A liquid detergent composition comprising a surfactant, an enzyme and a naphthalene boronic acid derivative enzyme stabilizer.

CROSS-REFERENCE TO RELATED APPLICATIONS 
This application is a 35 U.S.C. 371 national application of PCT/DK95/00168 
filed Apr. 25, 1995, which is incorporated herein by reference. 
FIELD OF INVENTION 
This invention relates to a liquid detergent composition comprising a 
surfactant, an enzyme and an improved enzyme stabilizer. 
BACKGROUND OF THE INVENTION 
Storage stability problems are well known with liquids containing 
enzyme(s). Especially in enzyme-containing liquid detergents a major 
problem, in particular if the detergent contains protease, is that of 
ensuring enzyme activity over time. 
The prior art has dealt extensively with improving the storage stability, 
for example by adding a protease inhibitor. 
Boric acid and boronic acids are known to reversibly inhibit proteolytic 
enzymes. A discussion of the inhibition of one serine protease, 
subtilisin, by boronic acid is provided in Molecular & Cellular 
Biochemistry 51, 1983, pp. 5-32. 
Boronic acids have very different capacities as subtilisin inhibitors. 
Boronic acids containing only alkyl groups such as methyl, butyl or 
2-cyclohexylethyl are poor inhibitors with methylboronic acid as the 
poorest inhibitor, whereas boronic acids bearing aromatic groups such as 
phenyl, 4-methoxyphenyl or 3,5-dichlorophenyl are very good inhibitors 
with 3,5-dichlorophenylboronic acid as a particularly effective one (see 
Keller et al, Biochem. Biophys. Res. Com. 176, 1991, pp. 401-405). 
It is also claimed that aryl boronic acids which have a substitution at the 
3-position relative to boron are unexpectedly good reversible protease 
inhibitors. Especially, acetamidobenzene boronic acid is claimed to be a 
superior inhibitor of proteolytic enzymes (see WO 92/19707). 
The inhibition constant (K.sub.i) is ordinarily used as a measure of 
capacity to inhibit enzyme activity, with a low K.sub.i indicating a more 
potent inhibitor. However, it has earlier been found that the K.sub.i 
values of boronic acids do not always tell how effective inhibitors are 
(see for instance WO 92/197077). 
SUMMARY OF THE INVENTION 
In this invention it is surprisingly found that naphthalene boronic acid 
derivatives have extraordinary good capacities as enzyme stabilizers in 
liquid detergents. 
Accordingly, the present invention relates to a liquid detergent 
composition comprising a surfactant, an enzyme and a naphthalene boronic 
acid derivative enzyme stabilizer of the following formula: 
##STR1## 
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 is 
the same or different and selected from hydrogen, C.sub.1 -C.sub.6 alkyl, 
substituted C.sub.1 -C.sub.6 alkyl, aryl, substituted aryl, hydroxy, 
hydroxyl derivative, halogen, amine, alkylated amine, amine derivative, 
nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, 
sulfonate or phosphonate. 
DETAILED DISCLOSURE OF THE INVENTION 
One embodiment of the present invention provides a liquid detergent 
composition comprising a surfactant, an enzyme and a naphthalene boronic 
acid derivative enzyme stabilizer of the following formula: 
##STR2## 
where R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 is 
the same or different and selected from hydrogen, C.sub.1 -C.sub.6 alkyl, 
substituted C.sub.1 -C.sub.6 alkyl, aryl, substituted aryl, hydroxy, 
hydroxyl derivative, halogen, amine, alkylated amine, amine derivative, 
nitro, thiol, thiol derivative, aldehyde, acid, acid salt, ester, 
sulfonate or phosphonate. 
A preferred embodiment of the present invention provides a liquid detergent 
composition comprising a surfactant, an enzyme and a naphthalene boronic 
acid derivative enzyme stabilizer of the formula disclosed above, wherein 
at least five of the seven groups (R.sub.1 -R.sub.7) are hydrogen. 
Preferred examples belonging to this group are dihalogennaphthalene 
boronic acids such as dichloronaphthalene boronic acids. 
A further preferred embodiment of the present invention provides a liquid 
detergent composition comprising a surfactant, an enzyme and a naphthalene 
boronic acid derivative enzyme stabilizer of the formula disclosed above, 
wherein at least six of the seven groups (R.sub.1 -R.sub.7) are hydrogen. 
Preferred examples belonging to this group are 
6-hydroxynaphthalene-2-boronic acid, naphthalene-2-boronic acid and 
naphthalene-1-boronic acid. 
Preparation of Naphthalene Boronic Acid Derivatives 
Naphthalene boronic acid derivatives may be prepared using methods well 
known to those skilled in the art, for example by using a Grignard 
preparation: 
The Grignard reagent is prepared by the slow dropwise addition of the 
appropriate bromonaphthalene starting material in sodium dried ether to 
magnesium turnings in sodium dried ether. The reaction is encouraged by 
the addition of a small iodine crystal. 
Trimethylborate or tri-n-butylborate in sodium dried ether is cooled to 
-70.degree. C. and the Grignard reagent is added dropwise over a period of 
2 hours while keeping the borate solution at -70.degree. C. and 
continuously agitating. 
The reaction mixture is allowed to warm to room temperature overnight 
whereupon it is hydrolysed by the dropwise addition of cold dilute 
sulphuric acid. The ether layer is separated and the aqueous layer 
extracted with ether. The ether containing fractions are combined and the 
solvent removed. The residue is made distinctly alkaline and any methanol 
or butanol so formed is removed. The alkaline solution is made acidic and 
cooled and the resulting crystals of desired boronic acid are removed by 
filtration. All products are preferably recrystallized from distilled 
water or some other appropriate solvent. 
The naphthalene boronic acids may also be prepared using either direct 
lithiation of the naphthalene and/or lithiation of the bromide. 
Any nuclear substitution or protection of functional groups may be achieved 
by using standard methods well known to those skilled in the art. 
Stabilizers 
According to the invention the liquid detergent composition may contain up 
to 500 mM of the stabilizer (the naphthalene boronic acid derivative), 
preferably the detergent composition may contain 0.001-250 mM of the 
stabilizer, more preferably the detergent composition may contain 
0.005-100 mM of the stabilizer, most preferably the detergent composition 
may contain 0.01-10 mM of the stabilizer. The naphthalene boronic acid 
derivative may be an acid or the alkali metal salt of said acid. 
Enzymes 
According to the invention the liquid detergent composition contains at 
least one enzyme. The enzyme may be any commercially available enzyme, in 
particular an enzyme selected from the group consisting of proteases, 
amylases, lipases, cellulases, oxidoreductases and any mixture thereof. 
Mixtures of enzymes from the same class (e.g. lipases) are also included. 
According to the invention a liquid detergent composition comprising a 
protease is preferred; more preferred is a liquid detergent composition 
comprising two enzymes in which the first enzyme is a protease and the 
second enzyme is selected from the group consisting of amylases, lipases, 
cellulases and oxidoreductases; even more preferred is a liquid detergent 
composition in which the first enzyme is a protease and the second enzyme 
is a lipase. 
The amount of enzyme used in the liquid detergent composition varies 
according to the type of enzyme(s). The amount of each enzyme will 
typically be 0.2-40 .mu.M, especially 0.4-20 .mu.M (generally 5-1000 mg/l, 
especially 10-500 mg/l) calculated as pure enzyme protein. 
Protease: Any protease suitable for use in a detergent composition can be 
used. Suitable proteases include those of animal, vegetable or microbial 
origin. Microbial origin is preferred. Chemically or genetically modified 
mutants are included. It may be a serine protease, preferably an alkaline 
microbial protease or a trypsin-like protease. Examples of alkaline 
proteases are subtilisins, especially those derived from Bacillus, e.g. 
subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and 
subtilisin 168 (described in WO 89/06279). Examples of commercial Bacillus 
subtilisins are Alcalase.RTM., Savinase.RTM., Esperase.RTM. and 
Durazym.RTM. products of Novo Nordisk A/S. Examples of trypsin-like 
pro-teases are trypsin (e.g. of porcine or bovine origin) and the Fusarium 
protease described in WO 89/06270. 
Amylase: Any amylase suitable for use in a detergent composition can be 
used. Suitable amylases include those of bacterial and fungal origin. 
Chemically or genetically modified mutants are included. Amylases include, 
for example, .alpha.-amylases obtained from a special strain of B. 
licheniformis, described in more detail in British Patent Specification 
No. 1,296,839. Particularly preferred is Termamyl.RTM., available from 
Novo Nordisk A/S. 
Lipase: Any lipase suitable for use in a detergent composition can be used. 
Suitable lipases include those of bacterial and fungal origin. Chemically 
or genetically modified mutants are included. Particularly preferred is 
lipase obtained by cloning the gene from Humicola lanuginosa and 
expressing the gene in Aspergillus oryzae as described in EP 0 258 068, 
available under the trade mark Lipolase.RTM. from Novo Nordisk A/S. 
Cellulase: Any cellulase suitable for use in a detergent composition can be 
used. Suitable cellulases include those of bacterial and fungal origin. 
Chemically or genetically modified mutants are included. Suitable 
cellulases are disclosed in U.S. Pat. No. 4,435,307. Particularly 
preferred is Celluzyme.TM. produced by a strain of Humicola insolens, 
available from Novo Nordisk A/S. 
Oxidoreductases: Any oxidoreductase suitable for use in a detergent 
composition, e.g., peroxidases and oxidases such as laccases, can be used 
herein. Suitable peroxidases herein include those of plant, bacterial and 
fungal origin. Chemically or genetically modified mutants are included. 
Examples of suitable peroxidases are those derived from a strain of 
Coprinus, e.g. C. cinerius or C. macrorhizus, or from a strain of 
Bacillus, e.g. B. pumilus, particularly peroxidase according to PCT/DK 
90/00260. 
Deterrents 
According to the invention the liquid detergent composition will beside 
enzyme(s) and stabilizer comprise a surfactant. The detergent composition 
may, e.g., be a laundry detergent composition or a dishwashing detergent 
composition. 
The detergent may be aqueous, typically containing up to 70% water and 
0-30% organic solvent, or nonaqueous. 
The detergent composition comprises one or more surfactants, each of which 
may be anionic, nonionic, cationic, or amphoteric (zwitterionic). The 
detergent will usually contain 0-50% of anionic surfactant such as linear 
alkylbenzene-sulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate 
(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), 
secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters, 
alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% of 
nonionic surfactant such as alcohol ethoxylate (AEO or AE), alcohol 
propoxylate, carboxylated alcohol ethoxylates, nonylphenol ethoxylate, 
alkylpolygly-coside, alkyldimethylamine oxide, ethoxylated fatty acid 
monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty 
acid amide (e.g. as described in WO 92/06154). 
Normally the detergent contains 1-65% of a detergent builder, but some 
dishwashing detergents may contain even up to 90% of a detergent builder, 
or complexing agent such as zeolite, diphosphate, triphosphate, 
phosphonate, citrate, nitrilotriacetic acid (NTA), 
ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid 
(DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered 
silicates (e.g. SKS-6 from Hoechst). 
The detergent builders may be subdivided into phosphorus-containing and 
non-phosphorous-containing types. Examples of phosphorus-containing 
inorganic alkaline detergent builders include the water-soluble salts, 
especially alkali metal pyrophosphates, orthophosphates, polyphosphates 
and phosphonates. Examples of non-phosphorus-containing inorganic builders 
include water-soluble alkali metal carbonates, borates and silicates as 
well as layered disilicates and the various types of water-insoluble 
crystalline or amorphous alumino silicates of which zeolites is the best 
known representative. 
Examples of suitable organic builders include alkali metal, ammonium or 
substituted ammonium salts of succinates, malonates, fatty acid malonates, 
fatty acid sulphonates, carboxymethoxy succinates, polyacetates, 
carboxylates, polycarboxylates, aminopolycarboxylates and polyacetyl 
carboxylates. 
The detergent may also be unbuilt, i.e. essentially free of detergent 
builder. 
The detergent may comprise one or more polymers. Examples are 
carboxymethylcellulose (CMC), poly(vinyl-pyrrolidone) (PVP), 
polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylates such 
as polyacrylates, polymaleates, maleic/acrylic acid copolymers and lauryl 
methacrylate/acrylic acid copolymers. 
The detergent composition may contain bleaching agents of the 
chlorine/bromine-type or the oxygen-type. The bleaching agents may be 
coated or encapsulated. Examples of inorganic chlorine/bromine-type 
bleaches are lithium, sodium or calcium hypochlorite or hypobromite as 
well as chlorinated trisodium phosphate. The bleaching system may also 
comprise a H.sub.2 O.sub.2 source such as perborate or percarbonate which 
may be combined with a peracid-forming bleach activator such as 
tetraacetylethylenediamine (TAED) or nonanoyloxybenzene-sulfonate (NOBS). 
Examples of organic chlorine/bromine-type bleaches are heterocyclic N-bromo 
and N-chloro imides such as trichloroisocyanuric, tribromoisocyanuric, 
dibromoisocyanuric and dichloroisocyanuric acids, and salts thereof with 
water solubilizing cations such as potassium and sodium. Hydantoin 
compounds are also suitable. The bleaching system may also comprise 
peroxyacids of, e.g., the amide, imide, or sulfone type. 
In dishwashing detergents the oxygen bleaches are preferred, for example in 
the form of an inorganic persalt, preferably with a bleach precursor or as 
a peroxy acid compound. Typical examples of suitable peroxy bleach 
compounds are alkali metal perborates, both tetrahydrates and 
monohydrates, alkali metal percarbonates, persilicates and perphosphates. 
Preferred activator materials are TAED or NOBS. 
The enzyme(s) of the detergent composition of the invention may 
additionally be stabilized using conventional stabilizing agents, e.g. a 
polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, 
lactic acid, boric acid, or a boric acid derivative such as, e.g., an 
aromatic borate ester, and the composition may be formulated as described 
in, e.g., WO 92/19709 and WO 92/19708. 
The detergent may also contain other conventional detergent ingredients 
such as, e.g., fabric conditioners including clays, deflocculant material, 
foam boosters/foam depressors (in dishwashing detergents foam depressors), 
suds suppressors, anti-corrosion agents, soil-suspending agents, 
anti-soil-redeposition agents, dyes, dehydrating agents, bactericides, 
optical brighteners, or perfume. 
The pH (measured in aqueous solution at use concentration) will usually be 
neutral or alkaline, e.g. in the range of 7-11. 
Particular forms of laundry detergent compositions within the scope of the 
invention include: 
1) An aqueous liquid detergent composition comprising 
______________________________________ 
Linear alkylbenzenesulfonate 
15-21% 
(calculated as acid) 
Alcohol ethoxylate (e.g. C.sub.12-15 
12-18% 
alcohol, 7 EO or C.sub.12-15 alcohol, 5 EO) 
Soap as fatty acid (e.g. oleic acid) 
3-13% 
Alkenylsuccinic acid (C.sub.12-14) 
0-13% 
Aminoethanol 8-18% 
Citric acid 2-8% 
Phosphonate 0-3% 
Polymers (e.g. PVP, PEG) 
0-3% 
Borate (as B.sub.4 O.sub.7) 
0-2% 
Ethanol 0-3% 
Propylene glycol 8-14% 
Enzymes (calculated as pure enzyme 
0.0001-0.1% 
protein) 
Minor ingredients (e.g. 
0-5% 
dispersants, suds suppressors, 
perfume, optical brightener) 
______________________________________ 
2) An aqueous structured liquid detergent composition comprising 
______________________________________ 
Linear alkylbenzenesulfonate 
15-21% 
(calculated as acid) 
Alcohol ethoxylate (e.g. C.sub.12-15 
3-9% 
alcohol, 7 EO, 
or C.sub.12-15 alcohol, 5 EO) 
Soap as fatty acid (e.g. oleic 
3-10% 
acid) 
Zeolite (as NaAlSiO.sub.4) 
14-22% 
Potassium citrate 9-18% 
Borate (as B.sub.4 O.sub.7) 
0-2% 
Carboxymethylcellulose 
0-2% 
Polymers (e.g. PEG, PVP) 
0-3% 
Anchoring polymers such as, e.g., 
0-3% 
lauryl methacrylate/acrylic acid 
copolymer; molar ratio 25:1; MW 
3800 
Glycerol 0-5% 
Enzymes (calculated as pure enzyme 
0.0001-0.1% 
protein) 
Minor ingredients (e.g. 
0-5% 
dispersants, suds suppressors, 
perfume, optical brighteners) 
______________________________________ 
3) An aqueous liquid detergent composition comprising 
______________________________________ 
Linear alkylbenzenesulfonate 
15-23% 
(calculated as acid) 
Alcohol ethoxysulfate (e.g. C.sub.12-15 
8-15% 
alcohol, 2-3 EO) 
Alcohol ethoxylate (e.g. C.sub.12-15 
3-9% 
alcohol, 7 EO, 
or C.sub.12-15 alcohol, 5 EO) 
Soap as fatty acid (e.g. lauric 
0-3% 
acid) 
Aminoethanol 1-5% 
Sodium citrate 5-10% 
Hydrotrope (e.g. sodium 
2-6% 
toluensulfonate) 
Borate (as B.sub.4 O.sub.7) 
0-2% 
Carboxymethylcellulose 
0-1% 
Ethanol 1-3% 
Propylene glycol 2-5% 
Enzymes (calculated as pure enzyme 
0.0001-0.1% 
protein) 
Minor ingredients (e.g. polymers, 
0-5% 
dispersants, perfume, optical 
brighteners) 
______________________________________ 
4) An aqueous liquid detergent composition comprising 
______________________________________ 
Linear alkylbenzenesulfonate 
20-32% 
(calculated as acid) 
Alcohol ethoxylate (e.g. C.sub.12-15 
6-12% 
alcohol, 7 EO, 
or C.sub.12-15 alcohol, 5 EO) 
Aminoethanol 2-6% 
Citric acid 8-14% 
Borate (as B.sub.4 O.sub.7) 
1-3% 
Polymer (e.g. maleic/acrylic acid 
0-3% 
copolymer, anchoring polymer such 
as, e.g., lauryl 
methacrylate/acrylic acid 
copolymer) 
Glycerol 3-8% 
Enzymes (calculated as pure enzyme 
0.0001-0.1% 
protein) 
Minor ingredients (e.g. hydro- 
0-5% 
tropes, dispersants, perfume, 
optical brighteners) 
______________________________________ 
5) Detergent formulations as described in 1)-4) wherein all or part of the 
linear alkylbenzenesulfonate is replaced by (C.sub.12 -C.sub.18) alkyl 
sulfate. 
6) Detergent formulations as described in 1)-5) which contain a stabilized 
or encapsulated peracid, either as an additional component or as a 
substitute for already specified bleach systems. 
7) Detergent composition formulated as a nonaqueous detergent liquid 
comprising a liquid nonionic surfactant such as, e.g., linear alkoxylated 
primary alcohol, a builder system (e.g. phosphate), enzyme and alkali. The 
detergent may also comprise anionic surfactant and/or a bleach system. 
Particular forms of dishwashing detergent compositions within the scope of 
the invention include: 
1) LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANT SYSTEM 
______________________________________ 
Nonionic surfactant 0-1.5% 
Octadecyl dimethylamine N-oxide 
0-5% 
dihydrate 
80:20 wt. C18/C16 blend of octadecyl 
0-4% 
dimethylamine N-oxide dihydrate and 
hexadecyldimethyl amine N-oxide 
dihydrate 
70:30 wt. C18/C16 blend of octadecyl 
0-5% 
bis (hydroxyethyl) amine N-oxide 
anhydrous and hexadecyl bis 
(hydroxyethyl) amine N-oxide 
anhydrous 
C.sub.13 -C.sub.15 alkyl ethoxysulfate with an 
0-10% 
average degree of ethoxylation of 3 
C.sub.12 -C.sub.15 alkyl ethoxysulfate with an 
0-5% 
average degree of ethoxylation of 3 
C.sub.13 -C.sub.15 ethoxylated alcohol with an 
0-5% 
average degree of ethoxylation of 12 
A blend of C.sub.12 -C.sub.15 ethoxylated 
0-6.5% 
alcohols with an average degree of 
ethoxylation of 9 
A blend of C.sub.13 -C.sub.15 ethoxylated 
0-4% 
alcohols with an average degree of 
ethoxylation of 30 
Sodium disilicate 0-33% 
Sodium tripolyphosphate 
0-46% 
Sodium citrate 0-28% 
Citric acid 0-29% 
Sodium carbonate 0-20% 
Sodium perborate monohydrate 
0-11.5% 
Tetraacetylethylenediamine (TAED) 
0-4% 
Maleic acid/acrylic acid copolymer 
0-7.5% 
Sodium sulphate 0-12.5% 
Enzymes 0.0001-0.1% 
______________________________________ 
2) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION 
______________________________________ 
Liquid nonionic surfactant (e.g. 
2.0-10.0% 
alcohol ethoxylates) 
Alkali metal silicate 
3.0-15.0% 
Alkali metal phosphate 
20.0-40.0% 
Liquid carrier selected from higher 
25.0-45.0% 
glycols, polyglycols, polyoxides, 
glycolethers 
Stabilizer (e.g. a partial ester of 
0.5-7.0% 
phosphoric acid and a C.sub.16 -C.sub.18 
alkanol) 
Foam suppressor (e.g. silicone) 
0-1.5% 
Enzymes 0.0001-0.1% 
______________________________________ 
3) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION 
______________________________________ 
Liquid nonionic surfactant (e.g. 
2.0-10.0% 
alcohol ethoxylates) 
Sodium silicate 3.0-15.0% 
Alkali metal carbonate 7.0-20.0% 
Sodium citrate 0.0-1.5% 
Stabilizing system (e.g. mixtures 
0.5-7.0% 
of finely divided silicone and low 
molecular weight dialkyl polyglycol 
ethers) 
Low molecule weight polyacrylate 
5.0-15.0% 
polymer 
Clay gel thickener (e.g. bentonite) 
0.0-10.0% 
Hydroxypropyl cellulose polymer 
0.0-0.6% 
Enzymes 0.0001-0.1% 
Liquid carrier selected from higher 
Balance 
lycols, polyglycols, polyoxides and 
glycol ethers 
______________________________________ 
4) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION 
______________________________________ 
C.sub.12 -C.sub.14 fatty acid 
0-0.5% 
Block co-polymer surfactant 
1.5-15.0% 
Sodium citrate 0-12% 
Sodium tripolyphosphate 
0-15% 
Sodium carbonate 0-8% 
Aluminum tristearate 0-0.1% 
Sodium cumene sulphonate 
0-1.7% 
Polyacrylate thickener 1.32-2.5% 
Sodium polyacrylate 2.4-6.0% 
Boric acid 0-4.0% 
Sodium formate 0-0.45% 
Calcium formate 0-0.2% 
Sodium n-decydiphenyl oxide 
0-4.0% 
disulphonate 
Monoethanol amine (MEA) 
0-1.86% 
Sodium hydroxide (50%) 1.9-9.3% 
1,2-Propanediol 0-9.4% 
Enzymes 0.0001-0.1% 
Suds suppressor, dye, perfumes, 
Balance 
water 
______________________________________ 
5) LIQUID AUTOMATIC DISHWASHING COMPOSITION 
______________________________________ 
Alcohol ethoxylate 0-20% 
Fatty acid ester sulphonate 
0-30% 
Sodium dodecyl sulphate 
0-20% 
Alkyl polyglycoside 0-21% 
Oleic acid 0-10% 
Sodium disilicate monohydrate 
18-33% 
Sodium citrate dihydrate 
18-33% 
Sodium stearate 0-2.5% 
Sodium perborate monohydrate 
0-13% 
Tetraacetylethylenediamine (TAED) 
0-8% 
Maleic acid/acrylic acid copolymer 
0-8% 
Enzymes 0.0001-0.1% 
______________________________________ 
6) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING PROTECTED BLEACH 
TICLES 
______________________________________ 
Sodium silicate 5-10% 
Tetrapotassium pyrophosphate 
15-25% 
Sodium triphosphate 
0-2% 
Potassium carbonate 
4-8% 
Protected bleach particles, e.g. 
5-10% 
chlorine 
Polymeric thickener 
0.7-1.5% 
Potassium hydroxide 
0-2% 
Enzymes 0.0001-0.1% 
Water Balance 
______________________________________ 
7) Automatic dishwashing compositions as described in 1) and 5), wherein 
perborate is replaced by percarbonate. 8) Automatic dishwashing 
compositions as described in 1), which additionally contain a manganese 
catalyst. The manganese catalyst may, e.g., be one of the compounds 
described in "Efficient manganese catalysts for low-temperature 
bleaching", Nature 369, 1994, pp. 637-639. 
Tests of Stabilizers 
According to the invention the effectiveness of each stabilizer may be 
tested in one or more of the following three tests: 
a) Storage Stability Test in Liquid Deterrent: Enzyme(s) and stabilizer are 
added to a liquid detergent formulation and stored at well defined 
conditions. The enzyme activity of each enzyme is determined as a function 
of time, e.g. after 0, 3, 7 and 14 days. 
To calculate the inhibition efficiency from the storage stability date a 
reaction mechanism is proposed. The following reactions give a relatively 
simple, but yet plausible, mechanism for a liquid detergent containing 
protease (P), lipase (L), and inhibitor (I): 
I) Autodigestion of protease: 
EQU P+P.fwdarw.D.sub.P +P 
II) Denaturation of protease: 
EQU P.fwdarw.D.sub.P 
III) Inhibition of protease: 
EQU P+I.revreaction.PI 
IV) Protease digestion of inhibited enzyme: 
EQU P+PI.fwdarw.P+D.sub.P +I 
V) Denaturation of inhibited enzyme: 
EQU PI.fwdarw.D.sub.P +I 
VI) Protease digestion of lipase: 
EQU P+L.fwdarw.P+D.sub.L 
VII) Denaturation of lipase: 
EQU L.fwdarw.D.sub.L 
where D.sub.P and D.sub.L are denatured (i.e. non-active) protease and 
lipase. 
From these reactions three coupled differential equations are derived 
describing the deactivation of P, L and PI. The reaction rate constants 
are derived from storage stability data by the use of a parameter 
estimation method (Gauss-Newton with the Levenberg modification). The 
storage stability data give the concentration of (P+PI) and L as a 
function of time. 
Reaction III is much faster than the other reactions and equilibrium is 
assumed in the calculations. Reaction IV is excluded from the system to 
reduce the number of parameters thereby describing the stability of the 
inhibited enzyme by only one reaction rate constant (from equation V). 
In all experiments there is a large surplus of inhibitor molecules compared 
to protease molecules, i.e. a constant concentration of inhibitor 
(corresponding to the added amount of inhibitor) is a reasonable 
assumption. 
The specific values of the reaction rate constants are somewhat sensitive 
to small variations in the data, but the sensitivity is reduced 
significantly by giving the results relatively to the value from Boric 
Acid. An improvement factor is thus derived: 
##EQU1## 
IF.sub.1 measures the inhibition efficiency given by the inhibition 
constants K.sub.i from reaction III. 
b) The "Milk" Test: In this test the stabilizer to be tested is compared 
with a reference inhibitor (boric acid). The test is described in details 
below: 
Preparation of "inhibitor" milk: 0.075 g of CaCl.sub.2 (dried fine-granular 
pure, Merck), 0.16 g of 3,3-dimethylglutaric acid (SIGMA) and 2.5 mmole of 
stabilizer/inhibitor are weighed out and dissolved in 50 ml of 
demineralised water. pH is adjusted to approx. 6.0 with NaOH. 6.0 g of 
skimmed milk powder (dehydrated, DIFCO Lab.) is weighed out in a 100 ml 
beaker, and the solution of salt+buffer+stabilizer/inhibitor is added. 
This mixture is stirred heavily for some minutes to be sure that all 
lumps, if any, are apart. Thereafter the mixture is stirred for 30 
minutes. pH is adjusted to 6.50 with NaOH. Skimmed milk from a bottle can 
be used instead of powder. Use milk from the same bottle for all 
stabilizers/inhibitors in one run. 
Preparation of the enzyme: Prepare a solution of approx. 30 KNPU/litre of 
Savinase.RTM. (available from Novo Nordisk A/S) in boric acid buffer (see 
below). The Savinase activity is determined relatively to an enzyme 
standard. A folder AF 220/1-GB describing the analytical method of 
determining the Savinase activity is available upon request to Novo 
Nordisk A/S, Denmark, which folder is hereby included by reference. 
Example: 1.0 g of 16 KNPU/g liquid Savinase is weighed out and 50 ml of 
boric acid buffer are added. The mixture is stirred for 15 minutes. 10 ml 
of this solution are filled into a 100 ml beaker, and boric acid buffer is 
added up to 100 ml. Thereafter the mixture is stirred for 15 minutes. 
Boric acid buffer: 2.5 g of boric acid (Merck) are dissolved in 500 ml of 
demineralised water. pH is adjusted to 9.0 with NaOH. 
The curdling: 10.0 ml of stabilizer/inhibitor are added to a test tube. 3 
test tubes of each stabilizer/inhibitor are made and placed in a 
30.degree. C. water bath. The test tubes are left in the water bath for 
one hour. 1.00 ml of Savinase solution is added to the test tube and the 
stop-watch is started. The tube is mixed for 10 seconds on the "vibrator" 
and thereafter placed in the water bath. When the curdling starts the 
stop-watch is stopped. The deviation between the curdling time for the 
three test tubes should not be more than approx. 10 seconds. How and when 
the curdling starts must be learned in practice and the same person should 
curdle all samples. The curdling time for the reference inhibitor (boric 
acid) should be around 3-4 minutes (if the curdling time is longer, a 
stronger protease solution should be used). The curdling time is approx. 
linear proportional to 1/ (protease activity). The result can be reported 
as an improvement factor IF defined by: (curdling time 
stabilizer)/(curdling time reference). 
c) Determination of K.sub.i : The inhibition constant K.sub.i may be 
determined by using standard methods, for reference see Keller et al, 
Biochem. Biophys. Res. Com. 176, 1991, pp.401-405; J. Bieth in 
Bayer-Symposium "Proteinase Inhibitors", pp. 463-469, Springer-Verlag, 
1974 and Lone Kierstein Hansen in "Determination of Specific Activities of 
Selected Deterrent Proteases using Protease Activity, Molecular Weights, 
Kinetic Parameters and Inhibition Kinetics", PhD-report, Novo Nordisk A/S 
and University of Copenhagen, 1991. 
The invention is further illustrated in the following examples which are 
not intended to be in any way limiting to the scope of the invention as 
claimed.

EXAMPLE 1 
Preparation of Naphthalene-1-Boronic Acid 
The Grignard reagent was prepared by the slow dropwise addition of 
1-bromonaphthalene (0.05 m) in sodium dried ether (50 ml) to magnesium 
turnings (0.05 m) in sodium dried ether (50 ml). The reaction was 
encouraged by the addition of a small iodine crystal. 
Trimethylborate (0.05 m) or tri-n-butylborate (0.05 m) in sodium dried 
ether was cooled to -70.degree. C. and the Grignard reagent was added 
dropwise over a period of 2 hours while keeping the borate solution at 
-70.degree. C. and continuously agitating. 
The reaction mixture was allowed to warm to room temperature overnight 
whereupon it was hydrolysed by the dropwise addition of cold dilute 
sulphuric acid (10%, 50 ml). The ether layer was separated and the aqueous 
layer extracted with ether. The ether containing fractions were combined 
and the solvent removed. The residue was made distinctly alkaline and any 
methanol or butanol so formed was removed. The alkaline solution was made 
acidic and cooled and the resulting crystals were removed by filtration. 
The combined boronic acid crystals were recrystallized from distilled 
water. C.sub.10 H.sub.9 BO.sub.2, mpt. 210.degree.-211.degree. C. 
Preparation of Naphthalene-2-Boronic Acid 
2-Naphthalene Boronic Acid was made in the same way as 1-Naphthalene 
Boronic Acid, only 2-bromonaphthalene was used instead of 
1-bromonaphthalene. C.sub.10 H.sub.9 BO.sub.2, mpt. 
258.degree.-259.degree.C. 
Protection of 6-hydroxy-2-bromonaphthalene in order to make 
6-hydroxynaphthalene-2-boronic acid 
6-hydroxy-2-bromonaphthalene (0.05M) was dissolved in dichloromethane (50 
ml). Dihydropyran (7.5 ml) was added along with a small amount of 
p-toluene sulphonic acid to catalyse the reaction. The mixture was stirred 
at 25.degree. C. for 3 hours. 
The solution was then basified by the addition of an ammonical methanol 
solution (1:5, 30 ml). The solvents were removed and the remaining brown 
residue was dissolved in dichloromethane (30 ml) and extracted with sodium 
carbonate solution (0.1M, 2.times.30 ml). 
The organic layer was dried over anhydrous sodium sulphate and the solvent 
removed. The resulting protected bromide was then used in the Grignard 
reaction to make 6-hydroxynaphthalene-2-boronic acid. 
EXAMPLE 2 
Determination of K.sub.i 
The inhibition constant K.sub.i for the inhibition of Alcalase and Savinase 
was determined using standard methods under the following conditions: 
Substrate: 
Succinyl-Alanine-Alanine-Proline-Phenylalanine-paranitro-anilide=SAAPFpNA 
(Sigma S-7388). 
Buffer: 0.1M Tris-HCl pH 8.6; 25.degree. C. 
Enzyme concentration in assay: 
Alcalase: 1.times.10.sup.-10 -3.times.10.sup.-10 M 
Savinase: 1.times.10.sup.-10 -3.times.10.sup.-10 M 
The initial rate of substrate hydrolysis was determined at nine substrate 
concentrations in the range of 0.01 to 2 mM using a Cobas Fara automated 
spectrophotometer. The kinetic parameters V.sub.max and K.sub.m were 
determined using ENZFITTER (a non-linear regression data analysis 
program). k.sub.cat was calculated from the equation V.sub.max =k.sub.cat 
.times.E.sub.o !. The concentration of active enzyme E.sub.o ! was 
determined by active site titration using tight-binding protein proteinase 
inhibitors. The inhibition constant K.sub.i was calculated from plots of 
K.sub.m /k.sub.cat as a function of the concentration of inhibitor. The 
inhibitors were assumed to be 100% pure and the molar concentrations were 
determined using weighing numbers and molecular weights. 
The results of the inhibition constants K.sub.i of the boronic and borinic 
acid derivative enzyme stabilizers tested are listed in 
TABLE 1 
______________________________________ 
The inhibition constants for the inhibition of 
Alcalase and Savinase by naphthalene boronic acids. Boric acid 
is included for comparison. 
K.sub.i K.sub.i 
Inhibitor Alcalase Savinase 
______________________________________ 
Boric acid 30 mM 20 mM 
Naphthalene-2- 0.4 mM 0.3 mM 
boronic acid 
Naphthalene-1- 0.5 mM 0.9 mM 
boronic acid 
6-hydroxy- 0.5 mM 0.6 mM 
naphthalene-2- 
boronic acid 
______________________________________ 
EXAMPLE 3 
Storage Stability Test in Liquid Detergent 
Naphthalene boronic acids were also tested in storage stability tests in 
liquid detergents using the method described previously under the 
following conditions: 
Deterrent base (US-type) 
______________________________________ 
% wt (as pure components) 
______________________________________ 
Nansa 1169/p 10.3 (Linear Alkylbenzene Sulfonate, LAS) 
Berol 452 3.5 (Alkyl Ether Sulfate, AES) 
Oleic acid 0.5 
Coconut fatty acid 
0.5 
Dobanol 25-7 6.4 (Alcohol Ethoxylate, AEO) 
Sodium xylene sulfonate 
5.1 
Ethanol 0.7 
MPG 2.7 (Mono Propylene Glycol) 
Glycerol 0.5 
Sodium sulfate 
0.4 
Sodium carbonate 
2.7 
Sodium citrate 
4.4 
Citric acid 1.5 
Water 60.8 
Enzyme dosage: 
1% w/w Savinase (14 KNPU/g) 
Enzyme Stabilizer Dosage: 
5 mmole/kg (for boric acid 160 mmole/kg) 
Storage: 0, 3, 7 and 14 days at 30.degree. C. 
______________________________________ 
The results of the inhibition effectiveness IF.sub.1 of the naphthalene 
boronic acid enzyme stabilizers tested are listed below. 
TABLE 2 
______________________________________ 
shows the results of different naphthalene boronic 
acids and the corresponding IF.sub.I . Boric acid is included for 
comparison. 
Improvement Factor 
Inhibitor IF.sub.I 
______________________________________ 
Boric acid 1 
Naphthalene-2-boronic acid 
30 
Naphthalene-1-boronic acid 
5 
6-hydroxynaphthalene-2- 
26 
boronic acid 
______________________________________ 
Comparing the results of Table 1 (K.sub.i Savinase) with the results of 
Table 2 it seems that the effect of a naphthalene boronic acid stabilizer 
in detergents can be predicted from the results obtained in buffer systems 
and vice versa.