Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W092/t30~3 2 1 0 0 ~ ~ ~ PCTtUS92tO019n
DETERGENT COMPOSITIONS WITH
HIGH A~llVllY CELLULASE AND SOFTENING CLAYS
Technical Field
The present invention relates to detergent
compositions comprising a high activity cellulase in
combination with a softening clay. In the detergent
compositions herein, the cellulase comprises a cellulase
of high activity defined by the C14CMC-method. Preferably
the detergent composition comprises a softening clay
together with a clay flocculating agent and in case of
liquid composition an anti-settling agent for the clay.
The present invention relates to detergent
compositions comprising a high activity cellulase in
combination with a softening clay. In the
Backqround of the Invention
The need for detergent compositions which exhibit
not only good cleaning properties, but also good
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W092/13053 PCT/US92tO019~
~o~S~ 4 2
fabric-softening performance, and other fabric care
benefits, is well-established in the art.
The efficiency of cellulolytic enzymes, i.e.
cellulases, in terms of textile cleaning and harshness-
reducing agent for fabrics has been recognized for some
time; GB-A-2,075,028, GB-A-2,095,275 and GB-A-
2,094,826, disclose detergent compositions with
cellulase for improved cleaning performance; GB-A-
l,368,599 discloses the use of cellulase for reducing
the harshness of cotton-containing fabrics; U.S.
4,435,307 teaches the use of a cellulolytic enzyme
derived from Humicola insolens as well as a fraction
thereof, designated ACXI, as a harshness-reducing
detergent additive.
EP-A-O 269 168 discloses optimized detergent
compositions containing cellulase, which are
formulated at a mild alkaline pH range and provide
combined fabric cleaning, fabric softening, and fabric
care performance.
In Wo 89109259 have been disclosed cellulase
preparations useful for reducing the harshness of
cotton-containing fabrics, comprising an endoglucanase
component with a high endoase activity and affinity
towards cellulose.
The practical exploitation of cellulases has
however, been set back by the fact that cellulase
preparations such as those disclosed in the above-
mentioned prior art documents, are complex mixtures,
of which only a certain fraction is effective in the
fabric-care context; it was thus difficult to
implement cost effective industrial production of
cellulase for the detergent industry; and large
quantities of such cellulase preparations would need
~UBglmlrE SHEE~
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2100SS4
to be applied, in order to obtain the desired effect
on fabrics.
Improvements in cellulase production also often have
not proven to be sufficiently identifiable in terms of
applicability in detergents. Defining a cellulase
selection criterium relevant for detergent application
of cellulase was made possible by the C14CMC-method
disclosed in EP-A-350 098. A minimum of 10% removal
of immobilized radioactive labelled carboxymethyl-
cellulose has now been found to provide high activity
cellulase. A preferred group of cellulase falling
under the high activity definition according to the
present invention has been disclosed in copending
Danish Patent Application No. : 1159/90 filed May 5,
1990. There is amongst others disclosed a cellulase
preparation consisting essentially of a homogeneous
endoglucanase component which is immunoreactive with a
monoclonal antibody raised against a partially
purified about 43kD cellulase derived from Humicola
insolens DM1800.
The finding that this particular endoglucanase
component of cellulase is advantageous for the
treatment of cellulose-containing materials now
permits to produce the cellulase cost-effectively,
e.g. by employing recombinant DNA techniques, and
allows to apply only a small quantity of the cellulase
preparation, and obtain the desired effect on fabrics.
EP-A-381 397 discloses the effect of low ionic-
strength on enzyme performance, in particular for
lipase. However, it has been surprisingly found, that
the effect of a compact matrix on the selected enzymes
of the present invention is much larger than what
could be expected from state of the art cellulases
such as disclosed in EP-A-381 397.
~WBSTm,rrE SHEE~
~'092/130~3 PCT/US92/00190
2~ 4
In EP-A-177 165 the use of softening clay
together with cellulase in detergent compositions has
been disclosed. The invention of this disclosure is
based on the lack of prior art disclosing the
combination of two principal softening or h~chness
reducing detergent compounds. EP-A-177 165 recognizes
that there is no reason to exclude clay or cellulase
from detergent compositions comprising the respective
other compound. However, EP-A-177 165 does not
recognize that for the selected group of highly active
cellulase according to the present invention, the
combination with clay is advantageous beyond the
additive performance which otherwise could be expected
of two softening ingredients.
Accordingly, it is an objective of the present
invention to provide detergent compositions comprising
a high activity cellulase and softening clay, which
detergent compositions exhibit an optimum softening
performance. An additional objective is to provide
such detergent compositions in liquid or granulate
form.
It is another objective of the present invention
to provide detergent compositions containing high
activity cellulase together with softening clay which
provide excellent colour rejuvenation and whiteness
maintenance for fabrics especially for those which
comprise cellulose fibres. An additional objective of
the present invention is to provide detergent
compositions which further exhibit good stain removal
and cleaning performance particularly at temperatures
of about 60 C or below.
It is a further object of the present invention to
provide such detergent compositions in a compact form,
having a relatively high density and containing a low
SIJBSrlTUI~ SHEE'F
F 2 1 o o 5 5 4
_ 5
amount of inorganic filler salt, which exhibit
optimum softening performanceO
It is yet another objective of the present
invention to provide liquid detergent compositions
comprising, in addition to the essential compounds
of the present invention, an anti-settling agent
to provide a storage stable clay suspension
matrix. An even further objective of the present
invention is to provide detergent compositions, be
it liquid or granular comprising in addition to
the essential compounds a clay flocculating agent
to additionally aid the softening clay deposition
on fibres.
Summary of the Invention
The present invention relates to a detergent
composition comprising a surface active agent, a
builder system, a softening clay, a clay
flocculating agent in an amount from about 0.005
to 20~ by weight of said clay, and a cellulase
characterized in that said cellulase provides at
least 10~ removal of immobilized, radio-active
labelled carboxylmethyl-cellulose according to the
C14CMC-method at 25x 10-6~ by weight of cellulase
protein in the laundry test solution.
Further preferred are such detergent composi-
tions in which the cellulase consists essentially
of a homogenous endoglucanase component which is
immuno-reactive with an anti-body raised against a
highly purified endoglucanase, being a cellulase
of about 43 Kd, derived from Humicola insolens,
DSM 1800 or a homologous to the about 43kD
cellulase. Additionally, detergent compositions
comprising softening clay and cellulase being an
endoglucanase enzyme with the aminoacid sequences
shown in the listings ID No. 2 and ID No. 4 or
homologues thereof have been found to provide the
desired synergetic fabric treatment
WO92/130~3 PCT/US92/0019n
2~o~ 6
benefits, particularly softening, according to the
present invention.
Definitions
Unless stated otherwise, the following
definitions will be used hereinafter :
- percentages are percent by weight
- softening refers to a range of fabric
treatments other than cleAn i ng; in
particular it includes softeni~g, colour
rejuvenation and whiteness maintenAnc~,
anti-wrinkling, anti-static and ease
of ironing treatments.
Detailed Description of the Invention
The present detergent compositions can be in
granular or liguid form. The form d~penA~ upon the
desired application for example as a softening-
through-the-wash detergent, at low or high
temperatures, ln an automatic washing machine or in a
semi-automatic hand washing procedure.
The desired form of the detergent will strongly
influence the selection and amounts of compounds of
surfactant, builder, cellulase, softeni~ clays and
especially optional ingredients for the particular
composition. For liquid compositions an anti-settling
agent for the softening clay is desirable and not
contradictive with flocculating agents used to aid
clay deposition on fibres for liquid or granular
compositions. However, the detailed description of all
individual compounds and the examples will enable the
man skilled in the art to formulate detergent
compositions according to the present invention.
,"
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WO92/13053 21 0 0 5 ~ 4 PCT/~IS92/0019~
SURFACTANT
A wide range of surfactants can be used in the
detergent compositions. A typical listing of anionic,
nonionic, ampholytic and zwitterionic classes, and
species of these surfactants, is given in US Patent
3,664,961 issued to Norris on May 23, 1972.
Mixtures of anionic surfactants are particularly
suitable herein, especially mixtures of sulphonate and
sulphate surfactants in a weight ratio of from 5:1 to
1:2, preferably from 3:1 to 2:3, more preferably from
3:1 to 1:1. Preferred sulphonates include alkyl
benzene sulphonates having from 9 to 15, especially 11
to 13 carbon atoms in the alkyl radical, and alpha-
sulphonated methyl fatty acid esters in which the
fatty acid is derived from a C12-C18 fatty source
preferably from a C16-Cl8 fatty source. In each
instance the cation is an alkali metal, preferably
sodium. Preferred sulphate surfactants are alkyl
sulphates having from 12 to 18 carbon atoms in the
alkyl radical, optionally in admixture with ethoxy
sulphates having from 10 to 20, preferably 10 to 16
carbon atoms in the alkyl radical and an average
degree of ethoxylation of l to 6. Examples of
preferred alkyl sulphates herein are tallow alkyl
sulphate, coconut alkyl sulphate, and Cl4_l5 alkyl
sulphates. The cation in each instance is again an
alkali metal cation, preferably sodium.
One class of nonionic surfactants useful in the
present invention are condensates of ethylene oxide
with a hydrophobic moiety to provide a surfactant
having an average hydrophilic-lipophilic balance (HLB)
in the range from 8 to 17, preferably from 9.5 to
13.5, more preferably from 10 to 12.5. The
hydrophobic (lipophilic) moiety may be aliphatic or
aromatic in nature and the length of the
polyoxyethylene group which is condensed with any
WBSllTUrE SHEE~:
WO92/130~3 ~ ~ PCT/US92/0019
~ 8
particular hydrophobic group can be readily adjusted
to yleld a water-soluble compound having the desired
degree of balance between hydrophilic and hydrophobic
elements.
Especially preferred nonionic surfactants of this
type are the C9-C15 primary alcohol ethoxylates
containing 3-8 moles of ethylene oxide per mole of
alcohol, particularly the C14-C15 primary alcohols
containing 6-8 moles of ethylene oxide per mole of
alcohol and the C12-C14 primary alcohols containing 3-
5 moles of ethylene oxide per mole of alcohol.
Another class of nonionic surfactants comprises
alkyl polyglucoside compounds of general formula
RO (cnH2no)tzx
wherein Z is a moiety derived from glucose; R is a
saturated hydrophobic alkyl group that contains from
12 to 18 carbon atoms; t is from 0 to 10 and n is 2 or
3; x is from 1.3 to 4, the compounds including less
than 10% unreacted fatty alcohol and less than 50%
short chain alkyl polyglucosides. Compounds of this
type and their use in detergent are disclosed in EP-B
0 070 077, 0 075 996 and 0 094 118.
Also suitable as nonionic surfactants are poly
hydroxy fatty acid amide surfactants of the formula
R2 - C - N - Z, wherein Rl is H,
Il I
O Rl
or Rl is Cl_4 hydrocarbyl, 2-hydroxy ethyl, 2-hydroxy
propyl or a mixture thereof, R2 is C5_31 hydrocarbyl,
and Z is a polyhydroxyhydrocarbyl having a linear
~JBSrlTUlE SHEE~
WO 92/13053 210 0 S ~ ~ PCr/~3S92/0019n
hydrocarbyl chain with at least 3 hydroxyls directly
connected to the chain, or an alkoxylated derivative
thereof. Preferably, Rl is methyl, R2 is a straight
C11_15 alkyl or alkenyl chain such as coconut alkyl or
mixtures thereof, and Z is derived from a reducing
sugar such as glucose, fructose, maltose, lactose, in
a reductive amination reaction.
A further class of surfactants are the semi-polar
surfactants such as amine oxides. Suitable amine
oxides are selected from mono C8-C20, preferably C10-
C14 N-alkyl or alkenyl amine oxides and propylene-1,3-
diamine dioxides wherein the remaining N positions aresubstituted by methyl, hydroxyethyl or hydroxypropyl
groups.
Another class of surfactants are amphoteric
surfactants, such as polyamine-based species.
Cationic surfactants can also be used in the
detergent compositions herein and suitable quaternary
ammonium surfactants are selected from mono C8-C16,
preferably C10-Cl4 N-alkyl or alkenyl ammonium
surfactants wherein remaining N positions are
substituted by methyl, hydroxyethyl or hydroxypropyl
groups.
Mixtures of surfactant types are preferred, more
especially anionic-nonionic and also anionic-nonionic-
cationic mixtures. Particularly preferred mixtures
are described in British Patent No. 2040987 and
European Published Application No. 0 087 914. The
detergent compositions can comprise from 1%-70% by
weight of surfactant, but usually the surfactant is
present in the compositions herein an amount of from
1% to 30%, more preferably from 10-25% by weight.
lTrUrE SHEE~
WO92/13053 PCT/US92/00190
QOS~ ~
BUILDER
Builder materials will typically be present at from
10% to 60% of the detergent compositions herein. The
compositions herein are free or substantially free of
phosphate-containing builders (substantially free
being herein defined to constitute less than 1% of the
total detergent builder system), and the builder
system herein consists of water-soluble builders,
water-insoluble builders, or mixtures thereof.
Water insoluble builders can be an inorganic ion
exchange material,commonly an inorganic hydrated
aluminosilicate material, more particularly a hydrated
synthetic zeolite such as hydrated Zeolite A, X, B or
HS.
Preferred aluminosilicate ion-exchange materials
have the unit cell formula
Mz [(A102)z (sio2)y] xH20
wherein M is a calcium-exchange cation, z and y are at
least 6; the molar ratio of z to y is from 1.0 to 0.5
and x-is at least 5, preferably from 7.5 to 276, more
preferably from 10 to 264. The aluminosilicate
materials are in hydrated form and are preferably
crystalline containing from 10% to 28%, more
preferably from 18% to 22% water.
The above aluminosilicate ion exchange materials are
further charaterized by a particle size diameter of
from 0.1 to 10 micrometers, preferably from 0.2 to 4
micrometers. The term "particle size diameter" herein
represents the average particle size diameter of a
given ion exchange material as determined by
conventional analytical techniques such as, for
example, microscopic determination utilizing a
scanning electron microscope. The aluminosilicate ion
~IJBSllTUrE SHEF~
WO92/13053 21 0 U ~5~ PCT/US92/0019~
excAange materials are further characterized by their
calcium ion exchange capacity, which is at least 200
mg equivalent of CaCO3 water hardness/g of
aluminosilicate, calculated on an anhydrous basis, and
which generally is in the range of from 300 mg eq./g
to 352 mg eq./g. The aluminosilicate ion exchange
materials herein are still further characterized by
their calcium ion exchange rate which is described in
detail in GB-l,429,143.
Aluminosilicate ion exchange materials useful in the
practice of this invention are commercially available
and can be naturally occurring materials, but are
preferably synthetically derived. A method for
producing aluminosilicate ion exchange materials is
discussed in US Patent No. 3,985,669. Preferred
synthetic crystalline aluminosilicate ion exchange
materials useful herein are available under the
designation Zeolite A, Zeolite B, Zeolite X, Zeolite
HS and mixtures thereof. In an especially preferred
embodiment, the crystalline aluminosilicate ion
exchange material is Zeolite A and has the formula
Nal2[(Al~2)12 (sio2)l2~ xH2O
wherein x is from 20 to 30, especially 27. Zeolite X
of formula Na86 [(Alo2)86(sio2)lo6] - lO
.276H20 is also suitable, as well as Zeolite HS of
formula Na6 [(A102)6(SiO2)6] 7 5 H2O)-
Another suitable water-insoluble, inorganic builder
material is layered silicate, e.g. SKS-6 (Hoechst).
SKS-6 is a crystalline layered silicate consisting of
sodium silicate (Na2Si2O5). The high Ca++/Mg++
binding capacity is mainly a cation exchange
mechanism. In hot water, the material becomes more
soluble.
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WO92/13053 PCT/US92/0019n
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12
The water-soluble builder can be a monomeric or
oligomeric carboxylate chelating agent.
Suitable carboxylates containing one carboxy group
include lactic acid, glycollic acid and ether
derivatives thereof as disclosed in Belgian Patent
Nos. 831,368, 821,369 and 821,370. Polycarboxylates
containing two carboxy groups include the water-
soluble salts of succinic acid, malonic acid,
(ethylenedioxy) diacetic acid, maleic acid,
diglycollic acid, tartaric acid, tartronic acid and
fumaric acid, as well as the ether carboxylates
described in German Offenlegenschrift 2,446,686, and
2,446,687 and U.S. Patent No. 3,935,257 and the
sulfinyi carboxylates described in Belgian Patent No.
840,623. Polycarboxylates containing three carboxy
groups include, in particular, water-soluble citrates,
aconitrates and citraconates as well as succinate
derivatives such as the carboxymethyloxysuccinates
described in British Patent No. 1,379,241,
lactoxysuccinates described in Netherlands Application
7205873, and the oxypolycarboxylate materials such as
2-oxa-1,1,3-propane tricarboxylates described in
British Patent No. 1,387,447.
Polycarboxylates containing four carboxy groups
include oxydisuccinates disclosed in British Patent
No. 1,261,829, 1,1,2,2-ethane tetracarboxylates,
1,1,3,3-propane tetracarboxylates and 1,1,2,3-propane
tetracarboxylates. Polycarboxylates containing sulfo
substituents include the sulfosuccinate derivatives
disclosed in British Patent Nos. 1,398,421 and
1,398,422 and in U.S. Patent No. 3,936,448, and the
sulfonated pyrolysed citrates described in British
Patent No. 1,082,179, while polycarboxylates
containing phosphone substituents are disclosed in
British Patent No. 1,439,000.
~UBSnTUrE SHEE~
WO92/13053 2 1 0 0 5 5 g PCT/~IS92tOOlg~
Alicyclic and heterocyclic polycarboxylates include
cyclopentane-cis,cis,cis-tetracarboxylates,
cyclopentadienide pentacarboxylates, 2,3,4,5-
tetrahydrofuran - cis, cis, cis-tetracarboxylates,
2,5-tetrahydrofuran -cis - dicarboxylates, 2,2,5,5-
tetrahydrofuran - tetracarboxylates, 1,2,3,4,5,6-
hexane -hexacarboxylates and and carboxymethyl
derivatives of polyhydric alcohols such as sorbitol,
mannitol and xylitol. Aromatic polycarboxylates
include mellitic acid, pyromellitic acid and the
phtalic acid derivatives disclosed in British Patent
No. 1,425,343.
Of the above, the preferred polycarboxylates are
hydroxycarboxylates containing up to three carboxy
groups per molecule, more particularly citrates.
Preferred builder systems for use in the present
compositions include a mixture of a water-insoluble
aluminosilicate builder such as zeolite A, and a
water-soluble carboxylate chelating agent such as
citric acid. Additionally, builder systems further
comprizing polycarboxylate polymers have been found
beneficial for the builder system but also for aiding
in the softening performance of detergent compositions
according to the present invention. Polycarboxylate
polymers have been disclosed in detail in the prior
art for example in EP-A-137 669.
Other builder materials that can form part of the
builder system for the purposes of the invention
include inorganic materials such as alkali metal
carbonates, bicarbonates, silicates, and organic
materials such as the organic phosphonates, amino
polyalkylene phosphonates and amino polycarboxylates.
WBSllTUrE SHEE~
W092/13053 55 4 14 PCT/US92/0019
CELLULASE
The activity of enzymes and particularly the activity
of cellulase enzyme has been defined for various
applications by different analytical methods. These methods
all attempt to provide a realistic assessment of the
expected in use performance or at least a measurement
correlating with the in use performance. As has been
detailed in European Patent Application EP-A-350098, many
of the methods, particularly these frequently used by
cellulase manufacturers, are not sufficiently correlated
with the in use performance of cellulase in laundry
detergent compositions. This is due to the various other
usage conditions for which these activity measurement
~nethoàs have been developed.
The method described in EP-A-350098, has been
developed to be and to have a predictive correlation for
the ranking of cellulase activity in laundry detergent
compositions.
The present invention therefore uses the method
disclosed in EP-A-350098 to screen cellulases in order to
distinguish cellulases which are useful in the present
invention and those which would not provide the objectives
of the present invention. The screening method,
hereinafter referred to as C14CMC-Method, which has been
adopted from the method disclosed in EP-A-350098, can be
described as follows :
Principle :
The principle of the C14CMC-Method for screening is to
measure at a defined cellulase concentration in a wash
solution the removal of immobilized carboxy methyl
cellulose (CMC) from a cloth substrate. The removal of CMC
is measured by radio-active labelling of some of the CMC by
~JBSrlTUrE SHEE~
WO92/13053 2 1 0 0 ~ ~ ~ PCT/US92/0019~
using C14 radio-active carbon. Simple counting of the
amount of radio-active C14 on the cloth substrate before
and after the cellulase treatment allows the evaluation of
the cellulase activity.
Sample Dre~aration :
CMC prep~ration : The radio-active CMC stock solution is
prepared according to Table I. The radio-active CMC can be
obtained by methods referred to in EP-A-350098.
F~bric substrates : The fabric substrates are muslin
cotton swatches having a size of S cm x 5 cm. They are
inocculated with 0.35 ml of the radio-active labelled CMC
stock solution in their center. The muslin cotton swatches
are then airdried.
Immobilization of CMC : To immobilize the radio-active
labelled CMC on the muslin cotton swatches, laundero-meter
equipment " Linitest Original Haunau " made by Original
Haunau, Germany, is used. A metal jar of the laundero-meter
is filled with 400 ml of hard water (4 mmol/liter of Ca++
ions). A maximum number of 13 swatches can be used per
jar. The jar is then incubated in a heat-up cycle from
20 C to 60OC over 40 minutes in the laundero-meter
equipment. After incubation the swatches are rinsed under
running city water for 1 minute. They are squeezed and
allowed to airdry for at least 30 minutes.
According to EP-A-350098 samples of the swatches with
immobilized radio-active CMC can also be measured as "blank
samples" without washing.
Sample treatment :
Laundry test solution : The laundry test solution is
prepared according to the composition of Table II. It is
balanced to pH 7.5. The laundry test solution is the basis
to which a cellulase test sample is added. Care should be
8uBsTmrrE SHEE~
WO92/13053 PCT/~'S92/00190
~lQQ~4 16
taken to not dilute the laundry test solution by adding
water to a 100~ balance prior to having determined the
amount of cellulase to be added. The amount of cellulase
which is used in this screening test should be added to
provide 25 x 1o~6 weight percent of cellulase protein in
the laundry test solution (equivalent to 0.25
milligram/liter at 14.5 ~C).
Wash procedure : The swatches thus inocculated with
radio-active labelled CMC are then treated in a laundry
simulation process. The laundry process is simulated in
the laundero-meter type equipment," Linitest, Original
Haunau", by Original Haunau, Haunau Germany. An individual
swatch is put into a 20 cm3 glass vial. The vial is filled
with 10 ml of the laundry test solution and then sealed
liquid tight. Up to S vials are put into each laundero-
meter jar. The jar is filled with water as a heat tranfer
medium for the laundering simulation. The laundering
simulation is conducted as a heat-up cycle from 20 C to
60 C over 40 minutes.
After the processing of the samples the vials are
submerged in cold water and subsequently each swatch is
taken out of its vial, rinsed in a beaker under running
soft water, squeezed and allowed to airdry for at least 30
minutes.
Measurement :
In order to measure radio-active labelled CMC removal,
a scintillation counter, for example, a LKB 1210 Ultrabeta
Scintillation Counter, is used. In order to obtain most
accurate results, the instruction manual for optimum
operation of the particular scintillation counter should be
followed. For example, for the LKB 1210 Ultrabeta
Scintillation Counter, the following procedure should be
followed. The swatch to be measured is put into a plastic
vial filled with 12 ml of scintillator liquid (e.g.
~UBSrlTU~ SHEE~
W092/13053 PCT/US92/0019~
2100SS4
17
scintillator 299 from Packard). The swatch is then allowed
to stabilize for at least 30 minutes. The vial is then put
into the LKB 1210 Ultrabeta Scintillation Counter and the
respective radio-activity counts for the swatch is
obtained.
In order to measure the amount of CMC removal due only
to the cellulase, a measurement of a swatch which has been
inocculated at the same time but has been treated in the
laundry test solution without cellulase, is neceCc~ry. The
activity of the cellulase is then expressed as percent of
radio-active labelled CMC removal. This percentage is
calculated by the following formula :
~ of radio-active CMC removal = X0 - XC x 100
XO
Wherein X0 is the radioactivity scintillation count of a
swatch treated with the laundry test solution
without cellulase
XC is the radioactivity scintillation count of a
swatch treated with the laundry test solution
containing the cellulase to be evaluated
8tatistical considerations, procedure confir~ation :
In order to provide statistically sound results,
standard statistical analysis should be employed. For the
given example, using the LKB 1210 Ultrabeta Scintillation
Counter, it has been found that a sample size of 3 swatches
for each radioactivity scintillation count can be used.
In order to confirm the procedure by internal
crosschecking, measurement and calculation of the "blank
sample" according to EP-A-3S0098 are recommended. This
will allow to detect and eliminate errors.
~IJBS~lTU~ S~EE~
WO92/13053 PCT/US92/0019~
21 9qSS~
Interpretation of results :
The described screening test does provide a fast,
unl~ue and reliable method to identify cellulases which
satisfy the activity criteria of the present invention
versus cellulases which are not part of the present
invention.
It has been found that a removal of 10% or more of the
immobilized radioactive labelled CMC according to the above
C14CMC-method, indicates that the respective cellulase
satisfies the requirements of the invention.
I' will be obvious to those skilled in the art that
removal percentages above 10% indicate a higher activity
for the respective cellulase. It therefore is contemplated
that cellulase providing above 25% or preferably above 50%
removal of radioactive labelled CMC, at the protein
concentration in the laundry test solution according to the
Cl4CMC-method, would provide indication of an even better
performance of the cellulase for use in laundry detergents.
It also has been contemplated that usage of higher
concentrations of cellulase for C14CMC-method, would
provide higher removal percentages. However, there exists
no linear proven correlation between cellulase
concentration and removal percentage obtained by it.
It also has been contemplated that usage of higher
concentrations of cellulase for C14CMC-method, would
provide higher removal percentages.
~IJBSrlllrrE SHEE F
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19
TABLE I : Radioactive C14 labelled CMC stock solution
(all percentages by weight of total solution)
Total CMC* 99.2 x 10-3%
(CMC should be detergent
grade CMC with a degree of
substitution from about 0.47
to about 0.7)
Ethanol 14985.12 x 10-3%
Deionized Water 84915.68 x 10-3%
Total : 100%
* Total CMC contains non-radio-active and radio-active CMC
to provide a radio-activity which allows sufficiently
clear readings on the scintillation counter used. For
example, the radio-active CMC can have an activity of
0.7 millicurie/g and be mixed with non-radio-active CMC
at a ratio of 1:6.7.
~J =E SHEFF
WO92/13053 PCT/~'S92/00190
2~005~ ~ 20
TABLE II : Laundry test solution
(all percentages by weight of total solution)
Linear C12 alkyl ber~ene sulphonic acid 0.110%
Coconut alkyl sl~lphate (TEA salt) 0.040%
~12-15 alcohol ethoxylate (E07) 0.100%
Coconut fatty acid 0.100%
Oleic acid 0.050%
Citric acid 0.010%
Tri~l,anola,.~ine 0.040%
Ethanol 0.060%
Propanediol 0.015%
Sodium hydroxide 0.030%
Sodium formate 0.010%
P,otease 0.006%
Water (2.5 mmd/liter Ca + + ) pH adjustment balance to 100%
agent (HCL or NaOH sdutions) and c 'lu'--e
~IJBSrlTUrE SHEE~
WO92/13053 PCT/US92/0019n
2100554
21
According tO the present invention, preferred
cellulases are those as described in Danish Patent
Application 1159/90. For example, a cellulase
preparation useful in the compositions of the
invention can consist essentially of a homogeneous
endoglucanase component, which is immunoreactive with
an antibody raised against a highly purified 43kD
cellulase derived from Humicola insolens, DSM 1800, or
which is homologous to said 43kD endoglucanase.
It should be stressed that all cellulase enzymes
according to the present invention have to meet the
criteria of the above mentioned screening test.
However, in the Danish Patent Application 1159/90
additional criteria are established allowing to
identify preferred cellulase enzymes in combination
with the present screening test.
Cellulase preparations particularly useful in the
compositions of the invention are those in which in
addition to the screening test, the endoglucanase
component exhibits a CMC-endoase activity of at least
about 50, preferably at least about 60, in particular
at least about 90 CMC-endoase units per mg of total
protein. In particular, a preferred endoglucanase
component exhibits a CMC-endoase activity of at least
100 CMC-endoase units per mg of total protein.
In the present context, the term "CMC-endoase
activity" refers to the endoglucanase activity of the
endoglucanase component in terms of its ability to
degrade cellulose to glucose, cellobiose and triose,
as determined by a viscosity decrease of a solution of
carboxymethyl cellulose (CMC) after incubation with
the cellulase preparation of the invention, as
described in detail below.
~UBSTITI.rrE SHEE~
WO92/13053 PCT/US92/0019n
5 5 '~ 22
The CMC-endoase (endoglucanase) activity can be
determined from the viscosity decrease of CMC, as
follows : A substrate solution is prepared, containing
35 g/l CMC (Hercules 7 LFD) in 0.1 M tris buffer at pH
sØ The enzyme sample to be analyzed is dissolved in
the same buffer. 10 ml substrate solution and 0.5 ml
enzyme solution are mixed and transferred to a
viscosimeter (e.g. Haake VT 181, NV sensor, 181 rpm),
thermostated at 40-C. Viscosity readings are taken as
soon as possible after mixing and again 30 minutes
later. The amount of enzyme that reduces the
viscosity to one half under these conditions is
defined as 1 unit of CMC-endoase activity, or
CEW/liter.
SDS polyacrylamide gel electrophoresis (SDS-PAGE) and
isoelectric focusing with marker proteins in a manner
known to persons skilled in the art were used to
determine the molecular weight and isolelectric point
(pI), respectively, of the endoglucanase component in
the cellulase preparation useful in the present
context. In this way, the molecular weight of a
specific endoglucanase component was determined to be
43kD. The isoelectric point of this endoglur~n~sG was
determined to be about 5.1.
The cellobiohydrolase activity may be defined as the
activity towards cellobiose p-nitrophenyl. The
activity is determined as lo~6 mole nitrophenyl
released per minute at 37-C and pH 7Ø The present
endoglucanase component was found to have essentially
no cellobiohydrolase activity.
The endoglucanase component in the cellulase
preparation herein has initially been isolated by
extensive purification procedures, i.a. involving
reverse phase HPLC purification of a crude H. insolens
cellulase mixture according to U.S. 4,435,307. This
~IJBSrlTUrE SHEE~
WO92/13053 PCT/US92/0019~
2100S~4
23
procedure ~as surprisingly resulted in the isolation
of a 43kD endoglucanase as a single component with
unexpectedly favourable properties due to a
surprisingly high endoglucanase activity.
Also, in addition to the screening test, the
cellulase enzymes useful in the present compositions
can further be defined as enzymes exhibiting
endoglucanase activity (in the following referred to
as an "endoglucanase enzyme"), which enzymes have the
amino acid sequence shown in the appended Sequence
Listing ID#2, or a homologue thereof exhibiting
endoglucanase activity.
In the present context, the term "homologue" is
intended to indicate a polypeptide encoded by DNA
which hybridizes to the same probe as the DNA coding
for the endoglucanase enzyme with this amino acid
sequence under certain specified conditions (such as
presoaking in 5xSSC and prehybridizing for 1 h at
40 C in a solution of 20% formamide, 5xDenhardt's
solution, 50 mM sodium phosphate, pH 6.8, and 50 ug of
denatured sonicated calf thymus DNA, followed by
hybridization in the same solution supplemented with
lO0 uM ATP for 18 h at 40-C). The term is intended
to include derivatives of the aforementioned sequence
obtained by addition of one or more amino acid
residues to either or both the C- and N-terminal of
the native sequence, substitution of one or more amino
acid residues at one or more sites in the native
sequence, deletion of one or more amino acid residues
at either or both ends of the native amino acid
sequence or at one or more sites within the native
sequence, or insertion of one or more amino acid
residues at one or more sites in the native sequence.
The endoglucanase enzyme herein may be one
producible by species of Humicola such as Humicola
JrE SHEE~
W092/130~3 21~QSs ~ PCT/~IS92/~l9n
24
insolens e.g. strain DSM 1800, deposited on October 1,
1981 at the Deutsche Sammlung von Mikroorganismen,
Mascheroder Weg lB, D-3300 Braunschweig, FRG, in
accordance with the provisions of the Budapest Treaty
on the International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure
(the Budapest Treaty).
In still a further aspect, the cellulase enzymes
useful herein can be defined, in addition to the
screening test, as endoglucanase enzymes which have
the amino acid sequence shown in the appended Sequence
Listing ID#4, or a homologue thereof (as defined
above) exhibiting endoglucanase activity. Said
endoglucanase enzyme may be one producible by a
species of Fusarium, such as Fusarium oxys~orum, e.g.
strain DSM 2672, deposited on June 6, 1983 at the
Deutsche Sammlung von Mikroorganismen, Mascheroder Weg
lB, D-3300 Braunschweig, FRG, in accordance with the
provisions of the Budapest Treaty.
Furthermore, it is contemplated that homologous
endoglucanases may be derived from other
microorganisms producing cellulolytic enzymes, e.g.
species of Trichoderma, Mvceliophthora, Phanerochaete,
SchizophYllum, Penicillium, Asper~illus, and
Geotricum.
For industrial production of the cellulase
preparation herein, however, it is preferred to employ
recombinant DNA techniques or other tD~hniques
involving adjustements of fermentations or mutation of
the microorganisms involved to ensure overproduction
of the desired enzymatic activities. Such methods and
techniques are known in the art and may readily be
carried out by persons skilled in the art.
SUBSllTUrE SHEE~
WO92/13053 21 0 0 5 5 4 PCT/US92/0019n
The endoglucanase component may thus be one which is
producible by a method comprising cultivating a host
cell transformed with a recombinant DNA vector which
carries a DNA sequence encoding said endoglucanase
component or a precursor of said endoglucanase
component as well as DNA sequences encoding functions
permitting the expression of the DNA sequence encoding
the endoglucanase component or precursor thereof, in a
culture medium under conditions permitting the
expression of the endoglucanase component or precursor
thereof and recovering the endoglucanase component
from the culture.
DNA constructs comprising a DNA sequence encoding an
endoglucanase enzyme as described above, or a
precursor form of the enzyme, include the DNA
constructs having a DNA sequence as shown in the
appended Sequence Listings ID#1 or ID#3, or a
modification thereof. Examples of suitable
mofidications of the DNA sequence are nucleotide
substitutions which do not give rise to another amino
acid sequence of the endogluc~nA~e, but which
correspond to the codon usage of the host organism
into which the DNA construct is introduced or
nucleotide substitutions which do give rise to a
different amino acid sequence and therefore, possibly,
a different protein structure which might give rise to
an endoglucanase mutant with different properties than
the native enzyme. Other examples of possible
modifications are insertion of one or more nucleotides
at either end of the sequence, or deletion of one or
more nucleotides at either end or within the sequence.
DNA constructs encoding endoglucanase enzymes useful
herein may be prepared synthetically by established
standard methods, e.g. the phosphoamidite method
described by S.L. Beaucage and M.H. Caruthers,
Tetrahedron Letters 22, 1981, pp. 1859-1869, or the
~UBSIlTUrE SHEE~
WO92/130S3 pcT/us92/ool9n
2~ Q ~55 ~ 26
method described by Matthes et al., EMBO Journal 3,
1984, pp. 801-805. According to the phosphoamidite
method, oligonucleotides are synthesized, e.g. in an
automatic DNA synthesizer, purified, annealed, ligated
and cloned in suitable vectors.
A DNA construct encoding the endoglucanase enzyme or
a precursor thereof may, for instance, be isolated by
establishing a cDNA or genomic library of a cellulase-
producing microorganism, such as Humicola insolens,
DSM 1800, and screening for positive clones by
conventional procedures such as by hybridization using
oligonucleotide probes sythesized on the basis of the
full or partial amino acid sequence of the
endoglucanase in accordance with st~n~rd techniques
(cf. Sambrook et al, Molecular Clonina : A Laboratory
Manual 2nd Ed. Cold Spring Harbor, 1989), or by
selecting for clones expressing the appropriate enzyme
activity (i.e. CMC-endoase activity as defined above),
or by selecting for clones producing a protein which
is reactive with an anti-body against a native
cellulase (endoglucanase)
Finally, the DNA construct may be of mixed
synthetic and genomic, mixed synthetic and cDNA or
mixed genomic and cDNA origin prepared by ligating
fragments of synthetic, genomic or cDNA origin (as
appropriate), the fragments corresponding to various
parts of the entire DNA construct, in accordance with
standard techniques. The DNA construct may also be
prepared by polymerase chain reaction using specific
primers, for instance as described in US 4,683,202 or
R. R. Saiki et al, Science 239, 1988, pp. 487-491.
Recombinant expression vectors into which the above
DNA constructs are inserted include any vector which
may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend
~U~STITUrE SHEE~
WO92/13053 PCT/~'S92/0019~
21005S4
27
on the host cell into which it is to be introduced.
Thus, the vector may be an autonomously replicating
vector, i.e. a vector which exists as an
extrachromosomal entity, the replication of which is
independent of chromosomal replication, e.g. a
plasmid. Alternatively, the vector may be one which,
when introduced into a host cell, is integrated into
the host cell genome and replicated together with the
chromosome(s) into wich it has been integrated.
In the vector, the DNA sequence encoding the
endoglucanase should be operably connected to a
suitable promoter and terminator sequence. The
promoter may be any DNA sequence which shows
transcriptional activity in the host cell of choice
and may be derived from genes encoding proteins either
homologous or heterologous to the host cell. The
procedures used to ligate the DNA sequences coding for
the endoglucanase, the promoter and the terminator,
respectively, and to insert them into suitable vectors
are well known to persons skilled in the art (cf., for
instance, Sambrook et al., OD. cit.).
Host cells which are transformed with the above DNA
constructs or the above expression vectors may be for
instance belong to a species of AsDergillus, most
preferably AsPergillys orYZae or AsDerq~illus niqer.
Fungal cells may be transformed by a process involving
protoplast formation and transformation of the
protoplasts followed by regeneration of the cell wall
in a manner known per se. The use of Aspergillus as a
host microorganism is described in EP 238 023 (of Novo
Industri A/S), the contents of which are hereby
incorporated by reference. The host cell may also be
a yeast cell, e.g. a strain of SaccharomYces
cerevisiae.
8IJBSlTllrrE SHEFF
WO92/13053 PCT/~IS92/0019n
~ 28
Alternatively, the host organism may be a bacterium,
in particular strains of strePtomYces and Bacillus,
and E. coli. The transformation of bacterial cells
may be performed according to conventional methods,
e.s. as described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor,
1989.
The screening of appropriate DNA sequences and
construction of vectors may also be carried out by
standard procedures, cf. Sambrook et al., o~.cit.
The medium used to cultivate the transformed host
cells may be any conventional medium suitable for
growing the host cells in question. The expressed
endoglucanase may conveniently be secreted into the
culture medium and may be recovered therefrom by well-
known procedures including separating the cells from
the medium by centrifugation or filtration,
precipitating proteinaceous components of the medium
by means of a salt such as ammonium sulphate, followed
by chromatographic procedures such as ion exchange
chromatography, affinity chromatography, or the like.
By employing recombinant DNA techniques as indicated
above, techniques of protein purification, techniques
of fermentation and mutation or other terhniques which
are well known in the art, it is possible to provide
endoglucanases of a high purity.
The level in the present composition of cellulase
described above should be such that the amount of
enzyme protein to be delivered in the wash solution is
from 0.005 to 40 mg/liter of wash solution, preferably
0.01 to 10 mg/liter of wash solution.
~UBSllTUrE SHEE~
WO92/13053 2 1 0 0 S S ~ PCT/~IS92/0019n
- 29
The softening clay
One essential component of the present detergent
compositions is a softening clay.
Any clay used in the art or mixtures thereof can be used in
the present invention. Preferred examples have been
disclosed in GB 1.400.8g8 or US 5.019.292.
Included among such clays are various heat-treated
kaolins and various multi-layer smectites. As known from
the art, preferred smectite clays exhibit a cation-exchange
capacity of at least 50 meq per 100 grams of clay, which
corresponds to a layer charge of 0.2 to 0.6.
Further preferred are clays which have a particle size
in the 5-50 micrometer range.
Additionally preferred smectite clays are hectorite
clays of the general formula
[(Mg3_xLix) Si4_yMeIIIyolo(oH2-zFz)]-(x+y)(x+y)Mn+
wherein y=0; or, if y $ 0, MeIII is Al, Fe, or B; Mn+
is a monovalent (n=l) or divalent (n=2) metal ion, for
example selected from Na, K, Mg, Ca, Sr. The value of
(x+y) is the layer charge of the hectorite clay. The
hectorite clays suitable for the detergent compositions of
the present invention have a layer charge distribution such
that at least 50% is in the range of from 0.23 to 0.31.
Preferred are hectorite clays of natural origin having
a layer charge distribution such that at least 65% is in
the range of from 0.23 to 0.31.
Specific non-limiting examples of fabric softening
smectite clay minerals are :
WBSlTlU~ SHEE~
W092/130~3 PCT/US92/OOl9~
~ 1~ 0 ~ o
Sodium Montmorillonite
Borck (R)
Volclay BC (R)
Gelwhite GP (R)
Thixo-Jel (R)
Ben-A-Gel (R)
Sodium Hectorite
Veegum F (R)
Laponite SP (R)
Sodium SaPonite
Barasym NAS 100 (R)
Calcium Montmorillonite
Soft Clark (R)
Gelwhite L (R)
Imvite K (R)
CSM-Clay (R) from Kimoulos
Lithium Hectorite
Barasym LIH 200 R
The amount of softening clay useful in the present
invention depends upon the form of the detergent
composition. In general, it can range from lower limits of
0.5%, 1% or 8% to upper limits of 50%, 20% or 15%.
~U~SmUrE SIIEE~
WO92/13053 PCT/~S92/0019~
2100~59
31
PREFERRED OPTIONAL INGREDIENTS
Clay flocculating agents are not commonly used in
rabric treatment compositions. On the co~trary, one is
inclined to use clay dispersants, which aid in removing
clay stains from fabrics. Clay flocculating agents are,
however, very well known in other industries like oil well
drilling, and for ore flotation in metallurgy. Most of
these materials are fairly long chain polymers and
copolymers derived from such monomers as ethylene oxide,
acrylamide, acrylic acid, dimethylamino ethyl methacrylate,
vinyl alcohol, viny~ pyrrolidone, ethylene imine. Gums,
like guar gum, are suitable as well.
Preferred are polymers of ethylene oxide, acryl amide,
or acrylic acid. It has been found that these polymers
dramatically enhance the deposition of a clay if their
molecular weights (weight average) are in the range of from
100,000 to 10 million. Preferred are such polymers having
a (weight average) molecular weight of from 150.000 to 5
million, more preferably from 150,000 to 800,000.
The most preferred polymer is poly-(ethylene-oxide).
Molecular weight distributions can be readily determined
using gel permeation chromatography, against standards of
poly-(ethylene-oxide) of narrow molecular weight
distributions.
The amount of clay flocculating agent, expressed as
percent of the clay, ranges from 0% to 20%. For clay
flocculating agents having a (weight average) molecular
weight of less than 800,000, the preferred amount is from
2% to 20% of the clay. For (weight average) molecular
weight above 800,000 the preferred amount is from 0.005% to
2% of the clay.
Another preferred optional ingredient is substituted
polysiloxane the amount of siloxane ranges from 0% to 50%
81J~S~llUrE SHEE~
WO92/13053 PCT/US92/0019n
S~4
by weight of the clay, preferably from 0.1% to 20%, most
preferably from 1.0% to 10%.
The siloxanes useful in the present invention can be
aescribed as softening, straight or branched, organo-
functional polydi-Cl_4-alkyl siloxane having the general
formula :
r
R R R R
R'- Si 0 Si _ o ~ Si O Si R'
R R R"n R
~ q2
y
--m
wherein R is Cl_4-alkyl;
R' is R or a polyether of (C2-3-oxides)1-50, with
a capping group of H or R:
R" is branched or straight Cl_4-alkyl;
ql and q2 are integers;
m and (ql + q2) are integers from 4 to 1700;
n is an integer from 0 to 6;
Y is a polyether of (C2_3-oxides)k, where k has an
average value from 7 to 100, with a capping group of H
or Cl_4-alkyl;
WBSllTUrE SHEE~
WO92/13053 2 1 0 0 S ~ 4 PCT/US92/0019~
33
or Y is :
I
N whereby X and V are selected from
,i\ -H;
/ \ -cl_30-alkyl~ -C-aryl;
/ \ -C5_6-cycloalkyl; -Cl_6-NH2;
X V -COR; with the proviso that the
nitrogen can be quaternized such as
to represent :
N W
X~ \V
whereby w can be selected from X and
v.
or Y is
H C T whereby T and P are selected from
-H,
-COOH, -CO-O-C1_2-alkyl, or
H C H epoxy-C5_6-cycloalkyl
Preferred siloxanes of said general formula are
characterized by
(ql + q2) being an integer from 50 to 1500 and
m being an integer from 4 to 100.
The most preferred siloxanes of said general formula
are characterized by either of the following
- R, R' is methyl and R~ is propyl and
(ql + q2) is 329 and m is 21 and n is 1 and
y is a polyether consisting of 12 ethyl oxides and an
acetic acid capping group or
~UBSlTI'UTE SHEE~
WO92/13053 PCT/~IS92/0019~
2~$~ 34
- R, R' is methyl and R" is propyl and
(ql + q2) is 485 and m is 15 and n is 1 and
y is a polyether consisting of 12 ethyl oxides and
acetic acid capping group or
- R, R' is methyl and R" is methyl-2-propyl
(q1 + q2) is 1470 and m is 30 and n is 1 and
y is an -(amino ethyl)amine
The detergent compositions of the present invention can
be provided in liquid form as an aqueous dispersion. If in
liquid form the detergent composition preferably further
comprises an antisettling agent together with a softening
clay, siloxane and clay flocculating agent.
A suitable antisettling agent must provide a fully
activated support matrix to suspend particles within the
liquid detergent composition.
Particles in this sense are granules or droplets of
suspendable size for the desired properties of the liquid
detergent composition. Usually the particle size will be
less than 200 micrometers. The individual particles can
comprise one or more of the essential or optional compounds
of the detergent composition.
Finally, an acceptable antisettling agent must not
adversely effect the viscosity, elasticity or aesthetics of
the product.
These antisettling agents, or mixtures thereof, are
used in the compositions of the present invention at levels
of from 0.25% to 5%.
Organophillic quaternized ammonium-clay compounds for
example of the Bentone R family of clays and also fumed
silicas are examples of antisettling agents suitable for
use in the present invention. Bentone R rheological
~JBSnT~rrE SHEE~
W092/13053 ~ 1 ~ 0 5 5 4 PCT/~IS92/0019~
additives are described as the products of a clay which
contains a negative layer-lattice and an organic compound
which contains a cation and at least one alkyl group
containing at least 10 carbon atoms. Bentones R have the
property of swelling in certain organic liquids.
Organophillic quaternized ammonium-clay compounds are
preferred antisettling agents as described in U.S. patent
~,~87,086.
Fumed silicas also provide excellent antisettling
characteristics to the compositions of the present
invention. Fumed silicas are generally defined as a
colloidal form of silica made by combustion of silicon
tetrachloride in a hydrogen-oxygen furnace. Fumed silicas
are normally used as thickener, thixotropic and reinforcing
agents in inks, resins, rubber, paints and cosmetics. CAB-
O-SIL(R) fumed silicas are suitable antisettling agents for
use in this invention. Mixtures of Bentone(R) clays, fumed
silicas or cellulosic suspending agents are also suitable
antisettling agents.
The rheological characteristics of the resulting liquid
compositions are very important to a commercially
acceptable product. A liquid which can be described as
stringy (i.e., elastic), thick or lumpy is undesirable.
The antisettling agents described above avoid these
undesirable rheological properties while maintAin;ng a
pourable, homogeneous product with good consumer appeal. A
viscosity in the range of from about 100 to about 1000
kg/(ms) is desirable.
It is also desirable for the liquid composition to
exhibit plastic rheology. Materials that exhibit plastic
flow characteristics will flow only after an applied
shearing stress exceeds a critical minimum value.
~U~STrllrlE SHEE~
W092/130~32~00~S 4 36 PCT/US92/U0190
OPTIONAL INGREDIENTS
The present compositions will typically include
optional ingredients that normally form part of
detergent compositions. Antiredeposition and soil
suspension agents, optical brighteners, bleaches,
bleach activators, suds suppressors, anticacking
agents, dyes and pigments are examples of such
optional ingredients and can be added in varying
amounts as desired.
Antiredeposition and soil suspension agents suitable
herein include cellulose derivatives such as
methylcellulose, carboxymethylcellulose and
hydroxyethylcellulose. These materials are normally
used at levels of from O.S% to 10% by weight, more
preferably from 0.75% to 8%, most preferably from 1%
to 6% by weight of the composition. They can be used
in granular detergent compositions but also to
supplement the above-mentioned suspending agents for
liquid detergent compositions.
Preferred optical brighteners are anionic in
character, examples of which are disodium 4,41-bis-(2-
diethanolamino-4-anilino -s- triazin-6-
ylamino)stilbene-2:21 disulphonate, ~isoAium 4, _ 41_
bis-(2-morpholino-4-anilino-s-triazin-6-
ylaminostilbene-2:21 - disulphonate, disodium 4,41
- bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:21
- disulphonate, monosodium 41,411 -bis-(2,4-dianilino-
s-triazin-6 ylamino)stilbene-2-sulphonate, disodium
4,41 -bis-(2-anilino-4-(N-methyl-N-2-
hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,21 -
disulphonate, disodium 4,41 -bis- (4-phenyl-2,1,3-
triazol-2-yl)-stilbene-2,21 disulphonate, disodium
4,41bis(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-
triazin-6-ylamino)stilbene-2,21disulphonate and sodium
~UBST~TI~ Sl~EE~
W092/13053 ' PCT/~IS92tO019n
210055~ '
37
2(stilbyl-411_(naphtho-11,21:4,5)-1,2,3 - triazole-
211-sulphonate.
Any particulate inorganic perhydrate bleach can be
used, in an amount of from 3% to 40% by weight, more
preferably from 8% to 2S% by weight and most
preferably from 12% to 20% by weight of the
compositions. Preferred examples of such bleaches are
sodium perborate monohydrate and tetrahydrate,
percarbonate, and mixtures thereof.
Another preferred separately mixed ingredient is a
peroxy carboxylic acid bleach percursor, commonly
referred to as a bleach activator, which is preferably
added in a prilled or agglomerated form in granular
detergents. Examples of suitable compounds of this
type are disclosed in British Patent Nos. 1586769 and
2143231 and a method for their formation into a
prilled form is described in European Published Patent
Application No. 0 062 523. Preferred examples of such
compounds are tetracetyl ethylene diamine and sodium
3, 5, 5 trimethyl hexanoyloxybenzene sulphonate.
Bleach activators are normally employed at levels of
from 0.5% to 10% by weight, more frequently from 1% to
8% and preferably from 2% to 6% by weight of the
composition.
Another optional ingredient is a suds suppressor,
exemplified by silicones, and silica-silicone
mixtures. Silicones can be generally represented by
alkylated polysiloxane materials while silica is
normally used in finely divided forms exemplified by
silica aerogels and xerogels and hydrophobic silicas
of various types. These materials can be incorporated
as particulates in which the suds suppressor is
advantageously releasably incorporated in a water-
soluble or water-dispersible, substantially non-
~UBSrlTUrE SHEFF
W092/13053 PCT/~'S92/0019~
s5~
surface-active detergent impermeable carrier.
Alternatively the suds suppressor can be dissolved or
dispersed in a liquid carrier and applied by spraying
on to one or more of the other components.
As mentioned above, useful silicone suds controlling
agents can comprise a mixture of an alkylated
siloxane, of the type referred to hereinbefore, and
solid silica. Such mixtures are prepared by affixing
the silicone to the surface of the solid silica. A
preferred silicone suds controlling agent is
represented by a hydrophobic silanated (most
preferably trimethyl-silanated) silica having a
particle size in the range from 10 millimicrons to 20
millimicrons and a specific surface area above 50 m2/g
intimately admixed with dimethyl silicone fluid having
a molecular weight in the range from about 500 to
about 200,000 at a weight ratio of silicone to
silanated silica of from about 1:1 to about 1:2.
A preferred silicone suds controlling agent is
disclosed in Bartollota et al. U.S. Patent 3,933,672.
Other-particularly useful suds suppressors are the
self-emulsifying silicone suds suppressors, described
in German Patent Application DTOS 2,646,126 published
April 28, 1977. An example of such a compound is DC-
544, commercially availably from Dow Corning, which is
a siloxane/glycol copolymer.
The suds suppressors described above are normally
employed at levels of from 0.001% to 2% by weight of
the composition, preferably from 0.01% to 1% by
weight. The incorporation of the suds mofidiers is
preferably made as separate particulates, and this
permits the inclusion therein of other suds
controlling materials such as C20-C24 fatty acids,
microcrystalline waxes and high MW copolymers of
ethylene oxide and propylene oxide which would
~UBSlTrUrE S~EE~
W092/13053 PCT/~IS92/0019n
39 21005~4
otherwise adversely affect the dispersibility of the
matrix. Techniques for forming such suds modifying
particulates are disclosed in the previously mentioned
Bartolotta et al U.S. Patent No. 3,933,672.
Other useful polymeric materials are the
polyethylene glycols, particularly those of molecular
weight 1000-10000, more particularly 2000 to 8000 and
most preferably about 4000. These are used at levels
of from 0.20% to 5% more preferably from 0.25% to 2.5%
by weight. These polymers as well as the previously
mentioned homo- or co-polymeric polycar~oxylate
polymers are valuable for improving whiteness
maintenance, fabric ash deposition, and cleaning
performance on clay, proteinaceous and oxidizable
soils in the presence of transition metal impurities.
Soil release agents useful in compositions of the
present invention are conventionally copolymers or
terpolymers of terephthalic acid with ethylene glycol
and/or propylene glycol units in various arrangements.
Examples of such polymers are disclosed in the
commonly assigned US Patent Nos. 4116885 and 4711730
and European Published Patent Application No. 0 272
033. A particular preferred polymer in accordance
with EP-A-O 272 033 has the formula
(C83(P~G~43)o 75~P08~o 2s[1-P0~2 .1~T-P~G~0.4 ]T~Po-8~o.25~ ~PI~G~43C83 ~0. 75
vbere P~G 1~ -~OC284)0-,PO 1~ ~OC3860~ eDt T 1~ ~pcOC61l4CO~.
Certain polymeric materials such as polyvinyl
pyrrolidones typically of MW 5000-20000, preferably
10000-15000, also form useful agents in preventing the
transfer of labile dyestuffs between fabrics during
the washing process.
Other fabric softening agents can also be
incorporated into detergent compositions in accordance
~UBSrllUrE SHEE~
WO92/130~3 PCT/US92/0019~
2~Qo~jS4
with the present invention. These agents may be
inorganic or organic in type. Organic fabric
softening agents include the water-insoluble tertiary
amines as disclosed in GB-A-1514276 and EP-B-O 011 340
and their combination with mono C12-C14 quaternary
ammonium salts are disclosed in EP-B-O 026 527 and EP-
B-o 026 528 and di-long-chain amides as disclosed in
EP-B-O 242 919. Other useful organic ingredients of
fabric softening systems include high molecular weight
polyethylene oxide materials as disclosed in EP-A-O
299 575 and 0 313 146.
Organic fabric softening agents such as the water-
insoluble tertiary amines or di-long-chain amide
materials are incorporated at levels of from 0.5% to
5% by weight, normally from 1% to 3% by weight whilst
the high molecular weight polyethylene oxide materials
and the water-soluble cationic materials are added at
levels of from 0.1% to 2~, normally from 0.15% to 1.5
by weight. For granular detergents these materials
are normally added to the spray dried portion of the
composition, although in some instances it may be more
convenient to add them as a dry mixed particulate, or
spray them as a molten liquid on to other solid
components of the composition.
Enzymes other than the specific cellulase
preparation herein can be present in the composition
herein, such as proteases, lipases and amylases.
MAKING PROCESS
Compositions according to the present invention,
depending on whether they are liquid or granular, can
be made via a variety of methods including liquid
mixing according to a temperature and pH time profile,
melting, dissolving, dry mixing, spray drying,
~JBSTITUIE SHEE~
WO92/13053 PCT/US92/0019n
41 ~1 ~0~Sl
agglomeration and granulation and combinations of any
of these techniques.
A preferred method of making granular detergent
compositions, particularly those having a high density
(compact) herein involves a combination of spray
drying, agglomeration in a high speed mixer and dry
mixing.
A first granular component containing a relatively
insoluble anionic surfactant is spray dried and part
of the spray dried product is diverted and subjected
to a low level of nonionic surfactant spray-on before
being reblended with the remainder. A second granular
component is made by dry neutralisation of an anionic
surfactant acid using sodium carbonate as the
neutralising agent in a continuous high speed blender
such as a Lodige KM mixer. The first and second
components together with other dry mix ingredients
such as the carboxylate chelating agent, inorganic
peroxyqen bleach, bleach activator, soil suspension
agent, silicate and the polycarboxylate polymer and
enzyme are then fed to a conveyor belt from which they
are transferred to a horizontally rotating drum in
which perfume and silicone suds suppressor are
sprayed-on to the product. In highly preferred
compositions, a further drum mixing step is employed
in which a low (approx. 2%) level of finely divided
crystalline aluminosilicate is introduced to increase
density and improve granular flow, characteristics.
For a preferred method of making the liquid
detergent compositions according to the present
invention, it has been found that liquid detergent
compositions are advantageously prepared when pH and
temperature are always kept constant or are reduced
during production of the liquid detergent.
~UBSIlTU~ Sl~EE~
W092/13053 PCT/US92/0019~
21~05~4 42
EXAMPLES
The following examples illustrate the invention and
facilitate its understanding.
The abbreviations for the individual ingredients
have the following meaning :
LAS: sodium salt of linear dodecyl benzene sulfonate
TAS: sodium salt of tallow alcohol sulfate
AS: sodium salt of alkyl (C14-C15) sulfate
AO: C12-C14 alkyl dimethylamine oxide
FA25E7: fatty alcohol (C12-C15) ethoxylated with about
7 moles of ethylene oxide
CAT: C12-C14 trimethyl(or dimethyl (hydroxyethyl))
ammonium chloride
Clay: montmorillonite clay
Zeolite 4A: sodium salt of zeolite 4A with average
particle size between 1 - 10 micrometer
SKS-6: crystalline layered silicate (Hoechst)
AA/MA: copolymeric polycarboxylate polymer of acrylic
acid and maleic acid
PAP: polyacrylic polymer, MW 1000 -> 10000
CMC: carboxymethylcellulose
Phosphonate: sodium salt of ethylenediamine
tetramethylene phosphonic acid
AOS : A-Olefin (C12-C18) sulfonate, sodium salt
NMN : N-methyl N-1-deoxyglycithyl (C12-C18) alkyl
amide
EDTA: sodium salt of ethylenediamine tetra acetate
PB1: NaB02.H202
PB4: NaB02.H202.3H20
TAED: tetra acetyl ethylene diamine
NOBS: - nonanoyl oxybenzene sodium sulfonate
P.A.: sulphonated zinc phthalocyanine
Silicate ( R = n ): SiO2 / Na20 = n
Amylase: Termamyl 60T ( Novo-Nordisk )
Lipase: Lipolase lOOT ( Novo-Nordisk )
~JBS~TI~ SHEE~
WO92/13053 PCT/~S92/0019n
21005~
43
Protease: savinase 4T ( Novo-Nordisk )
SP300 : Celluzyme SP300 - (prior art cellulase by Novo
Nordisk)
43kD : about 43kD cellulase according to the cellulase
defined in the present invention
SSS : Suds Suppressing System (silica/silicone
mixture)
NTA : Sodium salt of nitrilotriacetate
TAE-ll : Tallow alcohol ethoxylated with about ll
moles of ethylene oxide
DrMA : Ditallow methyl amine
CFA : Coconut fatty acid
HFA : Hydrogenated Cl6-22 fatty acid
To facilitate a softness comparison, the following
test procedure was used :
3.S kg of clean fabric laundry loads are washed in an
automatic drum washing machine MieleR 423 at 60-C for
l.5 hours. The hardness of the water was 2.5 mmol of
Ca2+/Mg2+ per liter and the composition concentration
was 0.7% in the wash liquid. For softness evaluation
swatches of terry towel softness tracers were added.
The softness tracers were line dried prior to
assessment of softness.
Evaluation of test results was done with two methods:
Comparative softness assessment was done by expert
judges using a scale of 0 to 4 panel-score-units
(PSU). In this scale 0 is given for no difference and
4 is given for maximum difference. In this scale 0 is
given for no difference and 4 is given for maximum
difference. Softness was assessed after eight wash
cycles on aged terry swatches, as defined below.
Another softness assessment is done by using a
laboratory softness measurement device, the Kawabata
KES - FBl machine, Kato Tech Corporation Ltd. Japan.
WBSTlllrrE S~EE~
W092/13053 21~ US~ PCT/~'S92/0019
44
In this machine the softness tracers are placed
between two clamps which are movable relative to each
other. Comparative softness was measured after eight
wash cycles by shear hysteresis at 5 degree angle (2HG
5). A decrease in shear hysteresis reflects increased
softness performance. Measurements are means of 3 user
aged terry swatches. User aged is defined by a minimum
of lO normal washes.
EXAMPLES I TO IV : A basic detergent composition is
evaluated to indicate the unexpected effect of higher-
than-additive softening performance of the selected
compositions vs. closest prior art which is considered
to be EP-A-177 165.
Examples I and II supports the prior art theory of not
creating an adverse effect by combining softening clay
with cellulase. However, the result also indicates
that this does not provide any benefit. In fact, since
the combination of clay and cellulase of this prior
art disclosure provides no benefit it may be
speculated that the ecological and economical burden
it creates, is the reason for its lack of commercial
success.
Examples III and IV, IV being according to the
invention, clearly indicate that the performance of
softening clay and cellulase provides more-than-
additive benefits.
From Example II the benefit of clay, in a prior art
cellulase environment can be found to be 0.3 PSU.
Turning to Example IV, which is tested in a 43kD
cellulase environment, the result jumps to more than 6
times that number, reaching 2 PSU.
Full test results of Examples I to IV can be found in
Table III. The detergent composition used is based on
composition I of Table V. In the last two columns of
~J~IllrrE SI-IEFF
WO92/13053 2 1 0 0 5 .~ ~ PCT/US92/0019~
Table III the dependence of the overall superiority of
the present invention on the selected cellulase in
combination with softening clay can be seen. However,
this is even clearer exemplified in the following
examples.
Examples V to VIII : A basic detergent composition,
composition II of Table V, is evaluated in above
described test procedure and measured with the
Kawabata machine.
The reduction, expressing the softening, of the
measurement for clay alone is 5%, for high activity
cellulase alone is 15%. The expected value for the
combination should therefore be about 20% which
surprisingly is surpassed by this combination and
reaches 39%, i.e. about twice the expected value.
Compositions III through XII of Table V provide
examples which include softening clay as well as high
activity cellulase and have been found to perform in
accordance with the objectives of the present
invention, providing especially good fabric treatment
performance.
Clay in these compositions was montmorillonite or
hectorite clay at levels of 10% for compositions III
to VI and IX, 12% for compositions VII, VIII and X and
8.6% for compositions XI and XII. Composition IX also
contained an anti-settling agent of the Bentone(R)
family such that settling of the clay is prevented.
The cellulase is generally added from O.Ol to lO.0,
prefer~bly o.l to 0.5, mg/liter of wash solution.
~U~E SHEE~
WO 92/13053 PCI /US92/00190
5 4 46
o
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~JBSllTUrE SHEE~
WO 92/13053 PCl'/l,'S92/0019~
2I Oo~
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~JBSTITtrrE SHEE~
WO 92/130~3 PCr/US92/0019~
5 ~ 48
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SrlTUrE SHEE~
WO 92/13053 PCJ/l,lS92/OOl91\
21~.554
49
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~JBSmUrE SHEEF
WO 92/13053 PCr/l 'S92/0019n
2~ 00~)S~ 50
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WO 92/13053 PCr/US92/00191~
21:~D~
51
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WO 92/130~3 PCr/US92/0019
2~0SS 4 52
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x 8 _ ~ c . . . -- ~ ~ -- -- '~
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WBSTrllrrE S~EE~
WO 92/13053 PCl/l~S92/00190
21~5~4
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~UBSTmrrE SHEE~
P ~ /US92/0019
W 0 92/13053
~4
~ooss~
SEQULNCE DESCRIPTION: SEQ IO NO:1:
GGATCCAAG ATG CGT TCC TCC CCC CTC CTC CCG TCC GCC GTT GTG GCC 48
Met Arg Ser Ser Pro Leu Leu Pro Ser Ala Val Val Ala
-21 -20 -15 -10
GCC CTG CCG GTG TTG GCC CTT GCC GCT GAT GGC AGG TCC ACC CGC TAC 96
Ala Leu Pro Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr
-5 1 S
TGG GAC TGC TGC M G CCT TCG TGC GGC TGG GCC AAG M G GCT CCC GTG 144
Trp Asp Cys Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val
10 15 20
AAC CAG CCT GTC TTT TCC TGC AAC GCC AAC TTC CAG CGT ATC ACG GAC 192
Asn Gln Pro Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp
25 30 35 ~0
TTC GAC GCC AAG TCC GGC TGC GAG CCG GGC GGT GTC GCC TAC TCG TGC240
Phe Asp Ala Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys
45 50 55
GCC GAC CAG ACC CCA TGG GCT GT6 AAC GAC GAC TTC GCG CTC GGT TTT288
Ala Asp Gln Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe
60 65 70
GCT GCC ACC TCT ATT GCC GGC AGC AAT GAG GCG GGC TGG TGC TGC GCC336
Ala Ala Thr Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala
75 80 85
TGC TAC GAG CTC ACC TTC ACA TCC GGT CCT GTT GCT GGC AAG AAG ATG 384
Cys Tyr Glu Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met
90 95 100
GTC GTC CAG TCC ACC AGC ACT GGC GGT GAT CTT GGC AGC AAC CAC TTC 432
Val Val Gln Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe
105 110 115 120
GAT CTC AAC ATC CCC GGC GGC GGC GTC GGC ATC TTC GAC GGA TGC ACT 480
Asp Leu Asn Ile Pro Gly Gly Gly Val Gly lle Phe Asp Gly Cys Thr
125 130 135
~UBSmUrE SHEFF
wO 92/130;3 21 0 0 ~ 5 4 pcr/~lss2/oo1sn
~CC CAG TTC GGC GGT CTG CCC G&C CAG CGC TAC GGC G&C ATC TCG rcc 528
Pro Gln Phe Gly Gly Leu Pro Gl~ Gln Arg Tyr Gly Gly Ile Ser Ser
140 145 150
CGC AAC GAG TGC GAT CGG TTC CCC GAC GCC CTC AAG CCC GGC TGC TAC 576
,4rg Asn Glu Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr
155 160 165
TGG CGC TTC GAC TGG TTC AAG AAC GCC GAC MT CCG ~GC TTC AGC TTC624
rrp Arg Phe Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe
170 175 180
CGT CAG GTC CAG TGC CCA GCC GAG CTC GTC GCT CGC ACC GGA TGC CGC 672
Arg Gln Val Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg
185 190 195 200
CGC AAC GAC GAC GGC AAC TTC CCT GCC GTC CAG ATC CCC TCC AGC AGC 720
Arg Asn Asp Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser
205 210 215
ACC AGC TCT CCG GTC AAC CAG CCT ACC AGC ACC AGC ACC ACG TCC ACC 768
Thr Ser Ser Pro Val Asn Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr
220 225 230
TCC ACC ACC TCG AGC CCG CCA GTC CAG CCT ACG ACT CCC AGC GGC TGC 816
Ser Thr Thr Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys
235 240 245
ACT GCT GAG AGG TGG GCT CAG TGC GGC GGC AAT GGC TGG AGC GGC TGC 864
Thr Ala Glu Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys
250 255 260
ACC ACC TGC GTC GCT GGC AGC ACT TGC ACG AAG ATT AAT GAC TGG TAC 912
Thr Thr Cys Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr
265 270 275 280
CAT CAG TGC CTG TAGACGCAGG GCAGCTTGAG GGCCTTACTG GTGGCCGCAA 964
~is Gln Cys Leu
285
CGAAATGACA CTCCCAATCA CTGTATTAGT TCTTGTACAT AATTTCGTCA TCCCTCCAGG 1024
GATTGTCACA TMATGCAAT GAGGMCAAT GAGTAC 1060
~UBSlTrU~ S~EE~
PCI /l,'S92/00l90
wo 92/t3053
56
~ooS~ 4
S~QUENCE DESCRIPTION: SEQ ID NO:2:
Met Arg Ser Ser Pro Leu Leu Pro Ser Ala Val Val Ala Ala Leu Pro
-21 -20 -15 -10
Val Leu Ala Leu Al~ ~la Asp Gly Arg Ser rhr Arg ~yr Trp Asp Cys
-5 1 5 10
Cys Lys Pro Ser Cys Gl y Trp Al a Lys Lys Al a Pro Val Asn Gl n Pro
Val Phe Ser Cys Asn Ala Asn Phe G)n Arg Ile Thr Asp Phe Asp Ala
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala ryr Ser Cys Ala Asp Gln
SO 55
~hr Pro ~rp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr
Ser lle Ala Gly 5er Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
Leu ~hr Phe ~hr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
~5 100 105
Ser ~hr Ser ~hr Gly Gly Asp Leu Gly Ser Asn His Phe Asp ~eu Asn
110 115 120
Ile Pro Gly Gly Gly Val Gly 1le Phe Asp Gly Cys ~hr Pro Gln Phe
125 ~30 135
Gly Gly Leu Pro Gly Gln Arg ~yr Gly Gly lle Ser Ser Arg Asn Glu
140 145 150 155
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys ~yr ~rp Arg Phe
160 165 170
Asp Trp Phe Lys Asn Al a Asp Asn Pro Ser Phe Ser Phe Arg Gl n Val
1~5 180 185
Gl n Cys Pro Al a Gl u Leu Val Al a Arg ~hr Gl y Cys Arg Arg Asn Asp
190 195 200
~SD Gl y Asn Phe Pro Al a ~Jal Gl n l l e Pro Ser Ser Ser ~hr Ser Ser
205 210 215
~UBSIlTUrE SHEFF
W O 92/130~3 PCT/~IS92/OOl9O
21Q0~4
Pro Val Asn Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr
220 22S 230 235
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
240 245 250
~rg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
255 260 265
Val Ala Gly Ser Thr Cys Thr ~ys lle Asn Asp Trp Tyr His Gln Cys
270 2~5 280
~eu
W~TrlrrE SHEFF
Pcr/~lss2/oo1 9n
W 0 92/13053
2~o~S 4
SE~UEHCE DESCRIPTIOH: SEQ ID NO:3:
G M TTCGCGG CCGCTCATTC ACTTCATTCA TTCTTTAG M TTACATACAC TCTCTTTCAA 60
AACAGTCACT CTTTAAAC M AAC M CTTTT GCAACA ATG CGA TCT TAC ACT CTT 114
Met Arg Ser Tyr Thr Leu
S
CTC GCC CTG GCC GGC CCT CTC GCC GTG AGT GCT GCT TCT GGA AGC GGT162
Leu Ala Leu Ala Gly Pro Leu Ala Val Ser Ala Ala Ser Gly Ser Gly
10 15 20
CAC ~CT ACT CGA TAC TGG GAT TGC TGC M G CCT TCT TGC TCT TGG AGC210
~is Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys Ser Trp Ser
25 30 35
GGA AAG GCT GCT GTC AAC GCC CCT GCT TTA ACT TGT GAT AAG M C GAC258
Gly Lys Ala Ala Yal Asn Ala Pro Ala Leu Thr Cys Asp Lys Asn Asp
~0 45 SO
AAC CCC ATT TCC MC ACC AAT GCT GTC AAC GGT TGT GAG GGT GGT GGT306
Asn Pro Ile Ser Asn Thr Asn Ala Val Asn Gly Cys Glu Gly Gly Gly
55 60 65 70
TCT GCT TAT GCT TGC ACC MC TAC TCT CCC TGG GCT GTC AAC GAT GAG354
Ser Ala Tyr Ala Cys Thr Asn Tyr Ser Pro Trp Ala Val Asn Asp Glu
75 80 85
CTT GCC TAC GGT TTC GCT GCT ACC AAG ATC TCC GGT GGC TCC GAG GCC 402
Leu Ala Tyr Gly Phe Ala Ala Thr Lys ~le Ser Gly Gly Ser Glu Ala
90 95 100
~IWBSTmrrE Sl~EFF
wO 92/130~3 21 D O ~ ~ ~ PCI/l1S92/0019(~
59
AGC TGG TGC TGT GCT TGC TAT GCT ~TG ACC ~TC ACC ACT GGC CCC G~C ~50
Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr Phe Thr Thr Gly Pro Val
105 110 llS
AAG GGC AAG MG ATG ATC GTC CAG TCC ACC AAC ACT GGA GGT GAT CTC 498
Lys Gly Lys Lys Met Ile Val Gln Ser Thr Asn Thr Gly Gly Asp Leu
120 125 130
GGC GAC AAC CAC TTC GAT CTC ATG ATG CCC GGC GGT GGT GTC GGT ATC 546
Gly Asp Asn His Phe Asp Leu Met Met Pro Gly Gly Gly Val Gly Ile
135 140 145 150
TTC GAC GGC TGC ACC TCT GAG TTC GGC AAG GCT CTC GGC GGT GCC CAG 594
Phe Asp Gly Cys Thr Ser Glu Phe Gly Lys Ala ~eu Gly Gl~r Ala Gln
155 160 165
TAC GGC GGT ATC TCC TCC CGA AGC GAA TGT GAT AGC TAC CCC GAG CT~ 642
Tyr Gly Gly Ile Ser Ser Arg Ser Glu Cys Asp Ser Tyr Pro Glu Leu
170 1~5 180
CTC AAG GAC GGT TGC CAC TGG CGA TTC GAC TGG TTC GAG AAC GCC GAC 690
Leu Lys Asp Gly Cys His Trp Arg Phe Asp Trp Phe Glu Asn Ala Asp
185 190 lgS
AAC CCT GAC TTC ACC TTT GAG CAG GTT CAG TGC CCC AAG GCT CTC CTC 738
Asn Pro Asp Phe Thr Phe Glu Gln Val Gln Cys Pro Lys Ala Leu Leu
200 205 210
GAC ATC AGT GGA TGC AAG CGT GAT GAC GAC TCC AGC TTC CCT GCC TTC 786
Asp Ile Ser Gly Cys Lys Arg Asp Asp Asp Ser Ser Phe Pro Ala Phe
215 220 225 230
AAG GTT GAT ACC TCG GCC AGC AAG CCC CAG CCC TCC AGC TCC GCT AAG 834
Lys Val Asp Thr Ser Ala Ser Lys Pro Gln Pro Ser Ser Ser Ala Lys
235 240 245
AAG ACC ACC TCC GCT GCT GCT GCC GCT CAG CCC CAG AAG ACC AAG GAT 882
Lys Thr Thr Ser Ala Ala Ala Ala Ala Gln Pro Gln Lys Thr Lys Asp
250 255 260
TCC GCT CCT GTT GTC CAG AAG TCC TCC ACC MG CCT GCC GCT CAG CCC 930
Ser Al a Pro Yal Val Gl n Lys Ser Ser Thr Lys Pro Al a Al a Gl n Pro
265 270 275
GAG CCT ACT AAG CCC GCC GAC AAG CCC CAG ACC GAC AAG CCT GTC GC; 97B
Glu Pro Thr Lys Pro Ala Asp Lys Pro Gln Thr Asp Lys Pro ~al Ala
280 285 290
ACC AAG CCT GCT GCT ACC AAG CCC GTC CAA CCT GTC AAC AAG CCC AAG 1026
Thr Lys Pro Ala Ala Thr Lys Pro ~lal Gln Pro Val Asn Lrs Pro Lys
~9~ 300 305 310
ACA ACC CAG AAG G~C CGT GGA ACC AAA ACC CGA GGA AGC TGC CCG GCC 107
~hr Thr Gln Lys '~al Arg ~ly Thr Lys Thr Arg Gly Ser Cys Pro A~a
~li 32~ 325
~JBSTITl~E Sl-IEE~
w O 92/13053 PCT/~S92/0019n
~oo~5~
AAG ACT GAC GCT ACC GCC AAG GCC TCC GTT GTC CCT GCT TAT TAC CAG 1122
Lys Thr Asp Ala Thr Ala Lys Ala Ser Val Val Pro Ala Tyr Tyr Gln
330 3~5 340
TGT GGT GGT TCC AAG TCC GCT TAr CCC AAC GGC AAC CTC GCT TGC GCT 1170
Cys Gly Gly Ser Lys Ser Ala Trr Pro Asn Gly Asn Leu Ala Cys Ala
3~5 350 355
ACT GGA AGC AAG TGT GTC AAG CAG AAC GAG TAC TAC TCC CAG TGT GTC 1218
Thr Gly Ser Lys Cys Val Lys G)n Asn Glu Tyr Tyr Ser Gln Cys Val
360 365 370
CCC AAC TAAATGGTAG ATCCATCGGT TGTGGAAGAG ACTATGCGTC TCAGAAGGGA 1274
Pro Asn
375
TCCTCTCATG AGCAGGCTTG TCATTGTATA GCATGGCATC CTGGACCAAG TGTTCGACCC 1334
TTGTTGTACA TAGTATATCT TCATTGTATA TATTTAGACA CATAGATAGC CTCTTGTCAG 1394
CGACAACTGG CTACAAAAGA CTTGGCAGGC TTGTTCAATA TTGACACAGT TTCCTCCATA 1454
lq73
~UBSlTrUrE SHEFF
n ~ c ~ PCr/US92/0019
W092/130~ bll3~J5.34
SEQUENCE DESCRIPTION: S~ ID NO:4:
Met Arg Ser Tyr Thr Leu Leu Ala Leu Ala Gly Pro Leu Ala Val Ser
Ala Ala Ser Gly Ser Gly ~1is Ser Thr Arg Tyr Trp Asp Cys Cys Lys
Pro Ser Cys Ser Trp Ser Gly Lys Al a Al a Val Asn Al a Pro Al a Leu
Tnr Cys Asp Lys Asn Asp Asn Pro I l e Ser Asn Thr Asn Al a Val Asn
Gly Cys Glu Gly Gly Gly Ser Ala Tyr Ala Cys Thr Asn Tyr Ser Pro
~5 80
Trp Ala Val Asn Asp Glu Leu Ala Tyr Gl~ Phe Ala Ala Thr Lys Ile
Ser Gly Gly Ser Glu Ala Ser Trp Crs Cys Ala Cys Tyr Ala Leu Thr
100 105 110
Phe Thr Thr Gly Pro Val Lys Gly Lys Lys Met lle Val Gln Ser Thr
115 120 125
Asn Thr Gly Gly Asp Leu Gly Asp Asn His Phe Asp Leu Met Het Pro
130 135 1~0
Gly Gly Gly Val Gly lle Phe Asp Gly Cys Thr Ser Glu Phe Gly Lys
145 150 155 160
Ala Leu Gly Gly Ala Gln Tyr Gly Gly lle Ser Ser Arg Ser Glu Cys
165 170 175
Asp Ser Tyr Pro Glu ~eu Leu Lys Asp Gly Cys His Trp Arg Phe Asp
180 185 190
Trp Phe Gl u Asn Al a Asp Asn Pro Asp Phe Thr Phe Gl u Gl n Val Gl n
195 200 205
~ys Pro Lys Ala Leu Leu ASD ll.e Ser Gly Cys Lys Arg Asp Asp Asp
210 215 220
Ser Ser Phe Pro Ala Phe Lys Val Asp Thr Ser Ala Ser Lys Pro Gln
223 230 235 240
lTUrE SHEE~
W O 92t130~3 P ~ /~'S92/0019
Pro Ser Ser Ser Ala Lys lys Thr Thr Ser Ala Ala Ala Ala Ala Gln
245 250 255
~ro Gln Lys Thr Lys Asp Ser Ala Pro Val Val Gln Lys Ser Ser Thr
26Q 265 270
Lys Pro Ala Ala Gln Pro Glu Pro Thr Lys Pro Ala Asp Lys Pro Gln
27S 280 285
fhr Asp Lys Pro Val Ala Thr Lys Pro Ala Ala Thr Lys Pro Val Gln
290 295 300
Pro Val Asn Lys Pro Lys Thr Thr Gln Lys Val Arg Gly Thr Lys Thr
305 310 315 320
~rg Gly Ser Cys Pro Ala Lys Thr Asp Ala Thr Ala Lys Ala Ser Val
325 330 335
~al Pro Ala Tyr Tyr Gln Cys Gly Gly Ser Lys Ser Ala Tyr Pro Asn
340 345 350
Gly Asn Leu Ala Cys Ala Thr Gly Ser Lys Cys Val Lys Gln Asn Glu
355 360 365
~yr Tyr Ser Gln Cys Val Pro Asn
370 375
rrE SHEE~
