Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPACT DETERGENT COMPOSITIONS ~ITH
HIGH A~l~Vl'l~ CELLULASE
Technical Field
The present invention concerns cellulase-cone~inine granular
detergent compositions which are in a "compact" form, i.e. they are of
a relatively high density and contain a relatively low amount o-f
inorganic filler salt, compared to conventional detergent compositions.
In the detergent compositions herein -ehe cellulase comprises a
cellulase of high activity defined by the C14CMC method described
herein. Preferably the cellulase is a specific single-c~ ,_ncnt
endoglucanase.
Background of the Invention
The need for detergent compositions which exhibit not only good
cleaning properties, but also good fabric-softening performsnce, and
other fabric care benefits, is well-established in the art.
The efficiency of cellulolvtic 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-1,368,599 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
detergen. addi.ive.
8UBS 111 ~JTE SHEET
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EP-A-0 269 168 published June 1, 1988, 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 published October 5, 1989, have been disclosed
cellulase preparations useful for reducing the harshness of
cotton-cont~;n;ng 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 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 published January 10,
1990. A minimum of 10% removal of immobilized radioactive
labelled carboxymethylcellulose has 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 EP 0 531 372, published March 17, 1993.
There is disclosed a cellulase preparation consisting essentially
of a homogeneous endoglucanase component which is ;mml]noreactive
with a monoclonal antibody raised against a partially purified
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.
~B
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On the other hand, a new generation of detergent compositions is now
being marketed, which can be best pictured as "compact detergents"
although they have been given a variety of trade names such as "Ultra~',
"Supra", "Micro" ... The particularity of such detergent compositions
is their relatively high density compared to conventional detergent
compositions, and their ability to achieve the same efficiency than
conventional detergent compositions by using a considerably lesser
amount of "compact" detergent composition. This particularity is best
reflected, in terms of composition, by a relatively low amount of
inorganic filler salt. The efficiency of such "compact" detergent
compositions is best achieved by eliminating the pre-wash cycle and by
using dispersing and diffusing devices, which are put directly in the
drum of the washing ~chine at the start of the main wsching cycle.
It is an object of the present invention to provide detergent
compositions in a compact form, having a relatively high density and
containing a low amount of inorganic filler salt, which exhibit optimum
cellulase efficiency.
In EP-A-381 397 has been disclosed the effect of low ionic-strength
on enz,vme performance, in particular lipase.
It has been surprisingly found however, that the effect of the
compact matrix on the selected enzymes of the present invention is much
higher than what could be expected from state of the art cellulases
such as disclosed in EP-A-381 397.
It is another object of the present invention to provide a method for
treating fabrics in a washing machine, comprising the utilization of
the present detergent compositions at low levels, for the main wash
cycle.
Summary of the Invention
The present invention relates to granular detergent compositions
containing a surface-active agent, a builder, an enzyme, and if desired
conventional additives, characterized in that the enzyme comprises a
cellulase preparation providing at leas. 1~ removal of immobilized
I ~ , . . .
SUBS 111 IJTE SHEET
4 7, ~
,~,
: radioactive labelled carboxymethylcellulose accordtng to the C14CMC-method,
at 25x106% by weight of cellulase protein in the laundry test solution.
Preferably, the cellulase comp~und consists essentially of a
homogeneous endoglucanase component which is immunoreactive with a monoclonal
antibody raised against a partially purified about z 43kD cellulase derived
from Humicola insolens, DSM 1800, or which is homologous to said - 43 kD
endoglucanase.
In a preferred embodiment the invention provides a granular deter~ent
composition comprising surface-active agent, builder and cellulase wherein
said cellulase consists essentially of a homogeneous endoglucanase component
which is immunoreactive with a monoclonal antibody raised against a partially
purified about 43 kD cellulase derived from Humicola insolens, DSM 1~00; said
granular deter~ent composition comprising no more than about 15% by weight of
inorganic filler salt, and said granular detergent composition having a
density of about 550 to about 950 g/liter of composition.
Detailed Description of the Invention
The present detergent compositions are in granular form and are
characterized by their density, which is hi~her than the density of
conventional detergent compositions. The density of the compositions herein
ranges from 550 to 950 g/liter, preferably 650 to 850 g~liter of composition,
measured at 20~C.
The "compact" form of the compositions herein is best reflected, in
terms of composition, by the amount of inorganic filler salt; inorganic filler
salts are conventional ingredients of detergent compositions in powder form.
In conventional detergent compositions, the filler salts are present in
substantial amounts, typically 17-35% by weight of the total composition.
In the present compositions, the filler salt is present in amounts not
exceedin~ 15% of the t~tal composition, preferably not exceeding 1~%, most
preferably not exceeding 5% by weight of the composition.
Inorganic filler salts, such as meant in the present compositions are
selected from the alkali and alkaline-earth metal salts of sulphates and
chlorides.
A preferred filler salt is sodium sulphate.
. . _ .. .. _
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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 prefera~ly
from 3:1 to 1:1. Preferred sulphonates Include alkyl benzene
sulphonates hsving 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-Clg fatty source preferably
from a C16-Clg 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 1 to 6. Examples of preferred alkyl
sulphates herein are tallow alkyl sulphate, coconut alkyl sulphate, and
C14 15 alkyl sulphates. The cation in each instance is again an alkali
metal cation, preferably sodium.
One class of nonionic surfac~ants useful in the present invention are
condensates of ethylene oxide with a hydrophobic moiety to provide a
surfactant having an a~erage h~d-ophilic-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 particular hydrophobic group can be readily
adjusted to yield 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
aiconols containing 3-5 moles o, ethylene oxide per mole or alconol.
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Another class of nonionic surfactants comprises alkyl polyglucoside
compounds of general formula
R0 (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 t~eir 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,
O Rl
Cl 4 hydrocarbyl, 2-l,~d~oxy ethyl, 2-hydroxy propyl or a mixture
thereof, R2 is C5 31 hydrocarbyl, and Z is a polyhydroxyhydrocarbyl
having a linear hydrocarbyl chain with at least 3 h~dLoA~ls directly
connected to the chain, or an alkoxylated derivative thereof.
Preferably, Rl is methyl, R2 is a straight Cll 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 Cg-C20,
preferably Clo-C14 N-alkyl or alkenyl amine oxides and propylene-1,3-
diamine dioxides wherein the remaining N positions are substituted bymethyl, hydroxyethyl or hydroxypropyl groups.
Another class of surfactants are amphoteric surfactants, such as
polvamine-based species.
Cationic surfactants can also be used in the detergent compositions
herein and suitable quaternary ammonium surfactants are selected from
mono Cg-C16, preferably Clo-C14 N-alkyl or alkenyl ammonium surfactants
wherein remaining N positions are substituted bv methyl, hydroxvethvl
o. hvdroxvproc~1 Eroups.
SU~ JTE SHEET
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ixtures of surfactant types are preferred, more especiallv anionic-
nonionic and also anionic-nonionic-cationic mixtures. Particularly
preferred mixtures are described in British Paeent 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 l~ to 30%, more preferably from lO-25~ by weight.
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 l~ 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 [(AlO2)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 l.0 to 0.5 and x is at least 5,
preferably from 7.5 to 276, more preferably from lO to 264. The
aluminosilicate materials are in hvdrated 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 O.l to lO 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 bv conventional analv~ical
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techniques such as, for example, microscopic determination utilizing a
scanning electron microscope. The aluminosilicate ion exchange
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-1,429,143.
Aluminosilicate ion e~chAn~e 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[(A1~2)12 (sio2)12] xH20
wherein x is from 20 to 30, especially 27. Zeolite X of formula Na86
[(A102)g6(SiO2)1o6] - 10
.276H20 is also suitable, as well as Zeolite HS of formula Na6
[(A102)6(si02)6] 7.5 H20).
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 (Na2Si205). The high Ca++/Mg++
binding capacity is mainly a cation exchange mechanism. In hot water~
the material becomes more soluble.
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
~eiEian Patent .~os. &31.368. 821.36~ and &2'.3'C. ?olycarboxvlates
SUBS 111 ~JTE SHEET
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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 sulfinyl
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 conr~ini~p 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.
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
carbox~methyl derivatives of polyhydric alcohols such as sorbitol,
mannitol and xylitol. Aromatic polvcarboxylates include mellitic acid.
pyromellitic acid and the phtalic acid derivatives disclosed in British
Patent No. 1,425.343.
Of the above, the preferred polvcarboxylates are hydroxycarboxvlates
containing up to three carboxy groups per molecule, more particularlv
citrates.
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Preferred builder systems for use in the present compositions incluae
a mixture of a water-insoluble aluminosilicate builder such as zeolite
A, and a water-soluble carboxylate chelating agent such as citric acid.
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.
Other suitable water-soluble organic salts are 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.
Polymers of this type are disclosed in GB-A-1,596,756. Examples of
such salts are polyacrylates of MW 2000-5000 and their copolymers with
maleic anhydride, such copolymers having a molecular weight of from
20,000 to 70,000, especially about 40,000.
CELLULASE
The activity of enzymes and particularly the activitv 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 bv 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 methods 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 activitv in
laundrv detergent compositions.
SU~ ITE SHEET
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1~ 20g95~3~
. .,_,
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 :
Princi~le :
The principle of the C14CMC-Method for screening is to measure at a
defined cellulase concentration in a wash solution the removal of
i obilized carboxy methyl cellulose (CMC) from a cloeh substrate. The
removal of CMC is measured by radio-active labelling of some of the CMC
by using C14 radio-active carbon. Simple counting of the amount of
radio-active C14 on the cloth substrate before and after the ce}lulase
treatment allows the evaluation of the cellulase activity.
Sample PreParation :
CMC preparatlon : 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.
Fabric substrates : The fabric substrates are muslin cotton
swatches having a size of 5 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 bv Ori~inal Haunau, Germanv, 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 cvcle from 20~C to
60~C over 40 minutes in the laundero-meter equipment. After incubation
the swatches are rinsed under running citv water for 1 minute. The~
~re saueezed and a,iowed to airdrv for ~. leas~ ~5 ~.inutes.
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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 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 10-6 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 ~ n~u, HA--nA~ 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 5 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 airdrv 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 oDtimum o~eration of the particular
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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. 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 necessary. The activity of the cellulase is then
expressed às 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
Statist$cal considerations, procedure confirmat1on :
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 bv internal crosschecking,
measurement and calculation of the ~blank sample" according to EP-A-
350098 are recommended. This will allow to detect and eliminate
errors.
Interpretation of results :
The described screening test does provide a fast, unique and reliable
methoa ~o identify cellulases which sa~isfy the activity criteria o-
SUBS I I ~ ~TE SHEET
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the present invention versus cellulases which are not part of thepresent invention.
It has been found that a removal of 10% or more of the immobilized
radioactive labelled CMC according to the above Cl4CMC-method,
indicates that the respective cellulase satisfies the requiremen~s of
the invention.
It 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 Cl4CMC-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 Cl4CMC-method, would provide higher removal percentages.
SUts~ TE SHEET
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~s
... .
TABLE I : Radioacti~e 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
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.~
TABLE II :Laundry test solution
(all percentages by weight of total solution)
Linear C12 alkyl benzene 0.110%
sulphonic acid
Coconut alkyl sulphate (TEA 0.040%
salt)
C12 15 alcohol ethoxylate (E07) 0.100%
Coconut fatty acid 0.100
Oleic acid 0.050%
Citric acid 0.010%
Triethanolamine 0.040%
Ethanol 0.060%
Propanediol 0.015%
Sodium hydroxide 0.030%
Sodium formate 0.010%
Protease 0.006%
Water (2.5 mmol/liter Ca++). pH balance to 100%
adjustment agent (HCL or NaOH
solutions) and cellulase
SUBS 111 ~JTE SHEET
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According to the present invention, preferred cellulases are
those as described in EP 0 531 372, published March 17, 1993. For
example, a cellulase preparation useful in the compositions of the
invention can co~sist essentially of a homogeneous endoglucanase
component, which is immunoreactive with an antibody raised against
a highly purified 43kD cellulase derived from Humicola insolens,
DSM1800, 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 No. 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-~n~o~.~e 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.
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 9Ø 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 m;x'ng and again 30
minutes later. The amount of enzyme
., ~., ~ ~,
W O 92/13057 P ~ /US92/00203
209~5~08 18
that reduces the viscosity to one half under these conditions is
defined as l unit of CMC-endoase activity.
SDS polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric
focusing with ~arker 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 weinht of a specific endoglucanase component was determined
to be 43kD. The isoelectric point of this endoglucanase _
was determined to be about 5.l.
The cellobiohydrolase activity may be defined as the activity towards
cellobiose p-nitrophenyl. The activity is determined as umole
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 procedure has
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 polvpeptide encoded by DNA which hvbridizes 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
?reh~b~idi_ing .or l h a~ 40~C in a solution of 20~ formamide.
SUt~;3 111 ~ITE SHEET
W O 92/13057 P ~ /US92/00203
,9 20995J8
.. , ~ .
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 100 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 acrd 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 insolens e.g. strain DSM 1800, deposited on
October 1, 1981 at the Deutsche Sa lung 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
Mikroorg~nis ~n, 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, M~celiophthora, Phanerochaete, Schizophvllum,
Penicillium, AsPer~illus, and Geotricum.
For industrial production of the cellulase preparation herein,
however, it is preferred to emplov recombinant DNA techniques or other
~echniques in:o;.in~ adiustements of 'ermen.ations o muta.ion o~ the
SU~ JTE SHEET
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2 U 9~ 20
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.
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 enceding 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#l 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
endoglucanase, 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. ;he
phosphoamidite method described bv S.L. Beaucage and M.H. Caruthers,
Tetrahedron Letters 22, 1981, pp. 1859-1869, or the 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.
SUBS 111 ~JTE SHEET
W O 92/13057 ~ O ~ P ~ /US92/00203
21~ ~
.~ .
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 bv
conventional procedures such as by hybridization using oligonucleotide
probes synthesized on the basis of the full or partial amino acid
sequence of the endoglucanase in accortance with standard techniques
(cf. Sambrook et al., Molecular Clonin~: A Laboratorv 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 antibody 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.K.
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
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
mav 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 activitv
in the host cell of choice and mav be derived from genes encodin~
proteins either homologous or heterologous .o the host cell. The
SU~;3 111 ~TE SHEET
22
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., op. 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 Aspergillus, most preferably Asperqillys orYzae or
Aspergillus 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 host cell may
also be a yeast cell, e.g. a strain of Saccharomyces cerevisiae.
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.g. as described in Sambrook et al.,
Molecular Cloninq:~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., op. 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
B.
W 0 92/l30~7 23 2 0 9 9 5~ J ~ PC~r/US92/00203
mutation or other techniques which are well known in the art, i 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 lO mg/liter of wash solution.
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, and homo- or co-polymeric polycarboxylic
acids or their salts. Polymers of this type include the polyacrylates
and maleic anhydride-acrylic acid copolymers previously mentioned as
builders, as well as copolymers of maleic anhydride with ethylene,
methylvinyl ether or methacrylic acid, the maleic anhydride
constituting at least 20 mole percent of the copolymer. These
materials are normally used at levels of from 0.5% to 10% by weight,
more preferably from 0.75~ to 8%, most preferably from 1% to 6% by
weight of the composition.
Preferred optical brighteners are anionic in character, examples of
which are disodium 4,41-bis-(2-diethanolamino-4-anilino -s- t:iazin-6-
ylamino)stilbene-2:21 disulphonate, disodium 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-
SU~ ITE SHEET
W O 92/13057 - PCT/US92/00203
2099~)8 24
hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2l - disulphonate.
disodium 4,41 -bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2
disulphonate, disodium 4,4lbis(2-anilino-4-(1-methyl-2-
hydroxyethylaminol-s-triazin-6-ylamino)seilbene-2,2ldisulphonate and
sodium 2(stilbyl-411-(naphtho-11,21:4,5)-1,2,3 - triazole-2ll-
sulphonate.
Any particulate inorganic perhydrate bleach can be used, in an amount
of from 3% to 40% by weight, more preferably from 8% to 25% 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. 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 64
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-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.
SUBS 111 ~JTE SHEET
W O 92/13057 PC~r/US92/00203
~a~s~
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 ran8e 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. V.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 otherwise adversely affect the
dispersibility of the matrix. Techniques for forming such suds
modifying particulates are disclosed in the previously mentioned
Bartolotta et al V.S. Patent No. 3,933,672.
Other useful polymeric materials are the polyethylene glycols.
particularly those of molecular weight 1000-10000, more particularlv
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 and the previously mentioned homo- or co-polymeric
polycarboxylate salts are valuable for improving whiteness maintenance,
fabric ash deposition. and cleaning performance on clav, proteinaceous
ana o.~idi-abie soiis i.. ~he presence c .~ansi~;on ~eta_ impu ;.ie-.
~;UBS 111 ~JTE SHEET
W 0 92/13057 2 0 ~ 9 ~ ~ 8 26 PC~r/US92/00203
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
(CH3(PEG)43)0 75(PoH)o.25[T-po)2.8(T-pEG)o.4]T(po
H)0.2s((pEG)4~cH3)o.75
where PEG is -(OC2H4)0-,PO is (OC3H60) and T is (pcOC6H4CO).
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.
Fabric softening agents can also be incorporated into detergent
compositions in accordance with the present invention. These agents-
may be inorganic or organic in type. Inorganic softening agents are
exemplified by the smectite clays disclosed in GB-A-1,400,898. 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.
Levels of smectite clay are normally in the range from 5% to 20~,
more preferably from 8% to 15% by weight with the material being added
as a dry mixed component to the remainder of the form~lation. 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% bv weight. These materials are normallv added to
SUBS 111 ~JTE SHEET
W O 92/13057 27 2 Q 9 9 5 3 8 P~/~ls92/00203
the sprav dried portion of the composition~ although in some instances
it mav be more convenient to add them as a dry mix~c partlcus~.e,-ar
spray them as a molten liquid on to other solid components of the
composition.
Enzvmes 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 can be made via a
variety of methods including dry mixing, spray drying, agglomeration
and granulation and combinations of any of these techniques.
PREFERRED MAKING PROCESS
A preferred method of making the compositions herein involves a
combination of spray drying, agglomeraeion 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 peroxygen bleach, bleach activator, soil
suspension agent, silicate and enzyme are then fed to a convevor bel.
from which they are transferred to a horizontally rotating drum in
which perfume and silicone suds suppressor are spraved 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
crvstalline aluminosilicate is introduced to increase densitv and
improve granular flow characteristics.
SU~ TE SHE~T
W 0 92/13057 X u ~ 28 P ~ /US92/00203
PROCESS OF WASHING
The compact detergent compositions herein have the ability to achieve
the same efficiency than conventional detergent compositions, when a
considerably lesser amount of composition herein, is used in the main
wash cycle of a washing machine.
Accordingly, in an other embodiment of the invention, it is herewith
provided for a process for washing fabrics in a washing machine wherein
an amount of from 15 to 170 g of a detergent composition ~ccording to
the present invention is used for the main wash cycle.
Typically, under European conditions, the recommended usage is from
80 to 140 g of detergent composition for the main wash cycle, without
the need of a pre-wash.
The detergent compositions herein are preferably delivered directly
to the drum and not indirectly via the outer casing of the machine.
This can most easily be achieved by incorporation of the composition in
a bag or container from which it can be released at the start of the
wash cycle in response to agitation, a rise in temperature or immersion
in the wash water in the drum. Such a container will be placed in the
drum, together with the fabrics to be washed. Alternatively the
washing machine itself may be adapted to permit direct addition of the
composition to the drum e.g. by a dispensing arrangement in the.access
door.
Products comprising a detergent composition enclosed in a bag or
container are usually designed in such a way that container integrity
is maintained in .he dry state ~o prevent egress of the contents when
dry, but are adapted for release of the container contents on exposure
to a washing environment, normally on immersion in an aqueous solution.
Usuallv the container will be flexible, such as a bag or pouch. The
bag may be of fibrous construction coated with a water impermeable
protective material so as to retain the contents, such as is disclosed
in European published Patent Application No. 0 018 678. Alternatively
:. mav be formed of a wate insoiuble svnthetic polvmeric material
SUts~ JTE SHEET
W O 92/13057 ~9~ P ~ ~US92/00203
provided with an edge seal or ciosure designed to rupture in aqueous
media as disclosed in European published Patent Application Nos. 0 Oll
500, 0 Oll 501, 0 Oll 502, and 0 Oll 968. A convenient form of water
frangible closure comprises a water soluble adhesive disposed along and
sealing one edge of a pouch formed of a water impermeable polymeric
film such as polyethylene or polypropylene.
In a variant of the bag or container product form, laminated sheet
products can be employed in which a central flexible layer is
impregnated and/or coated with a composition and then one or more outer
layers are applied to produce a fabric-like aesthetic effect. The.
layers may be sealed together so as to remain attached during use or
may separate on contact with water to facilitate the release of the
coated or impregnated material.
An alternative laminate form comprises one layer embossed or deformed
to provide a series of pouch-like containers into each of which the
detergent components are deposited in measured amounts, with a second
layer overlying the first layer and sealted thereto in those areas
between the pouch-like containers where the two layers are in contact.
The components may be deposited in particulate, paste or molten form
and the laminate layers should prevent egress of the contents of the
pouch-like containers prior to their addition to water. The layers may
separate or may remain attached together on contact with water, the
only requirement being that the structure should permit rapid release
of the contents of the pouch-like containers into solution. The number
of pouch-like containers per unit area of substrate is a matter of
choice but will normally vary between 500 and 25,000 per square metre.
Suitable materials which can be used for the flexible laminate layers
in this aspect of the invention include, among others, sponges, paper
and woven and non-woven fabrics.
However the preferred means of carrying out the washing process
according to the present invention includes the use of a reusable
dispensing device having walls that are permeable to liquid but
impermeable to the solid composition.
SU~;3~ JTE SHEET
W O 92/13057 . 2 0 9-9'~ ~ 8 30 P ~ /US92/00203
Devices of this kind are disclosed in European Patent Application
Publication Nos. 0 343 069 and 0 344 070. The latter Application
discloses a device comprising a flexible sheet in the form of a bag
extending from a support ring defining an orifice, the orifice being
adapted to admit to the bag sufficient product for one washing cycle in
a washing cycle. A portion of the washing medium flows through the
orifice into the bag, dissolves the product, and the solution then
passes outwardly through the orifice into the washing medium. The
support ring is provided with a masking arrangement to prevent egress
of wetted, undissolved, product, this arrangement typically comprising
radially extending walls extending from a central boss in a spoked-
wheel configuration, or a similar structure in which the walls have a
helical form.
EXAMPLES
The following examples illustrate the invention and facilitate its
underst~n~
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
A0: C12 - C14 alkyl dimethylamine oxide
FA45E7: fatty alcohol ( C14 - C15 ) ethoxylated with about 7 moles of
ethylene oxide
CAT: C12 alkyl trimethvl ammonium chloride
Clay: smectite clay
Zeolite 4A: sodium salt of zeolite 4A with average particle size
between 1 - 10 micrometer
SKS-6: crystalline layered silicate (Hoechst)
Copolymer AA~MA: copolymer of acrylic acid and maleic acid
PAA: polyacrylic acid MW 1000 -> 10000
CMC: carboxvmethylcellulose
SU~;3 111 ~JTE SHEET
W 0 92/130S7 P ~ /US92/00203
31 ; 20S~8
.,.,~. .
Phosphonate: sodium salt of ethylenediamine tetramethylene phosphonic
acid
EDTA: sodium salt of ethylenediamine tetra acetate
PBl: 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 )
Protease: Savinase 4T ( Novo-Nordisk )
SSS : Suds Suppressing System (silica/silicone mixture)
EXAMPLE I
Criticalit~ of the cellulase ~erformance Darameter of
claim 1
The following test was conducted :
Test conditions :
Washing temperature : 60~C (heat up cycle)
Washing time : 40 min.
pH - 7.5
Water hardness : 4 mmol/L
Detergent concentration : 1%
Detergent composition : cfr. EPA 350 098 ex. 1
Cellulases :
1) CelluzvmeR supplied by Novo Nordisk
- reference
2) 43kD endoglucanase
- cellulase according to the invention
Test Results :
~ C14-CMC Removal bv Cellulase
Detergent without cellulase (-reference) O
Detergent + Celluz~me~
0.25 mg protein/L below 3
0.9 mg pro~einiL lQ
SUBS 111 ~TE SHE~T
W O 92/13057 P ~ /US92/0020
' ''2 0 9 g 5 ~ 8 32
l.S mg protein/L 12.7
3.0 mg protein/L 17. 7
4.5 m~ protein/L 21.5
Detergent + 43kD endoglucanase
0.3 mg protein/L 20.3
0.25 mg protein/L 18.5
Discussion of the results :
The above data clearly demorstrate the criticality of the claimed
paraneter for the cellulases of the invention over the commercially
available Celluzyme.
EXAMPLE II.
The following base compositions were prepared :
COMPOSITIONS:
( all levels in ~ by weight )
Comp-ct Non-compact
Detergent Detergent
LAS 9.40 6.27
TAS 3.00 2.00
FA45E7 2.65 1.77
Na citrate/citric acid18.50 12.33
Zeolite 4A 32.65 21.77
Copolymer AA/MA 4.90 3.27
Phosphonate 0.19 0.13
Na carbonate 3.00 2.00
COMPOSITIONS:
( all levels in ~ by wei~ht ~
Compact Non-compact
Detergent Detergent
Silicate ( R 2 ) 2.90 1.93
Protease 1.62 1.08
Sulfate 4.50 30.00
SSS 0.40 0.27
~inors + water balance to 100
~er.si : (g, a 20~r 680 'll~
SU~ JTE SHEET
WO 92tl30~i7 PCl /US92/00203
33- '209~5~8
Recommended product usage
( g~wash ) 120 180
Color Reiuvenation Testin~
Test cond~tions :
Launderometer equipment
Washing temperature : 40CC
Washing time : 3h
Number of wash cycles : 2
pH - 8.2 non-compact detergent
8.5 compact detergent
Water hardness : 15gr./US gal.
Detergent concentration
0.75~ for non-compact detergent
0.66% for compact detergent
Test fabric : worn blue pyjama cotton
(90/lO cotton/Polyester)
Cellulases : 1) CelluzymeR supplied by Novo Nordisk
(- reference)
2)43kD endoglucanase - cellulase
according to the present invention
Wash test : Swatches of 8g of worn blue pyjama fabric were treated with
the different wash solutions. After tumble drying, the fabrics were
graded for colour clarification effects by direct comparison of the two
different detergent matrices at equal cellulase level. ~isual grading
bv expert judges using a 0 to 4 scale was preferred. (0 stands for no
difference and 4 stands for verv big difference.)
Test Results :
Ij Non-Compact Deter~ent
PSU m~ protein/PSU
N0 cellulase 0
Celluzvme
138 mg pro~ein/L + 2.3 60
SUBS 111 ~JTE SHEET
W O 92/13057 P ~ /US92/00203
2 0 g g S 0 ~ 34
43kD endoglucanase
18.6 mg protein/L + 2.2 8.5
II) Compact Deter~ent
PSU mg Drotein/PSU
N0 cellulase 0
Celluzyme
165 mg protein/L + 3.8 43
43kD endoglucanase
3.4 mg protein/ + 3.4 1.0
LSD (Least Significant Difference) - 0.5 PSU
From the m~ protein/PSU result, the following efficiency factors were
calculated :
Efficiency factor of 43kD endo~lucanase versus CelluzYme :
in Non ComPact Detergent in Compact Detergent
60/8.5 - 7 43/l.0 - 43
EfficiencY factor in Compact Deter~ent versus in Non ComPact DeterQent
of CelluzYme of 43kD endoglucanase
60/43 - 1.4 8.5/l - 8.5
Conclusions :
The above results show a cellulase selected accordin~ to the present
invention is 43 times more effective than a state-of-the-art cellulase
in the claimed compact matrix. Furthermore, the above results show
tha. the performance enhancement due to the claimed compac; matrix seen
with the selected cellulases is surprisingly much higher than what can
be obtained with a state-of-the-art cellulase.
SUt~;3 111 ~JTE SHEET
W O 92/13057 P ~ /~1S92/00203
20~9~
... .
EXAMPLE III.
CLAY SOIL REMOVAL TESTING
Cellulase enzymes also are very efficient in removing clav stains
from fabrics. This particular performance characteristic has been
checked for a 43kD endoglucanase in the two detergent compositions
given in example II.
Conditions:
Linitest equipment
60C wash ( heat up cycle )
Wash time: 40 min.
Water hardness: Brussels city water
Detergent concentrations:
0.66~ for the Compact detergent
l.O~ for the non compact detergent
Cellulase concentrations: l.55, 3.l0, 4,65 and 6.2mg enzyme protein / L
wash liquor.
Wash test:
Muslin cotton fabric was soiled with naturally-derived clays of two
different locations (US, UK). Cellulase performance was evaluated by
comparing the clay stains washed at equal cellulase level in the two
different detergent compositions. The visual grading scale used in
example II was again preferred.
Results:
Cellulase level: l.55 3 1 4 7 6 2
( mg enz. prot. / L wash liquor )
Compac. detergent
US clav + l.50 + 2.50 + 2.00 + l.50
UK clay + 0.50 + l.00 + l.50 + 2.50
SU~;3 ~ TE SHEET
W O 92/13057 2 U 9~ 9 ~ ~ 8 36 P ~ /VS92/00203
~on compact deter~ent
(-reference ) O O O O
LSD ( least significant difference ) - 0.42 at 95% confidence.
The clav stain removal performance of the cellulase selected accordin~
to the present invention, in the compact detergent composition of the
invention is significantly superior to the performance of the same
cellulase in the conventional, non compact detergent composition.
EXAMPLES IV-XI
The following compact detergent compositions are also prepared :
COMPACT ut I tl1GENT COMPOS~ IJS:
( all levels in 9~ bV w~iaht ~
EXAMPLE IV V Vl Vll Vlll IX X Xl Xll
LAS 9.40 t2.50 11.00 -- 7.58 7.58 8.20 6.50
TAS 3.00 --- -- -- 2.43 2.43 2.65 3.25 3.90
AS -- -- 4~80 12.00
FA45E7 2.65 2.00 4.00 1.00 5.11 5.11 3.15 2.20 6.00
CAT -- ~ -- -- 2.45
Coconut glwose amide --- 11.00
Tallow glucose amide -- -- -- 10.00
Na citrate/citric acld20.5029.50 18.00 18.00 -- 5.00 23.50 12.00 15.00
Zedite 4A 33.65 -- 32.00 32.5023.80 15.0 -- 16.00 20.00
SKS~ --- --- -- -- -- 12.50
Copdymer AA/MA 4 go 4.10 5.00 5.60 2.90 3.50 3.45 3.45
PAA
P~,osphonate 0.19 0.23 0.19 1.00 0.57 0.43 0.30
EDTA -- -- -- -- 0.25 -- -- 0.32 0.32
Na carbonate / bicarbonate 2.00 12.00 3.28 2.50 17.30 8.00 2.50 9.90 9.90Silicate ( R = 2 ) 3.00 4.20 3.00 2.00 2.00 2.50 2.30 2.50 2.50
CMC --- 0.15 ~ -- 0.48 0.34 0.25
Clay -- -- --- --- -- -- 12.00 8.60 8.60
PB1 --- -- -- -- 13.12 13.12 11.47 11.50
PB4 -- -- -- -- -- -- 3.55
Percarbona~e -- -- -- -- -- -- -- -- 12.00
TAED --- ~- -- -- 5.70 5.70 2.47 3.20
NOBs --- ---- -- -- -- -- 2.00
P A --- --- -- --- 0.002 0.002 -- 0.003 0.003Protease 1 62 1.30 1 20 1 60 1.35 1.35 1.05 1.40 1.40
Lipdase --- --- 0.40 0.30 -- 0.20 -- 0.30 0.30
Amylase 0.15 -- 0.20 0.30 -- 0.10
Sultate 2.54 3.79 2.38 2.45 1.50 1.50 2.23 3.~.5 3.45
8rightener _ 0.27 0.27 0.27 0.24 0.24 0.24 0.24 0.24
SSS 0.40 0.40 0.40 0.40 0.65 0.65 0.50 0.50 0.50
Minors + water balance to 1009~
~,~llulace at levels so as to deiiver 0.01 < X < 10 ma ertzVrne protein ~ wash liquor
SUB~ 111 ~JTE SHEET
P ~ /US92/00203
w 0 92/13057
'2'0'9g~
.~..~
SEQUENCE DESCRIPTION: 5EQ ID 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 TAC96
Ala Leu Pro Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr
5 - 1 5
TGG GAC TGC TGC AAG CCT TCG TGC GGC TGG GCC AAG AAG GCT CCC GTG144
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 GAC192
Asn Gln Pro Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp
25 30 35 40
TTC GAC GCC AAG TCC GGC TGC GAG CCG GGC GGT GTC GCC TAC rCG 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 GTG 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 GCC 336
Ala Ala Thr Ser lle 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
tys 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 ~is Phe
105 110 llS 120
GAT CTC AAC ATC CCC GGC GGC GGC GTC GGC ATC TTC GAC GGA TGC ACT 480
Asp Leu Asn lle Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys ~hr
12~ 130 135
SUts:~ ~11 UTE SHEET
W O 92/13057 P ~ /US92/00203
~ 209~0Y 38
ecc CAG TTC GGC GGT CTG CCC GGC CAG CGC TAC GGC GGC ATC TCG TCC 528
Pro Gln Phe Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser
140 145 150
CGC AAC GAG TGC GAT CGG TTC CCC GAC GCr CTC AAG CCC GGC TGC TAC 576
Arg Asn Glu Cys Asp Arg Pne PrG As~ Leu Lys Pro Gly ',s Tyr~
155 160 165
TGG CGC TTC GAC TGG TTC AAG AAC GCC GAC AAT CCG AGC TTC AGC TTC 624
Irp Arg Phe Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe
170 175 ]80
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 lle 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 TAr~CGC~GG GCAGCTTGAG GGCCTTACTG GTGGCCGCAA 964
~is Gln Cys Leu
285
CGAAATGACA CTCCC M TCA CTGTATTAGT TCTTGTACAT AATTTCGTCA TCCCTCCAGG 102q
GATTGTCACA TAAATGCAAT GAGGAACAAT GAGTAC 1060
SU~:3 111 ~JTE SHEET
WO 92/13057 PCI/US92/00203
39 20~9~8
SEQUENCE DESCRIPTION: SEQ I~ HO:2:
Met Arg Ser Ser Pro Leu Leu Pro Ser Al a Val Val Al a Al a Leu Pro
-21 -20 - 15 - 10
Val Leu Ala Leu Ala, ~la Asp Gly Arg Ser Thr Arg Tyr ~rp Asp Cys
-5 1 5 10
Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro
lS 20 ~5
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg lle ~hr Asp Phe Asp Ala
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln
S0 55
Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr
Ser Ile Ata Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln
100 105
Ser Thr Ser ~hr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn
110 115 120
Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe
125 130 135
Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly lle Ser Ser Arg Asn Glu
140 145 150 155
Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
160 165 170
Asp Trp Phe Lys Asn Al a Asp Asn Pro Ser Phe Ser Phe Arg Gl n Val
175 180 185
Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp
190 l9S 200
Asp Gly Asn Phe Pro Ala ~al Gln lle Pro Ser Ser Ser ~hr Ser Ser
20~ 21~ 215
SUB~ 111 ~JTE SHEET
PCT/US92/00203
W O 92/13057 ~ O~g ~ ~ 0 ~
w
Pro Va) Asn Gln Pro Thr Ser ~hr Ser Thr Thr Ser Thr Ser Thr Thr
220 225 230 235
Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu
240 24S 250
Arg Trp Al a Gl n Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys
255 260 265
Val Ala Gly Ser Thr Cys Thr Lys lle Asn Asp Trp Tyr ~is Gln Cys
270 275 280
Leu
SU8S I I t ~JTE SHE T
W 0 92/13057 41' ~iO 9 3
SE~UENCE DESCRIPTION: SEQ ID NO:3:
GA,ATTCGCGG CCGCTCATTC ACTTCATTCA TTCTTTAGAA TTACATACAC TCTCTTTCA~ 60
AACAGTCACT CTTTAAACAA AACAACTTTT GCAACA ATG CGA TCT TAC ACT CTT1l4
Met Arg Ser Tyr Thr Leu
CTC GCC CTG GCC GGC CCT CTC GCC GTG AG~ GCT GCT TCT GGA AGC GGTl62
Leu Ala Leu Ala Gly Pro Leu Ala Val Ser Ala Ala Ser Gly Ser Gly
10 ~ 15 20
CAC TCT ACT CGA TAC TGG GAT TGC TGC AAG CCT TCT TGC TCT TGG AGC-210
His Ser Thr Arg Iyr ~rp Asp Cys Cys Lys Pro Ser Cys Ser ~rp Ser
25 30 35
GGA AAG GCT GCT GTC AAC GCC CCT GCT TTA ACT TGT GAT AAG AAC GAC2S8
Gly Lys Ala Ala Val Asn A~a Pro Ala Leu Thr Cys Asp Lys Asn Asp
40 45 SO
AAC CCC ATT TCC AAC ACC AAT GCT GTC AAC GGT TGT GAG GGT GGT GGT306
Asn Pro lle Ser Asn Thr Asn Ala Val Asn Gly Cys Glu Gly Gly Gly
55 60 65 ~O
TC~ GC~ TAT GCT TGC ACC AAC TAC TC~ CCC TGG GCT GTC AAC GAT GAG 354
Ser Ala Tyr Ala Cys Thr Asn Iyr Ser Pro ~rp Ala Val Asn Asp Glu
~S 80 85
CTT GCC TAC GGT TTC GCT GCT ACC AAG ATC TCC GGT GGC TCC GAG GCC 402
Leu Ala Iyr Gly Phe Ala Ala Thr Lys lle Ser Gly Gly Ser Glu Ala
90 95 100
SUB~3 111 ~TE SHEET
WO 92/13057 ~ PCr/US92/00203
2099~0~ 4~
AGC TGG TGC TGT GCT TGC TAT GCT TTG ACC TTC ACC ACT GGC CCC GTC ~50
Ser Trp Crs Cys Ala Cys Tyr Ala Leu Thr Phe Thr Thr Gly Pro Val
105 110 1~5
AAG GGC MG A~AG ATG ATC GTC CAG TCC ACC AAC ACT GGA GGT GAT CTC 498
Lys Gly Lys Lys ~let Ile Val Gln Ser Thr Asn Thr Gly Gly Asp Leu
120 125 130
GGC GAC MC CAC TTC GAT C~C 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 C~C GGC GGT GCC CAG 594
Phe Asp Gly Cys Thr Ser Glu Phe Gly Lys Ala Leu Gly Gly Ala Gln .
155 160 165
TAC GGC GGT ATC TCC TCC CGA AGC GAA TGT GAT AGC TAC CCC GAG CTT 642
Tyr Gly Gly Ile Ser Ser Arg Ser Glu Cys Asp Ser Tyr Pro Glu Leu
170 175 180
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
185 190 195
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 Yal 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 lle 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
2l5 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 AAG CCT GCC GCT CAG CCC 930
Ser Ala Pro Val Val Gln Lys Ser Ser Thr Lys Pro Ala Ala Gln Pro
265 270 275
GAG CCT ACT AAG CCC GCC GAC AAG CCC CAG ACC GAC AAG CCT GTC GCC 978
Gl u Pro Thr Lys Pro Al a Asp Lys Pro Gl n Thr Asp Lys Pro Val Al a
280 285 290
ACC AAG CCT GCT GCT ACC AAG CCC GTC CAA CCT GTC AAC AA& CCC AAG 1û26
Thr Lys Pro Al a Al a Thr Lys Pro Val Gl n Pro Val Asn Lys Pro Lys
295 300 305 31C
ACA ACC CAG AAG GTC CG~ GGA AC. ~ ACC CGA GGA AGC TGC CCG GCC 10~4
Thr Thr Gl n Lys Yal Arg Gl ~ Thr Lys Thr Arg Gly Ser Cys Pro Al a
315 320 325
SUB~ JTE SHEET
WO 92/13057 43 2 0~ 95 D y PCT/US92/00203
..... .
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 A)a Tyr ~yr Gln
330 335 340
TGT GGT GGT TCC AAG TCC GCT TAT 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
345 350 355
ACT GGA AGC AAG TGT GTC AAG CAG AAC GAG TAC TAC TCC CAG TGT GTC 1218
Thr Gly Ser Lys Cys Val Lys Gln Asn Glu Tyr Tyr Ser Gln Cys Val
360 365 370
CCC ~AC TAAATGGTAG ATCCATCGGT TGTGGAAGAG ACTATGCGTC TCAr-~ACCC4 1274
Pro Asn
375
TCCTC~CA~G AGCAGGCTTG TCATTGTATA GCATGGCATC CTGGACCAAG TGTTCGACCC 1334
TTGTTGTACA TAGTATATCT TCATTGTATA TATTTAGACA CATAGATAGC CTCTTGTCAG 1394
CGACAACTGG CTACAAAAGA CTTGGCAGGC TTGTTCAATA TTGACACAGT TTCCTCCATA 1454
1473
SUBS 111 ~JTE SHEET
WO 92/13057 2 U 9 9 5 0 ~ 44 PCr/US92/00203
SE~UENCE DESC~iPTION: SEQ ID N0:4:
Met Arg Ser Tyr-Thr Leu Leu Ala Leu Ala Gly Pro Leu Ala Val Ser
lS
~la Ala Ser Gly Ser G)y His Ser Thr Arg Tyr Trp Asp Cys Cys Lys
~ro Ser Cys Ser Trp Ser Gly Lys Ala Ala Val Asn Ala Pro Ala Leu
- 40 45
Thr Cys Asp Lys Asn Asp Asn Pro I l e Ser Asn Thr Asn Al a- Val Asn
S0 SS 60
Gly Cys Glu Gly Gly Gly Ser Ala Tyr Ala Cys Thr Asn T~r Ser Pro
~0 ~S 80
~rp Ala Yal Asn Asp Glu Leu Ala Tyr Gly Phe Ala Ala Thr Lys l)e
9S
~er Gly Gly Ser Glli Ala Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr
100 105 110
Phe Thr Thr Gly Pro Val Lys Gly Lys Lys Het Ile Val Gln Ser Thr
115 120 125
Asn Thr Gly Gly Asp Leu Gly Asp Asn His Phe Asp Leu llet Met Pro
130 135 140
Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Ser Glu Phe Gly Lys
145 lS0 1SS 160
Ala Leu Gly Gly Ala Gln Tyr Gly Gly lle Ser Ser Arg Ser Glu Cys
165 170 ~7S
Asp Ser Tyr Pro Glu Leu Leu Lys Asp Gly Cys His Trp Arg Phe Asp
180 185 190
Irp Phe Glu Asn Ala Asp Asn Pro Asp Phe Thr Phe Glu Gln Val Gln
195 200 205
Cys Pro Lys Ala Leu Leu Asp lle Ser Gly Cys L~s Arg Asp Asp Asp
210 215 220
Ser Ser Pne Pro Al a Phe Lys ~al As~ Inr Ser Al a Ser Lys Pro Gl n
22. 230 2~5 240
SU~ JTE SHEET
W O 92/13057 PCT/US92/00203
4 2 09 9 ~ ~3 8
,.~, ~
Pro Ser Ser Ser Al a Lys Lys Thr Thr Ser Al a Al a Al a Al a Al a Gl n
245 250 255
Pro Gln Lys Thr Lys Asp Ser Ala Pro Val Val Gln Lys Ser Ser Thr
260 265 270
Lys Pro Al a Al a Gl n Pro Gl u Pro Thr Lys Pro Al a Asp Lys Pro Gl n
275 280 285
~hr Asp Lys Pro Val Al a Thr Lys Pro Al a Al .. Thr Lys Pro Val Gl n
290 295 300
Pro Va~ Asn Lys Pro Lys Thr Thr Gl n Lys Val Arg Gly Thr Lys Thr
305 310 315 - 324
Arg Gly Ser Cys Pro Ala Lys Thr Asp Ala Thr Ala Lys Ala Ser Val
325 330 335
Val Pro Al a Tyr Tyr Gl n Cys Gl y Gl y Ser Lys Ser Al a 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
Tyr Tyr Ser Gln Cys Val Pro Asn
3?0 3?5
SUBS 111 ~TE SHEET
