Note: Descriptions are shown in the official language in which they were submitted.
WO 94n9424 ~ 1 6 4 615 PCTAJS94/06522
SYNERGISTICALLY STABILIZED LIQUID ENZYMATIC CO~PnSITIONS
The present invention relates to novel formulations for
stabilizing at least one enzyme contained in a liquid
enzymatic composition. The unique rheological properties of
the components of these stabilizing formulations preferably
afford synergiRtic stabilizing capacity over other water-
based mixtures of enzymatic dispersions, even under
conditions of moderate to high heat and wide pH ranges. The
invention, therefore, also relates to stabilized liquid
enzymatic compositions. Additionally, the invention relates
to novel methods for the preparation of stabilized liquid
enzymatic compositions, and methods using the stabilizing
formulations with liquid enzymatic compositions.
The use of enzymes and liquid enzymatic compositions in
industry and in the commercial marketplace has grown rapidly
over the last several years. A~ is well-known, enzymes can
be acid, alkaline or neutral, depending upon the pH range in
which they are active. All of these types of enzymes are
contemplated to be useful in connection with the invention
discloRed herein.
Many enzymes and liquid enzymatic compositions have been
associated with liquid detergents and have shown utility as
solubilizing and cleaning formulations. In addition to their
association with liquid detergents, enzymes and liquid enzy-
matic compositions have also shown utility in a number of
different commercial and industrial sreas in which a wide
variety of enzyme classes are now used.
Proteases are a well-known class of enzymes frequently
utilized in a wide variety of industrial applications where
they act to hydrolyze peptide bonds in proteins and
proteinaceous substrates. Commercially, the greatest uses of
proteases are made in the laundry detergent industry, where
they help to le...~ve protein-based stains such as blood or egg
stains, and in the cheese-making industry, where they aid in
curdling milk. Proteases are also used as meat tenderizers,
for softening leather, for modifying food ingredients, and
WOg4~9~ PCT~S94/06522
2~6 461~ - 2 -
for flavor development. Liquid enzymatic compositions con-
t~ining alkaline proteases have also shown to be useful as
dispersants of bacterial films and algal and fungal mats in
cooling tower waters and metalworking fluid containment bays.
Proteases can be characterized as acid, neutral, or
alkaline proteases depending upon the pH range in which they
are active. The acid proteases include the microbial ren-
nets, rennin (chymosin), pepsin, and fungal acid proteases.
The neutral proteases include trypsin, papain, bromelain/
ficin, and bacterial neutral protease. The alkaline
proteases include subtilisin and related proteases.
Commercial liquid enzymatic compositions contAin;ng proteases
are available under the names Rennilase~,~"PTN~' (Pancreatic
Trypsin NOVO), "PEM" (Proteolytic Enzyme Mixture), Neutrase~,
Alcalase~, Esperase~, and Savinase'n which are all supplied by
Novo Nordisk Bioindustrials, Inc. of Danbury, CT. Another
commercial protease is available under the name HT-
Proteolytic supplied by Solvay Enzyme Products.
Amylases, another class of enzymes, have also been
utilized in many industrial and commercial processes in which
they act to catalyze or accelerate the hydrolysis of starch.
Amylases are used largely in the corn syrup industry for the
production of glucose syrups, maltose syrups, and a variety
of other more refined end products of starch hydrolysis such
as high fructose syrups. As a class they include ~-amylase,
~-amylase, amyloglucosidase (glucoamylase), fungal amylase,
and pullulanase. Commercial liquid enzymatic compositions
cont~in;~g amylases are available under the names BAN,
Termamyl~, AMG, Fungamyl~, and Promozyme'~, which are supplied
by Novo Nordisk, and Diazyme L-200, a product of Solvay
Enzyme Products.
Other commercially valuable enzyme classes are those
which affect the hydrolysis of fiber. These classes include
cellulases, hemicellulases, pectinases, and ~-glucanases.
Cellulases are enzymes that degrade cellulose, a linear
glucose polymer occurring in the cell walls of plants.
Hemicellulases are involved in the hydrolysis of
WOg4~94~ 216 4 ~ 15 PCT~S94/06522
-- 3 --
hemicellulose which, like cellulose, is a polysaccharide
found in plants. The pectinases are enzymes involved in the
degradation of pectin, a carbohydrate whose main component is
a sugar acid. ~-glucanases are enzymes involved in the
hydrolysis of ~-glucans which are also similar to cellulose
in that they are linear polymers of glucose. In a commercial
context, these enzymes have utility to a greater or lesser
degree in manufacturing processes dependent on fiber degra-
dation.
Cellulases have reported utility in the de-inking
process of old newsprint (ONP) wastepaper, eliminating the
need for any surfactants and alkaline chemicals. The enzymes
dislodge inks from fiber surfaces and disperse ink particles
to a finite size. See S. Say-Xyoun Ow, Biological De-Inking
Methods of Newsprint Wastepaper, World Pulp and Paper
Technology, pp. 63, 64 (1992). Collectively, cellulases
include endocellulase, exocellulase, exocello-biohydrolase,
mannase, and cellobiase. Commercial liquid enzymatic
compositions contAining cellulases are available under the
names Celluclast~ and Novozym~188 which are both supplied by
Novo Nordisk.
Hemicellulases are also used in the de-inking process to
dislodge ink particles from the fiber surface of ONP. See D.
Y. Prasad et al., Enzyme Deinking of Black and White
Letterpress Printed Newsprint Waste, Progress in Paper Recy-
cling, May 1992, pp. 21, 22. Additionally, hemicellulases,
such as the xylanases, are employed in the pulp bleaching
process. Xylanase pretreatment of kraft pulps has resulted
in major reductions in bleaching chemical requirements, such
as molecular chlorine, and has also improved pulp quality as
reflected by higher brightness ceilings. See D. J. Senior et
al., Reduction in Chlorine Use During Bleaching of Rraft Pulp
Following Xylanase Treatment, Tappi Journal (forthcoming
publication; aspects of the publication were presented at the
1991 International Pulp Bleaching Conference, Stockholm).
PULPZYM~ product, available from Novo Nordisk, and ECOPULP~
wog4ng~w PCT~S94/06522
~ 4 _
product, from Alko Biotechnology, are two examples of com-
mercially available liquid enzymatic compositions contAining
xylanase-based bleaching enzymes.
As a class, hemicellulases include hemicellulase mixture
and galactomannAnAse. Commercial liquid enzymatic composi-
tions containing hemicellulases are available as PULPZYM~
from Novo, ECOPULP~ from Alko Biotechnology and Novozym~280
and Gamanase~, which are both products of Novo Nordisk.
The pectinases are used commercially to weaken cell
walls and enhance extraction of fruit juice, as well as to
aid in decreasing viscosity and preventing gelation in these
extracts. Pectinases consist of endopolygalacturonase,
exopoly-galacturonase, endopectate lyase ~transeliminase),
exopectate lyase ttranseliminase), and endopectin lyase
(transeliminase). Commercial liquid enzymatic compositions
containing pectinases are available under the names Pectinex~
Ultra SP and Pectinex~*, both supplied by Novo Nordisk.
The ~-glucanases play an important role in the malting
and brewing industries where modification of barley cell
walls contA i n ing ~-glucans is necessary. ~-glucanases are
comprised of lichenase, lAminArinase, and exoglucanase.
Commercial liquid enzymatic compositions contAining ~-
glucanases are available under the names Novozym~234,
Cereflo~, BAN, Finizym~, and Ceremix~, all of which are
supplied by Novo Nordisk.
Two additional classes of industrially and commercially
useful enzymes are lipases and phospholipases. Lipases and
phospholipases are esterase enzymes which hydrolyze fats and
oils by attacking the ester bonds in these compounds. Li-
pases act on triglycerides, while phospholipases act on
phospholipids. In the industrial sector, lipases and
phospholipases represent the commercially available
esterases, and both currently have a number of industrial and
commercial applications.
In the pulp and paper industry, liquid enzyme
preparations containing lipases have proven to be
particularly useful in reducing pitch deposits on rolls and
WOg4~g4~ ~1 6 4 6 1~ PCT~S94/06522
other equipment during the production process. For example,
the treatment of unbleached sulfite pulp with lipases prior
to bleaching with chlorine to reduce the content of
chlorinated triglycerides, which are reportedly the cause of
pitch deposition during the paper manufacturing process, has
been reported. See K. Fischer and K. Messner, Reducing
Troublesome Pitch in Pulp Mills By Lipolytic Enzymes, Tappi
Journal, Feb. 1992, p. 130. Novo Nordisk markets two liquid
lipase preparations under the names Resinase~ A and Resinase~
A 2X, both of which, under certain conditions, reportedly
reduce pitch deposits significantly by breaking down wood
resins in pulp.
Another important use of lipases is to degrease hides
and pelts in the leather-making process. Alkaline lipases
are used in conjunction with special proteases and emulsify-
ing systems to aid degreasing, as well as to improve the
soaking and liming effect in leather-making. See J.
Christner, The Use of Lipases in the Beamhouse Processes, 87
J.A.L.C.A. 128 (1992).
Lipases have also been used for the development of fla-
vors in cheese and to improve the palatability of beef tallow
to dogs. In nonaqueous systems, lipases have been employed
to synthesize esters from carboxylic acids and alcohols.
Commercial liquid enzymatic compositions contAi~ing li-
pases are available. For example, such compositions are
available under the trade names Lipolase 100, Greasex 50L,
Palatase~A, Palatase~M, and Lipozyme~ which are all supplied
by Novo Nordisk.
With respect to the commercially useful phospholipases,
pancreatic phospholipase A2 has been used to convert lecithin
into lysolecithin. Lysolecithin reportedly is an excellent
emulsifier in the production of mayonnaise and the hAking of
bread. Commercially, phospholipase A2 is available in a
liquid enzymatic composition sold as LECITASE~ by Novo
Nordisk.
Another commercially valuable class of enzymes are the
isomerases which catalyze conversion reactions between
W094/29~ PCT~S94/065
~16 46~S - 6 -
isomers of organic compounds. The isomerases are particu-
larly important in the high fructose corn syrup industry.
For example, the aldose-ketose isomerase reaction, catalyzed
by glucose isomerase, involves the conversion of glucose to
fructose and is just one of three key enzyme reactions in the
industry. Sweetzyme~ product is a liquid enzymatic
composition cont~ining glucose isomerase which is supplied by
Novo Nordisk.
Redox enzymes are enzymes that act as catalysts in
chemical oxidationtreduction reactions and, consequently, are
involved in the breakdown and synthesis of many biochemicals.
Currently, many redox enzymes have not gained a prominent
place in industry since most redox enzymes require the
presence of a cofactor. However, where cofactors are an
integral part of an enzyme or do not have to be supplied,
redox enzymes are commercially useful, particularly in the
food processing industry.
The redox enzyme, glucose oxidase, is used to prevent
unwanted browning reactions affecting food color and flavor.
Glucose oxidase is also used as an "oxygen scavenger" to
prevent the development of off-flavors in juices and to
preserve color and stability in certain sensitive food
ingredients. The redox enzyme, catalase, has been utilized
to decompose residual hydrogen peroxide used as a sterilizing
agent. A third redox enzyme, lipoxidase (lipoxygenase),
found naturally in soya flour and not usually purified for
industrial use, is used in baking not only to obtain whiter
bread, but also to reverse the dough softening effects caused
by certain agents. Other redox enzymes have possible
applications ranging from the enzymatic synthesis of steroid
derivatives to use in diagnostic tests. These redox enzymes
include peroxidase, superoxide dismutase, alcohol oxidase,
polyphenol oxidase, xanthine oxidase, sulfhydryl oxidase, hy-
droxylases, cholesterol oxidase, laccase, alcohol
dehydrogenase, and steroid dehydrogenases.
When enzymes, such as those described above, are
prepared or sold for use in industrial processes, they
wog4ng4~ PCT~S94/065~
216461~
- 7 -
generally are formulated as water-based or aqueous liquid
enzymatic compositions designed for a particular process.
Water-based liquid enzymatic compositions may contain
additional solvents depending upon the particular enzyme or
use of the composition. These liquid enzymatic compositions,
however, have historically been plagued with problems such as
chemical instability which can result in the loss of
enzymatic activity, particularly upon storage. This critical
problem of loss of enzymatic activity upon storage has
particularly affected the liquid detergent industry.
It is not uncommon to have industrial products, such as
liquid enzymatic compositions, stored in warehouses in
various climates around the world where the product is
subjected to a temperature that may range from freezing to
above 50C for extended periods. After storage at
temperature extremes ranging from 0C to 50C for many
months, most liquid enzymatic compositions lose from 20 to
100 percent of their enzymatic activity due to enzyme
instability.
Various attempts have been made to stabilize enzymes
contained in liquid enzymatic compositions. Attempts to
increase the stability of liquid enzymatic compositions using
formulations containing alcohols, glycerols,
dialkylglycolethers, and mixtures of these and other
compounds have had only marginal success, even in moderate
storage temperature ranges.
In U.S. Patent No. 4,801,544, a system of ethylene gly-
col and ethoxylated linear alcohol nonionic surfactant with
hydrocarbon solvent was utilized as a stabilizer, and the
encapsulation of enzymes in micelles within the solvent/
surfactant mixture was described. The water content of the
composition was kept at less than 5 percent, and enzyme
stability was checked at 35, 70 and 100F.
The stabilization of an aqueous enzyme preparation using
certain esters has been described in U.S. Patent No.
4,548,727. The ester used as a stabilizer has the formula,
RCOOR', where R is an alkyl of from one to three carbons or
g4ng4~ PCT~S94/065
2l6 4~ 15 - 8 -
hydrogen, and R' is an alkyl of from one to six carbons. The
ester is present in the aqueous enzyme preparation in an
amount from 0.1 to about 2.5% by weight.
U.S. Patent No. 4,318,818 describes a stabilizing system
for aqueous enzyme compositions where the stabilizing system
comprises calcium ions and a low molecular weight carboxylic
acid or its salt. The pH of the stabilizing system is from
about 6.5 to about 10.
U.S. Patent No. 4,243,543 teaches the stabilization of
liquid proteolytic enzyme-con~A i n ing detergent compositions.
The detergent compositions are stabilized by adding an
antioxidant and a hydrophilic polyol to the composition while
stabilizing the pH of the composition.
U.S. Patent No. 4,169,817 teaches a liquid cle~ning
composition cont~ining stabilized enzymes. The composition
is an aqueous solution contAining from 10~ to 50% by weight
of solids and including detergent builders, surface active
agents, an enzyme system derived from Bacillus subtilis and
an enzyme stabilizing agent. The stabilizing agents comprise
highly water soluble sodium or potassium salts and/or water
soluble hydroxy alcohols and enable the solution to be stored
for extended periods without deactivation of the enzymes.
European Patent No. 0 352 244 A2 describes stabilized
liquid detergent compositions using an amphoteric surfactant.
The present invention provides a formulation capable of
synergistically stabilizing one or more enzymes contained in
a liquid enzymatic composition.
The invention, thus, also provides stabilized liquid
enzymatic compositions.
Additionally, the invention provides methods for the
preparation of stabilized liquid enzymatic compositions.
The various aspects of the invention can be broadly
accomplished by the use of a formulation for stabilizing a
liquid enzymatic composition comprising:
(a) at least one polymer selected from a
poly(cellulosic)ether, an acrylic polymer, and a polyamide,
WOg4~9424 ~15 ~ 6 15 PCT~S94/065~
(b) a C2-C6 polyhydric alcohol, and
(c) water,
wherein components (a) and (b) are present in an amount
effective to stabilize at least one enzyme contained in a
liquid enzymatic composition. More preferably, the polymer
(a) and the C2-C6 polyhydric alcohol (b) are present in a
combined amount synergistically effective to stabilize at
least one enzyme contained in a liquid enzymatic composition.
The inventive stabilizing formulation can be used with a
wide variety of enzymes utilized in liquid enzymatic
compositions performing a wide variety of functions. The
enzymes and classes of enzymes with which this stabilizing
formulation can be used include, but are not limited to,
those discussed above.
The invention also relates to a stabilized liquid
enzymatic composition comprising:
(a) at least one polymer selected from a
poly(cellulosic)ether, an acrylic polymer, and a polyamide,
(b) a C2-C6 polyhydric alcohol,
(c) water, and
(d) at least one enzyme;
wherein components (a) and (b) are present in an amount
effective to stabilize at least one enzyme in the liquid
enzymatic composition. More preferably, the polymer (a) and
the C2-C6 polyhydric alcohol (b) are present in a combined
amount synergistically effective to stabilize at least one
enzyme contained in the liquid enzymatic composition.
The invention further relates to a method for the
preparation of a stabilized liquid enzymatic composition by
combining an enzyme with the stabilizing formulation above.
Additionally, the invention relates to a method of using the
stabilizing formulation to stabilize a liquid enzymatic
formulation comprising the step of combining the stabilizing
formulation with a liquid enzymatic composition.
In a preferred embodiment, the invention provides a
formulation for stabilizing a liquid enzymatic composition
comprising:
W094/29424 ~16 4 ~15 PCT~S94/06522
-- 10 --
(a) at least one polymer selected from a
poly(cellulosic)ether, an acrylic polymer, and a polyamide,
(b) a C2-C6 polyhydric alcohol, and
(c) water,
wherein components (a) and (b) are present in a combined
amount synergistically effective to stabilize at least one
enzyme contained in a liquid enzymatic composition.
A water-soluble, or at least partially water-soluble,
polymer is used in a formulation for stabilizing a liquid
enzymatic composition or a stabilized liquid enzymatic
compositions of the invention. That is, the polymer must
have sufficient solubility to be miscible with water and form
a single phase. Having this solubility, t~he polymer should
not separate out when combined with the C2-C6 polyhydric
alcohol and water of a stabilizing formulation or with a
liquid enzymatic composition. The formulation is not
required to be a clear solution. Preferably, the formulation
is an emulsion having an apparent homogeneous texture.
In a formulation for stabilizing a liquid enzymatic
composition or stabilized liquid enzymatic composition of the
invention, the amount of polymer present also depends on the
molecular weight of the particular polymer used. The higher
the molecular weight of the polymer used, the lower the
amount of polymer generally required to stabilize an enzyme.
The polymer may preferably be used in amounts up to
about 50% by weight of the stabilizing formulation. More
preferably, the polymer is present from 0.05 to 30% by
weight, and most preferably from 1% to 10% by weight. In a
preferred embodiment, the polymer, of course, is present in
an amount that gives the desired synergistic stabilization of
a liquid enzymatic composition when combined with the
polyhydric alcohol in a water-based or aqueous formulation.
The polymer employed in the present invention is
selected from a poly(cellulosic)ether, an acrylic polymer,
and a polyamide. The polymer may be substituted or
unsubstituted. The polymer may have a slight ionic charge,
but is preferably non-ionic in nature.
W094/29424 216 ~ 61~ PCT~S94/06522
When the polymer employed is a poly(cellulosic)ether,
the polymer is preferably selected from
poly(carboxymethylcellulose)ether, poly(hydroxypropyl-
methylcellulose)ether,
poly(hydroxyethylmethylcellulose)ether,
poly(hydroxybutylmethylcellulose)ether, poly(hydroxypropyl-
cellulose)ether, and poly(ethylhydroxyethylcellulose)ether.
More preferably, the polymer is poly(carboxymethyl-
cellulose)ether. Certain poly(cellulosic)ethers uæed in the
present invention, such as poly(carboxymethylcellulose)ether,
are sold as salts, i.e. sodium salts, and possess a slight
anionic charge. Preferable poly(cellulosic)ethers are those
with molecular weights ranging from lS,000 to 100,000, but
more preferred are those with molecular weights ranging from
20,000 to 75,000.
The acrylic polymers used in ~he invention are
preferably polymers or copolymers of acrylic acid,
methacrylic acid or derivatives thereof such as esters or
salts of acrylic acids. Examples of preferred acrylic
polymers are the Rohm and Haas polymers Acrysol GS product (a
sodium polyacrylate polymer), Acrysol TI-935 product (an
acrylic polymer), and Acrylin 22 product (an acrylic
polymer). The molecular weight of the acrylic polymers can
range from approximately 5,000 to greater than 4,000,000.
Preferred acrylic polymers have molecular weights ranging
from 100,000 to greater than 4,000,000, but more preferred
are those with molecular weights ranging from 750,000 to
greater than 4,000,000. Particularly preferred are acrylic
polymers having molecular weights of 1,250,000 and 4,000,000.
Acrylic polymers having these various molecular weights are
available from Aldrich Chemical Company. Acrylic polymers
can possess an anionic charge. Preferred acrylic polymers
are those polymers or derivatives which have only a slight
anionic charge or no charge, most preferably, no charge.
When the polymer employed is a polyamide, most
preferably the polyamide is polyvinylpyrrolidone. Preferred
polyvinylpyrrolidones are those with molecular weights
W094~9~ PCT~S94/06522
2 L~ 4~1~
- 12 -
ranging from 5,000 to 400,000, but more preferred are those
with molecular weights ranging from 300,000 to 400,000, with
the higher molecular weights being preferred.
The stabilizing formulation also contains a C2-C6
polyhydric alcohol as a second component. The C2-C6
polyhydric alcohol acts synergistically with the polymer,
described above, to stabilize an enzyme in a liquid enzyme
composition. The C2-C6 polyhydric alcohol is preferably
selected from a glycol and a trihydric alcohol. More
preferably, the C2-C6 polyhydric alcohol is glycerol,
sorbitol, propylene glycol, butylene glycol, hexylene glycol,
or ethylene glycol. Most preferably, the C2-C6 polyhydric
alcohol is glycerol.
The stabilizing formulation contains the C2-C6
polyhydric alcohol in an amount sufficient, with the polymer,
to stabilize at least one enzyme in a liquid enzymatic
composition. Preferably, the C2-C6 polyhydric alcohol
present is about 0.50 to 60% by weight of the stabilizing
formulation, more preferably, about 5 to 50% by weight and,
even more preferably, between 10 and 30%. Most preferably, a
stabilizing formulation or enzymatic composition contains the
C2-C6 polyhydric alcohol in a combined amount with the
polymer to achieve synergistic stabilization.
The formulations of the invention are water-based or
aqueous formulations cont~ining sufficient water to allow the
polymer to be miscible with the formulation and not separate
out. Generally, the C2-C6 polyhydric alcohol is soluble in
water, but sufficient water should be present to allow the
formulation to form a single phase. As discussed above, the
formulation is not required to be a clear solution and
preferably is an emulsion having an apparent homogeneous
texture. Water-based formulations may contain additional
solvents other than water.
While the stabilizing formulations of the invention can
be prepared by mixing the components in any order, the
formulations are preferably prepared by adding the desired
amount of polymer to a C2-C6 polyhydric alcohol/water
W094/294~ 216 4 61~ PCT~S94/06522
mixture. The C2-C6 polyhydric alcohol/water mixture can be
prepared by means known in the art. For example, the mixture
can be prepared by simply mixing the desired polyhydric
alcohol with an appropriate amount of water, or diluting a
previously prepared mixture.
The preferred mixture of the C2-C6 polyhydric alcohol
and water may contain any percentage of C2-C6 alcohol
sufficient with the polymer discussed above to stabilize,
preferably synergistically, at least one enzyme in a liquid
enzymatic composition. Preferably, the mixture is 1-95% by
weight of the polyhydric alcohol, or water-soluble polymer
thereof; more preferably 10-50% by weight; and most
preferably, 30-50% by weight. When the polyhydric alcohol is
glycerol, the mixture is preferably a 50% by weight glycerol/
water mixture. Not being bound to a particular theory,
applicant believes that the mixture acts to wet the polymer
used in the present invention and the C2-C6 polyhydric
alcohol in the mixture, to synergistically stabilize an
enzyme.
To adequately stabilize an enzyme in a liquid enzymatic
composition according to the invention, the enzyme should
possess at least 90% activity after 30 days at 25C. The
examples below demonstrate the preferred synergistic
stabilization of various enzymes at 50C after 30 days.
The stabilizing formulation described here can be
employed with a wide variety of enzymes and industrial
processes or commercial products. The enzymes, industrial
processes and commercial products with which this stabilizing
formulation can be used include, but are not limited to,
those previously discussed.
The use of the stabilizing formulation to stabilize a
liquid enzymatic composition results in a second embodiment
of this invention, a stabilized liquid enzymatic composition.
Thus, the invention also relates to a stabilized liquid
enzymatic composition comprising at least one polymer
selected from a poly(cellulosic)ether, an acrylic polymer,
and a polyamide; a C2-C6 polyhydric alcohol; water; and at
W094~9U~ PCT~S94/06522
216 4~ 14 -
least one enzyme. The polymer and C2-C6 polyhydric alcohol
are present in a combined amount effective to stabilize,
preferably to synergistically stabilize, at least one enzyme
contained in the liquid enzymatic composition. Preferred
compositions according to the invention are capable of
developing greater viscosities than quantitatively
proportional aqueous mixtures with the same polymers.
The contemplated and preferred embodiments regarding the
polymer, the C2-C6 polyhydric alcohol, and water present in
this stabilized liquid enzymatic composition are the same as
those discussed above with respect to the stabilizing for-
mulation of this invention.
As with the stabilizing formulation,~the liquid
enzymatic composition of this invention can be practiced with
a wide variety of enzymes. These enzymes include, but are
not limited to, the enzyme classes and specific enzymes
heretofore discussed. Enzymes that may be used are derived
from animal, plant, fungal, bacterial, and synthetic sources.
Preferable water dispersible enzymes for this system are
proteases, including acid, alkaline, and neutral proteases,
which are widely used in the laundry detergent and cheese
making industries; amylases, including acid, alkaline, and
neutral amylases, used, for example, in the corn syrup in-
dustry; lipases, used in developing flavors in cheese, and in
the pulp and paper and leather making industries; cellulases,
and xylases.
Depending on the intended use, enzymes are often
packaged and sold in concentrated liquid enzymatic
compositions to be diluted prior to use. Enzymes can also be
supplied in powdered or desiccated form. The amount of
enzyme present after dilution of the concentrated enzyme
depends on the form in which the enzyme is supplied. In
general, the amount of enzyme preferably may range from 0.05
to 40% by weight of a concentrated liquid enzymatic composi-
tion, more preferably 0.5 to 25%, and most preferably lO to
20%. The stabilizing formulation of this invention is
specifically contemplated for use in concentrated liquid
wog4ng424 PCT~S94/~522
21~4615
_ 15 --
enzymatic compositions as well as with compositions already
diluted for use. The amount of stabilizing formulation
needed to stabilize, or to synergistically stabilize, a
concentrated solution can, and most likely will, differ from
that for a diluted composition. Determi~ing the appropriate
quantity of stabilizing formulation or its components can be
readily ascertained by one of ordinary skill in the art using
the method set out in the examples below. As known in the
art, the amount of enzyme present, however, is dependent upon
the activity of the particular enzyme and the desired end
use.
Depending upon the enzyme it contains and its intended
use, the pH of the final stabilized liquid~enzymatic
composition is preferably from 5.0 to 10.0, but more pref-
erably around 7Ø Most preferably, the system should be
allowed to seek its own pH, generally around neutral. But,
as understood in the art, adjustment of pH may be necessary
with a small amount of acidic or alkaline material.
The stabilized liquid enzymatic composition may be
water-based or aqueous and contain other solvents or
additives directed toward the use of the composition in a
particular industrial process. For example, the stabilized
liquid enzymatic composition can contain additives such as
surfactants, defoamers, and the like, as are known in the
art. With synergistic stabilization, such additives may be
added in amounts not interfering with the synergistic
stabilization of the liquid enzymatic composition. One
skilled in the art can readily determine such amounts.
Advantageously, when a stabilized liquid enzymatic
composition of the invention is used, the stabilizing
formulation may also act as a dispersant aid for the enzyme
in industrial process waters.
The invention also relates to a method for the
preparation of a stabilized liquid enzymatic composition
comprising the step of combining at least one enzyme with the
inventive stabilizing formulation. The invention further
relates to a method of using the stabilizing formulation to
wO94n9424 ~1~ 4 ~ 15 PCT~S94/~522
- 16 -
stabilize a liquid enzymatic composition comprising the step
of combining a liquid enzymatic composition with the
stabilizing formulation. Illustrative and preferred
components, as well as the amounts of the components used in
the method, are the same as discussed above.
One of ordinary skill would understand that the
components of the formulation for stabilizing a liquid
enzymatic composition or the stabilized liquid enzymatic
composition can be combined in any order or even
simultaneously. However, the following order is preferred:
(a) ~i~i~g a C2-C6 polyhydric alcohol with water,
(b) adding to the mixture prepared in step (a) at least
one polymer selected from a poly(cellulosic)ether, an acrylic
polymer, and a polyamide, and
(c) adding at least one enzyme to the mixture resulting
from step (b). The polymer and C2-C6 polyhydric alcohol are
preferably present in a combined amount synergistically
effective to stabilize at least one enzyme in the resulting
liquid enzymatic composition.
An alternative method for the preparation of a
stabilized liquid enzymatic composition comprises the steps
of:
(a) mixing a C2-C6 polyhydric alcohol with water,
(b) adding to the mixture prepared in step (a) at least
one polymer selected from a poly(cellulosic)ether, an acrylic
polymer, and a polyamide, and
(c) combining the formulation resulting from steps (a)
and (b) with a liquid enzymatic composition cont~i n i ng at
least one enzyme. The polymer and C2-C6 polyhydric alcohol
are preferably present in a combined amount synergistically
effective to stabilize at least one enzyme in the liquid
enzymatic composition.
In the methods according to the invention, the polymer
added in step (b) may be added alone, as an aqueous
dispersion, or as a solution where the polymer is dissolved
in water or a suitable organic solvent. When other additives
are to be included in the stabilized liquid enzymatic
W094~94~ ~16 4 6 1~ PCT~S94/~522
composition, such additives may be added at any time, but
preferably prior to step (c), or in a separate step after the
enzyme is added.
The following examples are given to illustrate the
invention. It should be understood, however, that the
invention is not to be limited to the specific conditions or
details set forth in these examples.
EXAMPLE 1
Synergism was demonstrated by testing glycerol,
designated as Compound A, and poly(carboxymethylcel-
lulose)ether, (CMC), having a 250,000 molecular weight,
designated as Compound B, or polyvinylpyrrolidone, (PVP),
having a 360,000 molecular weight, designated as Compound B'.
As illustrated by Tables 1 and 2, experiments were set up by
varying ratios of Compound A to Compound B or B' over a range
of concentrations and assaying various types of enzymes for
enhanced stabilization of enzymatic activity at 50C for 30
days. The concentration of each compound required for an
assay of 90% of the original enzymatic activity was taken as
an end point. The concentrations are expressed as percent by
weight of the cG...~oulld in the final composition including the
added enzyme with the balance being water. Water was added
to compound A, the glycerol to form a glycerol-water mixture.
End points for the compositions cont~i~ing Compound A and
Compound B or B' were then compared with the end points for
Compound A alone and Compound B or B' alone.
Synergism was determined by the method described by
Kull, F.C., Eisman, P.C., Sylwestrowicz, H.D., and Mayer,
R.L., Applied Microbiology 9: 538-541 (1961) employing the
ratio determined by
~+~
where Qa = Percentage (by weight) of aqueous mixture of
Compound A, acting alone, which produced an
end point,
W094n94~ PCT~S94/065~
216 46 15 18 -
Qb = Percentage (by weight) of aqueous mixture of
Compound B, acting alone, which produced an
end point,
QA = Percentage (by weight) of aqueous mixture of
Compound A to Compound B, which produced an
end point, and
QB = Percentage (by weight) of aqueous mixture of
Compound B to Compound A, which produced an
end point.
Where the sum of QA/Qa and QB/Qb is greater than one,
antagonism is indicated. If the sum is equal to one,
additivity is indicated. Where the sum is less than one,
synergism is demonstrated.
This procedure for demonstrating the synergism of the
compositions of this invention is a widely used and accepted
procedure. More detailed information is provided in the
article by Kull et al. Further information concerning this
procedure is contained in U.S. Pat. No. 3,231,509, the
disclosure of which is incorporated here by reference.
The results obtained, which are set forth in Tables 1
and 2, demonstrate the enhanced stabilization of the enzyme,
HT-Proteolytic L-175, an alkaline protease sold by Solvay
Enzymes, Inc. Using the method described by Rull et al., the
sums of QA/Qa + QB/Qb for all compositions cont~ining
glycerol (Compound A) and CMC (Compound B) were calculated.
Sample calculations are shown for some end points where
synergism was evident in this example. As set forth in
Table l and the sample calculations, these end point values
were 0.58, 0.67, 0.67 and 0.67, respectively, indicating the
existence of synergism. Likewise, the sums of QA/Qa + QB'/
Qb' for all compositions containing glycerol (Compound A) and
PVP (Compound B') were calculated. As set forth in Table 2
and the sample calculations, the end points were 0.85, 0.95,
0.65, 0.85, respectively, again indicating the existence of
synergism.
wO94n9424 216 ~ 6 15 PCT~S94/06522
-- 19 --
TABLE 1
Compound A (~ by weight)
0
Compound B
(% by weight)
6 + + + + + +*
3 + + + +* +*
2 + + +
+ +
O +
(1)(*) = End point of > 90% enzymatic activity after 30 days
at 50C
(2)(+) = > 90% enzymatic activity after 30 days at 50 C
(3)(-) = < 90% enzymatic activity rer--;ning after 30 days
at 50C
(4) Compound A = Glycerol
(5) Compound B = CMC
Calculations:
Qa Qb QA QB QA/Qa + QB/Qb
(%) 60 6 5 3 5/60 + 3/6 = 0.58
310/60 + 3/6 = 0.67
220/60 + 2/6 = 0.67
30 130/60 + 1/6 = 0.67
W094/2g424 ~ 1~16 1~ PCT~S94/06522
- 20 -
TABLE 2
Compound A (% by weight)
0
Compound B'
(% by weight)
+ + + + + +*
+ + + +* +*
+ + +
1 0 + + +
O +
l)(*) = End point of > 90% enzymatic activity after 30 days
at 50C
(2)(+) = ~ 90% enzymatic activity after 30 days at 50C3)(-) = < 90% enzymatic activity remaining after 30 days
at 50C
(4) Compound A = Glycerol
(5) Compound B' = PVP
alculations:
Qa Qb' QA QB'QA/Qa + QB'/Qb'
(%) S0 40 5 305/50 + 30/40 = 0.85
3010/50 + 30/40 = 0.95
1020/50 + 10/40 = 0.65
1030/50 + 10/40 = 0.85
wog4ng424 216 4 6 1~ PCT~S94/06522
EXAMPLE 2
Stabilizing formulations of the invention as in Example
1 were tested with another enzyme, Diazyme L-200, a
glucoamylase sold by Solvay Enzymes, Inc. The results, set
forth in Tables 3 and 4, also demonstrate enhanced
stabilization with this enzyme. The sums of QA/Qa + QB/Qb
for all compositions cont~;~ing glycerol (Compound A) and CMC
(Compound B) were calculated. Sample calculations are shown
for some end points where synergism was evident in this
example. As set forth in Table 3 and the sample
calculations, these end point values were 0.59, 0.67, 0.67,
and 0.84, respectively, indicating the existence of
synergism. Similarly, the sums of QA/Qa +~QB~/Qb~ for all
compositions containing glycerol (Compound A) and PVP,
(Compound B') were calculated. The end point values, as set
forth in Table 4 and the sample calculations, were 0.77,
0.53, 0.73, and 0.77, respectively, indicating the existence
of synergism.
W094~g424 2 ~ 15 PCT~S94/065
- 22 -
TABL~ 3
Compound A (% by weight)
0
Compound B
(% by weight)
6 + + + + + +*
3 + + + +* +*
2 + + +
O +
(1)(*) = End point of > 90% enzymatic activity after 30 days
at 50C
(2)(+) = > 90% enzymatic activity after 30 days at 50C
(3)(-) = < 90% enzymatic activity remaining after 30 days
at 50C
(4) Compound A = Glycerol
(5) Compound B = CMC
Calculations:
Qa Qb QA QB QA/Qa + QB/Qb
(~) 60 6 5 3 5/60 + 3/6 = 0.59
3 10/60 + 3/6 = 0.67
2 20/60 + 2/6 = 0.67
2 30/60 + 2/6 = 0.84
W094~9424 ~16 4 6 15 PCT~S94/065~
- 23 -
TABLE 4
Compound A (% by weight)
0
Compound B'
(% by weight)
+ + +
+ + + + +*
+ + +* +*
+ +
O +
1)(*) = End point of > 90% enzymatic activity after 30 days
at 50C
(2)(+) = > 90% enzymatic activity after 30 days at 50C3)(-) = < 90% enzymatic activity ro~~ining after 30 days
at 50C
(4) Compound A = Glycerol
(5) Compound B' = PVP
alculations:
Qa Qb' QA QB'QA/Qa + QB'/Qb'
(%) 50 30 5 205/50 + 20/30 = 0.77
1010/50 + 10/30 = 0.53
1020/50 + 10/30 = 0.73
30/50 +5/30 = 0.77
og4ng4W ~ ~4~ 15 PCT~S94/06522
- 24 -
EXAMPLE 3
Enzymes with esterase activity were tested with
stabilizing formulations according to the invention as in
Example 1. The results obtAine~ with the enzyme, Lipolase, a
lipase sold by Novo Nordisk Bioinduætries, Inc., are set
forth in Tables 5 and 6 and indicate the enhanced
stabilization of this enzyme. The sums of QA/Qa + QB/Qb for
all compositions contAini~g aqueous glycerol (Compound A) and
CMC (Compound B) were calculated. Sample calculations are
shown for some end points where synergism was evident in this
example. As set forth in Table 5 and the sample
calculations, these end point values were 0.43, 0.37, 0.57,
and 0.77, respectively, indicating the existence of
synergism. Similarly, the sums of QA/Qa + QB'/Qb' for all
compositions contAini~g glycerol (Compound A) and PVP
(Compound B') were calculated. The end point values, as
shown in Table 6 and the sample calculations, were 0.60,
0.70, 0.65, and 0.85, respectively, indicating the existence
of synergism.
wo 94ng424 ~16 4 615 PCT~S94/065~
- 25 -
TABLE S
Compound A (% by weight)
0
Compound B
(% by weight)
6 + + + + + +*
3 + + + + +
2 + + + + +*
+ + + +
O +
l)(*) = End point of > 90% enzymatic activity after 30 days
at 50C
(2)(+) = > 90% enzymatic activity after 30 days at 50C3)(-) = ~ gO% enzymatic activity remaining after 30 days
at 50C
(4) Compound A = Glycerol
(5) Compound B = CMC
alculations:
Qa Qb QA QBQA/Qa + QB/Qb
(~) 50 6 5 25/50 + 2/6 = 0.43
110/50 + 1/6 = 0.37
120/50 + 1/6 = 0.56
130/50 + 1/6 = 0.76
wog4ng4~ . PCT~S94/065~
21C46~15 26
TABLE 6
Compound A (% by weight)
0
Compound B'
(% by weight)
+ + + + *
+ + + + + +
+ + + +* +*
+ + +
O + -- _ _ _ _
(1)(*) = End point of > 90% enzymatic activity after 30 days
at 50C
(2)(+) = > 90% enzymatic activity after 30 days at 50C
(3)(-) = < 90% enzymatic activity r~-in;ng after 30 days
at 50C
(4) Compound A = Glycerol
(5) Compound B' = PVP
Calculations:
Qa Qb' QA QB'QA/Qa + QB'/Qb'
(%) 50 20 5 105/50 + 10/20 = 0.60
1010/50 + 10/20 = 0.70
520/50 + 5/20 = 0.65
30/50 +5/20 = 0.85
WOg4/29424 2 1 6 ~ 6 15 PCT~S94106522
- 27 -
BXAMPLE 4
- The hemicellulase, Pulpzyme HB, a xylanase sold by Novo
Nordisk Bioindustrials, Inc., was selected for enhanced
stability formulation testing as in Example l. The following
results obtained for this example are set forth in Tables 7
and 8. The sums of QA/Qa + QB/Qb for all compositions
cont~in;ng glycerol (Compound A) and CMC (Compound B) were
calculated. Sample calculations are shown for those end
points where synergism was evident in this example. As set
forth in Table 7 and the sample calculations, these end point
values were 0.43, 0.53, 0.57, and 0.77, respectively,
indicating the existence of synergism. Similarly, the sums
of QA/Qa + QB'/Qb' for all compositions cont~ining glycerol
(Compound A) and PVP (Compound B') were calculated. The end
point values were 0.35, 0.45, 0.65, and 0.85, respectively,
indicating the existence of synergism.
wOg4/29424 PCT~S94/06522
~164615
- 28 -
TABLE 7
Compound A (~ by weight)
0
Compound B
(% by weight)
6 + + + + + +*
3 + + + + +
2 + + + +* +*
+ + +
O +
(l)(*) = End point of ~ 90% enzymatic activity after 30 days
at 50C
(2)(+) = > 90% enzymatic activity after 30 days at 50C
(3)(-) = < 90% enzymatic activity r~m~ining after 30 days
at 50C
(4) Compound A = Glycerol
(5) Compound B = CMC
Calculations:
Qa Qb QA QBQA/Qa + QB/Qb
(%) 50 6 5 25/50 + 2/6 = 0.43
210/50 + 2/6 = 0.53
120/50 + 1/6 = 0.57
130/50 + 1/6 = 0.77
wog4ng424 PCT~S941065~
~1 646 1~ ~
- 29 -
TABLE 8
Compound A (% by weight)
0
Compound B'
(% by weight)
+ + +
+ + + + +
+ + + + +
+ +* +* +* +*
+
O +
(1)(*) = End point of ~ 90% enzymatic activity after 30 days
at 50C
(2)(+) = > 90% enzymatic activity after 30 days at 50C
(3)(-) = < 90% enzymatic activity remaining after 30 days
at 50C
(4) Compound A = Glycerol
(5) Compound B' = PVP
Calculations:
Qa Qb' QA QB'QA/Qa + QB'/Qb'
(%) 50 40 5 105/50 + 10/40 = 0.35
1010/50 + 10/40 = 0.45
1020/50 + 10/40 = 0.65
1030/50 + 10/40 = 0.85
