Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
5~
l C 72~8 (R)
Detergent composition
The present invention relates to detergent compositions
comprising a lipolytic enzyme. In particular the
present invention relates to liguid detergent
compositions comprising a lipolytic enzyme.
It has previously been suggested to incorporate
lipolytic enzymes into detergent compositions. For
example US 4,566,985 (Applied Biochemists, Inc)
describes the combined use of cellulase, protease,
amylase and lipase type enzymes in liquid detergent
compositions.
A problem in using lipolytic enzymes in detergent
compositions is often the occurrence of enzyme
instability, leading to a reduction in enzyme activity
upon use of the compositions, for example in the washing
of fabrics. These instability problems are especially
apparent in detergent compositions comprising lipolytic
enzymes in combination with proteolytic enzymes.
Surprisingly it has now been found that the stability of
lipolytic enzymes in detergent compositions can markedly
be improved by the use of a stabilising material which
is capable of reversibly forming a complex with the
active site of the lipolytic enzyme, said stabiliser-
enzyme complex being less prone to destabilisation
reactions.
Preferably the formation of a stabiliser-lipolytic
enzyme complex prevents the destabilisation reaction i.e
between lipolytic enzymes and proteolytic enzymes and/or
7~
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improves the resistance against denaturation and against
proteolytic breakdown of the lipolytic enzyme, such that
the half-life time of the lipase in the composition at
37C is increased by a least 25 %, more preferably at
least 50 %, most preferably at least 100 %~ For the
purpose of this invention, the half-life time of the
lipolytic enzyme is the time-period starting at the
moment of preparation of the detergent fGrmulation until
the moment that only 50% of the enzyme-activity is left.
Most preferably the stabilisation reaction is an
inhibitory reaction, whereby the stabiliser-lipolytic
enzyme complex is inactivated.
The reversibility of complex formation can generally be
effected by adding the stabilisation material in a
concentration of from 2 to 100 times the Ki, wherein the
Ki is the concentration at which 50 % of the lipase
enzymes have formed the complex~ More preferably the
concentration is from 5-25 times the Ki, most preferably
from 8 to 15 times. In use the detergent composition
will generally be diluted 50 to 500 times, especially
about 100 times, providing a concentration of stabiliser
such that the stabiliser-lipolytic enzyme complex will
be dissociated, thus providing active lipolytic enzymes
in the wash liquor.
Accordingly the present invention relates to a detergent
composition comprising a lipolytic enzyme and a
stabilising material capable of reversibly forming a
complex with the active site of the lipolytic enzyme,
therewith increasing the half-lifetime of the lipase
material.
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In a preferred embodiment of the invention the
stabiliser material is added to the detergent
composition in a concentration of from 2 to 100 times
the Ki.
For the purpose of the present invention any material
capable of reversible forming a complex with the active
site of the lipolytic enzyme may be used. In a preferred
embodiment of the invention, boronic acid materials are
used.
Surprisingly it has now been found that the stability of
lipolytic enzymes in detergent compositions can markedly
be improved by specific boronic acid derivatives as
stabilising m~terials. Boronic acid derivatives which
are especially advantageous for stabilising lipase
enzymes are of the following formula:
/ R2
R1---(Ph)n---B \ (I)
R3
wherein:
Ph is a phenyl group, optionally substituted by haloyen
groups or NH2 groups;
n is O or l;
R1 is Hydrogen, halogen, NH2 or a Cl_24 alkyl or alkenyl
group, Provided that when n is O then R1 contains at
least 3, most preferably 8-20 carbon atoms;
R2 and R3 are independently selected from -OH, -Cl, -F,
-Br and Cl_8 alkyl or alkenyl groups.
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4 ~ 7248 (R)
Accordingly a second embodiment of the invention relates
to a detergent composition comprising a lipolytic enzyme
in combination with one or more boronic acid derivatives
of formula I as indicated above.
Although applicants do not wish to be hound by any
theory, it is believed that the stabilisation of the
lipase enzyme by the boronic acid derivatives works via
lQ an inhibitory mechanism, whereby the boronic acid
derivative reacts with the active site o~ the lipolytic
enzyme, therewith protecting the enzyme against
destabilisation. In using the detergent composition,
generally a dilution step will take place, for example
by the addition of the detergent composition to the wash
liquor. It is believed that due to this dilution,
generally the protective boronic acid group will be
detached form the active site of the lipolytic enzyme,
therewith providing an active enzyme material under use
conditions. This mechanism of stabilisation is believed
to be especially advantageous if lipase enzymes are used
in combination with proteolytic enzymes, because the
boronic acid derivative tends to be bound to the active
site of the lipase, therewith preventing the
destabilisation reaction (= proteolytic breakdown) with
the proteolytic enzyme.
Preferred borcnic acid derivatives are of the formula I,
whereby at least of the groups R2 and R3 is -OH, most
preferably groups R2 and ~3 both are -OH or one of these
groups is -OH and the other is a Cl-8 alkyl or alkenyl
group.
;2 ~5~077
C 7248 (R)
For environmental reasons it is further preferred that
the in formula I n is 0 and Rl is a C3-C24 group.
Especially preferred boronic acid derivatives are of the
following formula;
OH
R4~ B ~ (II)
- R5
wherein:
R4 is a phenyl group or a C8_24 alkyl or alkenyl group;
R5 is a -OH or a Cl_8 alkyl or alkenyl group.
The level of boronic acid derivatives in the detergent
compositions according to the present invention may be
varied within a broad range, dependant on the level of
lipolytic enzyme and the reactivity of the boronic acid
derivative. Generally the level of boronic acid
derivatives will be from 0.00001 to 3 % by weight of the
composition, more preferred from 0.0001 to 1 %, most
preferred from 0.001 to 0.5 ~.
The added amount of lipolytic enzyme in the composition
is preferably from 50-30,000 LU/g of detergent
composition, whereby LU (lipase units) are defined as
they are in EP 258 068. The level of lipolytic enzymes
is often at least 100 LU/g, very usefully at least 250
LU/g, preferably less than 5000 LU/g, more preferably
less than 2000 LU/g or less than 1000 LUtg.
The lipolytic enzyme can be chosen from among a wide
range of lipases: in particular the lipases described in
for example the following patent specifications, EP 214
761 (NOVO), EP 258 068 (NOVO) and EP 305 216 (NOVO), and
especially lipolytic enzymes showing immunological
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cross-reactivity with antisera raised against lipases
from Thermomyces lanuginosus ATCC 22070, EP 205 208
(UNILEVER) and EP 206 390 (UNILEVER), and especially
lipases showing immunological cross-reactivity with
antisera raised against lipase from Chromobacter
viscosum var lipolyticum NRRL B-3673 , or against
lipase from Alcaligenes PL-679, ATCC 31371 and FERM-P
3783, also the lipases described in specifications Wo
87/00859 (Gist-Brocades), WO 89/09263 (Gist Brocades),
EP 331 376 (AMANO), DE 39 08 131 (Toyo Jozo) and EP 204
284 (SAPPORO BREWERIES). Suitable in particular are for
example the following commercially available lipase
preparations: Novo Lipolase, Amano Lipase CE, P, B, AP,
M-AP, AML and CES, and Meito lipases MY-30, OF, and PL,
also esterase MM. lipozym, SP 225, SP 285, Siaken
lipase, Enzeco lipase, Toyo Jozo lipase and Diosynth
lipase.
These commercially available enzymes are preferably used
at levels of from 0.01 to 10 ~ by weight of the
detergent composition, more preferred 0.05 to 5 %, most
preferred 0.1 to 2 %, whereby the levels relate to the
enzyme preparation in the form as supplied.
Also lipase enzymes produced by genetic engineering may
be used, such as for example described in WO 89~09263
(Gist-Brocades) and EP 218 272 (Gist-Brocades) as well
as EP 258 068 (Novo) and EP 305 216 (Novo).
As stated above the use of boronic acid derivatives for
the stabilisation of lipolytic enzymes is especially
advantageous for detergent compositions comprising
lipases in combination with proteolytic enzymes.
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Protease can preferably be included in the composition
in amounts of the order of about 1 to 100 GU/mg
detergent composition. Preferably the amount ranges from
2-50 and more preferably from 5 to 20 GU/mg. A GU is a
Glycine unit, defined as the proteolytic enzyme activity
which, under standard conditions, during a 15 minute
incubation at 40C, with N-acetyl casein as substrate,
produces an amount of NH2 groups equivalent to 1
micromole of glycine.
A preferred example of a protease enzyme which may be
used in compositions according to the invention is the
subtilisin variety sold as Savinase (TM of Novo-Nordisk
A/S) or Maxacal (TM of Gist-Brocades/IBIS) or as
Opticlean (ex MKC) or API22 (ex Showa Denko). Other
useful examples of proteases include Maxatase, Esperase,
Alcalase, proteinase K and subtilisin BPN', or variants
obtained by enzyme engineering.
Commercially available proteolytic enzymes are
preferably used at a level of from 0.01 to lO % by
weight of the composition, more preferably 0.05 to 2 %,
most preferred 0.1 to 2 %, whereby the levels relate to
the enzyme preparation in the form as supplied.
Protease with a high isoelectric point (e.g Savinase or
Maxacal) have been found more stable under the
conditions generally encountered in detergent
compositions of the invention. Particularly good results
have been obtained while using a hiqh pI protease (ie pI
above 9, say about 10) in a liquid detergent composition
of pH less than about 9.
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Detergent compositions of the invention may take any
suitable physical form such as powders, pastes, tablets
and liquids. A preferred embodiment of the present
invention relates to liquid detergent compositions, in
particular liquid detergent compositions comprising an
aqueous phase.
Compositions of the present invention also comprise
detergent active materials. In the widest definition the
detergent active materials in general, may comprise one
or more surfactants, and may be selected from anionic,
cationic, nonionic, zwitterionic and amphoteric species,
and (provided mutually compatible) mixtures thereof. For
example, they may be chosen from any of the classes,
sub-classes and specific materials described in "Surface
Active Agents" Vol. I, by Schwartz & Perry, Interscience
1949 and "Surface Active Agents" Vol. II by Schwartz,
Perry & Berch (Interscience 1958), in the current
edition of "McCutcheon's Emulsifiers & Detergents"
published by the McCutcheon division of Manufacturing
Confectioners Company or in Tensid-Taschenbuch", H.
Stache, 2nd Edn., Carl Hanser Verlag, Munchen & Wien,
19~1 .
Suitable nonionic surfactants include, in particular,
the reaction products of compounds having a hydrophobic
group and a reactive hydrogen atom, for example
aliphatic alcohols, acids, amides or alkyl phenols with
alkylene oxides, especially ethylene oxide either alone
or with propylene oxide. Specific nonionic detergent
compounds are alkyl tC6-C18) primary or secondary
linear or branched alcohols with ethylene oxide, and
products made by condensation of ethylene oxide with the
.eaction products of propylene oxide and
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ethylenediamine. Other so-called nonionic detergent
compounds include long chain tertiary amine oxides, long
chain tertiary phosphine oxides and dialkyl sulphoxides.
Also possible is the use of salting out resistant active
materials, such as for example described in EP 328 177,
especially the use of alkyl poly glycoside surfactants,
such as for example disclosed in EP 70 074.
Suitable anionic surfactants are usually water-soluble
alkali metal salts of organic sulphates and sulphonates
having alXyl radicals containing from about S to about
22 carbon atoms, the term alkyl being used to include
the alkyl portion of higher acyl radicals. Examples of
suitable synthetic anionic detergent compounds are
sodium and potassium alkyl sulphates, especially those
obtained by sulphating higher (C8-C18) alcohols produced
for example from tallow or coconut oil, sodium and
potassium alkyl (Cg-C20) benzene sulphonates,
particularly sodium linear secondary alkyl (C10-C15)
benzene sulphonates; sodium alkyl glyceryl ether
sulphates, especially those ethers of the higher
alcohols derived from tallow or coconut oil and
synthetic alcohols derived from petroleum; sodium
coconut oil fatty monoglyceride sulphates and
sulphonates; sodium and potassium salts o~ sulphuric
acid esters of higher (C8-C18) fatty alcohol-alkylene
oxide, particularly ethylene oxide, reaction products;
the reaction products of fatty acids such as coconut
fatty acids esterified with isethionic acid and
neutralised with sodium hydroxide; sodium and potassium
salts of fatty acid amides of methyl taurine; alkane
monosulphonates such as those derived by reacting alpha-
C 7248 ~R)
olefins (c8-C20) with sodium bisulphite and those
derived from reacting paraffins with SO2 and C12 and
then hydrolysing with a base to produce a random
sulphonate; and olefin sulphonates, which term is used
to describe the material made by reacting olefins,
particularly Cl~-C20 alpha-olefins, with SO3 and then
neutralising and hydrolysing the reaction product. The
preferred anionic detergent compounds are sodium (C11-
C15) alkyl benzene sulphonates and sodium or potassium
primary (C10-Cl8) alkyl sulphates.
It is also possible, and sometimes preferred, to include
an alkali metal soap of a fatty acid, especially a soap
of an acid having from 12 to 18 carbon atoms, for
example oleic acid, ricinoleic acid, and fatty acids
derived from castor oil, alkylsuccinic acid, rapeseed
oil, groundnut oil, coconut oil, palmkernel oil or
mixtures thereof. The sodium or potassium soaps of these
acids can be used.
In many (but not all) cases, the total detergent active
material may be present at from 2% to 60% by weight of
the total composition, for example from 5% to 50~ and
typically from 10% to 40% by weight. However, one
preferred class of compositions comprises at least 20%,
most preferably at least 25% and especially at least 30%
of detergent active material based on the weight of the
total composition.
Liquid detergent compositions of the invention may be
un-structured (isotropic) or structured. --
Structured liquids of the invention may be internally
structured whereby the structure is formed by the
detergent active materials in the composition or
11 C 7248 (R)
externally structured, whereby the structure is provided
by an external structurant. Preferably compositions of
the invention are internally structured. Most preferably
compositions of the invention comprise a structure of
lamellar droplets comprising surfactant materials.
Some of the different kinds of active-structuring which
are possible are described in the reference H.A. Barnes,
"Detergents", Ch.2. in K. Walters (Ed), "~heometry:
Industrial Applications", J. Wiley & Sons, Letchworth
1980. In general, the degree of ordering of such systems
increases with increasing surfactant and/or electrolyte
concentrations. At very low concentrations, the
surfactant can exist as a molecular solution, or as a
solution of spherical micelles, both of these being
isotropic. With the addition of further surfactant
and/or electrolyte, structured (antisotropic) systems
can form. They are referred to respectively, by various
terms such as rod-micelles, planar lamellar structures,
lamellar droplets and liquid crystalline PhaseS. Often,
different workers have used different terminology to
refer to the structures which are really the same. For
instance, in European patent specification EP-A-151
884, lamellar droplets are called "spherulites". The
presence and identity of a surfactant structuring system
in a liquid may be determined by means known to those
skilled in the art for example, optical techniques,
various rheometrical measurements, x-ray or neutron
diffraction, and sometimes, electron microscopy.
When the compositions are of lamellar droplet structure
then in many cases it is preferred for the aqueous
continuous phase to contain dissolved electrolyte. As
used herein, the term electrolyte means any ionic water
Q~77
12 c 7248 (R)
soluble material. However, in lamellar dispersions, not
all the electrolyte is necessarily dissolved but may be
suspended as particles of solid because the total
electrolyte concentration of the liquid is higher than
the solubility limit of the electrolyte. Mixtures of
electrolytes also may be used, with one or more of the
electrolytes being in the dissolved aqueous phase and
one or more being substantially only in the suspended
solid phase. Two or more electrolytes may also be
distributed approximately proportionally, between these
two phases. In part, this may depend on processing, e.g.
the order of addition of components. On the other hand,
the term "salts" includes all organic and inorganic
materials which may be included, other than surfactants
and water, whether or not they are ionic, and this term
encompasses the sub-set of the electrolytes (water
soluble materials).
The selection of surfactant types and their proportions,
in order to obtain a stable liquid with the required
structure will be fully within the capability of those
skilled in the art. However, it can be mentioned that an
important sub-class of useful compositions is those
where the detergent active material comprises blends of
different surfactant types. Typical blends useful for
fabric washing compositions include those where the
primary surfactant(s) comprise nonionic and/or a non-
alkoxylated anionic and/or an alkoxylated anionic
surfactant.
In the case of blends of surfactants, the precise
proportions of each component which will result in such
stability and viscosity will depend on the type(s) and
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13 C 7248 (R)
amount(s) of the electrolytes, as is the case with
conventional structured liquids.
Preferably though, the compositions contain from 0% to
60%, especially from 1- 60 %, more preferably 10 to 45%
of a salting-out electrolyte. Salting-out electrolyte
has the meaning ascribed to in specification EP-A-79
646, that is salting-out electrolytes have a lyotropic
number of less than 9.5. Optionally, some salting-in
electrolyte (as defined in the latter specification) may
also be included, provided it is of a kind and in an
amount compatible with the other components and the
composition is still in accordance with the definition -
of the invention claimed herein. Some or all of the
electrolyte (whether salting-in or salting-out), or any
substantially water insoluble salt which may be present,
may have detergency builder properties. In any event, it
is preferred that compositions according to the present
invention include detergency builder material, some or
all of which may be electrolyte. The builder material is
any capable of reducing the level of free calcium ions
in the wash liquor and will preferably provide the
composition with other beneficial properties such as the
generation of an alkaline pH, the suspension of soil
removed from the fabric and the dispersion of the fabric
softening clay material. Preferably the salting-out
electrolyte comprises citrate.
Examples of phosphorus-containing inorganic detergency
builders, when present, include the water-soluble salts,
especially alkali metal pyrophosphates, orthophosphates,
polyphosphates and phosphonates. Specific examples of
inorganic phosphate builders include sodium and
potassium tripolyphosphates, phosphates and
14 c 7248 (R)
hexametaphosphates. Phosphonate sequestrant builders may
also be used.
Examples of non-phosphorus-containing inorganic
detergency builders, when present, include water-
soluble alkali metal carbonates, bicarbonates, silicates
and crystalline and amorphous aluminosilicates. Specific
examples include sodium carbonate (with or without
calcite seeds), potassium carbonate, sodium and
- 10 potassium bicarbonates, silicates and zeolites.
Examples of organic detergency builders, when present,
include the alkaline metal, ammonium and substituted
ammonium polyacetates, carboxylates, polycarboxylates,
polyacetyl carboxylates and polyhydroxysulphonates.
Specific examples include sodium, potassium, lithium,
ammonium and substituted ammonium salts of
ethylenediaminetetraacetic acid, nitrilitriacetic acid,
oxydisuccinic acid, CMOS, TMS, TDS, melitic acid,
benzene polycarboxylic acids and citric acid.
Preferably the level of non-soap builder material is
from 0-50% by weight of the composition, more preferred
from 5-40%, most preferred 10-35%.
In the context of organic builders, it is also desirable
to incorporate polymers which are only partly dissolved,
in the aqueous continuous phase as described in EP
301.882. This allows a viscosity reduction (due to the
polymer which is dissolved) whilst incorporating a
sufficiently high amount to achieve a secondary benefit,
especially building, because the part which is not
dissolved does not bring about the instability that
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C 7248 (R)
would occur if substantially all were dissolved. Typical
amounts are from 0.5 to 4.5% by weight.
It is further possible to include in the compositions of
the present invention, alternatively, or in addition`to
the partly dissolved polymer, yet another polymer which
is suostantially totally soluble in the aqueous phase
and has an electrolyte resistance of more than 5 grams
sodium nitrilotriacetate in 100ml of a 5% by weight
aqueous solution of the polymer, said second polymer
also having a vapour pressure in 20% aqueous solution,
equal to or less than the vapour pressure of a ref~rence
2~ by weight or greater aqueous solution of polyethylene
glycol having an average molecular weight of 6000; said
second polymer having a molecular weight of at least
1000. Use of such polymers is generally described in our
EP 301,883. Typical levels are from 0.5 to 4.5% by
weight.
The viscosity of compositions according to the present
is preferably less than 1500 mPas, more preferred less
than 1000 mPas, especially preferred between 30 and 900
mPas at 21 s~1.
One way of regulating the viscosity and stability of
compositions according to the present invention is to
include viscosity regulating polymeric materials.
Viscosity and/or stability regulating polymers which are
preferred for incorporation in compositions according to
the invention include deflocculating polymers having a
hydrophilic backbone and at least one hydrophobic side
chain. Such polymers are for instance described in our
~5~(~7~
16 C 7248 (R)
co-pending European application EP 89201530.6
(EP 346 995).
Preferably the amount of viscosity regulating polymer is
from 0.1 to 5% by weight of the total composition, more
preferred from 0.2 to 2%.
Compositions of the invention may also comprise
materials for adjusting the pH. For lowering the pH it
is preferred to use weak acids, especially the use of
organic acids is preferred, more preferred is the use of
C1_8 carboxylic acids, most preferred is the use of
citric acid.
Apart from the ingredients already mentioned, a number
of optional ingredients may also be present, for example
bleach materials such as percarbonates or perborates,
bleach precursors such as TAED, lather boosters such as
alkanolamides, particularly the monoethanolamides
derived from palm kernel fatty acids and coconut fatty
acids, fabric softeners such as clays, amines and amine
oxides, lather depressants, inorganic salts such as
sodium sulphate, and, usually present in very minor
amounts, fluorescent agents, perfumes, germicides
colorants and other enzymes such as cellulases and
amylases.
Liquid compositions of the invention preferably comprise
from 10 -80 % by weight of water, more preferably from
15- 60%, most preferably from 20-50 %.
Liquid detergent compositions according to the invention
are preferably physically stable in that they show less
17 C 7248 (R)
than 2% by volume phase separation upon storage for 21
days after preparation at 25C.
In use the detergent compositions of the invention will
be diluted with wash water to form a wash liquor for
instance for use in a washing machine. The
concentration of liquid detergent composition in the
wash liquor is preferably from O.OS to 10 %, more
preferred from 0.1 to 3% by weight.
To ensure effective detergency, the liquid detergent
compositions preferably are alkaline, and it is
preferred that they should provide a pH within the range
of about 7.0 to 12, preferably about 8 to about 11, when
used in aqueous solutions of the composition at the
recommended concentration. To meet this requirement, the
undiluted liquid composition should preferably be of a
pH above 7, for example about pH 8.0 to about 12.5. It
should be noted that an excessively high pH, e.g. over
about pH 13, is less desirable for domestic safety. If
hydrogen peroxide is present in the liquid composition,
then the pH is generally from 7.5 to 10.5, preferably 8
to 10, and especial]y 8.5 to 10, to ensure the combined
effect of good detergency and good physical and chemical
stability. The ingredients in any such highly alkaline
detergent composition should, of course, be chosen for
alkaline sta~ility, especially for pH-sensitive
materials such as enzymes, and a particularly suitable
proteolytic enzyme. The pH may be adjusted by addition
of a suitable alkaline or acid material.
Compositions according to the invention may be prepared
by any method for the preparation of detergent
compositions. A preferred method for the preparation of
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liquid detergent compositions of the invention comprises
the addition of electrolyte materials -if any~ to the
water, followed by the addition of deflocculating
polymers -if any-, the detergent active materials, the
stabiliser material and the lipolytic enzyme as a
premix and finally the remaining ingredients The
lipolytic enzyme is preferably added as a premix with
the stabiliser after addition of all ingredients
(including the proteolytic enzyme if any).
The invention will now be illustrated by way of the
following Examples. In all Examples, unless stated to
the contrary, all percentages are by weight.
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EXAMPLE I
Determination of the Ki for the stabilising reaction
between several stabilising materials and lipolytic
enzymes.
The Ki of the stabilisation reaction between Lipolase TM
~ex NOVO) and the stabiliser materials as indicated
below was calculated from the Km values in the absence
and presence of stabiliser. The Km was determined by
variation of the concentration of substrate (=olive oil
emulsion, stabilised by gum arabic in a weight ratio of
1 : 1) both in the absence and presence of a
stabiliser/Lipolase premix. If present the stabiliser
was added as a premix with the lipolytic enzyme. The
Ki was calculated from the Km values.
The following results were obtained:
20 STABILISER _ Ki ~microM~ _
Butane boronic acid 67
- Phenyl boronic acid 20
p-Bromophenyl boronic acid 0.4
p,o Dichlorophenyl boronic acid 0.36
These results indicate that a stabiliser/lipolytic
enzyme complex can be formed at very low concentrations
of stabiliser material (micromolar ran~e)
~a~c5~
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EXAMPLE II
The capability of several stabilisers to form complexes
with Proteolytic enzymes was tested by mixing a premix
of Savinase TM and the stabiliser material to a 0.1
mg/ml solution of purified Lipolase TM (=substrate) in 2
g/l LAS, buffered with 10 m~ Tris/HCl. While keeping the
pH at 9.0 in a pH-stat apparatus, the alkali consumption
was measured. From this the Savinase activity was
calculated. A Savinase activity of 50 % was found at
about the concentrations of stabilisers as indicated in
the following table:
STABILISER 50 % Sav. Act at fmM)
15 Butane Boronic acid 9
Phenyl Boronic acid 5
p-Bromo phenyl Boronic acid 2
p,o Dichloro Boronic acid
These results indicate that although the stabiliser
materials are capable of forming a complex with
Savinase, this complex is only formed at relatively high
concentrations (millimolar range).
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EXAMPLE III
The stability of lipolytic enzymes in the presence of
proteolytic enzymes at various levels of stabilising
materials was determined as follows:
180 LU/ml Lipolase TN was incubated with 10,000 GU/ml
Savinase TM in 2 g/l LAS and 20 mM Tris/HCl at a pH of
9.0 in the presence of various concentrations of
stabilising materials. The residual Lipolase activity
after 30 minutes at 30C was determined.
When butane boronic acid was used (Ki = 67 micromolair)
as the stabilising material a steep increase in
stability of the Lipolytic enzyme was observed when
raising the concentration of stabiliser from 0 to 2
times the Ki. By further increasing the concentration of
stabiliser material a slight increase of the lipolytic
enzyme stability could be observed.
When p,o Dichlorophenylboronic acid ( Ki is 0.36
micromolair) was used as the stabilising material, again
a steep increase in stability of the lipolytic enzyme
was observed by raising the level of stabilising
material from 0 to about 2 times the Ki value. By
further increasing the stabiliser concentration a slight
increase of the stability of the lipolytic enzymes could
be observed.
These results indicate that if mixtures of Lipolytic and
proteolytic enzymes are used, stabilisation take place
at very low levels of stabiliser material, corresponding
to the Ki of the formation of a stabiliser/lipolytic
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22 C 7248 (R)
enzyme complex. The formation of this complex renders
it possible to stabilise lipolytic enzymes by using only
minor amounts of stabilising materials. Therefore if
mixtures of Lipolytic enzymes and Proteolytic enzymes
are used in combination wi~h micromolar concentrations
of stabilising materials, the stabilisation takes place
via an inhibitory reaction on the active site of the
Lipolytic enzyme and not via the formation of a
Proteolytic enzyme/stabiliser complex.
2~s~
23 C 7248 (~)
EXAMPLE IV
The following compositions where prepared by adding the
ingredients to the water in the following order:
electrolytes, actives (as a premix~, other ingredients
except for lipase and stabiliser which were added as a
premix as the final ingredient.
Ingredient (wt parts) A
10 Water 50
LAS 6.8
LES 4.8
Nonionic 4.8
zeolite 19
15 Sokolan CP-5 3
NaOH to pH 8.5
CaC12 0.15
Glycerol 8
Borax 5.7
20 Lipase 0.5 LU/mg
Savinase var
- stabiliser var
Material specification:
LAS: C12_14 Linear alkyl benzene sulphonate(Dobanic 113)
LES: mixtu.re Lauryl 3EO ether sulphate and Lauryl 7EO
ether sulphate in a weight ratio of 1 : 1.
Nonionic: 1.2% synperonic A3, 3.6% synperonic A7.
Zeolite: Wessalith P
Lipase: Lipolase SP~00 (ex NOVO)
Savinase: Savinase 16.0 L DX (ex NOVO)
Sample I contained lipase in the absence of savinase and
stabilisers, sample II contained Lipase and Savinase,
24 C 7248 (R)
but no stabiliser, the remaining samples contained
Lipase and Savinase and a stabiliser as indicated below.
The level of Savinase in samples II-VIII was 10 GU/mg,
the level of stabiliser in samples III-VIII was 0.25 ~.
The half life time of the Lipase enzyme at 37C was
determined for each sample the results were as follows:
SAMPLE_ _STABILISER _ to 5 ~37C. days)
I -- 21
10 II -- 11
III A 11
IV B 11
V C 20
VI D 25
15 VII E 28
VIII F 35
wherein:
A : iminodiacetic acid
B : picolinic acid
C : formula II, wherein R4 is phenyl and R5 is -OH;
D : formula I, wherein R1 is -H, x=1, Ph = meta NH2
benzene, R2 and R3 are -OH;
E : formula I, wherein R1 is -H, x=l, Ph = para Br-
benzene, R2 and R3 are -OH;
F : formula I, wherein R1 is -H, x=1, Ph = ortho, para
diCl benzene, R2 and R3 are -OH;
These results indicate that the half-life time of lipase
is markedly decreased by the addition of proteolytic
enzymes (samples I and II). The addition of two
carboxylic protease inhibitors, iminodiacetic acid and
picolinic acid did not result in an enhancement of
lipase stability (samples III and IV). If however
37~
C 7248 (R)
boronic acid derivatives like phenylboronic acid, m-
amino phenyl boronic acid, 2,4 dichloro phenyl boronic
acid and p-bromo phenyl boronic acid are added a clear
increase of the half life time of lipase can be
observed.
Other useful boronic acid derivatives are: octadecane
boronic acid and phenyl, butyl boronic acid.
7~
26 C 7248 (R)
EXAMPLE V
The following formulations were prepared by mixing the
ingredients in the order listed to water.
5 INGREDIENTS 1~ wt~ A B _ _ C
Sodium-citrate 2aq -- 15 7
Borax 1.0
Glycerol 3-5 ~~ ~~
Propyleneglycol 12 -- --
10 Dobanic 113 7.5 18 16
Synperonic A7 19 18 8
Soap 15 9 16
Tinopal CBS-X -- 0.1 --
Polymer 1) -- 1.0 __
15 Silicone Q2-3300 -- 0.1 --
Perfume -- 0.4 --
SXS -- -- 3
Triethanolamine -- -- 2
Monoethanolamine -- -- 2
20 Savinase TM ~GU/mg) 10 10 10
Lipolase TM (LU/mg) 3) 0.4 0.5 0.5
Stabiliser 2) 0/0.250/0.250/0.25
Water <---------balance-------->
1) Polymer All of EP 346,995.
2) O,p dichlorophenyl boronic acid.
3) Added as a premix with the stabiliser.
The half life time of the lipase was determined at 37C.
For composition A the half life time in the absence of
stabiliser was 1 day, in the presence of stabiliser 3
days. For composition B, the half life time in the
absence of stabiliser was 14 days, in the presence of
stabiliser 32 days. For composition C, the half-life
time in the absence of stabiliser was 1 day, in the
presence of stabiliser 2 days.
27 2~ f;;~1~7~7
Example VI
To determine the enhancement of lipase stability in an
isotropic liquid formulation using dichlorophenyl
boronic acid DCPBA, the following isotropic composition
was prepared
anionic surfactant 22.5%
nonionic surfactant 7.5%
Sodium hydroxide 2.0%
Propylene glycol 11.4%
Lipase 0.5 LU/mg
Savinase (1)
DCPBA (2)
Water to 100%
(1) 0 or 10 GU/mg
(2) DCPBA inoremental amounts 0.001% - 0.25%
-
Upon storage at 37 C over 50 hours, it was found that
concentrations as low as 0.001% yive a measurable
improvement in lipase stability. For the formulation
contaning the protease, the stability is increased by at
least a factor of three (50 hours, 0.25% DCPBA). In the
absence of the protease, the improvement is less but
still significant.
These results show that the lipolase stability
enhancement is obtained not only in structured liquid
compositions but also in isotropic liquids.
