JPH04283298A - Detergent composition - Google Patents

Abstract

PURPOSE: To obtain the title compsn. capable of extending the modification of lipase and the resistance to protein by adding lipase and a specific stabilizing substance.

CONSTITUTION: Lipase and a stabilizing substance capable of extending the half-life of a lipase substance reversibly forming a complex with the active site of lipase, pref., a stabilizing substance being a boric acid derivative represented by formula [Ph is phanyl (subsid. by at least one halogen and/or NH₂), n is 0-1, R¹ is H, halogen or NH₂ and, when n is 0, R¹ is 1-24C alkyl or alkenyl on condition that 3 or more C-atoms, pref. 8-20 C-atoms are contained and R²-R³ are OH, Cl, F, Br, 1-8C alkyl or alkenyl] are added.

COPYRIGHT: (C)1992,JPO

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to detergent compositions containing lipolytic enzymes. In particular, the present invention relates to liquid detergent compositions containing lipolytic enzymes.

0002

BACKGROUND OF THE INVENTION The incorporation of lipolytic enzymes into detergent compositions has previously been suggested. For example, U.S. Patent No. 4,566,985 (A
Biochemists, Inc. describes the use of cellulase, protease, amylase and lipase type enzymes in combination in liquid detergent compositions.

[0003] A problem with using lipolytic enzymes in detergent compositions is that the enzymes are often unstable, which makes them difficult to use during use of the compositions (eg, when washing textiles).
One of the reasons is that enzyme activity decreases. These instability problems are particularly acute in detergent compositions containing lipolytic enzymes together with proteolytic enzymes.

0004

SUMMARY OF THE INVENTION It has surprisingly been found that the stability of lipolytic enzymes in detergent compositions can be controlled by stabilizing substances that can reversibly form complexes with the active sites of lipolytic enzymes.
It has been newly discovered that considerable improvements can be made by using stabilizing materials. The stabilizer-enzyme complex is less susceptible to destabilizing reactions.

By forming a stabilizer-lipolytic enzyme complex,
Inhibiting the destabilizing reaction between lipolytic enzymes and proteolytic enzymes and/or increasing the resistance of lipolytic enzymes to denaturation and proteolysis, the half-life of the lipase in the composition at 37°C is at least It is preferred to improve the length by 25%, more preferably by at least 50%, and most preferably by at least 100%. In the present invention,
The half-life of a lipolytic enzyme is the period from when the detergent formulation is manufactured to when only 50% of the enzyme activity remains. The most preferred stabilizing reaction is an inhibition reaction, which inactivates the stabilizer-lipolytic enzyme complex.

Reversibility of complex formation is generally brought about by adding stabilizing substances at a concentration of 2 to 100 times the Ki (Ki is the concentration at which 50% of the lipase enzyme forms a complex). obtain. The concentration is more preferably 5 to 25 times Ki, most preferably 8 to 15 times Ki. In use, the detergent composition is typically diluted 50 to 500 times, particularly about 100 times, to a stabilizer concentration such that the stabilizer-lipolytic enzyme complex dissociates to provide active lipolytic enzymes in the wash liquor. .

The present invention therefore relates to a detergent composition comprising a lipolytic enzyme and a stabilizing substance capable of reversibly complexing with the active sites of the lipase substance, increasing the half-life of the lipase substance.

In one preferred embodiment of the invention, the stabilizing substance is added to the detergent composition at a concentration of 2 to 100 times Ki.

[0009] Any substance that can reversibly form a complex with the active site of a lipolytic enzyme may be used in the present invention. In a preferred embodiment of the present invention, boric acid (bo
(ronic acid) substances.

Surprisingly, it has now been found that the stability of lipolytic enzymes in detergent compositions is greatly improved by the use of certain boric acid derivatives as stabilizing substances. Boric acid derivatives that are particularly useful for stabilizing lipase enzymes have the formula: where Ph is a phenyl group optionally substituted with halogen or NH2, n is 0 or 1, and R
is hydrogen, halogen, NH2, or a C1-C24 alkyl or alkenyl group, provided that when n is 0 it contains at least 3, most preferably 8 to 20 carbon atoms; and R3 are independently -OH
, -Cl, -F, -Br and C1-C8 alkyl or alkenyl groups).

A second aspect of the invention therefore relates to a detergent composition containing a lipolytic enzyme and one or more boric acid derivatives of formula (I) as defined above.

[0012] Although there is no particular theoretical basis, Applicants believe that boric acid derivatives act through an inhibitory mechanism whereby boric acid derivatives react with the active sites of lipolytic enzymes, resulting in a destabilizing effect. It is believed that the lipase enzyme is stabilized by protecting the enzyme against. When using a detergent composition, a dilution step is usually carried out, for example by adding the detergent composition to the washing liquor. It is believed that such dilution generally removes the boric acid protecting group from the active site of the lipolytic enzyme, providing active enzyme material under the conditions of use. This stabilization mechanism is believed to be particularly useful when lipase enzymes are used in conjunction with proteolytic enzymes. This is because boric acid derivatives easily bind to the active site of lipase and prevent destabilizing reactions (=proteolytic reactions) with proteolytic enzymes from occurring.

Preferred boric acid derivatives have the formula (I) in which at least one of R2 and R3 is -OH, most preferably both R2 and R3 are -OH or one of them is -OH. and the other is a C1-C8 alkyl or alkenyl group).

For environmental reasons, it is more preferred that n in formula (I) be 0 and R1 be a C3 to C24 group.

Particularly preferred boric acid derivatives have the following formula (
II): (wherein R4 is a phenyl group or a C8-C24 alkyl or alkenyl group, and R5 is -OH or a C1-C8 alkyl or alkenyl group)
Represented by

The level of boric acid derivatives in the detergent compositions of the present invention can vary over a wide range depending on the level of lipolytic enzymes and the reactivity of the boric acid derivative. Typically the level of boric acid derivative is 0.00001-3% by weight of the composition;
0.0001 to 1% by weight is more preferable, and 0.001 to 1% by weight.
0.5% by weight is most preferred.

The amount of lipolytic enzyme added to the composition is preferably 50 to 30,000 LU (LU (lipase unit) is as defined in European Patent No. 258068) per 1 g of detergent composition. Levels of lipolytic enzymes are often at least 100 LU/g and at least 25
0LU/g is very useful, less than 5000LU/g is preferred, less than 2000LU/g or 1000LU
More preferably, it is less than /g.

[0018] Lipolytic enzymes include various lipases, especially European Patent No. 214761 (NOVO) and European Patent No. 25806.
No. 8 (NOVO) and No. 305216 (NOVO)
Lipases such as those described in the patent specification of
rmomyceslanuginosus ATCC
Lipolytic enzyme showing immunological cross-reactivity with antiserum against lipase derived from 22070 [European Patent No. 20
No. 5208 (UNILEVER) and No. 206390
UNILEVER], especially Chromobact
er viscosum var lipoly
ticum NRRLB-3673 or Alcaligenes PL-679, ATC
C 31371 and FERM-P3783, and WO 87/00859 (Gist-
Brocades), WO 89/09263 (Gi
st Brocades), European Patent No. 331376
No. (AMANO), West German Patent No. 3908131 (To
yo Jozo) and European Patent No. 204284 (S
The lipase may be selected from the lipases described in the specification of APPORO BREWEries. Particularly suitable are
For example, commercially available lipase preparations [Novo Lipola
se, Amano Lipase CE, P, B,
AP, M-AP, AML and CES, and Meito
lipases MY-30, OF and PL, esterase MM. Lipozym, SP 225,S
P 285, Siaken lipase, Enzec
o lipase, Toyo Jozo lipase and Diosynth lipase].

[0019] These commercially available enzymes are available at levels as low as 0.0% of the detergent composition, with respect to the as-supplied enzyme formulation.
1 to 10% by weight, more preferably 0.05 to 5% by weight,
Most preferably it is used at a level of 0.1-2% by weight.

Lipase enzymes produced by genetic engineering [for example, WO 89-09263 (Gist-B
rocades) and European Patent No. 218272 (Gi
St-Brocades), EP 258 068 (Novo) and EP 305 216 (Novo)] may also be used.

As mentioned above, the use of boric acid derivatives to stabilize lipolytic enzymes is particularly useful in detergent compositions containing lipases and proteolytic enzymes.

[0022] The protease is present in detergent composition 1 in the composition.
It may be formulated in an amount of about 1-100 GU per mg. This amount is preferably in the range of 2 to 50 GU/mg, and 5 to 20 GU/mg.
A range of mg is more preferred. GU is a glycine unit defined as a protease activity that produces an amount of NH2 group equivalent to 1 micromole of glycine under standard conditions of incubation with N-acetylcasein as a substrate at 40°C for 15 minutes. be.

A preferred example of a protease enzyme that can be used in the compositions of the invention is Savinase™ (N
ovo-Nordisk A/S), Maxaca
l (trademark) (manufactured by Gist-Brocades/IBIS), Opticlean (manufactured by MKC) or API22
(manufactured by Showa Denko). Examples of other useful proteases include Maxatase, Esperase, Al
calase, protease K and subtilisin BPN
' or variants obtained by enzyme engineering.

Commercially available proteolytic enzymes are available at levels as low as 0.01% of the composition for the as-supplied enzyme preparation.
Levels of ~10% by weight are preferred, with 0.05-2% by weight
is more preferable, and 0.1 to 2% by weight is most preferable.

It has been found that proteases with high isoelectric points (such as Savinase or Maxacal) are more stable under the conditions normally incorporated into the detergent compositions of the present invention. In particular, high pI proteases (
That is, a pI of 9 or greater, about 10) has been used in liquid detergent compositions with a pH of less than about 9 with good results.

The detergent compositions of the present invention may take any suitable physical form, such as powders, pastes, tablets and liquids. A preferred embodiment of the invention relates to liquid detergent compositions, particularly liquid detergent compositions comprising an aqueous phase.

The compositions of the invention also contain detergent actives. In the broadest definition, detergent actives may generally include one or more surfactants and may be selected from anionic, cationic, nonionic, zwitterionic and zwitterionic surfactants and compatible mixtures thereof. . For example, these are “Sur
face Active Agents”, Vol.
．． I, Schwartz and Perry, Inter
science1949, “Surface Act
ive Agents”, Vol. II, Schwa
rtz, Perry and Berch, Intersc.
1958, “McCutcheon's
Emulsifiers & Deterge
nts”, McCutcheon division
of Manufacturing Conf.
ctioners Company, or “
Tensid-Taschenbuch”, H. Sta.
che, 2nd edition. , Carl Hanser Verl
ag, Munchen & Wien, 1981.

Suitable nonionic surfactants are in particular reaction products of compounds containing hydrophobic groups and reactive hydrogen atoms, such as aliphatic alcohols, acids, amides or alkylphenols with alkylene oxides, especially ethylene oxide or ethylene oxide. - reaction product with propylene oxide). Certain nonionic detergent compounds are
Alkyl (C6-C18) primary or secondary linear or branched alcohol with ethylene oxide, the product being made by condensation of ethylene oxide with the reaction product of propylene oxide and ethylene diamine. Other so-called nonionic detergent compounds include long chain tertiary amine oxides, long chain tertiary phosphine oxides and dialkyl sulfoxides.

Salting-out resistant active substances (for example, European Patent No. 32
8177), in particular alkyl polyglycoside surfactants (such as those described in EP 70074).

Suitable anionic surfactants are water-soluble alkali metal salts of organic sulfuric and sulfonic acids, usually having alkyl groups containing from about 8 to about 22 carbon atoms.
The term alkyl is intended to include the alkyl portion of higher acyl groups). Examples of suitable synthetic anionic detergent compounds include sodium alkyl sulfates and potassium alkyl sulfates [particularly those obtained by sulfating higher (C8-C18) alcohols made, for example, from tallow or coconut oil]; C9-C20) Sodium and potassium benzenesulfonates [especially linear secondary alkyl (C
10-C15) Sodium benzenesulfonate]; Sodium alkyl glyceryl ether sulfate (especially ethers of higher alcohols derived from tallow or coconut oil and synthetic alcohols derived from petroleum); Coconut oil fat monoglyceride sodium sulfate and coconut oil fat monoglyceride sulfone acid sodium; higher (C8-C18) fatty alcohol-alkylene oxide (especially ethylene oxide)
Sodium and potassium salts of sulfate esters of reaction products; fatty acid reaction products such as coconut fatty acids esterified with isethionic acid and then neutralized with sodium hydroxide;
Sodium and potassium salts of fatty acid amides of methyltaurine; alkane monosulfonates [e.g. those derived from the reaction of α-olefins (C8-C20) with sodium bisulfite, and those derived from the reaction of paraffins with SO2 and Cl2; such as those derived by subsequent hydrolysis with base to produce random sulfonates]; as well as olefin sulfonates [this term refers to olefins (
In particular, it refers to a material produced by reacting a C10 to C20 α-olefin) with SO3, then neutralizing it, and hydrolyzing the reaction product. Preferred anionic detergent compounds include sodium (C11-C15) alkylbenzenesulfonate, and primary alkyl (C10-C18)
) Sodium or potassium alkyl sulfate.

Alkali metal soaps of fatty acids, especially acids having 12 to 18 carbon atoms, such as oleic acid, ricinoleic acid, and castor oil, alkyl succinic acids, rapeseed oil, peanut oil, coconut oil, palm kernel oil or their It is also possible, and sometimes preferred, to include soaps of fatty acids derived from mixtures and the like. Sodium or potassium soaps of these acids may be used.

In many (but not all) cases, the total detergent actives will be between 2 and 60% by weight of the total composition, such as 5% by weight of the total composition.
-50% by weight, usually 10-40% by weight. However, one preferred class of compositions comprises at least 20%, more preferably at least 25% and especially at least 30% of detergent actives, based on the total weight of the composition.

The liquid detergent composition of the present invention has non-structural [u
n-structured (isotropic)] or structured. The structured liquids of the present invention may be internally structured, with structure formed by detergent actives in the composition, or externally structured, with structure provided by external components. Preferably, the compositions of the invention are internal structural. Most preferably, the composition of the invention consists of a lamellar droplet structure comprising a surfactant.

[0034] Some of the class activity-structures are H. A. B
Arnes, “Detergents”, Ch. 2. K
．． Walters (ed.), “Rheometry: In
Industrial Applications”, J
．． Wiley & Sons, Letchworth 1
980. Typically, the degree of order of this system increases with increasing surfactant and/or electrolyte concentration. At very low concentrations, surfactants can exist as molecular solutions or as solutions of spherical micelles, both of which are isotropic. Further addition of surfactants and/or electrolytes may form structured (anisotropic) systems. These are referred to separately by various terms, such as rod-micelle, plate-lamellar structure, lamellar droplet and liquid crystalline phase. Different researchers often have different technical terms for this structure, but they actually refer to the same thing. For example, in European patent application EP-A-151884, lamellar droplets are referred to as "spherulites". The presence and identification of surfactant structural systems in liquids can be determined by methods known to those skilled in the art, e.g.
It may be determined by optical methods, various rheometry measurements, X-ray or neutron scattering, and sometimes electron microscopy, etc.).

When the composition has a lamellar droplet structure, it is often preferred that the aqueous continuous phase comprises a dissolved electrolyte. The term electrolyte as used herein refers to any ionic, water-soluble substance. However, in a lamellar dispersion, all of the electrolyte does not need to be dissolved, but may be suspended as solid particles since the electrolyte concentration of the liquid is higher than the solubility limit of the electrolyte. Mixtures of electrolytes may be used with one or more electrolytes in the dissolved aqueous phase and one or more electrolytes substantially only in the suspended solid phase. Two or more electrolytes may also be approximately evenly distributed between these two phases. This is the manufacturing method (
for example, the order of addition of ingredients, etc.). On the other hand “
The term ``salt'' refers to all organic and inorganic substances, whether ionic or not, other than surfactants and water;
is also included.

Selection of the type and proportion of surfactants to obtain a stable liquid with the required structure is
It is well within the ability of those skilled in the art. However, it can be pointed out that in an important subclass of useful compositions detergent actives contain mixtures of different types of surfactants. Typical mixtures useful in textile laundry compositions include one or more primary surfactants that are nonionic surfactants, and/or non-alkoxylated anionic surfactants, and/or alkoxylated anionic surfactants. Mixtures containing surfactants are included.

When mixtures of surfactants are used, the exact proportions of each component that provide the stability and viscosity described above will depend, as in conventional structural liquids, on the type and amount of electrolyte.

The composition of the invention preferably contains 0 to 60%
, especially 1-60%, more preferably 10-45% salting-out electrolyte. A salting-out electrolyte is an electrolyte with a syneresis number of less than 9.5, as defined in European Patent Application No. 79646. Possibly some salt-soluble electrolyte (as defined in the above specification), in a type and amount compatible with the other ingredients, also forms part of the claimed composition of the invention. It can be present in the composition of the present invention if used within the range of . Some or all of the electrolyte (whether salt-soluble or salt-out), or any substantially water-insoluble salts that may be present, may have detergency builder properties. in any case,
Preferably, the composition according to the invention contains detergency builder substances, some or all of which may be electrolytes. A builder substance is any substance capable of reducing the level of free calcium ions in the wash liquor, preferably achieving an alkaline pH, suspending the soil removed from the textile, and dispersing clay substances to soften the textile. It also imparts other beneficial properties to the composition, such as. Preferably, the salting-out electrolyte comprises citrate.

When phosphorous-containing inorganic detergency builders are used, examples thereof include water-soluble salts, especially alkali metal salts of pyrophosphoric acid, orthophosphoric acid, polyphosphoric acid and phosphonic acid. Inorganic phosphate builders include in particular the sodium and potassium salts of tripolyphosphoric acid, phosphoric acid and hexametaphosphoric acid. It is also possible to use phosphonate sequestrant builders.

When phosphorus-free inorganic detergency builders are used, examples include water-soluble alkali metal salts of carbonic acid, bicarbonic acid, silicic acid, and crystalline and amorphous aluminosilicic acids. Mention may be made, in particular, of sodium carbonate (with or without calcite seeds), potassium carbonate, sodium and potassium bicarbonate, sodium and potassium silicates, and zeolites.

When organic detergency builders are used, examples include alkali metal, ammonium and substituted ammonium salts of polyacetic acids, carboxylic acids, polycarboxylic acids, polyacetylcarboxylic acids and polyhydroxysulfonic acids. In particular, ethylenediaminetetraacetic acid, nitrilotriacetic acid, oxydisuccinic acid, CMOS, TMS,
Mention may be made of the sodium, potassium, lithium, ammonium and substituted ammonium salts of TDS, mellitic acid, benzene polycarboxylic acid and citric acid.

The level of non-soap builder material is preferably from 0 to 50%, more preferably from 5 to 40%, and most preferably from 10 to 35% by weight of the composition.

When using organic builders, it is also desirable to have polymers that are only partially soluble in the aqueous continuous phase, as disclosed in EP 301,882. Thereby it is possible to reduce the viscosity (due to partial dissolution of the polymer) and to add a sufficient amount of builder to achieve secondary benefits, in particular increased detergency, since the insoluble part of the polymer This is because it prevents instability that would occur if substantially all of the solution was dissolved. Typical polymer amounts are 0.5-4.5% by weight.

In place of or in addition to the partially soluble polymers described above, the compositions of the present invention may further contain other polymers that are substantially completely soluble in the aqueous phase; The second polymer is 5% by weight of the polymer.
It has an electrolyte tolerance in which more than 5 g of sodium nitrilotriacetate is present in 100 ml of aqueous solution, and its 20
% aqueous solution is lower than the vapor pressure of a standard aqueous solution containing 2% by weight or more of polyethylene glycol having an average molecular weight of 6000. The second polymer has a molecular weight of 1000 or more. The use of such polymers is disclosed in European Patent No. 301,883 in the name of the applicant. Typical levels of second polymer are 0.5-4.5% by weight.

The viscosity of the composition according to the invention is 21 s-1
preferably less than 1500 mPa, more preferably 10
00 mPa, particularly preferably from 30 to 900 mPa.

The viscosity and stability of the compositions according to the invention are adjusted, for example, by the addition of viscosity-modifying polymeric substances.

Preferred viscosity and/or stability modifying polymers for inclusion in compositions according to the invention include deagglomerated polymers having a hydrophilic backbone and at least one hydrophobic side chain. Such polymers are described, for example, in the applicant's co-pending European Patent Application No. 89201530.6 (European Patent No. 346,
No. 995).

The amount of the viscosity adjusting polymer is preferably 0.1 to 5% by weight of the total composition, more preferably 0.2 to 5% by weight of the total composition.
It is 2% by weight.

The compositions of the invention may also contain pH-adjusting substances. To lower the pH, weak acids, particularly organic acids, can be preferably used, with C1-C8 carboxylic acids being preferred, and citric acid being most preferred.

[0050] In addition to the components already mentioned, some optional components,
Bleach substances such as percarbonates and perborates, TA
Bleach precursors such as ED, foam promoters such as alkanolamides, especially monoethanolamide derived from palm kernel fatty acids and coconut fatty acids, textile softeners such as clays, amines and amine oxides, defoamers, It is also possible to include inorganic salts such as sodium sulfate, as well as fluorescent agents, fragrances, fungicides, colorants, other enzymes such as cellulases and amylases, etc., which are normally used only in small amounts.

[0051] The liquid composition of the present invention preferably contains 10 to
It contains 80% by weight water, more preferably 15-60% and most preferably 20-50% by weight.

Liquid detergent compositions according to the invention are preferably physically stable, ie exhibit less than 2% by volume phase separation when stored at 25° C. for 21 days after manufacture.

[0053] When used, the detergent composition of the present invention is diluted with wash water to form a wash solution for use in, for example, a washing machine. The concentration of the liquid detergent composition in the washing liquid is preferably 0.05 to 10% by weight, more preferably 0.1 to 3% by weight.

[0054] In order to ensure effective cleaning power,
The liquid detergent composition is preferably alkaline and has a concentration of about 7.0 to 1
2, particularly from about 8 to about 11. To that end, the pH of the undiluted liquid composition should preferably be greater than 7, such as from about 8.0 to about 12.5. Too high a pH, for example above about 13, is less desirable for the safety of household detergents. If hydrogen peroxide is present in the liquid composition, p
H is usually from 7.5 to 10.5, preferably from 8 to 10, especially from 8.5 to 10, thus ensuring that both good detergency and good physical and chemical stability are achieved. Naturally, whenever a detergent composition is highly alkaline in this way, its components, especially pH-sensitive substances such as enzymes, especially proteolytic enzymes, should be selected in such a way that they are alkaline-stable. be. The pH can be adjusted by adding suitable alkaline or acidic substances.

[0055] The compositions according to the invention may be made by any method of making detergent compositions. A preferred method of making the liquid detergent compositions of the present invention is to add electrolyte materials (if used) to water, then premix the deagglomerated polymer (if used), detergent actives, stabilizer materials and lipolytic enzymes. and then finally adding the remaining ingredients. Lipolytic enzymes are preferably added as a premix with stabilizers after addition of all ingredients (including proteolytic enzymes, if used).

[0056]

EXAMPLES The present invention will be illustrated by the following examples. In all examples, all percentages are by weight unless otherwise specified.

Example 1 Determination of Ki for the stabilization reaction between several stabilizer substances and lipolytic enzymes The Ki for the stabilization reaction between Lipolase (trademark) manufactured by NOVO and the following stabilizer substances was determined in the absence of a stabilizer. It was calculated from the Km values in the presence and absence of the compound. Stabilizer-Li
Km was determined by varying the concentration of the substrate (i.e., olive oil emulsion stabilized with gum arabic in a weight ratio of 1:1) both in the absence and in the presence of the polase premix. Stabilizers, if present, were added as a premix with lipolytic enzymes. Ki was calculated from the obtained Km value.

The following results were obtained.

Stabilizer
Ki (μM
) butaneboric acid
67 phenylboric acid
20p-bromophenylboric acid
0.4p,o-dichlorophenylboric acid
0.36 These results show that stabilizer-lipolytic enzyme complexes can be formed at very low stabilizer substance concentrations (in the micromolar range).

Example 2 A premix of Savinase™ and stabilizer material was prepared by adding 1 portion of purified Lipolase™ (substrate)
2g/l LAS buffered with 0mM Tris/HCl
The ability of several stabilizers to form complexes with proteolytic enzymes was tested by adding them to the resulting solution in an amount of 0.1 mg/ml. Alkali consumption was measured while maintaining the pH at 9.0 with a pH stat. Savinase activity was calculated from the obtained measured values. Approximately, 50% Sav at the stabilizer concentrations shown in the table below.
inase activity was obtained.

Stabilizer
Stabilizer concentration at 50% Savinase activity (mM) Butaneboric acid
9 Phenylboric acid
5p-bromophenylboric acid
2p,o
-dichlorophenylboric acid
1 From these results, the above stabilizer substance is Sa.
It is clear that complexes with vinase can be formed, but the stabilizer concentration during complex formation will be relatively high (in the millimolar range).

Example 3 The stability of lipolytic enzymes in the presence of proteolytic enzymes at various stabilizer substance levels was investigated as follows.

180LU/ml Lipolase (
2g/l LAS and 20mM Tris
10,000 GU/ml Savina in /HCl
se™ in which stabilizer substances were present at various concentrations and the pH was 9.0. Residual Lipolase activity was measured after 30 minutes at 30°C.

Using butaneboric acid as the stabilizer substance (K
i = 67 μM), and when the stabilizer concentration was increased from 0 to twice the Ki, a rapid increase in the stability of the lipolytic enzyme could be observed. Further increases in stabilizer concentration resulted in only a small increase in the stability of the lipolytic enzyme.

When p,o-dichlorophenylboronic acid is used as a stabilizer substance (Ki=0.36 μM), the stability of the lipolytic enzyme increases from 0 to about twice the Ki value. This led to a rapid increase. Further increases in stabilizer concentration resulted in only a small increase in the stability of the lipolytic enzyme.

These results demonstrate that stabilization using mixtures of lipolytic and proteolytic enzymes is achieved at very low stabilizer substance levels corresponding to the Ki of stabilizer-lipolytic enzyme complex formation. It is shown that. The formation of a stabilizer-lipolytic enzyme complex allows the lipolytic enzyme to be stabilized using only small amounts of stabilizer material. Therefore, if mixtures of lipolytic and proteolytic enzymes are used with stabilizer substances at concentrations in the micromolar range, stabilization is not via the formation of proteolytic enzyme-stabilizer complexes, but rather through the activity of the lipolytic enzymes. This is achieved through an inhibitory reaction at the site.

Example 4 The following ingredients were prepared in the following order: first the electrolyte, then the active substance (as a premix), then the other ingredients excluding lipase and stabilizer, and finally the premix of lipase and stabilizer. Several compositions were prepared by adding the solution to water.

Ingredients
Part by weight water
5
0LAS
6.8LES

4.8 Nonionic surfactant
4.8 Zeolite
19Sokolan CP-5
3NaOH
pH
CaCl2 until 8.5
0.15 glycerol
8 borax

5.7 lipase
0.5LU/mgSav
inase
various stabilizers
Various L
AS: C12-C14 linear alkylbenzene sulfonate (Dobanic113) LES: Mixture of lauryl 3EO ether sulfate and lauryl 7EO ether sulfate in a weight ratio of 1:1 Nonionic surfactant: Synperonic A3
1.2%, Synperonic A7 3.6%
Zeolite: Wessalith P lipase: Li
polase SP400 (manufactured by NOVO) Savinase: Savinase 16.0
LDX (manufactured by NOVO) Sample I contained lipase but no Savinase or stabilizer, and Sample II contained lipase and Savinase.
The other samples contained lipase, Savinase, and the stabilizer described below. The Savinase level for Samples II-VIII was 10 GU/mg and the stabilizer level for Samples III-VIII was 0.25%. When the half-life of lipase enzyme at 37°C was measured for each sample, the following results were obtained.

A: iminodiacetic acid B: picolinic acid C: compound of formula II (wherein R4 is phenyl, R5 is -O
H) D: Compound of formula I (wherein R1 is -H, x=1, Ph=metaNH2benzene, R2 and R3 are -OH) E: Compound of formula I (wherein R1 is -H, x=1 , Ph=para-Br-benzene, R2 and R3 are -OH) F: compound of formula I (wherein R1 is -H, x=1, Ph=ortho, para-diCl-benzene, R2 and R3 are -OH) The results show that the half-life of lipase is significantly shortened by the addition of proteolytic enzymes (see samples I and II). Two types of carboxyl protease inhibitors,
That is, the addition of iminodiacetic acid and picolinic acid did not improve the stability of lipase (see samples III and IV).
. However, when boric acid derivatives such as phenylboric acid, m-aminophenylboric acid, 2,4-dichlorophenylboric acid and p-bromophenylboric acid are added, a clear extension of the lipase half-life can be observed.

Useful boric acid derivatives include, in addition to those mentioned above, octadecaneboric acid and phenylbutylboric acid.

Example 5 A composition was prepared by incorporating the following ingredients into water in the order listed.

[0072] The half-life of lipase was determined at 37°C. For composition A, the lipase half-life was 1 day without the stabilizer and 3 days with the stabilizer. Composition B has a half-life of 14 days without stabilizer and 32 days with stabilizer.
It was day. The lipase half-life of Composition C was 1 day without the stabilizer and 2 days with the stabilizer.

Example 6 To investigate the improvement of lipase stability in an isotropic liquid composition using dichlorophenylborate DCPBA,
An isotropic composition having the following composition was prepared.

Anionic surfactant
22.5% nonionic surfactant
7.5%
Sodium hydroxide
2.0% propylene glycol 11.
4% lipase
0.5LU/mgSa
vinase
(1) DCPBA

(2) Water
1
00% (1); 0 or 10 GU/mg (2); Gradual increase from 0.001% to 0.25% When stored at 37°C for 50 hours, DC as low as 0.001%.
It was found that the PBA concentration significantly improves the lipase stability. For protease-containing compositions, lipase stability is improved by at least 3 times (50 hours, DCPBA
0.25%). The degree of stability improvement in the absence of protease is smaller but still significant.

These results show that improved Lipolase stability is achieved not only in structured liquid compositions, but also in isotropic liquids.

Claims (11)

[Claims] 1. A detergent composition comprising a lipolytic enzyme and a stabilizing substance capable of reversibly complexing with the active site of the lipolytic enzyme to extend the half-life of the lipase material. Claim 2: A lipolytic enzyme and a compound of formula (I) (wherein, P
h is a phenyl group optionally substituted with at least one halogen and/or NH2 group, n is 0 or 1, and R1 is hydrogen, halogen, NH2, or if n is 0 at least 3, most preferably 8
is a C1-C24 alkyl or alkenyl group containing ~20 carbon atoms, and R2 and R3 are independently -Ph, -Cl, -F, -Br and C1-C
8 alkyl or alkenyl groups). 3. A detergent composition according to claim 1, wherein the stabilizing substance is added to the detergent composition at a concentration of 2 to 100 times Ki. 4. The stabilizing substance has the formula (II) (wherein R
4 is a phenyl group or a C8-C24 alkyl or alkenyl group, and R5 is -OH or C1-C
The detergent composition according to any one of claims 1 to 3, wherein the detergent composition is a boric acid derivative of 8 alkyl or alkenyl group. 5. Detergent composition according to claim 1, comprising from 0.00001 to 3% by weight of stabilizing substances. 6. 50 to 30,000 per gram of detergent composition
A detergent composition according to one or more of claims 1 to 5, comprising 0 LU of lipolytic enzyme. 7. Claims 1 to 6 comprising a proteolytic enzyme, preferably in an amount of 1 to 100 GU per mg of detergent composition.
The detergent composition according to one or more of the above items. 8. In liquid form, preferably comprising: (a) 2 to 60% by weight of a detergent active substance; and (b) 0 to 6% of a salting-out electrolyte.
(c) 0-50% by weight of a non-soap builder substance; and (d) 10-80% by weight of water. 9. The liquid detergent composition of claim 8 having a viscosity of less than 1,500 mPas at 21s-1. 10. A liquid detergent composition according to claim 8 or 9, wherein the composition comprises a suspension of lamellar droplets. 11. A liquid detergent composition according to claim 8 or 9, wherein the composition is isotropic.