Note: Descriptions are shown in the official language in which they were submitted.
2~51677
Hen~ 5aA Dr. Wilk/Mj
Henkelstrasse 67
4000 Dusseldorf, 21.03.1989
Patent application
D 8535 ~?(_ I
A proce~_for the purification of enzyme solutions
This invention relates to a process for the purifica-
tion and particularly for the deodorization and decolora-
tion of enzyme solutions of the type formed by fermentation
of bacteria or fungi.
Numerous enzymes, particularly hydrolases, such as for
example proteases, amylases or lipases, are produced by
fermentation of microorganisms. Suitable microoganisms and
production processes are described, for example, in the
following patents and patent applications: DE 18 00 508, DE
22 24 777, DE 25 51 742, US 3,827,938, WO 88/01293, DE 18
07 185, US 3,74~,318, DE 23 34 463, DE 20 26 092, EP 0 232
169, EP 0 220 921, EP 0 247 647 and EP 0 246 678.
lS In the fermentation of the strains, enzyme solutions
containing colored and odoriferous substances as impurities
are obtained after removal of the biomass.
Impurities such as these are unacceptable in many
applications, for example where the enzyme solutions are
!. 20 used in liquid detergents. In the industrial production of
the enzymes, therefore, efforts are made to remove the
impurities by precipitation processes. ~owever, hitherto
known precipitation processes have the disadvantage that
considerable losses of yield have to be accepted to obtain
good color quality. In addition, where the basically
favorable bentonites are used for precipitation, filtration
problems arise.
On the other hand, it is known that certain stabiliz-
D 8535 2 ~05~677
ing additives may be added to enzyme solutions. It is
assumed that thes~ additives are capable of masking hydro-
lases and thus pr~venting self-attack, for example in the
case of proteases. Known masking agents of the type in
S question are boric acid and its salts, sodium sulfite,
polybasic carboxylic acids, hydroxycarboxylic acids or even
glycols or glycerol. Masking agents are ~nown~ for ex-
ample, from DE 31 25 533, EP 0 080 223, EP 0 080 748, EP 0
126 508, DE 34 18 295.
Against the background of this prior art, the problem
addressed by the present invention is to provide a process
which enables enzyme solutions to be freed from troublesome
colored and odoriferous impurities with only minimal losses
of yield, the precipitates obtained being easy to remove.
Accordingly, the present invention relates to a
process for the purification and deodorization of aque4us
enzyme solutions obtained by fermentation of bacteria or
fungi by n ~i~ preparation of a water-insoluble precipi-
tate, adsorption of unwanted impurities on the precipitate
and subsequent solid-liquid separation, characterized in
that at least one masXing agent selected from the group
consisting of
- acids of boxon and/or water-soluble salts thereof,
- sulfurous acid and~or water-soluble salts thereof,
- hydroxycarboxylic acids and/or water-soluble salts
; thereof,
- - polyhydric alcohols
is added to the enzyme solutions and a precipitate is
subsequently formed by adding two water-soluble, mutually
precipitating ionic compounds in any order, further adsor-
bents are optionally added and the solids ar~ subsequently
removed from the enzyme solution.
In a general embodiment, the process according to the
invention comprises adding a masking agent to an enzyme
solution obtained by fermentation and prepurified solely by
D 8535 3 20S~677
removal of the biomass and then further purifying the
enzyme solution by precipitation.
Suitable masking agents are, on the one hand, acids or
boron and sulfurous acids and also alkali metal salts
thereof. The quantities to be added are from 0.5 to 2~ by
weight, based on enzyme solution, larger quantities being
inappropriate primarily on economic grounds. Suitable
acids of boron are boric acid, metaboric acid and/or penta-
boric a~id. Accordingly, suitable alXali metal salts are,
in particular, sodium borate, sodium metaborate, borax or
sodium pentaborate. Sodium sulfite is also suitable.
Dicarboxylic acids and/or hydroxycarboxylic acids
containing 3 to 10 carbon atoms may be used as further
masking agents either together with or instead of the
masking agents mentioned above. Hydroxydicarboxylic acids,
particularly citric: acid, tartaric acid and their isomers,
are preferred. The quantity added is from 1 to 5% by
weight. In this case, too, larger additions are in-
appropriate primarily on economic grounds and not because
of any weakening of the technical effects.
Dihydric and/or trihydric alcohols containing 2 to 10
carbon atoms may be used as further masking agents either
together with or instead of the masking agents mentioned
above. These alcohols are used in quantities of S to 30%
by weight and more especially in quantities of 10 to 25% by
weight based on the enzyme solution. Preferred alcohols
are ethylene glycol, propylene glycol, butanediol and
glycerol, although it is also possible to use dimers and
trimers thereof, i.e. di- arld triethylene glycol and di-
and tripropylene glycol. Trimethylol propane and neopentyl
glycol may also be used.
In one preferred embodiment of the process according
to the invention a precipitate is formed in the enzyme
solution by reaction of a water-soluble calcium salt with
an alkali metal salt of a phosphoric acid. More particu-
2~5~7
D 8535 4
larly, the ratio of the calcium salt to the phosphoric acid
salt is selected so that the molar ratio of calcium to
phosporus is from 1.7 to 2.5:1. It has been found in this
regard that ~alcium phosphates having a predomlnantly
amorphous structure and a large surface are formed under
these conditions ~Ind represent favorable adsorbents for the
colored impurities to be precipitated. The quantity of
precipitant, based on enzyme solution, is normally from o.5
to 20% by weight.
In addition to this measure, further adsorbents may
optionally be added to the enzyme solution. Thus, it may
be desirable to use active carbon as a further adsorbent.
In another embodiment of the invention, it may also be
desirable to use layer silicates, particularly bentonite~,
as additional adsorbents. Where bentonites are used as
adsorbents, it is preferred to use acid-activated typ~s.
As already known, bentonites are solids that are difficult
to remove by filtration, particularly in the acidic rangé.
; However, it has been found in connection with the inventionthat the solids to be removed here are easy to filter by
virtue of their interaction. A particularly preferred
acid-activated bentonite has a montmorillonite index of 70
and a fineness of 93% < 100 ~m. The quantity of layer
silicates and/or active carbon is typically between 1 and
10% by weight, based on the enzyme solution.
The process according to the invention is suitable for
the deodorization of hydrolase solutions, particularly
solutions of proteases, amylases or lipases. Of the
protease solutions, solutions of alkaline proteases,
including for example solutions of subtilisin proteases,
such as subtilisin Carlsberg or subtilisin BPN', are
preferred. The enzyme solutions to be treated have solids
contents of 2 to 40% by weight and contain more than
- 10,000, preferably more than S0,000 and in particular, more
than 100,000 proteolytic units/g.
2~5~'77
D 8535
Enzyme solutions which have been obtained by removal
of the microorganisms produced during fermentation and, if
desired, by further removal of salts and, optionally, by
concentration through evaporation or thickening by membrane
processes are preferably used in the process according to
the invention. Enzyme solutions of the type obtained by
the processes according to DE 35 15 650 or DE 37 30 868 are
preferahly purified.
The advantages of the process according to the inven-
tion lie in the inexpensive removal of colored and odorif-
erous impurities with no significant losses of yield
coupled with good filterability of the precipitates.
~ ~ a m p 1 ~ ~
A protease solution was prepared by fermentation of
Bacillus lichenifoL~is in accordance with German patent DE
29 25 427 and was then concentrated in accordance w~th
German patent application DE 37 30 868. The enzyme solu-
tion thus obtained contained 200,000 protease units/g.
0.037 t boric acid was added to 3.625 t of the solu-
tion which was then stirred for 10 minutes at 20C. 1.8 t
water was then added, followed by heating to 30-C. 0.711
t calcium chloride (containing 2 mol water of crystalliza-
tion) in the form of a 35% solution was then added over a
period of 30 minutes (in all 2.03 t solution).
1.564 t of a 33~ solution of 0.516 t Na2HPO~ x 2H2O in
water was then added over a period of 70 minutes and the
temperature increased to 35-C, followed by stirring for 30
minutes at that temperature. During the precipitation, the
pH value fell and had to be readjusted to p~ 6 with 0.15 t
50% NaOH. 0.15 t of an acid-activated bentonite (Bentonit
S 650, a product of FRlNGS-Werke, montmorillonite index 70,
fineness 93% < 100 ~m) was then adde~ over a period of 5
minutes, followed by stirring for 30 minutes. Finally,
D 8535 6 2~5~6 77
0.15 t active carbon in 0.~5 t water was added over a
period of 5 minutes, followed by stirring for another 30
minutes. The solid precipitates were then separated off in
a membrane filter press or in an inverted filter centri-
fuge. The enzyme solution obtained had a favorable color
and odor. The degree of retention of the enzyme was 90 -
9~% .
Compariscn Example
The procedure was as in the Example, except that no
boric acid was added. To obtain the same color quality,
the quantity of calcium chloride and sodium hydrogen
phosphate had to be increased. The enzyme loss for the
same color quality of the enzyme solution was more than
20%.
