, or a regular,
compact or concentrated liquid. In one embodiment of the present
invention, the detergent composition is in a solid form.
[0167] Pouches can be configured as single or multicompartments. It
can be of any form, shape and material which is suitable for hold
the composition, e.g. without allowing the release of the
composition to release of the composition from the pouch prior to
water contact. The pouch is made from water soluble film which
encloses an inner volume. Said inner volume can be divided into
compartments of the pouch. Preferred films are polymeric materials
preferably polymers which are formed into a film or sheet.
Preferred polymers, copolymers or derivates thereof are selected
polyacrylates, and water soluble acrylate copolymers, methyl
cellulose, carboxy methyl cellulose, sodium dextrin, ethyl
cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose,
malto dextrin, poly methacrylates, most preferably polyvinyl
alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC).
Preferably the level of polymer in the film for example PVA is at
least about 60%. Preferred average molecular weight will typically
be about 20,000 to about 150,000. Films can also be of blended
compositions comprising hydrolytically degradable and water soluble
polymer blends such as polylactide and polyvinyl alcohol (known
under the Trade reference M8630 as sold by MonoSol LLC, Indiana,
USA) plus plasticisers like glycerol, ethylene glycerol, propylene
glycol, sorbitol and mixtures thereof. The pouches can comprise a
solid laundry cleaning composition or part components and/or a
liquid cleaning composition or part components separated by the
water soluble film. The compartment for liquid components can be
different in composition than compartments containing solids:
US2009/0011970 A1.
[0168] Detergent ingredients can be separated physically from each
other by compartments in water dissolvable pouches or in different
layers of tablets. Thereby negative storage interaction between
components can be avoided. Different dissolution profiles of each
of the compartments can also give rise to delayed dissolution of
selected components in the wash solution.
[0169] A liquid or gel detergent, which is not unit dosed, may be
aqueous, typically containing at least 20% by weight and up to 95%
water, such as up to about 70% water, up to about 65% water, up to
about 55% water, up to about 45% water, up to about 35% water.
Other types of liquids, including without limitation, alkanols,
amines, diols, ethers and polyols may be included in an aqueous
liquid or gel. An aqueous liquid or gel detergent may contain from
0-30% organic solvent. In one embodiment, the detergent composition
comprises a solvent.
[0170] A liquid or gel detergent may be non-aqueous.
[0171] The detergent composition may be formulated as a bar, a
homogenous tablet, and a tablet having two or more layers, a pouch
having one or more compartments, a regular or compact powder, a
granule, a paste, a gel, or a regular, compact or concentrated
liquid. The detergent composition can be a liquid detergent, a
powder detergent or a granule detergent.
[0172] The invention further concerns a liquid detergent
composition comprising a surfactant and a detergent builder in a
total concentration of at least 3% by weight, and a detergent
enzyme containing microcapsule, wherein the membrane of the
microcapsule is produced by cross-linking of a polybranched
polyamine having a molecular weight of more than 1 kDa. The
inventors have found, that encapsulating enzymes in a microcapsule
with a semipermeable membrane of the invention, and having a water
activity inside these capsules (prior to addition to the liquid
detergent) higher than in the liquid detergent, the capsules will
undergo a (partly) collapse when added to the detergent (water is
oozing out), thus leaving a more concentrated and more viscous
enzyme containing interior in the capsules. The collapse of the
membrane may also result in a reduced permeability. This can be
further utilized by addition of stabilizers/polymers, especially
ones that are not permeable through the membrane. The collapse and
resulting increase in viscosity will reduce/hinder the diffusion of
hostile components (e.g., surfactants or sequestrants) into the
capsules, and thus increase the storage stability of the enzyme in
the liquid detergent. Components in the liquid detergent that are
sensitive to the enzyme (e.g., components that act as substrate for
the enzyme) are also protected against degradation by the enzyme.
During wash the liquid detergent is diluted by water, thus
increasing the water activity. Water will now diffuse into the
capsules (osmosis). The capsules will swell and the membrane will
either become permeable to the enzyme so they can leave the
capsules, or simply burst and in this way releasing the enzyme. The
concept is very efficient in stabilizing the enzymes against
hostile components in liquid detergent, and vice versa also
protects enzyme sensitive components in the liquid detergent from
enzymes.
[0173] Examples of detergent components which are sensitive to, and
can be degraded by, enzymes include (relevant enzyme in
parenthesis): xanthan gum (xanthanase), polymers with ester bonds
(lipase), hydrogenated castor oil (lipase), perfume (lipase),
methyl ester sulfonate surfactants (lipase), cellulose and
cellulose derivatives (e.g. CMC) (cellulase), and dextrin and
cyclodextrin (amylase).
[0174] Also, sensitive detergent ingredients can be encapsulated,
and thus stabilized, in the microcapsules of the invention.
Sensitive detergent ingredients are prone to degradation during
storage. Such detergent ingredients include bleaching compounds,
bleach activators, perfumes, polymers, builder, surfactants,
etc.
[0175] Generally, the microcapsules of the invention can be used to
separate incompatible components/compounds in detergents.
[0176] Addition of the microcapsules to detergents can be used to
influence the visual appearance of the detergent product, such as
an opacifying effect (small microcapsules) or an effect of
distinctly visible particles (large microcapsules). The
microcapsules may also be colored.
[0177] The microcapsules can be used to reduce the enzyme dust
levels during handling and processing of enzyme products.
[0178] Unless otherwise indicated, all percentages are indicated as
percent by weight (')/0 w/w) throughout the application.
[0179] Microcapsule: The microcapsules are typically produced by
forming water droplets into a continuum that is non-miscible with
water--i.e., typically by preparing a water-in-oil emulsion--and
subsequently formation of the membrane by interfacial
polymerization via addition of a cross-linking agent. After
eventual curing the capsules can be harvested and further rinsed
and formulated by methods known in the art. The capsule formulation
is subsequently added to the detergent.
[0180] The payload, the major membrane constituents and eventual
additional component that are to be encapsulated are found in the
water phase. In the continuum is found components that stabilize
the water droplets towards coalescence (emulsifiers, emulsion
stabilizers, surfactants etc.) and the cross linking agent is also
added via the continuum.
[0181] The emulsion can be prepared be any methods known in the
art, e.g., by mechanical agitation, dripping processes, membrane
emulsification, microfluidics, sonication etc. In some cases simple
mixing of the phases automatically will result in an emulsion,
often referred to as self-emulsification. Using methods resulting
in a narrow size distribution is an advantage.
[0182] The cross-linking agent(s) is typically subsequently added
to the emulsion, either directly or more typically by preparing a
solution of the crosslinking agent in a solvent which is soluble in
the continuous phase. The emulsion and cross-linking agent or
solution hereof can be mixed by conventional methods used in the
art, e.g., by simple mixing or by carefully controlling the flows
of the emulsion and the cross-linking agent solution through an
in-line mixer.
[0183] In some cases, curing of the capsules is needed to complete
the membrane formation. Curing is often simple stirring of the
capsules for some time to allow the interfacial polymerization
reaction to end. In other cases the membrane formation can be
stopped by addition of reaction quencher.
[0184] The capsules may be post modified, e.g., by reacting
components onto the membrane to hinder or reduce flocculation of
the particles in the detergent as described in WO 99/01534.
[0185] The produced capsules can be isolated or concentrated by
methods known in the art, e.g., by filtration, centrifugation,
distillation or decantation of the capsule dispersion.
[0186] The resulting capsules can be further formulated, e.g., by
addition of surfactants to give the product the desired properties
for storage, transport and later handling and addition to the
detergent. Other microcapsule formulation agents include rheology
modifiers, biocides (e.g., Proxel), acid/base for adjustment of pH
(which will also adjust inside the microcapsules), and water for
adjustment of water activity.
[0187] The capsule forming process may include the following steps:
[0188] Preparation of the initial water and oil phase(s), [0189]
Forming a water-in-oil emulsion, [0190] Membrane formation by
interfacial polymerization, [0191] Optional post modification,
[0192] Optional isolation and/or formulation, [0193] Addition to
detergent.
[0194] The process can be either a batch process or a continuous or
semi-continuous process.
[0195] A microcapsule according to the invention is a small aqueous
sphere with a uniform membrane around it. The material inside the
microcapsule is referred to as the core, internal phase, or fill,
whereas the membrane is sometimes called a shell, coating, or wall.
The microcapsules of the invention have diameters between 0.5 .mu.m
and 2 millimeters. Preferably, the mean diameter of the
microcapsules is in the range of 1 .mu.m to 1000 .mu.m, more
preferably in the range of 5 .mu.m to 500 .mu.m, even more
preferably in the range of 10 .mu.m to 500 .mu.m, even more
preferably in the range of 50 .mu.m to 500 .mu.m, and most
preferably in the range of 50 .mu.m to 200 .mu.m. Alternatively,
the diameter of the microcapsules is in the range of 0.5 .mu.m to
30 .mu.m; or in the range of 1 .mu.m to 25 .mu.m. The diameter of
the microcapsule is measured in the oil phase after polymerization
is complete. The diameter of the capsule may change depending on
the water activity of the surrounding chemical environment.
[0196] Microencapsulation of enzymes, as used in the present
invention, may be carried out by interfacial polymerization,
wherein the two reactants in a polymerization reaction meet at an
interface and react rapidly. The basis of this method is a reaction
of a polyamine with an acid derivative, usually an acid halide,
acting as a crosslinking agent. The polyamine is preferably
substantially water-soluble (when in free base form). Under the
right conditions, thin flexible membranes form rapidly at the
interface. One way of carrying out the polymerization is to use an
aqueous solution of the enzyme and the polyamine, which are
emulsified with a non-aqueous solvent (and an emulsifier), and a
solution containing the acid derivative is added. An alkaline agent
may be present in the enzyme solution to neutralize the acid formed
during the reaction. Polymer (polyamide) membranes form instantly
at the interface of the emulsion droplets. The polymer membrane of
the microcapsule is typically of a cationic nature, and thus
bind/complex with compounds of an anionic nature.
[0197] The diameter of the microcapsules is determined by the size
of the emulsion droplets, which is controlled, for example by the
stirring rate.
[0198] Emulsion: An emulsion is a temporary or permanent dispersion
of one liquid phase within a second liquid phase. The second liquid
is generally referred to as the continuous phase. Surfactants are
commonly used to aid in the formation and stabilization of
emulsions. Not all surfactants are equally able to stabilize an
emulsion. The type and amount of a surfactant needs to be selected
for optimum emulsion utility especially with regard to preparation
and physical stability of the emulsion, and stability during
dilution and further processing. Physical stability refers to
maintaining an emulsion in a dispersion form. Processes such as
coalescence, aggregation, adsorption to container walls,
sedimentation and creaming, are forms of physical instability, and
should be avoided. Examples of suitable surfactants are described
in WO 97/24177, page 19-21; and in WO 99/01534.
[0199] Emulsions can be further classified as either simple
emulsions, wherein the dispersed liquid phase is a simple
homogeneous liquid, or a more complex emulsion, wherein the
dispersed liquid phase is a heterogeneous combination of liquid or
solid phases, such as a double emulsion or a multiple-emulsion. For
example, a water-in-oil double emulsion or multiple emulsion may be
formed wherein the water phase itself further contains an
emulsified oil phase; this type of emulsion may be specified as an
oil-in-water-in oil (o/w/o) emulsion. Alternatively, a water-in-oil
emulsion may be formed wherein the water phase contains a dispersed
solid phase often referred to as a suspension-emulsion. Other more
complex emulsions can be described. Because of the inherent
difficulty in describing such systems, the term emulsion is used to
describe both simple and more complex emulsions without necessarily
limiting the form of the emulsion or the type and number of phases
present
[0200] Polyamine: The rigidity/flexibility and permeability of the
membrane is mainly influenced by the choice of polyamine. The
polyamine according to the invention is a polybranched polyamine.
Each branch, preferably ending with a primary amino group serves as
a tethering point in the membrane network, thereby giving the
favorable properties of the invention. A polybranched polyamine
according to the present invention is a polyamine having more than
two branching points and more than two reactive amino groups
(capable of reacting with the crosslinking agent, i.e., primary and
secondary amino groups). The polybranched polyamine is used as
starting material when the emulsion is prepared--it is not formed
in situ from other starting materials. To obtain the attractive
properties of the invention, the polybranched structure of the
polyamine must be present as starting material.
[0201] There is a close relation between number of branching points
and number of primary amines, since primary amines will always be
positioned at the end of a branch: A linear amine can only contain
two primary amines. For each branching point hypothetically
introduced in such a linear di-amine will allow one or more primary
amine(s) to be introduced at the end of the introduced branch(es).
In this context we understand the primary amino group as part of
the branch, i.e., the endpoint of the branch. For example, we
consider both tris(2-aminoethyl)amine and 1,2,3-propanetriamine as
molecules having one branching point. For the invention the
polyamine has at least four primary amines. Branching points can be
introduced from an aliphatic hydrocarbon chain as in the previously
stated examples or from unsaturated carbon bonds, such as in, e.g.,
3,3'-diaminobenzidine, or from tertiary amino groups, such as in
N,N,N',N'-tetrakis-(2-aminoethyl)ethylenediamine.
[0202] In addition to the number of branching points, we have found
that the compactness of the reactive amino groups is of high
importance. A substance such as, e.g.,
N,N,N',N'-tetrakis-(12-aminododecyl)ethylenediamine would not be
suitable. Neither would a peptide or protein, such as an enzyme, be
suitable for membrane formation. Thus, the polybranched polyamine
is not a peptide or protein.
[0203] In an embodiment, the reactive amino groups constitute at
least 15% of the molecular weight of the polybranched polyamine,
such as more than 20%, or more than 25%. Preferably, the molecular
weight of the polybranched polyamine is at least 1 kDa; more
preferably, the molecular weight of the polybranched polyamine is
at least 1.3 kDa.
[0204] In a preferred embodiment, the polybranched polyamine is a
polyethyleneimine (PEI), and modifications thereof, having more
than two branching points and more than two reactive amino groups;
wherein the reactive amino groups constitute at least 15% of the
molecular weight of the PEI, such as more than 20%, or more than
25%. Preferably, the molecular weight of the PEI is at least 1
kDa.
[0205] Combinations of different polybranched polyamines may be
used for preparing the microcapsule according to the invention.
[0206] The advantageous properties (e.g., enzyme storage stability,
reduced enzyme leakage, reduced in-flux of detergent ingredients)
of the microcapsule of the invention may be improved by adding one
or more small amines with a molecular weight of less than 1 kDa.
The small amine is preferably substantially water-soluble (when in
free base form) and can be a material such as ethylene diamine,
hexamethylene diamine, hexane diamine, diethylene tetramine,
ethylene tetramine, diamino benzene, piperazine, tetramethylene
pentamine or, preferably, diethylene triamine (DETA). The small
amines may be added in an amount of up to 50%, preferably up to
40%, up to 30%, up to 20%, up to 10%, or up to 5%, by weight of the
total content of small amine and polybranched polyamine, when
preparing the microcapsule of the invention.
[0207] Crosslinking agent: The crosslinking agent as used in the
present invention is a molecule with at least two groups/sites
capable of reacting with amines to form covalent bonds.
[0208] The crosslinking agent is preferably oil soluble and can be
in the form of an acid anhydride or acid halide, preferably an acid
chloride. For example, it can be adipoyl chloride, sebacoyl
chloride, dodecanedioc acid chloride, phthaloyl chloride,
terephthaloyl chloride, isophthaloyl chloride, or trimesoyl
chloride; but preferably, the crosslinking agent is terephthaloyl
chloride or trimesoyl chloride.
Liquid Detergent Composition
[0209] In one embodiment of the present invention, the detergent is
in a liquid form. The liquid detergent composition may comprise a
microcapsule, and thus form part of, any detergent composition in
any form, such as liquid and powder detergents, and soap and
detergent bars.
[0210] In one embodiment, the invention is directed to liquid
detergent compositions comprising a microcapsule, as described
above, in combination with one or more additional cleaning
composition components.
[0211] The microcapsule, as described above, may be added to the
liquid detergent composition in an amount corresponding to from
0.0001% to 5% (w/w) active enzyme protein (AEP); preferably from
0.001% to 5%, more preferably from 0.005% to 5%, more preferably
from 0.005% to 4%, more preferably from 0.005% to 3%, more
preferably from 0.005% to 2%, even more preferably from 0.01% to
2%, and most preferably from 0.01% to 1% (w/w) active enzyme
protein.
[0212] The liquid detergent composition has a physical form, which
is not solid (or gas). It may be a pourable liquid, a paste, a
pourable gel or a non-pourable gel. It may be either isotropic or
structured, preferably isotropic. It may be a formulation useful
for washing in automatic washing machines or for hand washing. It
may also be a personal care product, such as a shampoo, toothpaste,
or a hand soap.
[0213] The liquid detergent composition may be aqueous, typically
containing at least 20% by weight and up to 95% water, such as up
to 70% water, up to 50% water, up to 40% water, up to 30% water, or
up to 20% water. Other types of liquids, including without
limitation, alkanols, amines, diols, ethers and polyols may be
included in an aqueous liquid detergent. An aqueous liquid
detergent may contain from 0-30% organic solvent. A liquid
detergent may even be non-aqueous, wherein the water content is
below 10%, preferably below 5%.
[0214] Detergent ingredients can be separated physically from each
other by compartments in water dissolvable pouches. Thereby
negative storage interaction between components can be avoided.
Different dissolution profiles of each of the compartments can also
give rise to delayed dissolution of selected components in the wash
solution.
[0215] The detergent composition may take the form of a unit dose
product. A unit dose product is the packaging of a single dose in a
non-reusable container. It is increasingly used in detergents for
laundry. A detergent unit dose product is the packaging (e.g., in a
pouch made from a water soluble film) of the amount of detergent
used for a single wash.
[0216] Pouches can be of any form, shape and material which is
suitable for holding the composition, e.g., without allowing the
release of the composition from the pouch prior to water contact.
The pouch is made from water soluble film which encloses an inner
volume. Said inner volume can be divided into compartments of the
pouch. Preferred films are polymeric materials preferably polymers
which are formed into a film or sheet. Preferred polymers,
copolymers or derivates thereof are selected polyacrylates, and
water soluble acrylate copolymers, methyl cellulose, carboxy methyl
cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose,
hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates,
most preferably polyvinyl alcohol copolymers and, hydroxypropyl
methyl cellulose (HPMC). Preferably the level of polymer in the
film for example PVA is at least about 60%. Preferred average
molecular weight will typically be about 20,000 to about 150,000.
Films can also be a blend compositions comprising hydrolytically
degradable and water soluble polymer blends such as polyactide and
polyvinyl alcohol (known under the Trade reference M8630 as sold by
Chris Craft In. Prod. Of Gary, Ind., US) plus plasticizers like
glycerol, ethylene glycerol, Propylene glycol, sorbitol and
mixtures thereof. The pouches can comprise a solid laundry cleaning
composition or part components and/or a liquid cleaning composition
or part components separated by the water soluble film. The
compartment for liquid components can be different in composition
than compartments containing solids (see e.g., US
2009/0011970).
[0217] The choice of detergent components may include, for textile
care, the consideration of the type of textile to be cleaned, the
type and/or degree of soiling, the temperature at which cleaning is
to take place, and the formulation of the detergent product.
Although components mentioned below are categorized by general
header according to a particular functionality, this is not to be
construed as a limitation, as a component may comprise additional
functionalities as will be appreciated by the skilled artisan.
Laundry Soap Bars
[0218] The DNase of the invention may be added to laundry soap bars
and used for hand washing laundry, fabrics and/or textiles. The
term laundry soap bar includes laundry bars, soap bars, combo bars,
syndet bars and detergent bars. The types of bar usually differ in
the type of surfactant they contain, and the term laundry soap bar
includes those containing soaps from fatty acids and/or synthetic
soaps. The laundry soap bar has a physical form which is solid and
not a liquid, gel or a powder at room temperature. The term solid
is defined as a physical form which does not significantly change
over time, i.e. if a solid object (e.g. laundry soap bar) is placed
inside a container, the solid object does not change to fill the
container it is placed in. The bar is a solid typically in bar form
but can be in other solid shapes such as round or oval.
[0219] The laundry soap bar may contain one or more additional
enzymes, protease inhibitors such as peptide aldehydes (or
hydrosulfite adduct or hemiacetal adduct), boric acid, borate,
borax and/or phenylboronic acid derivatives such as
4-formylphenylboronic acid, one or more soaps or synthetic
surfactants, polyols such as glycerine, pH controlling compounds
such as fatty acids, citric acid, acetic acid and/or formic acid,
and/or a salt of a monovalent cation and an organic anion wherein
the monovalent cation may be for example Na.sup.+, K.sup.+ or
NH.sub.4.sup.+ and the organic anion may be for example formate,
acetate, citrate or lactate such that the salt of a monovalent
cation and an organic anion may be, for example, sodium
formate.
[0220] The laundry soap bar may also contain complexing agents like
EDTA and HEDP, perfumes and/or different type of fillers,
surfactants e.g. anionic synthetic surfactants, builders, polymeric
soil release agents, detergent chelators, stabilizing agents,
fillers, dyes, colorants, dye transfer inhibitors, alkoxylated
polycarbonates, suds suppressers, structurants, binders, leaching
agents, bleaching activators, clay soil removal agents,
anti-redeposition agents, polymeric dispersing agents, brighteners,
fabric softeners, perfumes and/or other compounds known in the
art.
[0221] The laundry soap bar may be processed in conventional
laundry soap bar making equipment such as but not limited to:
mixers, plodders, e.g a two stage vacuum plodder, extruders,
cutters, logo-stampers, cooling tunnels and wrappers. The invention
is not limited to preparing the laundry soap bars by any single
method. The premix of the invention may be added to the soap at
different stages of the process. For example, the premix containing
soap, DNase, optionally one or more additional enzymes, a protease
inhibitor, and a salt of a monovalent cation and an organic anion
may be prepared and the mixture is then plodded. The DNase and
optional additional enzymes may be added at the same time as the
protease inhibitor for example in liquid form. Besides the mixing
step and the plodding step, the process may further comprise the
steps of milling, extruding, cutting, stamping, cooling and/or
wrapping.
Formulation of Enzyme in Co-Granule
[0222] The DNase may be formulated as a granule for example as a
co-granule that combines one or more enzymes. Each enzyme will then
be present in more granules securing a more uniform distribution of
enzymes in the detergent. This also reduces the physical
segregation of different enzymes due to different particle sizes.
Methods for producing multi-enzyme co-granulates for the detergent
industry are disclosed in the IP.com disclosure
IPCOM000200739D.
[0223] Another example of formulation of enzymes by the use of
co-granulates are disclosed in WO 2013/188331, which relates to a
detergent composition comprising (a) a multi-enzyme co-granule; (b)
less than 10 wt zeolite (anhydrous basis); and (c) less than 10 wt
phosphate salt (anhydrous basis), wherein said enzyme co-granule
comprises from 10 to 98 wt % moisture sink component and the
composition additionally comprises from 20 to 80 wt % detergent
moisture sink component.
WO 2013/188331 also relates to a method of treating and/or cleaning
a surface, preferably a fabric surface comprising the steps of (i)
contacting said surface with the detergent composition as claimed
and described herein in an aqueous wash liquor, (ii) rinsing and/or
drying the surface.
[0224] The multi-enzyme co-granule may comprise a DNase and (a) one
or more enzymes selected from the group consisting of first-wash
lipases, cleaning cellulases, xyloglucanases, perhydrolases,
peroxidases, lipoxygenases, laccases and mixtures thereof; and (b)
one or more enzymes selected from the group consisting of
hemicellulases, proteases, care cellulases, cellobiose
dehydrogenases, xylanases, phospho lipases, esterases, cutinases,
pectinases, mannanases, pectate lyases, keratinases, reductases,
oxidases, phenoloxidases, ligninases, pullulanases, tannases,
pentosanases, lichenases glucanases, arabinosidases, hyaluronidase,
chondroitinase, amylases, and mixtures thereof.
Methods and Uses of Detergent Composition
[0225] The invention further concerns a method for laundering a
textile. Accordingly, one aspect of the present invention concerns
a method for preventing, reducing or removing biofilm from a
textile or fabric comprising; [0226] (i) contacting the a textile
or fabric with a wash liquor comprising the detergent composition
according to any one of claims 1 to 15, [0227] (ii) subjecting said
textile to at least one washing cycle, [0228] (iii) optionally
rinsing said textile.
[0229] In a preferred embodiment, the textile is at least partly
coated with a biofilm.
[0230] In one embodiment, the temperature of the wash liquor is in
the range of 5.degree. C. to 95.degree. C., such as in the range of
10.degree. C. to 80.degree. C., for example in the range of
10.degree. C. to 70.degree. C., such as in the range of 10.degree.
C. to 60.degree. C., for example in the range of 10.degree. C. to
50.degree. C., such as in the range of 15.degree. C. to 40.degree.
C., for example in the range of 20.degree. C. to 30.degree. C. In
one embodiment the temperature of the wash liquor is 30.degree.
C.
[0231] The pH of the liquid solution is in the range of 1 to 11,
such as in the range 5.5 to 11, such as in the range of 7 to 9, in
the range of 7 to 8 or in the range of 7 to 8.5. In one embodiment
of the present invention, the concentration of the polypeptide
having deoxyribonuclease activity in the wash liquor is within the
range of 0.001 ppm to 100 ppm.
[0232] In some aspects, the invention relates to a method for
preventing, reducing or removing biofilm from a textile or fabric
comprising;
[0233] (i) contacting the a textile or fabric with a wash liquor
with a detergent composition, comprising (a) a polypeptide having
deoxyribonuclease activity, wherein the concentration of the DNase
in the detergent is in the range of about 0.001-2 ppm enzyme
protein, optionally form about 0.01-0.2 ppm enzyme protein,
optionally from about 0.01-0.02 ppm enzyme protein and
[0234] (b) at least one surfactant, wherein the total amount of
surfactant(s) in said composition is in the range of 3.6 w/w % to
28.5 w/w %, optionally in the range of 3 w/w % to 30 w/w %,
optionally in the range of 5 w/w % to 20 w/w %, optionally in the
range of 10 w/w % to 20 w/w % or optionally in the range of 15 w/w
% to 20 w/w %, optionally in the range of w/w % 10 to 15 w/w % or
optionally below 20 w/w %, such as below 15 w/w % but more than 0
w/w %.
[0235] (ii) subjecting said textile to at least one washing
cycle,
[0236] (iii) optionally rinsing said textile.
[0237] In a preferred embodiment, the textile is at least partly
coated with a biofilm.
[0238] In one embodiment, the temperature of the wash liquor is in
the range of 5.degree. C. to 95.degree. C., such as in the range of
10.degree. C. to 80.degree. C., for example in the range of
10.degree. C. to 70.degree. C., such as in the range of 10.degree.
C. to 60.degree. C., for example in the range of 10.degree. C. to
50.degree. C., such as in the range of 15.degree. C. to 40.degree.
C., for example in the range of 20.degree. C. to 30.degree. C. In
one embodiment the temperature of the wash liquor is 30.degree.
C.
[0239] The pH of the liquid solution is in the range of 1 to 11,
such as in the range 5.5 to 11, such as in the range of 7 to 9, in
the range of 7 to 8 or in the range of 7 to 8.5.
[0240] In one embodiment of the invention, the method for
laundering a textile or fabric further comprises draining of the
wash liquor or part of the wash liquor after completion of a wash
cycle. The wash liquor can then be re-used in a subsequent wash
cycle or in a subsequent rinse cycle. The textile or fabric may be
exposed to the wash liquor during a first and optionally a second
or a third wash cycle. In one embodiment the textile or fabric is
rinsed after being exposed to the wash liquor. The textile or
fabric can be rinsed with water or with water comprising a
conditioner.
[0241] A further aspect of the present invention concerns the use
of the detergent composition of the present invention for
preventing, reducing or removing biofilm from a textile or a
fabric.
Composition of Model Detergent A (Liquid)
[0242] Ingredients: 12% LAS, 11% AEO Biosoft N25-7 (NI), 7% AEOS
(SLES), 6% MPG (monopropylene glycol), 3% ethanol, 3% TEA, 2.75%
cocoa soap, 2.75% soya soap, 2% glycerol, 2% sodium hydroxide, 2%
sodium citrate, 1% sodium formiate, 0.2% DTMPA and 0.2% PCA (all
percentages are w/w).
Composition of Model Detergent B (Liquid)
[0243] Ingredients: 7.2% LAS, 6.6% AEO, 7% AEOS (SLES), 6% MPG
(monopropylene glycol), 3% ethanol, 3% TEA, 2.75% cocoa soap, 2.75%
soya soap, 2% glycerol, 1.2% sodium hydroxide, 2% sodium citrate,
1% sodium formiate, 0.2% DTMPA and 0.2% PCA (all percentages are
w/w).
Composition of Model Detergent T (Powder)
[0244] Ingredients: 11% LAS, 2% AS, 2% soap, 3% AEO, 15.15% sodium
carbonate, 3% sodium slilcate, 18.75% zeolite, 0.15% chelant, 2%
sodium citrate, 1.65% AA/MA copolymer, 2.5% CMC, 44% sodium
sulphate and 0.5% SRP (all percentages are w/w).
Composition of Model Detergent V (Powder)
[0245] Ingredients: 7% LAS, 1% AS, 1% soap, 3% AEO, 15% sodium
carbonate, 3% sodium slilcate, 19% zeolite, 0.15% chelant, 2%
sodium citrate, 1.65% AA/MA copolymer, 2.5% CMC, 44% sodium
sulphate and 0.5% SRP (all percentages are w/w).
Composition of Model Detergent N (Liquid)
[0246] Ingredients: 5.3% LAS, 10.7% AEOS, 1% soap, 5.3% non-ionic
surfactants 2% sodium citrate, 0.4% TEA, 0.73 NaOH, 0.02%
CaCl.sub.2 add water to 100% (all percentages are w/w).
Composition of Model Detergent O (Liquid)
[0247] Ingredients: 4% LAS, 8% AEOS, 1% soap, 4% nonionic
surfactants, 2% sodium citrate, 0.4% TEA, 0.60% NaOH, 0.02%
CaCl.sub.2, 0.1% preservatives add water to 100% (all percentages
are w/w).
Composition of Model Detergent P (Liquid)
[0248] Ingredients: 2.4% LAS, 4.8% AEOS, 1% soap, 2.4% nonionic
surfactants 2% sodium citrate (trisodium citrate dehydrate), 0.4%
TEA, 0.02% CaCl.sub.2 (*2H.sub.2O), 0.1%, pH adjusted to 8 with
NaOH, add water to 100% (all percentages are w/w).
PREFERRED EMBODIMENTS
[0249] This section describes non-limiting embodiments of the
present invention.
Paragraph 1. A detergent composition comprising;
[0250] (i) a polypeptide having deoxyribonuclease activity,
[0251] (ii) at least one surfactant,
[0252] wherein the total amount of surfactant(s) in said
composition is in the range of 3.6 w/w % to 28.5 w/w %.
Paragraph 2. The detergent composition of paragraph 1, wherein said
at least one sucfactant is selected from the group consisting of
anionic surfactant, cationic surfactant and non-ionic surfactant.
Paragraph 3. The detergent composition of paragraph 1 or 2, wherein
said total amount of surfactant(s) including soap ifoen or more
soap is present in the detergent is in the range of about 2 w/w %
to about about 35 w/w %, optionally in the range of about 3 w/w %
to 30 w/w %, optionally in the range of about 5 w/w % to about 20
w/w %, optionally in the range of about 10 w/w % to about 20 w/w %
or optionally in the range of about 15 w/w % to about 20 w/w % or
optionally below 20 w/w %, such as below 15 w/w % but more than 0
w/w %. Paragraph 4. The detergent composition of paragraph 3,
wherein said composition comprises at least one anionic surfactant
and the amount of said anionic surfactant(s) in said composition is
in the range of 2.5 w/w % to 19.6 w/w %, optionally in the range of
about 5 w/w % to about 20 w/w %, optionally in the range of about 5
w/w % to about 15 w/w % or optionally in the range of about 5 w/w %
to about 10 w/w %. Paragraph 5. The detergent composition according
to any one of the preceding paragraphs, wherein said composition
comprises linear alkylbenzene sulfonate (LAS).
[0253] Paragraph 6. The detergent composition according to
paragraph 5, wherein the amount of linear alkylbenzene sulfonate
(LAS) in said composition is in the range of 1.2 w/w % to 9.6 w/w
%, optionally in the range of about 2 w/w % to about 10 w/w %.
Paragraph 7. The detergent composition according to any one of the
preceding paragraphs, wherein said composition comprises at least
one alkyl ethoxysulfate (AEOS). Paragraph 8. The detergent
composition according to paragraph 7, wherein the amount of said
least one alkyl ethoxysulfate (AEOS) in said composition is in the
range of 0.7 w/w % to 9.6 w/w %, optionally in the range of about 1
w/w % to about 10 w/w %. Paragraph 9. The detergent composition
according to any one of the preceding paragraphs, wherein said
composition comprises at least one non-ionic surfactant and the
amount of said non-ionic surfactant(s) in said composition is in
the range of 1.1 w/w % to 8.8 w/w %, optionally in the range of
about 2 w/w % to about 10 w/w %. Paragraph 10. The detergent
composition according to paragraph 9, wherein said least one
non-ionic surfactant is AEO Biosoft N25-7. Paragraph 11. The
detergent composition according to any one of the preceding
paragraphs further comprising a builder. Paragraph 12. The
detergent composition according to any one of the preceding
paragraphs, wherein said composition is in a solid form. Paragraph
13. The detergent composition according to any one of the preceding
paragraphs, wherein said composition is in a liquid form. Paragraph
14. The detergent composition according to paragraph 13, wherein
said detergent composition comprises a solvent. Paragraph 15. The
detergent composition according to any one of the preceding
paragraphs, wherein the said polypeptide having deoxyribonuclease
activity is selected from the group consisting of a polypeptide
having at least 85% sequence identity to the mature polypeptide of
SEQ ID NO: 1, a polypeptide having at least 85% sequence identity
to the amino acid sequence set forth in SEQ ID NO: 2, a polypeptide
having at least 85% sequence identity to the amino acid sequence
set fort in SEQ ID NO: 3, a polypeptide having at least 85%
sequence identity to the mature polypeptide of SEQ ID NO: 4, a
polypeptide having at least 85% sequence identity to the mature
polypeptide of SEQ ID NO: 5 and a polypeptide having at least 85%
sequence identity to the mature polypeptide of SEQ ID NO: 6.
Paragraph 16. The detergent composition according to any one of the
preceding paragraphs, wherein the said polypeptide having
deoxyribonuclease activity is selected from the group consisting of
a polypeptide comprising or consisting of the mature polypeptide of
SEQ ID NO: 1, a polypeptide comprising or consisting of the amino
acid sequence set forth in SEQ ID NO: 2, a polypeptide comprising
or consisting of the amino acid sequence set fort in SEQ ID NO: 3,
a polypeptide comprising or consisting of the mature polypeptide of
SEQ ID NO: 4, a polypeptide comprising or consisting of the mature
polypeptide of SEQ ID NO: 5 and a polypeptide comprising or
consisting of the mature polypeptide of SEQ ID NO: 6. Paragraph 17.
The detergent composition according to any one of the preceding
paragraphs, wherein the concentration of said polypeptide having
deoxyribonuclease activity in a wash dose of said detergent
composition is within the range of 0.001 ppm to 100 ppm, optionally
in the range of about 0.001-2 ppm enzyme protein, optionally from
about about 0.01-0.2 ppm enzyme protein or optionally from about
0.01-0.02 ppm enzyme protein. Paragraph 18. A method for
preventing, reducing or removing biofilm from a textile or fabric
comprising; [0254] (i) contacting the a textile or fabric at least
partly coated with a biofilm with a wash liquor comprising the
detergent composition according to any one of paragraphs 1 to 17,
[0255] (ii) subjecting said textile to at least one washing cycle,
[0256] (iii) optionally rinsing said textile. Paragraph 19. The
method according to paragraph 18, wherein the temperature of said
wash liquor is in the range of 5.degree. C. to 95.degree. C., such
as in the range of 10.degree. C. to 80.degree. C., for example in
the range of 10.degree. C. to 70.degree. C., such as in the range
of 10.degree. C. to 60.degree. C., for example in the range of
10.degree. C. to 50.degree. C., such as in the range of 15.degree.
C. to 40.degree. C., for example in the range of 20.degree. C. to
30.degree. C. Paragraph 19. The method according to paragraph 17 or
18, wherein the pH of the liquid solution is in the range of 1 to
11, such as in the range 5.5 to 11, such as in the range of 7 to 9,
in the range of 7 to 8 or in the range of 7 to 8.5. Paragraph 20.
The method according to any one of paragraphs 17 to 19, wherein the
concentration of said polypeptide having deoxyribonuclease activity
in the wash liquor is within the range of 0.001 ppm to 100 ppm,
optionally in the range of about 0.001-2 ppm enzyme protein,
optionally from about about 0.01-0.2 ppm enzyme protein, optionally
from about about 0.2-0.5 ppm enzyme protein or optionally from
about 0.01-0.02 ppm enzyme protein. Paragraph 21. Use of the
detergent composition according to any one of paragraphs 1 to 17
for preventing, reducing or removing biofilm from a textile or a
fabric.
Wash Assays
Mini Launder-O-Meter (MiniLOM) Model Wash System
[0257] MiniLOM is a modified mini wash system of the
Launder-O-Meter (LOM), which is a medium scale model wash system
that can be applied to test up to 20 different wash conditions
simultaneously. A LOM is basically a large temperature controlled
water bath with 20 closed metal beakers rotating inside it. Each
beaker constitutes one small washing machine and during an
experiment, each will contain a solution of a specific
detergent/enzyme system to be tested along with the soiled and
unsoiled fabrics it is tested on. Mechanical stress is achieved by
the beakers being rotated in the water bath and by including metal
balls in the beaker.
[0258] The LOM model wash system is mainly used in medium scale
testing of detergents and enzymes at European wash conditions. In a
LOM experiment, factors such as the ballast to soil ratio and the
fabric to wash liquor ratio can be varied. Therefore, the LOM
provides the link between small scale experiments, such as AMSA and
mini-wash, and the more time consuming full scale experiments in
front loader washing machines.
[0259] In miniLOM, washes are performed in 50 ml test tubes placed
in Stuart rotator.
Terg-O-Timeter (TOM) Wash Assay
[0260] The Tergo-To-Meter (TOM) is a medium scale model wash system
that can be applied to test 12 different wash conditions
simultaneously. A TOM is basically a large temperature controlled
water bath with up to 12 open metal beakers submerged into it. Each
beaker constitutes one small top loader style washing machine and
during an experiment, each of them will contain a solution of a
specific detergent/enzyme system and the soiled and unsoiled
fabrics its performance is tested on. Mechanical stress is achieved
by a rotating stirring arm, which stirs the liquid within each
beaker. Because the TOM beakers have no lid, it is possible to
withdraw samples during a TOM experiment and assay for information
on-line during wash.
[0261] The TOM model wash system is mainly used in medium scale
testing of detergents and enzymes at US or LA/AP wash conditions.
In a TOM experiment, factors such as the ballast to soil ratio and
the fabric to wash liquor ratio can be varied. Therefore, the TOM
provides the link between small scale experiments, such as AMSA and
mini-wash, and the more time consuming full scale experiments in
top loader washing machines.
[0262] Equipment: The water bath with 12 steel beakers and 1
rotating arm per beaker with capacity of 500 or 1200 mL of
detergent solution. Temperature ranges from 5 to 80.degree. C. The
water bath has to be filled up with deionised water. Rotational
speed can be set up to 70 to 120 rpm/min.
[0263] Set temperature in the Terg-O-Tometer and start the rotation
in the water bath. Wait for the temperature to adjust (tolerance is
+/-0.5.degree. C.). All beakers shall be clean and without traces
of prior test material.
[0264] The wash solution with desired amount of detergent,
temperature and water hardness is prepared in a bucket. The
detergent is allowed to dissolve during magnet stirring for 10 min.
Wash solution shall be used within 30 to 60 min after
preparation.
[0265] 800 ml wash solution is added into a TOM beaker. The wash
solution is agitated at 120 rpm and optionally one or more enzymes
are added to the beaker. The swatches are sprinkled into the beaker
and then the ballast load. Time measurement starts when the
swatches and ballast are added to the beaker. The swatches are
washed for 20 minutes after which agitation is terminated. The wash
load is subsequently transferred from the TOM beaker to a sieve and
rinse with cold tap water. The solid swatches are separated from
the ballast load. The soil swatches are transferred to a 5 L beaker
with cold tap water under running water for 5 minutes. The ballast
load is kept separately for the coming inactivation. The water is
gently pressed out of the swatches by hand and placed on a tray
covered with a paper. Another paper is placed on top of the
swatches. The swatches are allowed to dry overnight before
subjecting the swatches to analysis, such as measuring the color
intensity using a Color Eye as described herein.
EXAMPLES
Example 1
Materials and Methods
Isolating Laundry Specific Bacterial Strains
[0266] One strain of Brevundimonas sp. isolated from laundry was
used in the present example. The Brevundimonas sp. was isolated
during a study, where the bacterial diversity in laundry after
washing at 15, 40 and 60.degree. C., respectively, was
investigated. The study was conducted on laundry collected from
Danish households. For each wash, 20 g of laundry items (tea towel,
towel, dish cloth, bib, T-shirt armpit, T-shirt collar, socks) in
the range 4:3:2:2:1:1:1 was used. Washing was performed in a
Laundr-O-Meter (LOM) at 15, 40 or 60.degree. C. For washing at 15
and 40.degree. C., Ariel Sensitive White & Color was used,
whereas WFK IEC-A* model detergent was used for washing at
60.degree. C. Ariel Sensitive White & Color was prepared by
weighing out 5.1 g and adding tap water up to 1000 ml followed by
stirring for 5 minutes. WFK IEC-A* model detergent (which is
available from WFK Testgewebe GmbH) was prepared by weighing out 5
g and adding tap water up to 1300 ml followed by stirring for 15
min. Washing was performed for 1 hour at 15, 40 and 60.degree. C.,
respectively, followed by 2 times rinsing with tap water for 20 min
at 15.degree. C.
[0267] Laundry was sampled immediately after washing at 15, 40 and
60.degree. C., respectively. Twenty grams of laundry was added 0.9%
(w/v) NaCl (1.06404; Merck, Damstadt, Germany) with 0.5% (w/w)
tween 80 to yield a 1:10 dilution in stomacher bag. The mixture was
homogenized using a Stomacher for 2 minutes at medium speed. After
homogenization, ten-fold dilutions were prepared in 0.9% (w/v)
NaCl. Bacteria were enumerated on Tryptone Soya Agar (TSA) (CM0129,
Oxoid, Basingstoke, Hampshire, UK) incubated aerobically at
30.degree. C. for 5-7 days. To suppress growth of yeast and moulds,
0.2% sorbic acid (359769, Sigma) and 0.1% cycloheximide (18079;
Sigma) were added. Bacterial colonies were selected from countable
plates and purified by restreaking twice on TSA. For long time
storage, purified isolates were stored at -80.degree. C. in TSB
containing 20% (w/v) glycerol (49779; Sigma).
Preparation of Biofilm Swatches
[0268] In the present study, one strain of Brevundimonas sp. was
used. Brevundimonas sp. was pre-grown on Tryptone Soya Agar (TSA)
(pH 7.3) (CM0131; Oxoid Ltd, Basingstoke, UK) for 2-5 days at
30.degree. C. From a single colony, a loop-full was transferred to
10 mL of TSB (Tryptone Soya broth, Oxoid) and incubated for 1 day
at 30.degree. C. with shaking (240 rpm). After propagation,
Brevundimonas sp. was pelleted by centrifugation (Sigma Laboratory
Centrifuge 6K15) (3000 g at 21.degree. C. in 7 min) and resuspended
in 10 mL of TSB diluted twice with water. Optical density (OD) at
600 nm was measured using a spectophometer (POLARstar Omega (BMG
Labtech, Ortenberg, Germany). Fresh TSB diluted twice with water
was inoculated to an OD600 nm of 0.03, and 20 mL was added into a
petridish (diameter 8.5 cm), in which a swatch of polyester (WFK
30A) measuring 5 cm.times.5 cm were placed. After incubation (24 h
at 15.degree. C. with shaking (100 rpm), swatches were rinsed twice
with 0.9% (w/v) NaCl.
Washing Experiment
[0269] Model detergent A wash liquor (100%) was prepared by
dissolving 3.33 g/l of model detergent A containing 12% LAS, 11%
AEO Biosoft N25-7 (NI), 7% AEOS (SLES), 6% MPG, 3% ethanol, 3% TEA
(triethanolamine), 2.75% cocoa soap, 2.75% soya soap, 2% glycerol,
2% sodium hydroxide, 2% sodium citrate, 1% sodium formiate, 0.2%
DTMPA and 0.2% PCA (all percentages are w/w) in water with hardness
15.degree. dH. Wash liquors containing 80, 60, 40, 20 and 10% of
model detergent A were prepared by diluting model detergent A wash
liquor (100%) in water with hardness 15.degree. dH. TOM beakers
were added model detergent A wash liquor (1000 ml) and then pigment
soil (Pigmentschmutz, 09V, wfk, Krefeld, Germany) (0.7 g/L). In
washes with DNase, Aspergillus oryzae DNase (0.5 ppm) were added to
the wash liquor. Five rinsed swatches with Brevundimonas sp. and
mixed textile giving a total weight of 10 g were added to the TOM
beakers and washing were carried out for 30 min at 30.degree. C. at
110 rpm. After washing, swatches with Brevundimonas sp. were rinsed
twice in tap water and dried on filter paper over night. Color
difference (L values) was measured using a Color Eye (Macbeth Color
Eye 7000 reflectance spectrophotometer). The measurements were made
without UV in the incident light and the L value from the CIE Lab
color space was extracted.
TABLE-US-00001 TABLE 1 Dose of deter- gent Type of L-value.sub.with
DNase- (%) textile L-value.sub.with DNase L-value.sub.without DNase
L-value.sub.without DNase 100 Polyester 87.6 85.8 1.7 80 Polyester
87.5 86.0 1.5 60 Polyester 87.4 85.3 2.1 40 Polyester 86.0 84.1 1.9
20 Polyester 86.5 85.3 1.2 10 Polyester 85.6 83.2 2.4
Example 2
[0270] Preparation of Swatches with Biofilm
[0271] Brevundimonas sp. (isolated as described in Example 1) was
pre-grown on Tryptone Soya Agar (TSA) (pH 7.3) (CM0131; Oxoid Ltd,
Basingstoke, UK) for 2-5 days at 30.degree. C. From a single
colony, a loop-full was transferred to 10 mL of TSB and incubated
for 1 day at 30.degree. C. with shaking (240 rpm). After
propagation, Brevundimonas sp. was pelleted by centrifugation
(Sigma Laboratory Centrifuge 6K15) (3000 g at 21.degree. C. in 7
min) and resuspended in 10 mL of TSB diluted twice with water.
Optical density (OD) at 600 nm was measured using a spectophometer
(POLARstar Omega (BMG Labtech, Ortenberg, Germany). Fresh TSB
diluted twice with water was inoculated to an OD.sub.600 nm of
0.03, and 1.6 mL was added into each well of a 12-well polystyrene
flat-bottom microplate (3512; Corning Incorporated, Corning, N.Y.,
USA), in which a round swatch (diameter 2 cm) of sterile Polyester
WFK30A was placed. After incubation (24 h at 15.degree. C. with
shaking (100 rpm), swatches were rinsed twice with 0.9% (w/v)
NaCl.
Wash Experiment
[0272] Wash liquors of liquid model detergent A, liquid model
detergent B, powder model detergent T, powder model detergent V,
liquid model detergent N, liquid model detergent 0 and liquid model
detergent P were prepared by weighing out and dissolving detergents
in water with water with hardness 15.degree. dH. Dosing of model
detergent A and B was 3.33 g/L, whereas dosing of model detergent T
and V was 5.30 g/L. The AEO Biosoft N25-7 (NI) (0.16 g/l) component
of model detergent T and V was added separately. Dosing of model
detergent 0, P and V was 5.30 g/L and water hardness was 15.degree.
dH. Pigment soil (Pigmentschmutz, 09V, wfk, Krefeld, Germany) (0.7
g/L) was added to the wash liquors, and 10 ml was added to a 50 ml
test tube in which five rinsed swatches with Brevundimonas sp.
biofilm and five sterile polyester (WFK30A) swatches were placed.
In washes, where A. oryzae DNase (mature polypeptide of SEQ ID NO
1) was included, DNase (0.5 ppm) was added to wash liquors. In
washes, where T. harzianum DNase (mature polypeptide of SEQ ID NO
4) was included, DNase (0.5 ppm) was added to wash liquors. In
washes, where B. licheniformis DNase (mature polypeptide of SEQ ID
NO 5) was included, DNase (0.5 ppm) was added to wash liquors. In
washes, where B. subtilis DNase (mature polypeptide of SEQ ID NO 6)
was included, DNase (0.5 ppm) was added to wash liquors. Test tubes
were placed in a Stuart rotator (Mini LOM) for 1 hour at 30.degree.
C. Swatches were rinsed twice with tap water and dried on filter
paper over night. Color difference (L values) was measured using a
Color Eye (Macbeth Color Eye 7000 reflectance spectrophotometer).
The measurements were made without UV in the incident light and the
L value from the CIE Lab color space was extracted. The color
difference (L value, L*) represents the darkest black at L*=0, and
the brightest white at L*=100. Data is represented as Delta L
values meaning the L value of the swatch washed with DNase minus
the L value of swatch washed without DNase.
TABLE-US-00002 TABLE 2 Deep cleaning by A. otyzae DNase in liquid
and powder detergents with reduced level of surfactants.
.DELTA.L.sub.Biofilm .DELTA.L.sub.Sterile .sub.swatches
.sub.swatches Surfactant L.sub.Biofilm (L.sub.(DNase)-
L.sub.Sterile (L.sub.(DNase)- Detergent Format level DNase
.sub.swatches L.sub.(noDNase)) .sub.swatches L.sub.(noDNase)) Model
Liquid Normal + 87.5 6.6 87.7 4.0 detergent A Model Liquid Normal -
80.9 83.7 detergent A Model Liquid Low + 88.4 6.1 88.9 4.0
detergent B Model Liquid Low - 82.3 84.9 detergent B Model Powder
Normal + 88.9 5.2 86.2 4.4 detergent T Model Powder Normal - 83.7
81.8 detergent T Model Powder Low + 88.5 4.3 87.7 2.4 detergent V
Model Powder Low - 84.2 85.3 detergent V
[0273] For clarity the surfactant level of the table above could be
explained as if normal=100 and low=60, then the surfactant level is
reduced by 40%.
TABLE-US-00003 TABLE 3 Sur- Sur- factant factant
.DELTA.L.sub.Biofilm .DELTA.L.sub.Sterile level level .sub.swatches
.sub.swatches with without L.sub.Biofilm (L.sub.(DNaseAo)-
L.sub.Sterile (L.sub.(DNaseAo)- Detergent Format soap soap DNase
.sub.swatches L.sub.(no DNase)) .sub.swatches L.sub.(no DNase))
Model Liquid 33.5 28.0 + 88.2 4.6 88.2 0.9 detergent A Model Liquid
33.5 28.0 - 83.7 87.3 detergent A Model Liquid 22.1 16.6 + 88.8 3.8
89.3 0.6 detergent B Model Liquid 22.1 16.6 - 85.0 88.7 detergent B
Model Powder 17.0 15.0 + 89.6 5.2 87.3 0.6 detergent T Model Powder
17.0 15.0 - 84.4 86.7 detergent T Model Powder 10.2 9.0 + 88.1 4.0
88.8 3.0 detergent V Model Powder 10.2 9.0 - 84.1 85.8 detergent V
Model Liquid 22.3 21.3 + 85.3 4.6 85.1 2.7 detergent N Model Liquid
22.3 21.3 - 80.7 82.4 detergent N Model Liquid 17.0 16.0 + 85.0 3.0
85.7 1.6 detergent O Model Liquid 17.0 16.0 - 82.0 84.2 detergent O
Model Liquid 10.6 9.6 + 87.2 4.3 87.1 2.6 detergent P Model Liquid
10.6 9.6 - 82.9 84.5 detergent P Sur- Sur- factant factant
.DELTA.L.sub.Biofilm .DELTA.L.sub.Sterile level level .sub.swatches
.sub.swatches with without L.sub.Biofilm (L.sub.(DNaseTh)-
L.sub.Sterile (L.sub.(DNaseTh)- Detergent Format soap soap DNase
.sub.swatches L.sub.(no DNase)) .sub.swatches L.sub.(no DNase))
Model Liquid 33.5 28.0 + 86.5 2.9 88.2 0.9 detergent A Model Liquid
33.5 28.0 - 83.7 87.3 detergent A Model Liquid 22.1 16.6 + 89.1 4.1
90.2 1.6 detergent B Model Liquid 22.1 16.6 - 85.0 88.7 detergent B
Model Powder 17.0 15.0 + 87.0 2.6 87.0 0.3 detergent T Model Powder
17.0 15.0 - 84.4 86.7 detergent T Model Powder 10.2 9.0 + 87.2 3.1
87.9 2.1 detergent V Model Powder 10.2 9.0 - 84.1 85.8 detergent V
Model Liquid 22.3 21.3 + 84.8 4.0 84.9 2.5 detergent N Model Liquid
22.3 21.3 - 80.7 82.4 detergent N Model Liquid 17.0 16.0 + 85.1 3.1
86.3 2.1 detergent O Model Liquid 17.0 16.0 - 82.0 84.2 detergent O
Model Liquid 10.6 9.6 + 86.8 3.9 87.0 2.5 detergent P Model Liquid
10.6 9.6 - 82.9 84.5 detergent P Sur- Sur- factant factant
.DELTA.L.sub.Biofilm .DELTA.L.sub.Sterile level level .sub.swatches
.sub.swatches with without L.sub.Biofilm (L.sub.(DNaseBl)-
L.sub.Sterile (L.sub.(DNaseBl)- Detergent Format soap soap DNase
.sub.swatches L.sub.(no DNase)) .sub.swatches L.sub.(no DNase))
Model Liquid 33.5 28.0 + 87.9 4.3 87.8 0.5 detergent A Model Liquid
33.5 28.0 - 83.7 87.3 detergent A Model Liquid 22.1 16.6 + 88.1 3.1
88.8 0.1 detergent B Model Liquid 22.1 16.6 - 85.0 88.7 detergent B
Model Powder 17.0 15.0 + 88.2 3.8 87.0 0.3 detergent T Model Powder
17.0 15.0 - 84.4 86.7 detergent T Model Powder 10.2 9.0 + 88.5 4.4
86.5 0.7 detergent V Model Powder 10.2 9.0 - 84.1 85.8 detergent V
Model Liquid 22.3 21.3 + 84.9 4.2 85.5 3.1 detergent N Model Liquid
22.3 21.3 - 80.7 82.4 detergent N Model Liquid 17.0 16.0 + 85.1 3.1
85.9 1.7 detergent O Model Liquid 17.0 16.0 - 82.0 84.2 detergent O
Model Liquid 10.6 9.6 + 86.9 4.1 87.1 2.6 detergent P Model Liquid
10.6 9.6 - 82.9 84.5 detergent P Sur- Sur- factant factant
.DELTA.L.sub.Biofilm .DELTA.L.sub.Sterile level level .sub.swatches
.sub.swatches with without L.sub.Biofilm (L.sub.(DNaseBs)-
L.sub.Sterile (L.sub.(DNaseBs)- Detergent Format soap soap DNase
.sub.swatches L.sub.(no DNase)) .sub.swatches L.sub.(no DNase))
Model Liquid 33.5 28.0 + 87.5 3.8 87.6 0.3 detergent A Model Liquid
33.5 28.0 - 83.7 87.3 detergent A Model Liquid 22.1 16.6 + 89.4 4.4
89.4 0.8 detergent B Model Liquid 22.1 16.6 - 85.0 88.7 detergent B
Model Powder 17.0 15.0 + 88.0 3.6 87.3 0.5 detergent T Model Powder
17.0 15.0 - 84.4 86.7 detergent T Model Powder 10.2 9.0 + 86.6 2.4
85.3 -0.5 detergent V Model Powder 10.2 9.0 - 84.1 85.8 detergent V
Model Liquid 22.3 21.3 + 83.0 2.3 84.8 2.4 detergent N Model Liquid
22.3 21.3 - 80.7 82.4 detergent N Model Liquid 17.0 16.0 + 86.4 4.3
86.7 2.6 detergent O Model Liquid 17.0 16.0 - 82.0 84.2 detergent O
Model Liquid 10.6 9.6 + 86.5 3.6 86.6 2.0 detergent P Model Liquid
10.6 9.6 - 82.9 84.5 detergent P
[0274] In table 3 Results of washes with detergents comprising
different level of surfactants, where A. oryzae (L.sub.(DNaseAo),
T. harzianum (L.sub.(DNaseTh), B. licheniformis (L.sub.(DNaseBI))
and B. subtilis (L.sub.(DNaseBs)) DNase (0.5 ppm) was added to wash
liquors or not added.
[0275] Firstly, table 3 shows that all the tested DNases have
effect on biofilm swatch tested and that the effect is maintained
when the amount of surfactant is reduced e.g. performance of the
DNases in detergents with surfactant level (total amount of
surfactant with soap) about 30 e.g. 33.5 (Model A) is compared to
performance of the DNases in detergents with surfactant level
(total amount of surfactant with soap) about 17 (Model T). A
specific example the DNase from Bacillus subtilis (DNaseBs) mature
polypeptide of SEQ ID NO 6 have comparable performance in Model
detergent 0 (total amount of surfactant with soap 17) and Model P
(total amount of surfactant with soap about 11 (10.6). In other
words the surfactant level does not influence the performance of
the DNases and the effect of the DNases is maintained even in
detergents with surfactant levels (total amount with soap) of about
11.
Sequence CWU 1
1
71243PRTAspergillus
oryzaeSIGNAL(1)..(22)PROPEP(23)..(37)CHAIN(38)..(243)mature
polypeptide 1Met Gln Leu Thr Lys Ser Leu Leu Val Phe Ala Leu Tyr
Met Phe Gly 1 5 10 15 Thr Gln His Val Leu Ala Val Pro Val Asn Pro
Glu Pro Asp Ala Thr 20 25 30 Ser Val Glu Asn Val Ala Leu Lys Thr
Gly Ser Gly Asp Ser Gln Ser 35 40 45 Asp Pro Ile Lys Ala Asp Leu
Glu Val Lys Gly Gln Ser Ala Leu Pro 50 55 60 Phe Asp Val Asp Cys
Trp Ala Ile Leu Cys Lys Gly Ala Pro Asn Val 65 70 75 80 Leu Gln Arg
Val Asn Glu Lys Thr Lys Asn Ser Asn Arg Asp Arg Ser 85 90 95 Gly
Ala Asn Lys Gly Pro Phe Lys Asp Pro Gln Lys Trp Gly Ile Lys 100 105
110 Ala Leu Pro Pro Lys Asn Pro Ser Trp Ser Ala Gln Asp Phe Lys Ser
115 120 125 Pro Glu Glu Tyr Ala Phe Ala Ser Ser Leu Gln Gly Gly Thr
Asn Ala 130 135 140 Ile Leu Ala Pro Val Asn Leu Ala Ser Gln Asn Ser
Gln Gly Gly Val 145 150 155 160 Leu Asn Gly Phe Tyr Ser Ala Asn Lys
Val Ala Gln Phe Asp Pro Ser 165 170 175 Lys Pro Gln Gln Thr Lys Gly
Thr Trp Phe Gln Ile Thr Lys Phe Thr 180 185 190 Gly Ala Ala Gly Pro
Tyr Cys Lys Ala Leu Gly Ser Asn Asp Lys Ser 195 200 205 Val Cys Asp
Lys Asn Lys Asn Ile Ala Gly Asp Trp Gly Phe Asp Pro 210 215 220 Ala
Lys Trp Ala Tyr Gln Tyr Asp Glu Lys Asn Asn Lys Phe Asn Tyr 225 230
235 240 Val Gly Lys 2206PRTAspergillus oryzaeCHAIN(1)..(206)mature
polypeptide 2Ala Leu Lys Thr Gly Ser Gly Asp Ser Gln Ser Asp Pro
Ile Lys Ala 1 5 10 15 Asp Leu Glu Val Lys Gly Gln Ser Ala Leu Pro
Phe Asp Val Asp Cys 20 25 30 Trp Ala Ile Leu Cys Lys Gly Ala Pro
Asn Val Leu Gln Arg Val Asn 35 40 45 Glu Lys Thr Lys Asn Ser Asn
Arg Asp Arg Ser Gly Ala Asn Lys Gly 50 55 60 Pro Phe Lys Asp Pro
Gln Lys Trp Gly Ile Lys Ala Leu Pro Pro Lys 65 70 75 80 Asn Pro Ser
Trp Ser Ala Gln Asp Phe Lys Ser Pro Glu Glu Tyr Ala 85 90 95 Phe
Ala Ser Ser Leu Gln Gly Gly Thr Asn Ala Ile Leu Ala Pro Val 100 105
110 Asn Leu Ala Ser Gln Asn Ser Gln Gly Gly Val Leu Asn Gly Phe Tyr
115 120 125 Ser Ala Asn Lys Val Ala Gln Phe Asp Pro Ser Lys Pro Gln
Gln Thr 130 135 140 Lys Gly Thr Trp Phe Gln Ile Thr Lys Phe Thr Gly
Ala Ala Gly Pro 145 150 155 160 Tyr Cys Lys Ala Leu Gly Ser Asn Asp
Lys Ser Val Cys Asp Lys Asn 165 170 175 Lys Asn Ile Ala Gly Asp Trp
Gly Phe Asp Pro Ala Lys Trp Ala Tyr 180 185 190 Gln Tyr Asp Glu Lys
Asn Asn Lys Phe Asn Tyr Val Gly Lys 195 200 205 3204PRTAspergillus
oryzaeCHAIN(1)..(204)mature polypeptide 3Lys Thr Gly Ser Gly Asp
Ser Gln Ser Asp Pro Ile Lys Ala Asp Leu 1 5 10 15 Glu Val Lys Gly
Gln Ser Ala Leu Pro Phe Asp Val Asp Cys Trp Ala 20 25 30 Ile Leu
Cys Lys Gly Ala Pro Asn Val Leu Gln Arg Val Asn Glu Lys 35 40 45
Thr Lys Asn Ser Asn Arg Asp Arg Ser Gly Ala Asn Lys Gly Pro Phe 50
55 60 Lys Asp Pro Gln Lys Trp Gly Ile Lys Ala Leu Pro Pro Lys Asn
Pro 65 70 75 80 Ser Trp Ser Ala Gln Asp Phe Lys Ser Pro Glu Glu Tyr
Ala Phe Ala 85 90 95 Ser Ser Leu Gln Gly Gly Thr Asn Ala Ile Leu
Ala Pro Val Asn Leu 100 105 110 Ala Ser Gln Asn Ser Gln Gly Gly Val
Leu Asn Gly Phe Tyr Ser Ala 115 120 125 Asn Lys Val Ala Gln Phe Asp
Pro Ser Lys Pro Gln Gln Thr Lys Gly 130 135 140 Thr Trp Phe Gln Ile
Thr Lys Phe Thr Gly Ala Ala Gly Pro Tyr Cys 145 150 155 160 Lys Ala
Leu Gly Ser Asn Asp Lys Ser Val Cys Asp Lys Asn Lys Asn 165 170 175
Ile Ala Gly Asp Trp Gly Phe Asp Pro Ala Lys Trp Ala Tyr Gln Tyr 180
185 190 Asp Glu Lys Asn Asn Lys Phe Asn Tyr Val Gly Lys 195 200
4205PRTTrichoderma harzianumSIGNAL(1)..(17)CHAIN(18)..(205)mature
polypeptide 4Met Lys Leu Ser Ile Ser Val Ala Leu Thr Ser Ala Ile
Ala Val Leu 1 5 10 15 Ala Ala Pro Ala Pro Met Pro Thr Pro Pro Gly
Ile Pro Thr Glu Ser 20 25 30 Ser Ala Arg Thr Gln Leu Ala Gly Leu
Thr Val Ala Val Ala Gly Ser 35 40 45 Gly Thr Gly Tyr Ser Arg Asp
Leu Phe Pro Thr Trp Asp Ala Ile Ser 50 55 60 Gly Asn Cys Asn Ala
Arg Glu Tyr Val Leu Lys Arg Asp Gly Glu Gly 65 70 75 80 Val Gln Val
Asn Asn Ala Cys Glu Ser Gln Ser Gly Thr Trp Ile Ser 85 90 95 Pro
Tyr Asp Asn Ala Ser Phe Thr Asn Ala Ser Ser Leu Asp Ile Asp 100 105
110 His Met Val Pro Leu Lys Asn Ala Trp Ile Ser Gly Ala Ser Ser Trp
115 120 125 Thr Thr Ala Gln Arg Glu Ala Leu Ala Asn Asp Val Ser Arg
Pro Gln 130 135 140 Leu Trp Ala Val Ser Ala Ser Ala Asn Arg Ser Lys
Gly Asp Arg Ser 145 150 155 160 Pro Asp Gln Trp Lys Pro Pro Leu Thr
Ser Phe Tyr Cys Thr Tyr Ala 165 170 175 Lys Ser Trp Ile Asp Val Lys
Ser Phe Tyr Lys Leu Thr Ile Thr Ser 180 185 190 Ala Glu Lys Thr Ala
Leu Ser Ser Met Leu Asp Thr Cys 195 200 205 5142PRTBacillus
licheniformisSIGNAL(1)..(33)CHAIN(34)..(142)mature polypeptide 5Met
Ile Lys Lys Trp Ala Val His Leu Leu Phe Ser Ala Leu Val Leu 1 5 10
15 Leu Gly Leu Ser Gly Gly Ala Ala Tyr Ser Pro Gln His Ala Glu Gly
20 25 30 Ala Ala Arg Tyr Asp Asp Ile Leu Tyr Phe Pro Ala Ser Arg
Tyr Pro 35 40 45 Glu Thr Gly Ala His Ile Ser Asp Ala Ile Lys Ala
Gly His Ser Asp 50 55 60 Val Cys Thr Ile Glu Arg Ser Gly Ala Asp
Lys Arg Arg Gln Glu Ser 65 70 75 80 Leu Lys Gly Ile Pro Thr Lys Pro
Gly Phe Asp Arg Asp Glu Trp Pro 85 90 95 Met Ala Met Cys Glu Glu
Gly Gly Lys Gly Ala Ser Val Arg Tyr Val 100 105 110 Ser Ser Ser Asp
Asn Arg Gly Ala Gly Ser Trp Val Gly Asn Arg Leu 115 120 125 Ser Gly
Phe Ala Asp Gly Thr Arg Ile Leu Phe Ile Val Gln 130 135 140
6136PRTBacillus subtilisSIGNAL(1)..(26)CHAIN(27)..(136)mature
polypeptide 6Met Lys Lys Trp Met Ala Gly Leu Phe Leu Ala Ala Ala
Val Leu Leu 1 5 10 15 Cys Leu Met Val Pro Gln Gln Ile Gln Gly Ala
Ser Ser Tyr Asp Lys 20 25 30 Val Leu Tyr Phe Pro Leu Ser Arg Tyr
Pro Glu Thr Gly Ser His Ile 35 40 45 Arg Asp Ala Ile Ala Glu Gly
His Pro Asp Ile Cys Thr Ile Asp Arg 50 55 60 Asp Gly Ala Asp Lys
Arg Arg Glu Glu Ser Leu Lys Gly Ile Pro Thr 65 70 75 80 Lys Pro Gly
Tyr Asp Arg Asp Glu Trp Pro Met Ala Val Cys Glu Glu 85 90 95 Gly
Gly Ala Gly Ala Asp Val Arg Tyr Val Thr Pro Ser Asp Asn Arg 100 105
110 Gly Ala Gly Ser Trp Val Gly Asn Gln Met Ser Ser Tyr Pro Asp Gly
115 120 125 Thr Arg Val Leu Phe Ile Val Gln 130 135 7269PRTBacillus
lentus 7Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala
Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala
Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile
Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln
Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala
Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser
Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Ser Gly Ser
Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly
Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120
125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly
130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser
Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val
Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln
Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val
Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly
Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu
Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245
250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260
265
* * * * *