Note: Descriptions are shown in the official language in which they were submitted.
11~1088
ueous Colllpositions Contalnin~
Stabilized Enz~mes
I. Description:
Background of the Invention
This invention relates to long term stabili- ~
zation of an enzyme contained in an aqueous compo-
sition by a salt of a lower molecular weight organic
acid.
The de~irability of using enzymes of the proteo-
lytic and alpha amylolytic type in cleaning
compositions is well known. These enzymes are
useful for tlleir ability to reduce macromolecules
such as proteins and starches into smaller
molecules so that they can be readily washed away
by detergents and/or water. Specifically, the
proteolytic enzymes are useful in breaking down
prot~ins and the alpha amylolytic enzymes are
useful in breaking down carbohydrates. Detergent
compositions containing these enzymes have a wide
variety of uses in that they are capable of
removin~ proteinaceous and starchy stains such
as eg~ stains, blood stains, qravy stains and the
like.
1151088
Detergent compositions containing enzymes have been
commerically available in dry powdered form. However, there are
inherent problems with these compositions. First, they must be
stored in such a way as to be protected from humidity and high
heat to ensure enzyme stability. Second, these dry powdered
compositions are not well suited for several useful applications
such as spot cleaners, laundry pre-soaks and pre-spotters which
require direct application to the stained surface. For those and
other applications it is desirable to have a liquid enzyme
composition. Further for economic as well as processing consid-
erations it is advantageous to include significant amounts of
water in liquid enzyme compositions. However, there is an
inherent problem in adding significant amounts of water to an
enzyme containing composition. This is due to the fact that
enzymes are inherently unstable in the presence of water
- resulting in a rapid decrease of enzymatic activity, i.e.,
the ability of the enzyme to effectively reduce macromolecules
into smaller molecules. It is speculated that the loss of
enzymatic activity is due to the hydrolizing action of water
on the enzyme.
Further decreases in enzymatic activity will also
result from exposing the aqueous enzyme containing compositions
to temperatures in excess of 70C. In fact, if these
compositions are exposed to these temperatures for more than
a few hours, complete deactivation will occur.
-` ilS1088
The~efore, in order to h~ve an ~queous based
enzyme containing composition which is suitable
for the uses described above, it is clear that the
enzyme must not only remain stable in water, i.e.
retain its enzymatic activity, but it must also be
capable of maintainin~ such stability for extended
periods of time at elevated temperatures. For, it
is not uncommon to have commercial products stored ~
in warehouses for a period of time before being - _
sold to consumers, where the temperatures during
storage far exceeds those of normal room temperature.
Various attempts have been made to stabilize
enzymes contained in acqueous compositions. The
following are exemplary of these. r
U.S. Patent 3,296,094 to Cayle utilizes a
partially hydrolyzed and solubilized collagen, and
glycerol to stabilize an aqueous proteolytic
enzyme composition. The amount of glycerol
required for stabilization in this composition is
between 35% to 60~ by weight of the total compo-
sition.
U.S. Patent 3,557,002 to McCarthy utilizes a
monohydroxy alcohol ox an alkoxy alcohol to
stabilize a ~roteolytic enzyme. Although the
amount of alcohol used in this composition is less
than t}lat used in Cayle the residual acti~ity of
`- `` 1151088
the enzyme of thi~ composition decr~ases after
long p~riod~ of st.orage at relatively high tempe- ~
ratures. ~ -
U.S. Patent 4,16~,817 to Weber utilizes
either water soluble salts such as sodium or
potassium sulfates or chlorides and/or glycerol or
alkylene glycols to stabilize a proteolytic
enzyme in compositions containing ionic builders
and surfactants. Again, significant amounts of
glycerol and/or other solids are required to
maintain long term enzyme stability in these
compositions.
U.S. Patent 3,682,842 to Innerfield utilizes r
an enzyme-ion binding agent such as trichloroacetic
acid or tungstic acid; a salt, such as sodium
chloride or ammonium sulfate; an organic solvent
such as ethanol; and an anionic surfactant to
stabilize a mixture of protolytic and amylolytic r
enzymes.
U.S. Patent No. 3,676,374 to Zaki et al.
utilizes a mixture of alkane sulfonates or alpha-
olefin sulfonate compounds, along with an alkyl
alkyleneoxy hydro~yl or sulfate compounds to
stabilize a proteolytic enzyme in a liquid deter-
gent composition containing water. Additionally,
. various stabilizing agents can be employed with
these compositions such as the water-soluble
calcium and magnesium chloride lactates and acetates.
- 1151088
--5--
In my United States Patent No. 4,243,546 I dis-
closed that enzymes could be stabilized in an aqueous
medium for long periods of time by adding to the compo-
sition an alkanolamine and an organic acid. I have
now found that enzyme s~ability can be maintained in
an aqueous medium containing small amounts of
alcohol by adding an alkanolammonium salt of a
lower molecular weisht organic acid or ~y adding
an ammonium or alkali metal salt of such-acids.
Summary of the Invention
It is an object of this invention to provide
aqueous based compositions containing stabilized
enzymes which are suitable for use as
where the enzymes will be stabilized, i.e. maintain
their activity, for long periods of time. It is a
further object of this invention to provide such
stability by using small amounts of a relatively
inexpensive stabilizing agent.
` The compositions of this invention require
; only minor amounts of an enzyme stabilizing agent
- 25 and an alcohol to achieve superior long term
enzyme stabiiity which will be maintained even at
elevated temperatures. These compositions are
particularly effective as cleaning preparations in
a wide range of applications.
Thé compositions of this invention are
comprised of the following in~redien~s (all
amounts given below and throughout this applica-
tion are on a weight basis):
.,` ~
:
:
1151088
-- 6
a) from about 0.01% to 15% of an enzyme
stabilizing agent which is an alkali
metal, ammonium, or alkanolammonium salt
of a lower molecular weight organic acid
selected from the group consisting of
formic, acetic, propionic, butyric and
valeric acids;
b) from about 0.006% to about 5% of an
enzyme selected from the group consisting
of proteases, alpha amylases and mixtures
thereof;
c) from about 1% to about 25% of an alcohol
selected from the group consisting of
alcohols having the formula ROH wherein
R is an alkyl residue of one to six
carbon atoms; and
d) from about 10~ to about 9o% water.
Detailed Description of the
Preferred Embodiment
In accordance with the present invention, it has
been found that the alkali metal, alkanolammonium, or ammonium
salts of certain lower molecular weight organic acids, can in
the presence of a lower molecular weight monohydroxy alcohol
stabilize proteolytic or amylolytic enzymes or mixtures thereof
in an aqueous medium. Further, it has been found that the
enzyme thus stabilized will retain its activit~ for an extended
period of time, in the order of one year to eighteen months.
`,,~,j,~,, ~
~ . .
` - ~151088
--7--
The main ingredi~nts of the compositions of
this invention are water, enzymes, stabilizing
agents and an alcohol~
Water can comprise from about 10% to about
90Qo of the total composition of the present invention.
Preferably water will be present in amounts ranging
from about 40% to about 90% by weight. Although
not mandatory deionized water is preferred for usé
herein.
The enzymes which are stabilized by and
therefore suitable for use in the present invention
are the proteases, the alpha amylases and mixtures
Gf proteases and alpha amylases. r
The proteases which are deriv~d from bacterial
or fungal sources can be classified into three
different categories: acidic, neutral, and
alkaline proteases. All of which are useful r
herein. Proteaes derived from plant and animal
sources although not readily classifiable into the
above recited categories are also useful herein.
These enzymes are active in pH's ranging from
about 3 to about ll. Optimum activity of these
enzymes is generally exhibited in the pH range of
about 6 to about 10. The proteases catalyze the
hydrolysis of the peptide linkages of proteins,
.
^` 1151088
polypeptides and other related compounds. By
breaking the peptide bonds of proteins, free amino
and carboxy groups a~re formed which are short
chain molecules that can easily be washed away by
water and/or a detergent. All categories of
proteases enumerated above are useful in this
invention, however, those having optimum activity
in pH's ranging from about 6 to about 9 are preferred.
An example of a preferred protease is a serine
protease.
The alpha amylases exhibit optimum activity
in the acidic pH ranges. These enzymes catalize
reactions which break starch molecules into shorter
chain molecules that are readily washed away by
detergents and/or water. The alpha amylases may
be obtained from animal sources, cereal grains, or
bacterial or fungal sources.
The enzyme ingredient of the present invention
can be conveniently added in the form of a commercial
enzyme preparation. These are generally sold in a
dry powder, solution, or slurry form and are
comprised of from about 2% to about 80% of active
enzymes in combination with an inert carrier such
as sodium or calcium sulfate, sodium chloride,
glycerol, nonionic surfactants, or mixtures thereof
as the remaining 20~ to 98~. Examples of commercial
protease preparations which are suitable for use
in the compositions of this invention include
Savinase, Savinase 8.0 Slurry, Esperase, Esperase
8.0 Slurry and Alcalase from Novo Industri A/S,
Copenhagen, Denmark; and High Alkaline Protease,
*Trademarks
.
"` 1151088
Maxatase P , and Alkaline Protease* 201 P from G.B.
Fermentation Inc., Des Plaines, Ill. Examples of com-
mercial alpha amylase preparation which can be used
herein include Amalase THC* from G.B. Fermentation Inc.,
Des Plaines, Ill.; and Termamyl 60L, and Termamyl* 60G
from Novo Industri A/S, Denmark. An example of a com-
mercial enzyme preparation containing a mixture of alpha
amylases and alkaline proteases which can be used herein
is Maxatase* from G.B. Fermentation Inc.
The commercial enzyme preparation preferred for
use herein is Savinase 8.0 Slurry from Novo Industries,
an alkaline proteolytic enzyme preparation obtained from
the genus Bacillus Subtilis containing about 6% by weight
of the enzyme and having an activity of 8 kilo novo
units.
.
Compositions of this invention will stabilize from
about 0.006% to about 5.0% of an active enzyme. The
amount of enzyme which is preferred is from about 0.006%
to about 2.5% by weight.
The stabilizing agents which stabilize the
enzymes described above are the alkali metals,
ammonium, and alkanolammonium salts of lower
molecular weight organic acids such as formic,
*Trademarks
. 1
D
1151088
--10--
acetic, propionic, butyric and valeric acids.
These agents can be used in effective amounts, '~-
ranging from about 0.01% to about 15% by weight of
the composition. The preferred ranges for these
5 agents are from about 2~ to about 10% by weight of
the composition, while the most preferred range is
from about 2% to about 6%.
As set forth above, alkanolammonium compounds
can be used to form the stabilizing salts of the
10 instant invention. Examples of such useful compounds
are monoethanolammonium, diethanolammonium, and
triethanolammonium.
The fourth main ingredient of the composition
of this invention is alcohol which acts to enhance
15 enz~ne stability and which also aids in lowering
the viscosity and preventing skinning in the
compositions of the instant invention. The
alcohols which are suitable for use herein are
those having the formula of ROH where R is an r
20 alkyl residue having from one to six carbon atoms
in either the branched or straight chain configurations.
The amount of alcohol which is suitable for use in
the composition of this invention ranges from
about 1% to about 25% by weight of the composition.
25 The amount which is preferred is form about 4% to
about 8% by weight of the composition.
;
,
r
'` ~151088
In addition to the essential ingredients described
` above the compositions of this invention can contain other
ingredients such as surfactants of either the nonionic or
anionic type, organic solvents, solubilizing compounds and
perfumes.
Inclusion of a surfactant of either the nonionic or
anionic type is advantageous in that they tend to enhance
the enzymatic stability of these compositions, however, more
importantly they significantly improve the detergent charac-
teristics of these compositions. The nonionics or anionics
may be utilized in amounts ranging from about 1% to about
55% and preferably from about 10% to about 30% by weight of
the total composition.
Thus in a preferred aspect the present invention
provides such aqueous based compositions further comprising
by weight from about 1% to about 55% of an anionic sur-
factant, a nonionic surfactant, or a mixture of an anionic
and a nonionic surfactant.
Examples of suitable nonionics include:
(1) Ethoxylated fa*ty alcohols - having the formula:
.
RO-(CH2CH2O)nH where R is from 8 to 18 carbon atoms and n
is an integer of from 1 to 500.
Examples of these are:
(a) the condensation product of 1 mole of an
aliphatic alcohol, having from 12 to 13
carbon atoms in either a straight or
branched chain configuration, with an
average of 6.5 moles of ethylene oxide;
'
. .
. .
.~. - - - .
:
.
-
- 115~088
-12-
(b) tlle condensatio~ product of 1 mole of an
aliphatic alcohol, having from 12 to 15
carbon atoms in either a straight or
branched chain configuration, with 9
moles of ethylene oxide; and
(c) the condensation product of 1 mole of an
aliphatic alcohol, having between 12 and
15 carbon atoms in either the straight
or branched chain configuration, with 3
moles of ethylene oxide.
Examples of (a), (b) and (c) are commercially
available from the Shell Oil Company under the
trade names of Neodol,* Neodol 23-6.5, Neodol 25-9,
and Neodol*25-3 respecti~ely.
(2) Ethoxylated fatty acids - having the
formula:
O
- R-~-O- (CH2CH20)nH where R and n are as in
(1) .
* Trademarks
.
, j, ~.
.. . . . ..
1151088
-13-
(3) Ltl20xylated alkyl phenols - having th~
~ormul~: ~
R - ~ ~ (~H2CH20)nH where R is an
al~yl radical having from 6 to 16 carbons and n is
an integer from 1 to 500.
Examples of suitable anionics include:
(1) Soaps - having the formula: R - ~-OX,
where X is sodium, potassium or ammonium and R is a
fatty acid radical either saturated or unsaturated
,10 branched or straight chain configuration having
from 10 to 18 carbon a,toms.
(2) Alkyl benzene sulfonates - having the
formula: ~
R - ~ -S03X, where X is ammonium, tri-
ethanol-ammonium, sodium or potassium and R is an
alkyl radical having from 8 to 18 carbon atoms.
(3) HydroxY alkane sulfonates - having the
formula: OIH
R - CH-CH2-CH2 S03X where X is as in (2)
and R is an alkyl radical having from 10 to 15
-, carbon atoms.
: (4) Sulfonated fatt~ acids - having the
formula: CH3(1H2)n COOH ,,
S03X where X is as in (2) and
25 n lS an integer between 12 snd 18.
~'
.
1151088
~5) Sulfonated nonionics - having the formula: _
~ O - (CH2CH2O) H
where X is as in (2~ and n is an integer
from 8 to 16 where R is as in 1.
(6) ~atty alcohol sulfates - having the
formula: CH3(CH2) C~2O - SO X where X is as i
(2) and n is an integer from 8 to 16.
(7) Sulfated nonionics - having the formula:
.
RO - (CH2CH2O)n SO3X where X is as in (2), R is
an alkyl radical having from 12 to 18 carbon atoms
and n is an integer from 1 to 50. r
r
(8) Mono- and di-esters of sodium sulfo-
-
succinates - having the formula:
~ ,1
Rl-O-C-CH -CH2- CO-R
S03Na
where Rl is either sodium, hydrogen or an alkyl
radical having from 1 to 12 carbon atoms. R2 is
an alkyl radical having from 1 to 12 carbon atoms.
The surfactants which are preferred are the
nonionics of the ethoxylated fatty alcohol type.
The compositions of this invention can also
contain organic solvents such as the isoparafinic
mixtures of petroleum distillates. These may be
added in amounts of up to 75% by weight with about
10% to about 40% by weight being the amount
preferred. r
`` .`` ~151088
-15-
Co~positions containing the oryanic solvents
set forth above can also contain solubilizing ç
compounds. Examples of: such compounds are the
sodium salts of benzene sulfonate, toluene sulfonate,
S and xylene sulfonate. These agents can be added
in amounts of up to about 10% by weight, however
- about 3% to about 6% by weight of these agents is
the preferred amount for inclusion.
In addition to the various ingredients recited
above the compositions of this invention can also
contain optical brighteners, fabric softeners,
anti-static agents, anti-redeposition agents and
small amounts of perfume and dye.
The pH of these compositions will generally
be around 7, however depending on the enzyme being
used the pH can be raised by adding sodium hydroxide
or lowered by adding acetic acid.
The various components of the enzyme containing
compositions can be mixed together in any order.
However, it is preferred that the salt, alcohol
and water mixture be prepared first and the enzymes
added thereto to prevent any degredation or deactivation
of the enzyme. The optional components such as
the surfactants can be added at any time.
There are a variety of uses for the compositions
of this invention. For example they may be used
as spot removers. They may also be used in home
laundering operations as pre-soaks and as laundry
additives for use during the wash cycle of an
automatic washer.
~151088 ' `
-16-
Tl~ followin~ e.~mples illustrate the invention.
Example 1
The followiny compositions were prepared and
stored in closed-glass containers at 100F for the
indicated periods of time. It is estimated that
one week's storage at 100F is equa1 to between
about 2 to 3 months of storage at room temperature.
The pH of each of the following compositions
was about 7.
` -` 115~088
--17--
3 o "
o a~
,,~ o O ~ ~ O
O O Q O O
I~ ~Pu~ ~ ~ q~ o u~ o o ~ ~
r
- 3 ~~.
I~ o ~ a~ oo ~
o u~ o o u ~r co Ll~
U~U~ ''
3 ~ .
er ~~ ~ ~ ~ O U~ ~ O
N N r-l ~O 1~ ~ O 1` 1~ ~ r--
3 "~
N ~ o o
u~ o o~
~ ~o r~ o ~ ~ ,, ~, ;
N ~,)
O C ul IQ 4 4 4
O O ~ -1 X ~ ~
O O ~ u~ ~ ~ O ~:J H ~ Icl 3 F~ ~ .¢ 3 ,¢ ~:2
` 1151088
1. Nonionic surfactant comprised of an etho~ylated
alcohol where one mole of aliphatic alcohol having
from 12 to 15 çarbon atoms was ethoxylated with
an average of 9 moles of ethylene oxide.
2. Nonionic surfactant comprised of an ethoxylated
alcohol where one mole of aliphatic alcohol
having from 12 to 13 carbon atoms was ethoxylated
with an average of 6.5 moles of ethylene
oxide.
3. A commercial alkaline proteolytic enzyme
preparation available from Novo Industries
containing 6% active enzymes with an activity
of 8.0 Kilo Novo protease units.
4. Percent remaining activity was determined
by Tri-nitro-benzene sulfonate method using casein
as a substrate. Activity values are subject
to an experimental error of + 5%.
5. Sample numbers 8 and 9 are not in accordance
with the present invention and have been
included for the purpose of comparison only.
Review of this data shows that the enzyme will
deactivate fairly rapidly as demonstrated by samples
8 and 9 when a stabilizing agent of the present
invention is not used. Additionally, as demonstrated
by sample No. 10, as little as 2% of one of the
stabilizing agents of this inv~ntion provides e~cellent
long term stability.
r
~, - ilS1088
. ,
-19-
E~le 2
A composition in accordance with the present
invention was pre~ared, containing 4% by weight of
sodium acetate, an enzyme stabilizer salt; 1% by
weight of SAVINf~SE 8.0 Slurry, a commercial
enzyme preparation available from Novo Industries;
7% by weight of ethanol; 15% by weight of a nonionic S
surfactant which is.the reaction product of one
mole of an aliphatic alcohol having from 12 to 15
carbon atoms with 9 moles of ethylene oxide,
available from the Shell Oil ~ompany under the
tradename of Neodol 25-~; 15~ by weight of a
nonionic surfactant which is the reaction product
of one mole of an aliphatic alcohol having from 12 r
to 13 carbon atoms.with 6.5 moles of ethylene
oxide; 0.25% by weight of a perfume; 0.0l~ by
weight of a dye; and 57.74~ by weight of deionized
water. This composition was then evaluated for
stain removal activity as a prespotter by applying ..
a portion of the composition onto two stained
cloths; one cloth containing a stain which was
comprised of blood, milk and ink, and a second
cloth containing a stain which was dirty motor
oil. After the cloths were treated with the composition
they were washed in a commercial detergent composition
available from the Procter & Gamble Company under
the trademark of Extra Action Tide. Following the
wash the stained cloths were allowed to dry and
were then analyzed by a Gardener Tri-stimulus
color difference meter to determine the percent of
the stain which was removed. The percentage of
stain removal achieved by prespotting with this
composition is given below in column l.
`` ~15108B
.~ .
--~o--
;
For purpose of com~arison,. ~tained clo~lls
having the same stain as thos~ used above were
washed in ~tra ~ctioin Tide, and then analyzed.
The results of this comparative test are given in
S the table below in column 2.
Column 1 2
_
Prespotter This in- . None
vention
S tain
Blood
Mil~ & . L
Ink 65.0* 43.2
Dirty
Motor t
15 Oil 28.4 17.1
*all entries in table represent percent stain
removal.
~151088
From the above data it is apparent that the
compositions of this invention are effective prespotters.
Example 3
The composition of this inYention which was used
as a prespotter in Example 2 was tested as a laundry additive.
Again, two stained cloths, one containing a blood, milk and ink
stain and a second containing a dirty motor oil stain, were
washed separately with approximately 1.2 grams of a commerical
detergent composition, Extra Action Tide and approximately 0.75
grams of the composition of Example 2. These washes were
conducted in a tergotometer which holds approximately 1 liter
of water. After being laundered these stained swatches were
allowed to dry and then tested for the percentage of stain
removal again by using a Gardener Tristimulus color difference
meter. The results of this anaylsis are given in the table
below in column 1.
Again, for the purposes of comparison, stained
clothes having the stains recited above were washed separately
in a tergotometer with Extra Action Tide and with a commerical
laundry additive containing enzymes BIZ and Extra Action Tide.
These were also analyzed for percent stain remoyal by the
method recited above. The results of this analysis are given
in columns 2 and 3, respectively.
_,
.` 1151088
. -22-
Column 1 2 3
Additive Composition None BIZ
of this in-
vention
Stain
Blood
Milk ~
Ink 44.2* 20.4 8.6
Dirty
Motor
Oil 17.8 13.4 13.9
*all results in table are expressed in terms of
percent stain removal.
-
From the above data, it is apparent that the r
compositions of this invention are also well suited
for use as laundry additives.
Having described some typical embodiment of
this invention it is not my intent to be limited to
the specific details set forth herein. Rather, I
wish to reserve to myself any variations or modifica-
tions that may appear to those skilled in the art and
fall within the scope of the following claims.
