Liquid enzymatic detergent composition

The solubility of an enzyme in a liquid detergent can be improved by chemical modification of free primary amino groups in the enzyme while still preserving the enzyme activity. The modification preferably involves aldehyde treatment, acylation, or alkylation of the amino groups.

TECHNICAL FIELD 
This invention relates to a liquid enzymatic detergent composition and to 
an enzymatic detergent additive in the form of a stabilized liquid for use 
therein. The invention also relates to a method of increasing the 
solubility of an enzyme, particularly in the preparation of a liquid 
enzymatic detergent or a liquid enzymatic detergent additive. 
BACKGROUND ART 
Enzymes are commonly incorporated in liquid detergents to improve 
detergency. The most commonly used detergent enzymes are proteases, mainly 
alkaline Bacillus proteases such as subtilisin Carlsberg. The prior art 
deals extensively with the formulation of liquid enzymatic detergents, 
particularly with improving the stability of the enzyme during storage. 
One recent example is EP 352,244. 
In general, the enzyme should be fully dissolved in the liquid detergent to 
prevent phase separation during storage and to ensure that the enzyme 
activity is immediately available during washing. 
We have found that in some liquid detergents, the enzyme is not fully 
soluble and may precipitate. It is the object of the invention to provide 
a liquid enzymatic detergent composition with improved enzyme solubility, 
an enzymatic detergent additive for use therein, and a method of improving 
the solubility of an enzyme without serious loss of enzyme activity. 
DISCLOSURE OF INVENTION 
We have, surprisingly, found that by chemical modification of free primary 
amino groups in an enzyme, the solubility of the enzyme can be improved 
while the enzyme activity is preserved. The modification preferably 
involves aldehyde treatment, acylation or alkylation. 
It is known that aldehydes can react with primary amino groups in enzymes, 
and treatment of enzyme with glutaraldehyde has been widely used for 
immobilization of enzymes, i.e. for preparing a water insoluble enzyme 
product (e.g. U.S Pat. No. 3,980,521, JP-B 50-037274). It is also known 
from DE-A 2,919,622 that a water-insoluble product can be obtained by 
treatment of a protease with a polysaccharide derivative containing 
aldehyde groups such as dialdehyde starch. Further, it is known from J. 
Boudrant et al., Biotech. and Bioeng., XVIII, 1719-34 (1976) that aldehyde 
treatment of a typical detergent enzyme converts part of the enzyme 
protein into an insoluble fraction. GB 1,280,497 discloses treatment of 
enzyme with e.g. glutaraldehyde and removal of insoluble residue to 
improve the stability of the enzyme when added to a solution of powder 
detergent. There is, however, no suggestion in the prior art that such 
treatment of an enzyme can be used to increase its solubility. 
Accordingly, the invention provides a liquid enzymatic detergent 
composition and an enzymatic detergent additive in the form of a 
stabilized liquid, each characterized by comprising dissolved therein an 
enzyme containing chemically modified primary amino groups. 
Further, the invention provides a method of increasing the solubility of an 
enzyme containing free primary amino groups, said method comprising 
chemical modification of part or all of said primary amino groups.

DETAILED DESCRIPTION OF INVENTION 
Enzyme 
Besides the terminal amino group, most enzymes, in particular most 
detergent enzymes, contain lysine with free primary amino (--NH.sub.2) 
groups. The invention may be used for modifying any enzyme, provided it 
contains free primary amino groups, preferably at least 2, and most 
preferably at least 4 free primary amino groups per molecule. The enzyme 
typically has molecular weight 20,000-100,000, especially 20,000-60,000, 
and isoelectric point 7-12. The enzyme may be a protease, an amylase, a 
lipase, a peroxidase or a cellulase, especially a subtilisin (an alkaline 
Bacillus protease), e.g. Subtilisin Carlsberg, derived from B. 
licheniformis (e.g. Alcalase.RTM., product of Novo Nordisk A/S), 
Savinase.RTM. (product of Novo Nordisk, produced from alkalophilic 
Bacillus according to U.S. Pat. No. 3,723,250) or a mutant subtilisin such 
as those described in WO 89/06279 or DK 0541/90. Subtilisin Carlsberg has 
molecular weight 27,000, pl around 8.3 and the molecule contains 10 
primary amino groups (the N-terminal and 9 lysine groups). The detergent 
of the invention typically contains 2-40 .mu. mole of modified enzyme per 
kg. 
Chemical modification 
According to the invention, the enzyme may be modified by any method acting 
on primary amino groups, particularly by reaction with an aldehyde, by 
acylation or by alkylation. 
The enzyme is preferably treated in aqueous solution at a concentration of 
0.1-5 mM. The enzyme to be treated is preferably largely free from other 
proteins and other compounds with primary amino groups as these may 
interfere by reacting with the modifying reactant. In general, the amount 
of the modifying reactant may correspond to 0.1-3 reactive groups per 
primary amino group, preferably 0.2-2 and most preferably 0.4-1.4. 
When using a monofunctional reactant, it may be preferable to use an excess 
of reactant, e.g. 1-5 times of the stoichiometric. 
When using a bifunctional (or polyfunctional) reactant it may be 
preferable, in order to improve the solubility, to minimize formation of 
oligomers by intermolecular reactions. The formation of oligomers may be 
minimized by using a relatively low concentration of enzyme, e.g. 0.1-1 
mM, and/or by using essentially the stoichiometric amount or less of the 
bi/polyfunctional reactant, e.g. 10-100% of the stoichiometric. If 
oligomers precipitate after the treatment, this precipitate may be removed 
(e.g. by centrifugation) to obtain modified enzyme consisting mostly of 
monomer. 
Aldehyde treatment may be made using an aliphatic or aromatic mono- or 
dialdehyde, e.g. formaldehyde, glutaraldehyde or o-phthalic aldehyde. The 
treatment is preferably carried out at pH in the range 5-10 and a 
temperature of 0.degree.-70.degree. C., conveniently near ambient 
temperature. The mixture of enzyme and aldehyde should be left until the 
reaction is essentially complete (as indicated by stable pH), typically 
0.5-8 hours. 
Acylation may be made by use of an anhydride of a mono- or dicarboxylic 
acid, e.g. acetic, propionic, butyric, valeric, caprylic, maleic or 
succinic anhydride. 
Alkylation of primary amino groups may be achieved e.g. by reductive 
treatment with an aldehyde, such as those mentioned above. An example is 
reductive methylation. 
Detergent additive 
The enzymatic detergent additive of the invention contains an enzyme 
stabilizer to improve the storage stability. Many such enzyme stabilizers 
are known in the art, e.g. a polyol such as propylene glycol, typically 
used in an amount of 20-70%, e.g. according to U.S. Pat. Nos. 4,543,333 or 
4,497,897. 
Detergent 
The invention is particularly applicable to the formulation of liquid 
detergent where it is desired to incorporate enzyme at a concentration 
that exceeds the solubility of the unmodified enzyme. 
The invention particularly relates to a homogeneous concentrated liquid 
detergent with water content of 20-50% (hereinafter, all percentages are 
by weight), especially 40-50%. The pH of the detergent may be 6-10.5, 
especially 7-9. 
The detergent may contain 4-25% of C.sub.10 -C.sub.16 alkyl or alkenyl 
succinate, especially 5-15% of said succinate. The detergent may further 
contain other builders, e.g. 0.5-6% citrate. The cation may be sodium, 
potassium, ammonium or mono-, di- or triethanol-ammonium. 
The detergent may additionally contain 5-30%, especially 10-20% of anionic 
surfactant, such as alkyl benzene sulphonate, alpha-olefin sulphonate, 
dialkyl sulfosuccinate, alkyl sulphate, alkyl ethoxy sulphate, fatty acid 
soap or a combination of two or more of these. In each of these, the alkyl 
group may be linear C.sub.10 -C.sub.18, and the cation may be sodium, 
potassium, ammonium or mono, di- or triethanol-ammonium. 
The detergent may also contain 3-35%, especially 5-15% of nonionic 
surfactant, such as polyethoxylated alcohol or alkyl phenol. 
The detergent may contain 2-20%, especially 2-10% of solvent, such as 
C.sub.1 -C.sub.4 alcohol or polyol, e.g. ethanol or glycerol or a 
combination of two or more solvents. 
The detergent may additionally contain 0.5-5% of one or more electrolytes, 
such as borate, carbonate, formate or chloride of sodium or potassium. 
Specific examples of detergents according to the invention are obtained 
from the compositions given in EP 200,263, EP 212,723 and EP 223,306 (said 
three publications incorporated herein by reference) by including 12-16 
.mu.mole/kg, e.g. 15 .mu.mole/kg of modified enzyme according to the 
invention, and optionally substituting dodecyl-succinate for 
dodecenyl-succinate. 
EXAMPLES 
Example 1 
Phthaldialdehyde was added to a solution of enzyme (Alcalase) to prepare an 
aqueous system containing 63 mg/g (2.3 .mu.mole/g) of enzyme and 1 mg/g 
(7.5 .mu.mole/g) of aldehyde, i.e. a ratio of 0.65 aldehyde groups per 
primary amino group. The system also contained 25% of propylene glycol, 
0.2% of calcium ions and 1.8% of formate ions. The pH was adjusted to 10 
and the phthaldialdehyde reacted with the enzyme solution for approx. 5 
hours. Afterwards the solution was filtered, pH adjusted to 5.5. 
By measuring the protease activity before treatment with phthaldialdehyde 
and after treatment and pH adjustment, it was found that there had been no 
detectable loss of activity. 
0.6% of this solution was added to a commercial liquid detergent ("Ariel" 
(batch 279A42), marketed by Procter & Gamble in Denmark). After storage 
for 6 weeks at both 25.degree. C. and 35.degree. C. no enzyme deposit was 
observed in the detergent. As a reference the same Alcalase which has not 
been treated with the aldehyde was added to the detergent (the same amount 
of active enzyme as for the aldehyde treated enzyme was added). After four 
days at 35.degree. C. and one week at 25.degree. C. needle-shaped enzyme 
deposit was observed. 
Example 2 
As Example 1 except that phthaldialdehyde was exchanged with 10 .mu.mole/g 
of glutaraldehyde, i.e. a molar ratio of 0.87. After adding to the 
detergent the system was observed for 4 weeks and no enzyme deposit was 
found neither at 25.degree. C. nor at 35.degree. C. 
Example 3 
Glutaraldehyde was added to a solution of enzyme (Alcalase) to prepare an 
aqueous solution containing 87 mg/g (3.2 .mu.mole/g) of enzyme and 1.16 
mg/g (11.6 .mu.mole/g) of aldehyde, i.e. a ratio of 0.73 aldehyde groups 
per primary amino group. The pH was kept at 7.5 throughout the treatment. 
The treatment was terminated when base consumption ceased, and the 
solution was filtered. 
A formulation containing 25% propylene glycol, 0.2% Ca, 1.8% formate and pH 
5.5 was prepared. 0.6% of this formulation was added to the same detergent 
as in Example 1, and after storage at 35.degree. C. and 25.degree. C. for 
3 weeks no crystalline enzyme deposit was observed. 
The molecular weight of the glutaraldehyde-treated Alcalase was determined 
by SDS-PAGE together with untreated Alcalase. The molecular weights of the 
two enzymes were found to be exactly the same, approx. 27,000. 
Example 4 
Glutaraldehyde was added to a solution of enzyme (Alcalase) to prepare an 
aqueous solution containing 60 mg/g (2.2 .mu.mole/g) of enzyme and 0.90 
mg/g (9.0 .mu.mole/g) of aldehyde, i.e. a ratio of 0.82 aldehyde groups 
per primary amino group. The pH was kept at 7.5 throughout the treatment. 
The treatment was terminated when base consumption ceased. 
A detergent with the following composition was used: 
______________________________________ 
Linear Alkyl Sulphonate (NANSA 1169/P) 
9.65% 
Alcohol Ethoxylate (Dobanol 25-7) 
10.00% 
2-dodecenyl succinic anhydride 
13.60% 
Citric acid 0.85% 
Oleic acid 3.65% 
Diethylenetriamine pentamethylene 
0.80% 
phosphonic acid 
Coconut alkyl sulphate TEA salt 
3.30% 
Ethanol, 96% 3.00% 
1,2-propanediol 1.70% 
Monoethanolamine 0.50% 
Sodium formate 0.95% 
Calcium chloride (CaCl.sub.2, 2H.sub.2 O) 
0.06% 
NaOH 4.15% 
Water balance to 100% 
pH 7.6 
______________________________________ 
The mixing procedures for the detergent, which take place at 
80.degree.-85.degree. C., are as follows: 
I: The following components are mixed in the mentioned order: 
LAS, 1,2-propanediol, ethanol, coconut alkyl sulphate TEA salt and 
monoethanolamine. Afterward the NaOH and 2-dodecenyl succinic anhydride 
are added simultaneously. One half of the water, alcohol ethoxylate. 
II: The other half of the water, sodium formate, calcium chloride, 
phosphonate and citric acid are mixed. 
III: I+II+oleic acid are mixed. 
0.6% of the aldehyde treated enzyme was added to the detergent. As 
reference, 0.6% of untreated enzyme was added to the same detergent. 
After three days at 35.degree. C. needle-shaped precipitate was observed in 
the reference detergent. No precipitate was observed in the detergent with 
the aldehyde-treated Alcalase. 
Storage stability was determined by measuring residual enzyme activity at 
various times during storage at controlled temperature. No difference was 
seen between the detergent with modified enzyme and reference detergent. 
Comparative washing tests showed the detergent with modified enzyme and the 
reference detergent to be equally effective for removal of proteinaceous 
soiling. 
Example 5 
1 g protease (Alcalase) was dissolved in 25 ml 0.1M borate buffer pH 7 at 
0.degree. C. 700 .mu.l of a 100 mg/ml succinic anhydride solution in 
acetone was added portion wise (theoretically 2.1 equivalent 
anhydride/--NH.sub.2 lysine group). pH was adjusted to 7 with 2M NaOH 
after each addition of succinic anhydride. The reaction was followed on an 
MonoS ion exchange. The reaction mixture was dialysed against water when 
no more free enzyme could be detected. The mixture was then freeze dried. 
A stabilized liquid formulation was prepared, containing 55.55 mg modified 
enzyme per g, 25% propylene glycol, 0.2% calcium, 1.8% formic acid at pH 
5.5. 0.6% of this formulation was added to the detergents described in 
Examples 1 and 4. 
A reference was made by treating the enzyme in the same way, but without 
addition of succinic anhydride. 0.8% of the reference formulation was 
added to the detergents. 
In the reference detergents, needle-shaped deposit was observed after 10 
days storage at 35.degree. C. and one month at 25.degree. C. No deposit 
was observed in the detergents with modified enzyme according to the 
invention. 
Example 6 
10 g of an enzyme stock solution containing 55.5 mg pure enzyme (Alcalase), 
25% propylene glycol, 0.2% calcium and 1.8% formic acid was adjusted from 
pH 5.2 to pH 8.0. 2 g of a 200 mM NaBH.sub.3 CN solution was added. 73.97 
mg of a 37% HCHO solution was then added portion wise (corresponding to 5 
mole equivalents HCHO per mole lysine amino group). The reaction was 
conducted at room temperature for 2 hours. The pH was the adjusted to 5.2 
with formic acid. No enzymatic activity was lost during the procedure. 80 
mg of this formulation was added to 10 g of the detergents described in 
Examples 1 and 4. As reference, 80 mg of the enzyme stock solution added 
to 10 g of the same detergents. 
After 1 week, needle-shaped enzyme deposit was observed in the reference 
detergents, whereas no deposit could be observed in the detergents with 
modified enzyme according to the invention. 
Example 7 
10 g of an enzyme stock solution containing 55.5 mg of pure enzyme 
(Alcalase), 25% propylene glycol, 0.2% Ca, 1.8% formic acid, was adjusted 
from 5.2 to 8.0. 1 g of a 200 mM NaBH.sub.3 CN solution was added. 33.33 
mg of a 50% Glutaraldehyde solution was then added portion wise 
(Corresponding to 0.91 mole glutaraldehyde per mole lysine group). The 
reaction was conducted at room temperature for 2 hrs. The pH was then 
adjusted to 5.2 with formic acid. No enzymatic activity was lost during 
the procedure. To 10 g of the detergent described in example 1, 80 mg of 
the above formulation was added. As reference, 80 mg of the enzyme stock 
solution added to 10 g of the same detergent. 
After 3 weeks, needle-shaped enzyme deposit was observed in the reference 
detergent, whereas no deposit could be observed in the detergent 
containing modified enzyme according to the invention. 
Example 8 
Glutaraldehyde was added to a solution of protease (Savinase.RTM.) to 
prepare an aqueous solution containing 45 mg/g (1.6 .mu.mole/g) of 
Savinase.RTM. and 0.32 mg/g (3.2 .mu.mole/g) of aldehyde (i.e. a ratio of 
aldehyde molecules to primary amino groups of 0.33). The solution also 
contained 35% propylene glycol, 0.7% Ca.sup.++ and 2% formate. pH was 
adjusted to 8.0 and the solution was left to react at room temperature for 
five hours. 
When 0.2% of the treated Savinase.RTM. was added to a clear detergent of 
essentially the same composition as the detergent in Example 1 a slight 
haziness was observed after 1 week at 35.degree. C., whereas the untreated 
enzyme caused a slight precipitate (same formulation, dosage and storage 
conditions). When stored at 4.degree. C. for 4 weeks the same pattern was 
observed: The treated enzyme gave a slight haziness in the detergent, but 
the untreated gave a marked precipitation.