U.S. patent number 3,819,528 [Application Number 05/232,300] was granted by the patent office on 1974-06-25 for stabilized aqueous enzyme compositions.
This patent grant is currently assigned to The Procter & Gamble Company. Invention is credited to Jim S. Berry.
United States Patent |
3,819,528 |
Berry |
June 25, 1974 |
**Please see images for:
( Certificate of Correction ) ** |
STABILIZED AQUEOUS ENZYME COMPOSITIONS
Abstract
Aqueous amylolytic enzyme-containing compositions comprising
water, amylolytic enzyme, a water-soluble calcium salt, an organic
co-stabilizing agent selected from aliphatic glycols and
1,3-propanediol and, optionally, a nonionic or zwitterionic
detergent are disclosed. The compositions, useful as
starch-degrading compositions, are stabilized substantially against
loss of amylolytic enzyme activity during storage.
Inventors: |
Berry; Jim S. (Springfield
Twsp., Hamilton County, OH) |
Assignee: |
The Procter & Gamble
Company (Cincinnati, OH)
|
Family
ID: |
26925851 |
Appl.
No.: |
05/232,300 |
Filed: |
March 6, 1972 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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786432 |
Dec 23, 1968 |
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Current U.S.
Class: |
510/393; 435/188;
510/281; 510/284; 510/530; 510/321 |
Current CPC
Class: |
B01J
31/003 (20130101); C11D 3/38618 (20130101); C11D
3/38663 (20130101); C12N 9/96 (20130101) |
Current International
Class: |
C12N
9/96 (20060101); B01J 31/00 (20060101); C11D
3/38 (20060101); C11D 3/386 (20060101); C07g
007/02 (); C11d 007/42 (); C11d 007/50 () |
Field of
Search: |
;195/63,68 ;424/94
;252/89,132,135,546,398,403,DIG.12,153 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
"Stabilities of Enzymes in Polyhydric Alcohols" by Yasumatsu et
al., Agr. Biol. Chem., Vol. 29, No. 7, pp. 665-671, 1965. .
"The Role of Enzymes in Detergent Products" by H. E. Worne,
Detergent Age, September 1968, Pages 19-22 & 81. .
"Stabilization of Enzyme Activity by an Organic Solvent" by S.
Takemori et al., Nature, Vol. 215, July 1967, Pages
417-419..
|
Primary Examiner: Guynn; Herbert B.
Assistant Examiner: Albrecht; Dennis L.
Attorney, Agent or Firm: Witte; Richard C. Allen; George W.
Filcik; Julius P.
Parent Case Text
This is a continuation of application Ser. No. 786,432, filed Dec.
23, 1968, now abandoned.
Claims
What is claimed is:
1. A stabilized aqueous enzyme composition consisting essentially
of by weight of the composition:
1. from about 65 percent to about 97 percent water;
2. from about 0.001 percent to about 1 percent amylolytic
enzyme;
3. from about 0.001 percent to about 1 percent with respect to
calcium ion of a water-soluble enzyme-stabilizing calcium salt;
4. from about 2 percent to about 27 percent of an organic
co-stabilizing compound selected from the group consisting of
aliphatic glycols having the formula
HO[CH.sub.2 CH.sub.2 O].sub.x H
wherein x is from about 2 to about 200; and 1,3-propanediol;
and
5. from 0 to about 15 % of a detergent selected from the group
consisting of nonionic detergents and zwitterionic detergents.
2. The composition of claim 1 wherein the co-stabilizing compound
is an aliphatic alcohol having the formula
HO[CH.sub.2 CH.sub.2 O].sub.x H
wherein x is from about 2 to about 200; and wherein the amylolytic
enzyme is an .alpha.-amylase characterized by amylolytic activity
in the pH range of from about 4.5 to about 10 and at a temperature
of from about 60.degree.F. to about 150.degree.F.
3. The composition of claim 2 wherein the .alpha.-amylase is
derived from Bacillus subtilis.
4. The composition of claim 3 wherein the .alpha.-amylase is
present in an amount of about 0.01 to about 0.5 percent.
5. The composition of claim 4 wherein the calcium ion is present in
an amount of about 0.005 to 0.05 percent and the organic
co-stabilizing compound is present in an amount of from about 5
percent to about 20 percent.
6. The composition of claim 5 wherein the organic costabilizing
compound is selected from the group consisting of diethylene
glycol; triethylene glycol; and glycols of the formula
HO[CH.sub.2 CH.sub.2 O].sub.x H
wherein the average value of x is from 4 to about 80.
7. The composition of claim 6 wherein from about 4 to about 10
percent of a nonionic or zwitterionic detergent is present.
8. The composition of claim 7 wherein the detergent is selected
from the group consisting of condensation products of 1 mole of
aliphatic alcohol having from eight to 22 carbon atoms with from 5
to 40 moles of ethylene oxide; 3-(N,N-dimethyl-N-alkylammonio)
propane-1-sulfonate wherein the alkyl has from eight to 22 carbon
atoms; and
3-(N,N-dimethyl-N-alkylammonio)-2-hydroxypropane-1-sulfonate
wherein the alkyl has from eight to 22 carbon atoms.
9. The composition of claim 8 wherein the detergent is
3-(N,N-dimethyl-N-coconutalkylammonio)-2-hydroxypropane-1-sulfonate
and the organic co-stabilizing compound is triethylene glycol.
10. The composition of claim 8 wherein the calcium salt is selected
from the group consisting of calcium acetate, calcium sulfate and
calcium chloride.
11. A stabilized aqueous enzyme composition consisting essentially
of by weight of the composition:
1. from about 65 to about 97 percent water;
2. from about 0.001 to about 1 percent amylolytic enzyme;
3. from about 0.001 to about 1 percent with respect to caclium ion
of a water-soluble, enzyme-stabilizing calcium salt;
4. from about 2 to about 27 percent of a 1,3-propanediol
co-stabilizing compound; and
5. from 0 to about 15 percent of a detergent selected from the
group consisting of nonionic detergents and zwitterionic
detergents.
12. The composition of claim 11 wherein the amylolytic enzyme is an
.alpha.-amylase characterised by amylolytic activity in the pH
range of from about 4.5 to about 10 and at a temperature of from
about 60.degree.F. to about 150.degree.F.
13. The Composition of claim 12 wherein the .alpha.-amylase is
derived from Bacillus subtilis.
14. The composition of claim 13 wherein the .alpha.-amylase is
present in an amount of from about 0.01 to about 0.5 percent.
15. The composition of claim 14 wherein the calcium ion is present
in an amount of from about 0.005 to 0.05 percent and the organic
co-stabilizing compound is present in an amount of from about 5 to
about 20 percent.
16. The composition of claim 15 wherein from about 4 to about 10
percent of a detergent selected from the group consisting of
nonionic and zwitterionic detergents is present.
17. The composition of claim 16 wherein the detergent is selected
from the group consisting of condensation products of 1 mole of
aliphatic alcohol having from eight to 22 carbon atoms with from 5
to 40 moles of ethylene oxide; 3-(N,N-dimethyl-N-alkylammonio)
propane-1-sulfonate wherein the alkyl has from eight to 22 carbon
atoms; and
3-(N,N-dimethyl-N-alkylammonio)-2-hydroxypropane-1-sulfonate
wherein the alkyl has from eight to 22 carbon atoms.
18. The composition of claim 17 wherein the detergent is
3-(N,N-dimethyl-N-coconutalkylammonio)-2-hydroxypropane-1-sulfonate.
19. The composition of claim 17 wherein the calcium salt is
selected from the group consisting of calcium acetate, calcium
sulfate and calcium choride.
Description
FIELD OF THE INVENTION
This invention relates to aqueous amylolytic enzymecontaining
compositions useful in the degradation of starchy materials and
characterized by stabilization against loss of amylolytic
activity.
The use of amylolytic enzymes in the alteration and/or degradation
of starchy materials is known. For example, U.S. Pat. No. 2,607,359
(Aug. 19, 1962) describes compositions containing an amylolytic
enzyme useful in facilitating the removal of porous materials such
as wallpapers, labels and casein type pastes from surfaces to which
the porous materials are held by a starch-containing adhesive.
Similarly, Jaag in Seifen, Ole, Fette, Wachse 88, No. 24, pp.
789-793, (Nov. 1962) describes the use of amylolytic enzymes in
laundry formulations. These enzymes aid in the laundry process by
attacking starchy soils and stains found on soiled fabrics and
decomposing and/or altering them so as to render them more
removable during laundering. Enzymatic materials are expensive and
powerful materials which must be judiciously formulated and used.
These enzymes when employed in aqueous compositions are unstable
and suffer appreciable destruction during long periods of storage
as evidenced by substantial loss in starch-degrading and/or soil-
and stain-removing efficacy. The loss in amylolytic activity is
particularly severe under conditions of high temperature.
Furthermore, aqueous laundering solutions containing amylolytic
enzymes often contain additional components desirable in the
laundering process but which have an adverse effect on the
amylolytic enzyme. Proteolytic enzymes, for example, while useful
in providing proteinaceous soil- and stain-removing properties in
laundering compositions, often tend to have such an adverse
degrading effect on amylolytic enzymes.
Attempts have been made in the art to provide amylolytic
enzyme-containing compositions wherein the enzymatic activity is
preserved by the incorporation of a stabilizing agent. These
attempts have generally involved incorporation in such compositions
of water-soluble calcium salts. A trade bulletin describing
bacterial amylases derived from Bacillus subtilis, published by
Daiwa Kasei K. K. of Osaka, Japan, describes the stabilization of
bacterial amylase with calcium and sodium ions. Similarly, Hamada
et al., Agr. Biol. Chem., 31, No. 1, pp. 1-6 (1967), describe the
stabilizing effect of calcium ions on .alpha.-amylase. The
employment of calcium salts to impede loss of amylolytic activity,
particularly at high temperatures, has not been entirely
satisfactory, particularly over extended periods of storage at high
temperatures. Accordingly, there has been a need for amylolytic
enzyme-containing compositions having improved amylolytic enzyme
stability.
It is therefore an object of this invention to provide stabilized
aqueous amylolytic enzyme-containing compositions which retain
substantially their amylolytic activity upon storage.
It is another object of this invention to provide aqueous
amylolytic enzyme-containing compositions stabilized substantially
against loss of activity by the presence of minor amounts of
enzyme-stabilizing compounds.
Other objects of this invention will be obvious from consideration
of the invention which is more fully described hereinafter.
SUMMARY OF THE INVENTION
These and other objects of the present invention re achieved by the
provision of aqueous amylolytic enzyme-containing compositions
containing minor amounts of calcium ion and certain organic
co-stabilizing agents. The aqueous compositions of the present
invention can additionally contain nonionic or zwitterionic
detergent components to enhance the stability of the amylolytic
enzymes in the aqueous compositions of this invention and to
enhance the detergent properties of these compositions. The present
invention is based in part on the discovery that extended periods
of stabilization can be achieved by introducing into aqueous
enzyme-containing compositions a source of calcium ion and an
organic compound selected from the group consisting of aliphatic
glycols and 1,3-propane diol.
The stabilized aqueous enzyme-containing compositions of this
invention comprise
1. from about 65 to about 97 percent water;
2. from about 0.001 to about 1 percent amylolytic enzyme;
3. from about 0.001 to about 1 percent with respect to calcium ion
of a water-soluble enzyme-stabilizing calcium salt;
4. from about 2 to about 27 percent of an organic compound selected
from the group consisting of aliphatic glycols having the
formula
HO[CH.sub.2 CH.sub.2 O].sub.x H
wherein x is from about 1 to about 200; and 1,3-propane diol;
and
5. from 0 to about 15 percent of a detergent selected from the
group consisting of nonionic detergents and zwitterionic
detergents.
The amylolytic enzymes which can be stabilized in aqueous solution
by the action of the hereinbefore described combination of calcium
ion and organic compound are known materials and can be of fungal,
plant, animal or bacterial origin. Suitable amylolytic enzymes
include the .alpha.-amylases which are particularly well suited for
breaking down starch molecules as they attack the .alpha..sub.1,4
-glycosidic linkages in starch. The degraded short chains are
easily removed from their environment with water or aqueous
solutions of detergents. Examples of suitable amylolytic enzymes
include the .alpha.-amylases of mold origin including those derived
from Aspergillus oryzae, Aspergillus niger, Aspergillus alliaceus,
Aspergillus wentii, and Pencillium glaucum. The .alpha.-amylases
derived from cereal grains, pancreatic sources and such bacteria as
Bacillus subtilis Bacillus macerans, Bacillus mesentericus and
Bacillus thermophilus are also useful herein. These enzymes are
active in the pH range of from about 4.5 to about 10 and at
temperatures from about 60.degree.F. to about 150.degree.F. Optimum
activity of these .alpha.-amylases is generally exhibited in the pH
range of from about 5.5 to about 7.5.
Preferred amylolytic enzymes herein are the .alpha.-amylases
derived from the bacterial organism Bacillus subtilis. These
amylases provide excellent desizing and starch digestive properties
and are especially useful in the laundering of textile materials
containing soils and stains of a starchy nature.
The amylolytic enzymes useful herein can be employed in a pure
state. Generally they are employed in the form of a powdered
commercially available preparation wherein the amylolytic enzyme is
present in an amount of from about 2 to about 80 percent of the
preparation. The remaining portion, i.e. about 20 to about 98
percent, comprises inert vehicle such as sodium sulfate, calcium
sulfate, sodium chloride, clay or the like. In preparing the
stabilized aqueous starch-degrading compositions of this invention,
such commercial enzyme preparations are admixed with water and the
remaining components of the compositions. The active enzyme content
of these commercial enzyme compositions is the result of
manufacturing methods employed and is not critical herein so long
as the finished compositions of this invention have the hereinafter
specified enzyme content. Insoluble inert materials are generally
removed from the compositions of this invention to provide aqueous
compositions which are clear and substantially free of precipitated
deposits. Specific examples of commercial enzyme preparations
suitable for use herein and the manufacturers thereof include:
Diasmen .alpha.-amylase (Daiwa Kasei K. K. Tokyo, Japan); Rapidase
.alpha.-amylase THC-25 (Rapidase, Seclin, France); Novo Bacterial
.alpha.-amylase (Novo Industri, Copenhagen, Denmark); Wallerstein
.alpha.-amylase (Wallerstein Company, Staten Island, New York);
Rhozyme-33 and Rhozyme H-39 (Rohm & Haas, Philadelphia,
Pennsylvania).
Preferred herein is a powdered enzyme preparation containing
.alpha.-amylase and a mixture of alkaline and neutral proteases
available as CRD-Protease (or Monsanto DA-10) from Monsanto
Company, St. Louis, Missouri. This composition contains about 3
.alpha.-amylase and is useful herein to provide the compositions of
the invention with amylolytic and proteolytic enzyme activity.
Mixtures of proteases and .alpha.-amylases are preferred herein and
include the enzyme preparations described in U.S. Pat. No.
3,031,380 to Minagawa et al. (Apr. 24, 1962).
The amount of amylolytic enzyme employed in the compositions of
this invention can vary depending upon the activity of the enzyme
or enzyme preparation, conditions of pH and the intended use of the
compositions. When the stabilized aqueous compositions of this
invention are employed as spot removers, they should contain an
amount of amylolytic enzyme sufficient to remove the starchy soils
and stains normally encountered in a laundering situation. Normally
the compositions of this invention are prepared to contain from
about 0.001 to about 1 percent enzyme by weight of the aqueous
composition on a pure enzyme basis. For best results, the
compositions preferably contain from about 0.01 to about 0.5
percent amylolytic enzyme. When a commercially available powdered
enzyme preparation is employed as the source of amylolytic enzyme,
the compositions of this invention contain from about 0.1 to about
4.0 percent of the powdered amylolytic enzyme-containing
preparation as it is available in commercial form e.g., containing
from about 2 to about 80 percent active enzyme. It will be
appreciated that when such preparations are employed herein, the
amount of the preparation required to provide aqueous compositions
having desirable levels of amylolytic activity will depend on the
activity level of the enzyme-containing preparation employed. The
precise amounts of such materials employed can be readily
determined by methods known in the art so long as the stabilized
compositions of the invention provide an amount of amylolytic
enzyme activity sufficient to provide desirable levels of starch
degrading properties.
As used herein, amylolytic activity refers to the tendency of an
amylolytic enzyme to perform the desired function of catalytic
alteration and/or degradation of starchy materials. Stability, as
used herein, refers to the tendency of an amylolytic enzyme to
retain its enzymatic activity. The activity level of amylolytic
enzyme suitable herein can be determined by numerous methods. A
suitable method is the 3,5-dinitrosalicylate assay method. In
accordance with this method, a sample of amylase is allowed to
catalyze the hydrolysis of the 1,4-.alpha.-glycosidic bonds of
starch and glycogens for five minutes at a temperature of
37.degree.C. at a pH of 6.0. The reaction is terminated by the
addition of buffered sodium 3,5-dinitrosalicylate, the color is
developed and the amount of maltose determined by
spectrophotometric response and comparison with solutions of
analytical grade maltose hydrate. The amylase has one activity unit
for each 0.4 mg. of maltose hydrate produced during hydrolysis
under the specified conditions. The amylase activity method is well
known and is described with particularly in P. Bernfeld, Methods in
Enzymol. Vol I. p. 149 (1955).
As hereinbefore described, the present invention is based in part
upon the surprising discovery that extended periods of enzyme
stabilization can be achieved by incorporating into aqueous enzyme
solutions a combination of water-soluble calcium salt and organic
co-stabilizing compound hereinbefore described. The water-soluble
salts of calcium include, for example, calcium chloride, calcium
acetate, calcium citrate, calcium glycerol phosphate, calcium
gluconate, calcium glucoheptanate, calcium lactate, calcium
levulinate, calcium lactobionate, calcium malate, calcium
lactophosphate, calcium succinate, calcium maleate, and calcium
sulfate. The stabilized compositions of the invention are prepared
to contain from about 0.001 to about 1 percent of the stabilized
composition with respect to the calcium ion. Preferably from about
0.005 to about 0.05 percent with respect to the calcium ion, is
employed for best stabilization. As described hereinbefore, certain
of the commercially available enzyme preparations suitable herein
contain, in addition to active enzyme, certain inert materials
including for example, calcium chloride or calcium sulfate. When
such an enzyme preparation is employed as the source of amylolytic
enzyme, an amount of calcium ion is also incorporated thereby.
Additional calcium ion is conveniently provided by the addition of
one or more of the calcium salts hereinbefore described so as to
provide a level of calcium ion within the hereinbefore described
range. Preferred calcium salts include calcium acetate, calcium
sulfate and calcium chloride.
The organic co-stabilizers which in concert with calcium ion
provide enhanced amylolytic enzyme activity include the aliphatic
glycols and 1,3-propanediol. The aliphatic glycols employed herein
have the formula
HO[CH.sub.2 CH.sub.2 O].sub.x H
wherein x is from 1 to about 200, and include ethylene glycol and
the polyethylene glycols. The polyethylene glycols useful herein
are those wherein x in the hereinbefore described formula ranges
from 2 to about 200 and include diethylene glycol, triethylene
glycol and the corresponding polymers of ethylene oxide wherein the
average number of oxyethylene groups ranges upward from 4
(tetraethylene glycol) to about 200.
The aliphatic glycols useful herein range in consistency from light
liquids to white waxy solids and dissolve in water to form clear
solutions. Preferred aliphatic glycols herein include diethylene
glycol and triethylene glycol. Also preferred are the polyethylene
glycols wherein the average value of x is from 4 to about 80. These
polyethylene glycols have average molecular weights of about 200 to
about 3500 and are commercially available under the trade
designation "Carbowax" with numerical designation referring to
average molecular weight, e.g. 200, 400, 600, 1000, or the like.
The upward numerical gradation corresponds to increasing molecular
weight, increasing melting point and decreasing water-solubility.
Mixtures of aliphatic glycols of the invention can be employed
herein.
It has been found that 1,3-propanediol also provides an amylolytic
enzyme-stabilizing effect as hereinbefore described. This compound
is a preferred co-stabilizing agent herein and provides excellent
stabilizing effects.
The organic amylolytic enzyme co-stabilizing compounds of this
invention are employed in minor but effective amounts ranging from
about 2 to about 27 percent by weight of the composition.
Preferably the stabilized compositions are prepared to contain from
about 5 to about 20 percent by weight of the co-stabilizing agent.
The latter range is preferred from the standpoint of optimum
stabilizing effects, particularly over long storage periods at high
temperatures.
While the mechanism by which the calcium salts and organic
co-stabilizing agent hereinbefore described coact to protect
amylolytic enzymes against loss of activity is not precisely known,
the combination of salt and organic compound provides levels of
enzyme stability substantially greater than can be achieved by
conventional calcium stabilization alone. This stabilization effect
is observed even in the presence of proteases which tend to exert a
harmful denaturing effect on .alpha.-amylases.
Water-soluble nonionic and zwitterionic detergents can be employed,
as optional ingredients, in the compositions of this invention.
These detergents enhance considerably the storage stability of the
amylolytic enzymes employed herein and significantly improve the
detergent characteristics of the composition. Because of these
useful characteristics it is preferred to include nonionic and
zwitterionic detergents in the aqueous enzyme compositions of the
invention. The nonionics and zwitterionics can be utilized herein
in amounts ranging from 0 to about 15 percent, and preferably from
4 to 10 percent, by weight of the enzyme-containing
composition.
Examples of suitable nonionics for use herein include:
1. The polyethylene oxide condensates of alkyl phenols, e.g., the
condensation products of alkyl phenols having an alkyl group
containing from about six to 12 carbon atoms in either a straight
chain or branched chain configuration with ethylene oxide, the said
ethylene oxide being present in amounts equal to 5 to 25 moles of
ethylene oxide per mole of alkyl phenol. The alkyl substituent in
such compounds may be derived from polymerized propylene,
diisobutylene, octene or nonene, for example. 2. Those nonionic
synthetic detergents derived from the condensation of ethylene
oxide with the product resulting from the reaction of propylene
oxide and ethylene diamine. For example, compounds containing from
about 40 to about 80 percent polyoxyethylene by weight and having a
molecular weight of from about 5,000 to about 11,000 resulting from
the reaction of ethylene oxide groups with a hydrophobic base
constituted of the reaction product of ethylene diamine and excess
propylene oxide, said base having a molecular weight of the order
of 2,500 to 3,000 are satisfactory. 3. The condensation product of
1 mole of aliphatic alcohols having from eight to 22 carbon atoms,
in either straight chain or branched chain configuration, with from
5 to 40 moles of ethylene oxide, e.g., a coconut alcohol-ethylene
oxide condensate having from 5 to 40 moles of ethylene oxide per
mole of coconut alcohol, the coconut alcohol fraction having from
10 to 14 carbon atoms. 4. The unsubstituted amides and the
monoethanol and diethanol amides of fatty acid having acyl moieties
of from about eight to about 22 carbon atoms. These acyl moieties
are normally derived from naturally occurring glycerides (e.g.,
coconut oil, palm oil, soybean oil and tallow), but can be derived
synthetically (e.g., by the oxidation of petroleum, or by
hydrogenation of carbon monoxide by the Fischer-Tropsch process).
5. Long chain tertiary amine oxides corresponding to the following
general formula ##SPC1##
wherein R.sup.1 is an alkyl radical of from about eight to about 22
carbon atoms, R.sup.2 and R.sup.3 are each methyl, ethyl or
hydroxyethyl radicals, R.sup.4 is ethylene, and n equals from 0 to
about 10. The arrow in the formula is a conventional representation
of a semi-polar bond. Specific examples of amine oxide detergents
include: dimethyldodecylamine oxide and
bis-(2-hydroxyethyl)-dodecylamine oxide.
6. Long chain tertiary phosphine oxides corresponding to the
following general formula
RR'R"P .fwdarw.O
wherein R is an alkyl, alkenyl or monohydroxyalkyl radical ranging
from 10 to 22 carbon atoms in chain length and R' and R" are each
alkyl or monohydroxyalkyl groups containing from one to three
carbon atoms. The arrow in the formula is a conventional
representation of a semi-polar bond. Examples of suitable phosphine
oxides are found in U.S. Pat. No. 3,304,263 which issued Feb. 14,
1967, and include: dimethyldodecylphosphine oxide and
bis-(2-hydroxyethyl)dodecylphosphine oxide.
7. Long chain sulfoxides having the formula ##SPC2##
wherein R.sup.5 is an alkyl radical containing from about 10 to
about 22 carbon atoms, from 0 to about 5 ether linkages and from 0
to about 2 hydroxyl substituents, at least one moiety of R.sup.5
being uninterrupted by ether linkages and containing from about 10
to about 18 carbon atoms, and wherein R.sup.6 is an alkyl radical
containing from one to three carbon atoms and from 0 to 2 hydroxyl
groups. Specific examples of these sulfoxides are: dodecyl methyl
sulfoxide and 3-hydroxy tridecl methyl sulfoxide.
The zwitterionic synthetic detergents suitable for use herein can
be broadly described as derivatives of aliphatic quaternary
ammonium, phosphonium and sulfonium compounds, in which the
aliphatic radical may be straight chain or branched, and wherein
one of the aliphatic substituents contains from about eight to 22
carbon atoms and one contains an anionic water solubilizing group,
e.g., carboxy, sulfo, sulfato, phosphato or phosphono. Examples of
compounds falling within this definition are
3-(N,N-dimethyl-N-hexadecylammonio) propane-1-sulfonate and
3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxypropane-1-sulfonate.
For more examples of zwitterionic synthetic detergents, see Diehl
and Smith, "Laundering Fabrics in Cold Water Containing a Synthetic
Detergent Composition," Canadian Patent No. 708,147 issued Apr. 20,
1965 at page 6, lines 1-22. This disclosure is specifically
incorporated herein by reference.
Mixtures of various nonionic detergents or mixtures of nonionic
detergents and zwitterionic detergents can be employed. Preferred
herein are the condensation products of 1 mole of aliphatic alcohol
having eight to 22 carbon atoms with from 5 to 40 moles of ethylene
oxide, e.g. tallow alcohol ethoxylated with 11 or 30 moles of
ethylene oxide and coconut alcohol ethoxylated with 6 moles of
ethylene oxide. Also preferred are the
3-(N,N-dimethyl-N-alkylammonio)-2-hydroxypropane-1-sulfonates
wherein the alkyl has from eight to 22 carbon atoms, e.g.
3-(N,N-dimethyl-N-coconutalkylammonio)-2-hydroxypropane-1-sulfonate
and the 3-(N,N-dimethyl-N-alkylammonio) propane-1-sulfonates
wherein the alkyl has from eight to 22 carbon atoms, e.g.
3-(N,N-dimethyl-N-tallowalkylammonio) propane-1-sulfonate. These
compounds in addition to providing amylase stability per se enhance
the stabilization of calcium and organic costabilizing compound. In
addition they provide excellent detergency properties.
The stable compositions of the present invention are prepared to
contain from about 65 to about 97 percent by weight of water.
Preferably from about 72 to about 95 percent is employed.
Demineralized water is preferred, although not mandatory for use
herein.
The various components of the enzyme compositions of this invention
can be mixed together in any order. However, it is preferred that a
stabilizer-water mixture be prepared first and the enzymes added
thereto to prevent any degradation or deactivation which might
occur by adding the enzyme to water which does not contain the
enzyme-stabilizing combination of the invention. The optional
detergent components can be added at any time.
The pH of the stabilized aqueous enzyme compositions of this
invention generally ranges from about 5.0 to 10.0 and preferably
ranges from about 6.5 to about 8.5. Maximum stabilizing affects are
obtained in the preferred pH range. The pH can be raised with a
base, e.g., sodium or potassium hydroxide, or lowered with an acid,
e.g., hydrochloric acid.
It is also preferred, although not mandatory, that a preservative
be added to the compositions to prevent bacterial and fungal
growth. Phenyl mercuric acetate which is generally utilized herein
in amounts ranging from about 10 to about 40 parts per million of
the compositions is an effective preservative. Any preservative
compatible with the components of the compositions can be utilized
herein.
The stabilized aqueous compositions of this invention can also
contain any of the usual detergent adjuvents, diluents and
additives so long as they do not substantially interfere with the
activity of the enzymatic components. For example, perfumes,
anti-tarnishing agents, inert salts such as sodium sulfate,
anti-redeposition agents, bacteriostatic agents, dyes, fluorescers,
suds builders, suds depressors, and the like, can be utilized
herein without detracting from the advantageous properties of these
compositions. It is preferred that the compositions of the present
invention contain in addition certain proteolytic enzymes. These
enzymes include the alkaline proteases, neutral proteases, and acid
proteases which aid materially the removal of proteinaceous soils
and stains from laundered textiles. The employment of proteolytic
enzymes in combination with the amylolytic enzymes of the present
invention is preferred from the standpoint of facilitating the
removal of a broad spectrum of varied soils and stains. The
preferred proteolytic enzymes are the subtilisins, obtained from
the bacterial organism, Bacillus subtilis. When proteolytic enzymes
are included in the compositions of the present invention, it is
desirable to include any of the known proteolytic
enzyme-stabilizing materials known in the art to thereby enhance
proteolytic enzyme activity upon storage. Suitable proteolytic
enzyme stabilizing materials are described for example in Ser. No.
683,196, entitled "Stabilized Aqueous Enzyme Preparation" filed
Nov. 15, 1967 by Charles Bruce McCarty.
The compositions of this invention can be employed as spot
removers, detergent additives or as detergent cleaning compositions
per se. These compositions can be packaged in spray-type bottles
and conveniently used to remove relatively small spots from fabrics
or can be employed in larger quantities as additives to other
detergent compositions. These compositions can be substituted for
hypochlorite bleaches as they remove any of the stains which these
bleaches remove, do not weaken textile fibers, and do not attack or
degrade fluorescers and whiteners. With the addition of optional
nonionic and/or zwitterionic detergents, these compositions can be
utilized per se as excellent cleaning compositions under a variety
of washing conditions.
EXAMPLES
The following examples merely serve to illustrate the invention in
specific detail and when read in conjunction with the foregoing
description will aid in determining the full scope of the present
invention. The examples are merely illustrative and are not
intended to restrict this invention. All parts, percentages and
ratios set forth herein are by weight unless otherwise
indicated.
The following compositions were prepared and stored in closed glass
bottles for the lengths of time indicated in Table I. Each
composition containined 10 percent by weight of the organic
co-stabilizing agent; 1 percent Monsanto CRD-Protease (a
commercially available mixture of proteases and amylases derived
from Bacillus subtilis); and 89 percent of an aqueous stock
solution containing 0.01 percent calcium acetate monohydrate and
0.29 percent sodium chloride. The amylolytic activity of each
composition was measured at the stated intervals by the assay
method hereinbefore described.
Control samples stored under identical conditions were also
evaluated for retention of enzymatic activity. In Control-1, no
organic co-stabilizing agent was employed. In the case of
Control-2, no organic co-stabilizing agent was present and the
stock solution was replaced with distilled water, i.e., no calcium
acetate monohydrate or sodium chloride was present. The results are
tabulated as follows:
Table I
__________________________________________________________________________
Percent Remaining Activity Organic Co- After Storage at
100.degree.F. for Example % Ca.sup.+.sup.+ Stabilizing Agent Weeks
__________________________________________________________________________
2 4 6 8 Control-1 0.0023 None 54 34 29 17 Control-2 None None 0* --
-- -- 1 0.0021 Ethylene Glycol 70 51 34 28 2 0.0021 Diethylene
Glycol 71 64 44 37 3 0.0021 1,3-propanediol 60 47 43 35
__________________________________________________________________________
*3 days
The following stabilized compositions, Examples 4to 29, were
prepared. In each example, the water (containing calcium acetate
monohydrate and sodium chloride) organic co-stabilizing agent and
ethanol (lwhere employed) were thoroughly mixed, the nonionic or
zwitterionic was added and the enzyme was added last. The enzymes
employed were Alcalase (a proteolytic enzyme preparation having a
crystalline enzyme content of about 6 percent and derived from
Bacillus subtilis); and/or Monsanto CRD-Protease (a mixture of
proteolytic and amylolytic enzymes derived from Bacillus subtilis).
Ethanol, where present, was employed as a stabilizer for the
proteolytic enzyme. In each example, an aqueous stock solution,
hereinbefore described, was employed in an amount to bring the
balance of the composition to 100 percent. The compositions were
stored for the periods of time indicated in Table II at a
temperature of 100.degree.F. and their amylolytic activity
evaluated as hereinbefore described. The compositions of Examples 4
to 29 perform well as spot removers, as additives to detergent
compositions and as laundry detergents per se.
Table II
__________________________________________________________________________
% Remaining % Enzyme After Storage Mon- at 100.degree.F. for % %
Alca- santo % Surfactant* Weeks Ex. Ca.sup.+.sup.+ Ethanol %
Organic Co-stabilizer lase CRD A B C D E 2 4 6 8
__________________________________________________________________________
4 0.0017 10 Diethylene Glycol (10%) 0.5 0.5 5 - - - - 100 100 88
100 5 0.0019 5 Diethylene Glycol (5%) 0.5 0.5 5 - - - - 100 79 50
50 6 0.0017 10 Triethylene Glycol (10%) 0.5 0.5 5 - - - - 100 83 58
63 7 0.0019 5 Triethylene Glycol (5%) 0.5 0.5 5 - - - - 98 70 53 30
8 0.0017 10 Polyethylene Glycol 380 (10%) 0.5 0.5 5 - - - - 90 83
45 33 9 0.0019 5 Polyethylene Glycol 380 (5%) 0.5 0.5 5 - - - - 82
61 31 11 10 0.0017 10 Polyethylene Glycol 4000 (10%) 0.5 0.5 5 - -
- - 70 42 18 7 11 0.0019 5 Polyethylene Glycol 4000 (5%) 0.5 0.5 5
- - - - 79 32 18 4 12 0.0020 - Triethylene Glycol (10%) 0.5 0.5 - -
- - - 90 100 100 100 13 0.0020 - Polyethylene Glycol 4000 (10%) 0.5
0.5 - - - - - 78 87 83 82 14 0.0019 10 Diethylene Glycol (10%) 0.5
0.5 - 5 - - - 68 75 59 59 15 0.0017 10 Diethylene Glycol (10%) 0.5
0.5 - - 5 - - 95 77 45 54 16 0.0017 10 Triethylene Glycol (10%) 0.5
0.5 - - - 5 - 100 85 75 74 17 0.0017 10 Polyethylene Glycol 380
(10%) 0.5 0.5 - - - 5 - 91 73 64 65 18 0.0017 10 Polyethylene
Glycol 4000 (10%) 0.5 0.5 - - - 5 - 87 69 57 64 19 0.0020 -
Diethylene Glycol (10%) 0.5 0.5 - - - - - 96 73 73 75 20 0.0018 -
Diethylene Glycol (10%) - 1.0 10 - - - - - 95 - 89 21 0.0018 -
Diethylene Glycol (10%) - 1.0 - 10 - - - - 87 - 72 22 0.0018 -
Diethylene Glycol (10%) - 1.0 - - 10 - - - 59 - 49 23 0.0018 -
Diethylene Glycol (10%) - 1.0 - - - 10 - - 51 - 36 24 0.0018 -
Diethylene Glycol (10%) - 1.0 - - - - 10 - 48 - 30 25 0.0018 -
1,3-Propanediol (10%) - 1.0 10 - - - - - 89 - 81 26 0.0018 -
1,3-Propanediol (10%) - 1.0 - 10 - - - - 77 - 72 27 0.0018 -
1,3-Propanediol (10%) - 1.0 - - 10 - - - 86 - 65 28 0.0018 -
1,3-Propanediol (10%) - 1.0 - - - 10 - - 100 - 55 29 0.0018 -
1,3-Propanediol (10%) - 1.0 - - - - 10 - 100 - 74
__________________________________________________________________________
*A--Tallow alcohol ethoxylated with 11 moles of ethylene oxide.
*B--Tallow alcohol ethoxylated with 30 moles of ethylene oxide.
*C--Coconut alcohol ethoxylated with 6 moles of ethylene oxide.
*D--HAPS - 3-(N,N-dimethyl-N-alkylammonio)2-hydroxy
propane-1-sulfonate wherein the alkyl group is derived from
middle-cut coconut alcohol: 2% C.sub.10 ; 66% C.sub.12 ; 23%
C.sub.14 and 9% C.sub.16. *E--3-(N,N-dimethyl-N-tallowalkylammonio)
propane-1-sulfonate.
EXAMPLE 30
A stabilized aqueous enzyme composition is formulated according to
this invention from the following components:
Component Weight % ______________________________________
.alpha.-Amylase (Diasmen, Daiwa Kasei KK) 1 Calcium Chloride 0.01
Polyethylene Glycol 4000 10 Alcalase (6% proteolytic enzyme) 1
Ethanol 10 Water 78 ______________________________________
This composition can be employed without dilution as a soil- and
stain-removing composition to remove or facilitate removal of
starchy and proteinaceous matter from textile materials. The soil-
and stain-removing efficacy is demonstrated even after extended
periods of storage (8 weeks) at elevated temperature
(120.degree.F.). The composition of this example can be employed as
an additive to commercial detergent formulations. When about 1.2
ml. of the composition is added per gallon of washing solution,
excellent soil- and stain-removing properties are demonstrated.
EXAMPLE 31
Similar results are obtained when the following organic
co-stabilizing compounds are employed in lieu of the co-stabilizing
compounds employed in Examples 1 to 29 in that the amylolytic
enzyme is stabilized in aqueous solution: ethylene glycol;
diethylene glycol; triethylene glycol; tetraethylene glycol;
polyethylene glycol 200; polyethylene glycol 300; polyethylene
glycol 380; polyethylene glycol 600; polyethylene glycol 1000;
polyethylene glycol 1500; polyethylene glycol 4000; polyethylene
glycol 6000; and 1,3-propanediol.
Similar results are obtained when the following amylolytic enzymes
are employed in lieu of those employed in Examples 1 to 29 in that
the .alpha.-amylase is stabilized in aqueous solution: Diasmen
.alpha.-amylase; Rapidase .alpha.-amylase THC-25; Novo Bacterial
.alpha.-amylase; Wallerstein .alpha.-amylase; Rhozyme-33 and
Rhozyme H-39.
Similar results are obtained when the following nonionic and
zwitterionic detergents are substituted for the tallow alcohol
ethoxylates, coconut alcohol ethoxylate,
3-(N,N-dimethyl-N-middlecut-coconut-alkylammonio)2-hydroxypropane-1-sulfon
ate and 3-(N,N-dimethyl-N-tallowalkylammonio) propane-1-sulfonate
employed in Examples 4 to 11, 14 to 18 and 20 to 29 in that the
stabilization by calcium ion and organic co-stabilizing compound is
enhanced and excellent cleaning properties are provided: decyl
phenol ethoxylated with 20 moles of ethylene oxide per mole of
decyl phenol, hexadecanoic amide, hexadecanoic diethanol amide,
dimethyldodecylamine oxide, dimethyldodecylphosphine oxide, and
dodecyl methyl sulfoxide, the condensation product of ethylene
oxide with the condensation product of propylene oxide with
propylene glycol, the ethylene oxide portion of the compound being
50 percent of the total weight of the compound and the total
molecular weight of the compound being about 1700; the condensation
product of ethylene oxide with the condensation product of
propylene oxide and ethylene diamine wherein the product contains
about 65 percent polyethylene oxide by weight and the total
molecular weight of the compound is 6000.
Similar results are obtained when the calcium acetate monohydrate
of Examples 1 to 29 is replaced with the following calcium salts in
amounts providing an equal amount of calcium ion in that
stabilization of amylolytic enzyme in aqueous solution is observed:
calcium chloride; calcium citrate; calcium glycerol phosphate;
calcium gluconate; calcium glucoheptanate; calcium lactate; calcium
levulinate; calcium lactobionate; calcium malate; calcium
lactophosphate; calcium succinate; calcium maleate; and calcium
sulfate.
* * * * *