Note: Descriptions are shown in the official language in which they were submitted.
20~0~1~
.
Henkel KGaA Dr. Wilk/Mj/ah
Henkelstra~e 67
4000 Dusseldorf, 06.06.1989
Patent Application
D 866a f~
. :
A liouid enzyme concentrate
This invention relates to a liquid enzyme concentrate
which may be used as a stock solution for the production of
liquid detergents or the like.
It is known that enzymes, particularly proteases, are
used in detergents, including liquid detergents. For use
in solid detergents, the enzymes are made up into solid
granular preparations. With the advances being made by
liquid detergents, there is also a need for such prepa~-
ations in order to be able to produce storable stock
solutions which may then be added to the liquid detergent
in a subsequent process step.
However, enzyme solutions of hydrolases, particularly
proteases are not storable per s This applies in partic-
ular to enzvme solutions which are to be used for liquid
detergents because, in addition to impurities, substances
which have a stabilizing effect would also appear to be
removed during the purification of the enzvme solutions.
Numerous proposals have already been put forward in
the specialist literature with a view to stabilizing
enzymes in liquid detergents. However, since the enzyme
content of liquid detergents is very low compared with
; other substances, such as surfactants for example, so that
totally different conditions prevail, the results of these
proposals are not applicable to enzyme concentrates.
Thus, US-PS 3,781,212 describes a liquid detergent in
which a polyol is used in conjunction with boric acid to
. : ' .
,' ' . ; . '.'' ' ~' . ' ' : : ' '
. .
20S0~1 ~
D 8668 2
stabilize the enzyme. The ratio of polyol to boric acid is
said to be less than l, i.e. boric acid is used in excess
compared with the polyol.
DE-OS 31 25 533 describes an aqueous builder-contain-
ing liquid detergent containing enzymes, washing-active
substances, a polyol and boric acid or salts thereof in a
ratio by weight of polyol to boric acid of more than 1, one
part crosslinked polyacrylate also being present. In
addition to other polyols, propane-1,2-diol is also men-
tioned and, in addition to other boron compounds, boric
acid and salts thereof are also mentioned. The quantity of
polyol must amount to between 100 and 200% by weight of the
quantity of boric acid and preferably amounts to between
100 and 160% by weight of the quantity of boric acid. How-
ever, the hydrolase content of liquid detergents is, typic-
ally, only about 0.1% by weight protein and, at best, up to
0.5% by weight.
; There is nothing in the German patent application
cited above to suggest how a stable liquid concentrate
- 20 could be obtained. Accordingly, the problem addressed by
the present invention is to provide such a liquid concen-
trate.
The present invention relates to a liquid enzyme con-
centrate based on a hydrolase solution having an enzyme
content of 30,000 to 400,000 hydrolase units which is
; obtained by fermentation and which is freed from the cellmass and at least partly from soluble impurities, con-
taining
` - 1.5 to 20% by weight hydrolase,
- 20 to 40% by weight propylene glycol and
- 0.5 to 5% by weight boric acid and/or soluble salts
thereof and, optionally,
; - 0.5 to 5% by weight salts of mono- to tribasic car-
boxylic acids containing up to 6 carbon atoms.
In one preferred embodiment of the invention, the
.
' ~
D 8668 3 2~0~1~
liquid enzyme concentrates according to the invention are
concentrated, at least partly purified enzyme solutions ob-
tained by fermentation which, apart from the stabilizers
used in accordance with the invention and the substances
present or formed during the fermentation process, contain
no other constituents.
According to the invention, a combination of propylene
glycol and boric acid and/or salts thereof is used as the
stabilizer. The quantity of propylene glycol (propane-1,2-
diol) is from 20 to 40% by weight and more especially from
- 20 to 30% by weight. The quantity of boric acid is from
0.5 to 5% by weight and more especially from 1 to 3% by
weight.
The percentages by weight in respect of the boric acid
are based on the acid, even if it is present in the form of
a solution of its salts so that larger quantities by weight
are present.
Suitable salts of mono- to tribasic carboxylic acids
containing up to 6 carbon atoms are the soluble alkali
metal or ammonium salts of the corresponding carboxylic
acids. Thus, it is possible to use salts of formic acid,
propionic acid, malonic acid, maleic acid or fumaric acid.
Typical concentrations of the salts, based on the prepar-
ation as a whole, are from 0.5 to 5% by weight. For
example, favorable results have been obtained with 1% by
weight formic acid or 1% by weight maleic acid, used in
either case in the form of the sodium salt.
Since a number of hydrolase solutions are unstable at
certain pH values, the boric acid is preferably used in the
form of a salt, i.e. the enzyme solutions are preferably
adjusted to a pH value of 6 to 8 before, after or during
the addition of the boric acid. Suitable boric acid salts
are the alkali metal or ammonium salts, particularly the
sodium or potassium salt.
According to the invention, the starting enzyme
: ~, . . . .
, ,
;' . . -: . :
: . ' , ' ~ : '
,
D 8668 4 2 ~
solution may be an enzyme solution of the type formed in
the fermentation of yeast or bacteria capable of forming
hydrolases. Solutions containing proteases, particularly
subtilisin proteases, as hydrolases are preferred. Of
these proteases, those based on subtilisin Carlsberg or
subtilisin BBN' are preferred.
To produce the enzyme concentrates according to the
invention, the cell components are removed from the fer-
menter solutions and the enzymes are subsequently enriched.
This may be done, for example, by a combination of micro-
and ultrafiltration or even by precipitation and floccula-
tion steps, optionally with subsequent concentration of the
aqueous solution by evaporation.
Propane-1,2-diol and the boric acid are added to the
purified enzyme solutions thus obtained. To ensure that
the pH does not fall below a critical value, which with
some hydrolases is only at pH 4, the desired pH of 6 to 8
may be established by addition of sodium hydroxide before
or during addition of the boric acid.
In addition, the preparations according to the inven- -
tion may contain preservatives. In many cases, however,
; this is not necessary because the combination of propylene
glycol and boric acid itself has a preserving effect.
Formaldehyde is one example of a suitable preservative.
The invention is based on the synergistic stabilizing
effect of boric acid and propylene glycol on enzymes of the ~-
type in question which provides particularly favorable
stability values within the stated limits, as can be seen
from the following Examples.
Example~
Example 1
A solution of a subtilisin Carlsberg protease having
~ an activity of 130 EP units/g was incubated with quantities
; 35 of 0, 20, 30 and 50~ by weight propylene glycol and sub-
.. .
D 8668 5 20~0~1 ~
jected an accelerated ageing test at 60'C. The following
residual activities were measured after 24 hours:
Without 20% by weight 30% by weight 50% by weight
propylene propylene propylene propylene
glycol glycol glycol glycol
Below 5 23 25 16
Example 2
The procedure was as in Example 1 except that 1, 3 and
5% by weight sodium metaborate were also added for 20% by
weight propylene glycol. The following residual activities
in percent were measured after 24 hours:
1% metaborate 50%
3% metaborate 46%
5% metaborate 34%.
Example 3
The procedure was as before using
1~ by weight sodium metaborate,
30% by weight propylene glycol and
1% by weight formic acid or maleic acid.
After storage for 100 minutes at 60-C, no reduction in
activity was observed whereas, without the addition of the
carboxylic acid salts, there was a reduction in activity to
84%.
In every case, enzyme activity was measured by the so-
called EPE method described in German patent application DE
37 34 047. In principle, this method is based on cleavage
of a phenolic ester bond of N-CBZ aminoacid p-nitrophenyl
ester at 25-C and at pH 6.0, the concentration of the p-
nitrophenol formed being determined photometrically at 340
nm.
':
, .
,
,
.
- ~ -
-~
