U.S. patent number 3,717,550 [Application Number 05/075,764] was granted by the patent office on 1973-02-20 for liquid compositions of bacterial protease and/or amylase and preparation thereof.
This patent grant is currently assigned to Pabst Brewing Company. Invention is credited to Jack Ziffer.
United States Patent |
3,717,550 |
Ziffer |
February 20, 1973 |
LIQUID COMPOSITIONS OF BACTERIAL PROTEASE AND/OR AMYLASE AND
PREPARATION THEREOF
Abstract
Liquid concentrates of bacterial protease and/or amylase of
enhanced stability are prepared. Bacterial protease and/or amylase
liquid compositions free of turbidity and a process of producing
them are provided. These compositions have advantages over powdered
enzymes in avoiding atmospheric contamination and are especially
useful in detergents and in textile treatment.
Inventors: |
Ziffer; Jack (Milwaukee,
WI) |
Assignee: |
Pabst Brewing Company
(Milwaukee, WI)
|
Family
ID: |
22127829 |
Appl.
No.: |
05/075,764 |
Filed: |
September 25, 1970 |
Current U.S.
Class: |
435/188; 435/202;
510/530; 8/138; 435/220 |
Current CPC
Class: |
C11D
3/38663 (20130101); D06L 1/14 (20130101) |
Current International
Class: |
D06L
1/00 (20060101); D06L 1/14 (20060101); C11D
3/38 (20060101); C11D 3/386 (20060101); C07g
007/02 () |
Field of
Search: |
;195/68,63,62,65,66
;252/DIG.12 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Yasumatsu et al. Agricultural and Biological Chemistry vol. 29, No.
7 pp. 665-671 (1965) Vol..
|
Primary Examiner: Shapiro; Lionel M.
Claims
The invention is hereby claimed as follows:
1. A liquid composition consisting essentially of:
Component (A), fermentation soluble solids consisting essentially
of an enzyme from the group consisting of bacterial protease and
bacterial amylase and mixtures thereof, and
Component (B), a liquid from the group consisting of water, a
normally liquid, water miscible polyhydric alcohol, and both water
and a normally liquid, water miscible polyhydric alcohol, in which
said enzyme is soluble to the extent of at least 32% fermentation
soluble solids by weight at 4.4.degree.C., said composition
containing a concentration of said components sufficient to
maintain an enzyme activity of at least 50% when said composition
is stored in a container under atmospheric pressure for six days at
42.degree.C., Component (A) constituting at least 32% by weight of
the solids content of said composition, on a dry basis.
2. A composition as claimed in claim 1 in which the protease
activity is at least 5,000 PV units per gram at 4.4.degree.C.
3. A composition as claimed in claim 1 in which the protease
activity is 5,000 to 2,000,000 PV units per gram at
4.4.degree.C.
4. A composition as claimed in claim 1 in which the protease
activity is 30,000 to 600,000 PV units per gram at
4.4.degree.C.
5. A composition as claimed in claim 1 in which the amylase
activity is at least 10,000 DV units per gram at 4.4.degree.C.
6. A composition as claimed in claim 1 in which the amylase
activity is 10,000 to 1,000,000 DV units per gram at
4.4.degree.C.
7. A composition as claimed in claim 1 in which the amylase
activity is 30,000 to 400,000 DV units per gram at
4.4.degree.C.
8. A composition as claimed in claim 1 in which the solids content,
on a dry basis, of the enzyme is within the range of 32% by weight
to saturation.
9. A composition as claimed in claim 1 in which the solids content,
on a dry basis, of the enzyme is within the range of 32 to 43.5
percent by weight.
10. A composition as claimed in claim 1 in which component (A)
consists essentially of at least 32 percent by weight, on a dry
basis, of the composition of bacterial protease, and component (B)
consists essentially of glycerol.
11. A composition as claimed in claim 1 in which component (A)
consists essentially of at least 32 percent by weight, on a dry
basis, of the composition of bacterial amylase, and component (B)
consists essentially of glycerol.
12. A composition as claimed in claim 1 in which component (A)
consists essentially of at least 32 percent by weight, on a dry
basis, of the composition of both bacterial protease and bacterial
amylase, and component (B) consists essentially of glycerol.
13. A composition as claimed in claim 1 in which said enzyme is
present in a concentration of at least 32 percent by weight, on a
dry basis, and said liquid is from the group consisting of glycerol
and water.
14. A process of clarifying an aqueous solution of an enzyme from
the group consisting of bacterial protease, bacterial amylase and
mixtures thereof which comprises adding to a turbid aqueous
solution in which the concentration of said enzyme is at least 32
percent by weight, on a dry basis, a sufficient amount of a liquid,
water miscible polyhydric alcohol to reduce the turbidity.
15. A process as claimed in claim 14 in which said polyhydric
alcohol is glycerol.
16. A process as claimed in claim 14 in which said enzyme is
bacterial protease.
17. A process as claimed in claim 14 in which said aqueous solution
containing said polyhydric alcohol is concentrated under conditions
facilitating the elimination of at least a part of the water
without removing said polyhydric alcohol.
18. A process as claimed in claim 17 in which said concentration is
carried out under subatmospheric pressure conditions.
19. A process as claimed in claim 14 in which said enzyme is
bacterial amylase.
20. A process as claimed in claim 14 in which said enzyme is a
mixture of bacterial protease and bacterial amylase.
Description
BACKGROUND OF THE INVENTION
In recent years the use of enzymes, especially bacterial protease
and bacterial amylase, for presoak and detergent washing products
has greatly increased. These enzymes are now being produced in the
form of powders in very high potency concentrations and handling
problems have developed as a result of these high potency levels.
Many individuals have found these enzymes to be a source of
irritation. The problem is particularly acute in the manufacturing
and handling of the enzymes themselves and also in the handling of
the enzymes in plants where they are formulated into various forms
of detergent products. In order to alleviate the problem it has
heretofore been proposed to produce a dust-free powdered enzyme
product.
Another possible solution of the problem would be the provision of
the enzymes in the form of a liquid composition. When attempts are
made to do this, however, other problems arise. One of these
problems is the instability of the enzymes which causes the enzyme
activity of a liquid product to be reduced in storage. Another
problem arises due to the fact that the enzymes as normally
produced form turbid solutions in water containing suspended
particles. It would, therefore, be desirable to solve the dusting
problem which is a source of irritation to individuals by providing
a liquid composition containing enzymes which retain a relatively
high activity when the composition is stored prior to use. In
addition, It would be desirable to provide high potency liquid
concentrates of bacterial protease and/or amylase which are free of
turbidity. These liquid concentrates of bacterial protease and/or
amylase of enhanced stability would avoid the source of irritation
caused by the dusting of powdered enzymes and they could be used in
the manufacture of various detergent products. In addition, liquid
enzyme compositions free from turbidity would be acceptable for use
as such in household detergent formulations and also as enzyme
compositions for the treatment of textiles, for example, in the
de-sizing of textile fibers.
OBJECTS
One of the objects of the present invention is to provide new and
improved liquid concentrates of bacterial protease and/or bacterial
amylase of enhanced stability.
Another object of the invention is to provide liquid compositions
containing bacterial protease and/or bacterial amylase which are
free of turbidity.
Still a further object of the invention is to provide a new and
improved process for producing liquid compositions containing
bacterial protease and/or bacterial amylase.
Another object of the invention is to provide new and improved
liquid compositions containing bacterial protease and/or bacterial
amylase in combination with surfactants. Other objects will appear
hereinafter.
BRIEF SUMMARY OF THE INVENTION
In accordance with the invention liquid compositions are prepared
consisting essentially of: Component (A), fermentation soluble
solids consisting essentially of an enzyme from the group
consisting of bacterial protease and bacterial amylase and mixtures
thereof, and Component (B), a liquid from the group consisting of
water, a normally liquid water miscible polyhydric alcohol, and
both water and a normally liquid water miscible polyhydric alcohol,
in which said enzyme is soluble to the extent of at least 32
percent by weight fermentation soluble solids at 4.4.degree.C.
(40.degree.F), said composition containing a concentration of said
components sufficient to maintain an enzyme activity of at least 50
percent when said composition is stored in a container under
atmospheric pressure for 6 days at 42.degree.C.
The compositions of the invention will normally have a protease
activity of at least 5,000 PV units per gram at 4.4.degree.C.,
preferably within the range of 5,000 to 2,000,000 PV units per gram
at 4.4.degree.C., and generally within the range of 30,000 to
600,000 PV units per gram at 4.4.degree.C. The amylase activity can
be up to 1,000,000 DV units per gram at 4.4.degree.C., is
preferably at least 10,000 DV units per gram at 4.4.degree.C., and
is generally within the range of 30,000 to 400,000 DV units per
gram at 4.4.degree.C. The enzymes in the liquid compositions can
consist essentially of bacterial protease or bacterial amylase or
both bacterial protease and bacterial amylase in the above
described proportions.
The liquid compositions can be used as such for washing or they can
be mixed with other ingredients for the preparation of detergent
compositions. Thus, they can be mixed with various types of
compatible surfactants to form liquid concentrates which can be
added to wash water in predetermined amounts.
It has been found that where Component (B) is water, the stability
of the enzymes is enhanced by maintaining a relatively high enzyme
concentration, namely, a concentration of at least 32 percent by
weight at 4.4.degree.C. (40.degree.F.). Where a polyhydric alcohol
such as glycerol, either alone or mixed with water, constitutes the
liquid phase, the retained activity of the enzymes over a period of
time is enhanced even at enzyme concentrations below 32 percent by
weight. Furthermore, polyhydric alcohols, for example, glycerol,
propylene glycol, ethylene glycol, diethylene glycol and higher
liquid homologues, have the property of removing the turbidity
which is normally present in concentrated bacterial protease and
bacterial amylase solutions in water. Hence, the use of polyhydric
alcohols in the preparation of these liquid compositions makes it
possible to produce clear liquid compositions which do not leave
deposits when employed in washing procedures and in textile
treatment and are therefore more acceptable for such uses.
DETAILED DESCRIPTION OF THE INVENTION
In preparing compositions in accordance with the invention several
different methods may be used. The usual procedure involves first
preparing an aqueous concentrate by adding bacterial protease and
bacterial amylase to water and agitating the resultant composition
at room temperature (20.degree.-32.degree.C.) so as to dissolve the
enzyme. The composition is then filtered and the filter cake washed
with water. The combined filtrate and wash water after
concentration is a turbid solution, probably containing colloidal
protein solid in suspension. In accordance with the invention it
has been found that the retained enzyme activity of this solution
on standing is enhanced by increasing the proportion of enzyme
solids in the solution to the point where they are at least 32
percent by weight, based on the weight of the dry solids, and
preferably within the range of 32 percent to saturation. In most
cases this range will be from 32 to 45 percent by weight.
Surprisingly, when a liquid polyhydric alcohol is added to the
previously described turbid enzyme solution it clarifies the
solution. Furthermore, when the polyhydric alcohol is glycerol, the
enzyme solution is not only clarified but the retained enzyme
activity on storage is greatly enhanced, even when the
concentration of enzyme solids is less than 32 percent by weight.
Thus, the concentration of enzyme solids can be within the range of
2 to 60 percent by weight and is usually within the range of 32 to
43.5 percent by weight. Where glycerol is employed the composition
may contain 40 to 98 percent by weight glycerol. Various types of
compositions in which the liquid phase consists essentially of both
water and a polyhydric alcohol can be prepared as hereinafter
described.
As previously indicated, these liquid enzyme compositions can be
employed as such in washing by adding them to the wash water in the
proper proportions. In most cases the proportions should be
sufficient to give an enzyme concentration of 200-2,000 PV
units/liter and 0-500 DV units/liter in a water having a pH within
the range of 8 to 12. Compatible surfactants can also be added to
the liquid enzyme concentrates. For most purposes the surfactant is
either a nonionic or an anionic surfactant. It may also be an
amphoteric surfactant. Any of the compatible surfactants described
in McCutcheon's Detergents and Emulsifiers, 1969 Annual, can be
employed. Examples of such surfactants are Alrosperse DC, Triton
QS-15, QS-30, BG-5, and X-102. It is usually preferable to carry
out routine experiments in order to determine the compatibility of
a particular surfactant. Some of these surfactants are compatible
with some polyhydric alcohols but not with others.
The invention will be further illustrated but is not limited by the
following examples in which the quantities are stated in parts by
weight unless otherwise specified.
EXAMPLE 1
A 20 percent (w/v) solution of Al.sub.2 (SO.sub.4).sub.3.sup..
18H.sub.2 O was slowly added to an agitated bacterial protease
fermentation whole culture (500 gal., 10,720 PV units per ml., 20.3
.times. 10.sup.9 PV units) while maintaining the whole culture pH
at 6.4 by the simultaneous addition of Na.sub.2 SO.sub.3 (42.5
lb.). The amount of Al.sub.2 (SO.sub.4).sub.3.sup.. 18H.sub.2 O
added was 1 percent (w/v) based on the whole culture. Coagulants
Nalco 674 (120 grams, as a 0.25 percent (w/v) solution) and
Purifloc C-31(5,025 grams, as a 27 percent (w/v) solution were then
added, together with filteraid Chem-Flo 325 CK (290 lbs.). The
mixture (pH 5.3) was adjusted to pH 6.4 with NaOH and filtered on a
plate and frame press. The resultant filtered cake was washed with
water and the combined clear solution of filtrate plus wash
evaporated in vacuo to 83.6 gal. (pH 6.3, 18.1 .times. 10.sup.9 PV
units) for a filtration and concentration recovery of 89.2 percent.
Sodium sulfate (7,000 grams), sodium sulfite (1,600 grams) and
sodium bisulfite (200 grams) were added to a portion of the
concentrate warmed to 45.degree.C. (40,000 ml., pH6.4, 25 percent
dry solids, 22.40 .times. 10.sup.8 PV units) and the mixture
filtered on a small Dorr-Oliver rotary filter using a Na.sub.2
SO.sub.4 wash. The total PV units recovered in the filtered cake
(4,794 grams) and heel totaled 21.94 .times. 10.sup.8 PV units for
a filtration recovery of 97.9 percent. The overall recovery from
the whole culture was 86.7 percent.
EXAMPLE 2
The bacterial protease filtered cake obtained in Example 1 after
precipitation with Na.sub.2 SO.sub.4, Na.sub.2 SO.sub.3 and
NaHSO.sub.3 was used to prepare a series of aqueous concentrates.
Distilled water (7,200 ml.) was added to a portion of the filtered
cake (1,765 grams, 680 .times. 10.sup.6 PV units) and the mixture
agitated at room temperature for 1.5 hours. The mixture was then
filtered on a Buchner funnel using a Filteraid FW-20 pre-coat and
the filtered cake washed with water (3,000 ml.). The combined
filtrate and washed totaled 11,840 ml. and contained 620 .times.
10.sup.6 PV units, for a protease enzyme recovery of 91.2 percent.
Portions of the combined filtrate and wash were evaporated in vacuo
to varying concentrations of dry solids. The resultant concentrates
were visually tan turbid solutions, appearing to contain colloidal
protein solid in suspension. Aliquots of these concentrates were
tested at 4.4.degree.C. (40.degree.F.) and 42.degree.C.
(107.6.degree.F.) for storage stability in containers at
atmospheric pressure. The results obtained are shown below.
Protease Activity (PV/Gm) After 36 Days at Indicated % Dry Solids
Temperature % Protease in Concentrate 4.4.degree.C. 42.degree.C.
activity remaining
__________________________________________________________________________
31.4 308,000 180,000 58.4 33.8 334,000 217,000 65.0 37.8 392,000
270,000 68.9 43.5 425,000 362,000 85.2
EXAMPLE 3
Using the procedures described in Examples 1 and 2, an aqueous
concentrate was prepared having a per cent dry solids of 32 percent
and a protease enzyme activity of 360,000 PV units per gram. The
resultant concentrate was a tan turbid solution containing
suspended solid.
A portion of the aqueous concentrate (100 grams) was added to
distilled water (20 grams). The resultant mixture was still a tan
turbid solution containing suspended solid. Aliquots of this
solution (without any further evaporation) were tested at
4.4.degree.C. and 42.degree.C. for storage stability.
A portion of the aqueous concentrate (100 grams) was added to
propylene glycol (20 grams, reagent grade) resulting in an almost
complete clearing of the turbidity of the aqueous concentrate.
Aliquots of this solution (without any further evaporation) were
tested at 4.4.degree.C. and 42.degree.C. for storage stability.
A portion of the aqueous concentrate (100 grams) was added to
propylene glycol (50 grams, reagent grade) and the mixture
evaporated in vacuo to 100 grams final weight. The resultant
product was a clear solution. Aliquots of this solution were tested
at 4.4.degree.C. and 42.degree.C. for storage stability.
A portion of the aqueous concentrate (100 grams) was added to
propylene glycol (50 grams, reagent grade) and the mixture
evaporated in vacuo to 100 grams. An additional 20 grams of
propylene glycol was then added to the clear solution resulting in
a final total weight of 120 grams, of which 70 grams constituted
propylene glycol. Aliquots of this clear solution were tested at
4.4.degree.C. and 42.degree.C. for storage stability.
A portion of the aqueous concentrate (200 grams) was added to
ethylene glycol (100 grams, reagent grade) and the mixture
evaporated in vacuo to 200 grams final weight. The resultant
product was a clear solution. Aliquots of this solution were tested
at 4.4.degree.C. and 42.degree.C. for storage stability.
Additional ethylene glycol (20 grams) was added to a portion of the
above ethylene glycol-aqueous solution (100 grams). Aliquots of
this clear mixture (without any further concentration) were tested
at 4.4.degree.C. and 42.degree.C. for storage stability.
A portion of the aqueous concentrate (200 grams) was added to
diethylene glycol (100 grams) and the mixture evaporated in vacuo
to 200 grams final weight. The resultant product was a clear
solution. Aliquots of this solution were tested at 4.4.degree.C.
and 42.degree.C. for storage stability.
Additional diethylene glycol (20 grams) was added to a portion of
the above diethylene glycol-aqueous solution (100 grams). Aliquots
of this clear mixture (without any further concentration were
tested at 4.4.degree.C. and 42.degree.C. for storage stability.
A portion of the aqueous concentrate (200 grams) was added to
glycerol (100 grams, U.S.P. grade) and the mixture evaporated in
vacuo to 200 grams final weight. The resultant product was a clear
solution. Aliquots of this solution were tested at 4.4.degree.C.
and 42.degree.C. for storage stability.
Additional glycerol (20 grams) was added to a portion of the above
glycerol-aqueous solution (100 grams). Aliquots of this clear
mixture (without any further concentration) were tested at
4.4.degree.C. and 42.degree.C. for storage stability.
The data obtained for these products are as follows:
Protease activity PV/Gm. remaining after storage at indicated
temperatures Poly- % Poly- % Non- % Protease hydride hydric Alcohol
Activity alcohol Alcohol in Dry Storage, Remain- used Product
Solids Days 4.4.degree.C. 42.degree.C. ing
__________________________________________________________________________
None None 32 14 350 K 192 K 54.9 None None 14 .7 304 K 96 K 31.6
propylene glycol 16.7 26.7 7 320 K 200 K 62.5 propylene glycol 50
32 14 380 K 140 .8 K 37.1 propylene glycol 58 26.7 7 282 K 125 K
44.3 ethylene glycol 50 32 14 375 K 125 .4 k 33.4 ethylene glycol
58 26.7 7 275 k 95 k 34.5 diethylene glycol 50 32 13 375 k 98 .4 k
26.2 diethylene glycol 58 26.7 6 282 .5 k 207 .5k 73.5 glycerol 50
32 6 340 k 325 k 95.6 21 340 k 315 k 92.6 43 355 k 286 .4 k 80.7
glycerol 58 26.7 6 302 .5 k 275 k 90.9 21 300 k 252 84.0 43 310 k
280 .3 k 90.4
__________________________________________________________________________
EXAMPLE 4
Using the procedures described in Examples 1 and 2, an aqueous
protease concentrate was prepared having a per cent dry solids of
38.3 percent and a protease enzyme activity of 355,000 PV units per
gram. The resultant concentrate was a tan turbid solution
containing suspended solid. Aliquots of this solution were tested
at 4.4.degree.C. and 42.degree.C. for storage stability.
A portion of the aqueous concentrate (200 grams) was added to
glycerol (100 grams, U.S.P. grade) and the mixture evaporated in
vacuo to 200 grams final weight. The resultant product was a clear
solution. Aliquots of this solution were tested at 4.4.degree.C.
and 42.degree.C. for storage stability.
The data obtained for these products are shown below:
Protease Activity PV/Gm. Remaining after Storage at Indicated
Temperatures % protease % % Non- activity glycerol glycerol
storage, remain- in product dry solids days 4.4.degree.C.
42.degree.C. ing
__________________________________________________________________________
None 38.3 21 367,500 328,000 89.3 50 38.3 20 400,000 400,000 100.0
__________________________________________________________________________
EXAMPLE 5
Using the procedures described in Examples 1 and 2, an aqueous
concentrate was prepared having a per cent dry solids of 32 percent
and a protease enzyme activity of 350,000 PV units per gram. The
resultant concentrate was a tan turbid solution containing
suspended solid.
A portion of the aqueous concentrate (400 grams) was added to
glycerol (128 grams, U.S.P. grade) and the mixture evaporated in
vacuo to 294 grams. The resultant product was a clear solution.
Aliquots of this solution were tested at 4.4.degree.C. and
42.degree.C. for storage stability.
A portion of the aqueous concentrate (100 grams) was added to
glycerol (55 grams, U.S.P. grade) and the mixture evaporated in
vacuo to 100 grams. The resultant product was a clear solution.
Aliquots of this solution were tested at 4.4.degree.C. and
42.degree.C. for storage stability.
A portion of the aqueous concentrate (100 grams) was added to
glycerol (60 grams, U.S.P. grade) and the mixture evaporated in
vacuo to 100 grams. The resultant product was a clear solution.
Aliquots of this solution were tested at 4.4.degree.C. and
42.degree.C. for storage stability.
A portion of the aqueous concentrate (120 grams) was added to
glycerol (53 grams, U.S.P. grade) and the mixture evaporated in
vacuo to 107 grams. The resultant product was a clear solution.
Aliquots of this solution were tested at 4.4.degree.C. and
42.degree.C. for storage stability.
A portion of the aqueous concentrate (120 grams) was added to
glycerol (48 grams, U.S.P. grade) and the mixture evaporated in
vacuo to 98 grams. The resultant product was a clear solution.
Aliquots of this solution were tested at 4.4.degree.C. and
42.degree.C. for storage stability.
The data obtained for these products are shown below:
Protease Activity PV/Gm. Remaining After Storage At Indicated
Temperatures % Protease % non- activity % glycerol glycerol
storage, remain- in product Dry solids days 4.4.degree.C.
42.degree.C. ing
__________________________________________________________________________
43.5 43.5 34 425,000 312,500 73.5 55 32 34 328,000 280,000 85.4 60
32 34 322,000 316,000 98.1 50 36 34 372,500 368,000 98.8 50 38 34
402,500 360,000 89.4
__________________________________________________________________________
EXAMPLE 6
Using the procedures described in Examples 1 and 2, an aqueous
concentrate was prepared having a percent dry solids of 38.9
percent and a protease enzyme activity of 381,000 PV units per
gram. The resultant product was a tan turbid solution containing
suspended solids. Aliquots of this solution were tested at
4.4.degree.C. and 42.degree.C. for storage stability.
A portion of the aqueous concentrate (1,000 grams) was added to
glycerol (500 grams, U.S.P. grade) and the mixture evaporated in
vacuo to 1,000 grams. The resultant product was a clear solution.
Aliquots of this solution were tested at 4.4.degree.C. and
42.degree.C. for storage stability.
Portions of the glycerol-aqueous concentrate were then further
diluted with additional glycerol in the glycerol concentration
range of 50-99 percent of the total solution. The resultant
products were clear solutions. Aliquots of these solutions were
tested at 4.4.degree.C. and 42.degree.C. for storage stability.
The data obtained for these products are shown below:
Protease Activity PV/Gm. Remaining after Storage % Non- at
Indicated Temperatures % Glyc- Glycerol Storage, % Protease erol in
Dry % days Activity Re- Product Solids Water 4.4.degree.C.
42.degree.C. maining
__________________________________________________________________________
None 38.9 61.1 15 367,500 354,000 96.3 50.0 38.9 11.1 15 367,500
360,000 98.0 83.3 13.0 3.7 13 128,000 126,700 98.9 90.0 7.8 2.2 13
74,800 94.1 95.0 3.9 1.1 13 39,000 32,200 82.6 97.4 2.0 0.6 13
19,000 15,500 81.6 98.7 1.0 0.3 9,750 4,750 48.7
EXAMPLE 7
Using the salt treatment and precipitation procedure described in
U.S. Ser. No. 849,148, filed Aug. 11, 1969, a filtered precipitated
bacterial amylase wet cake was obtained assaying 128,000 DV units
per gram. Tap water (4,000 ml.) was added to a portion of the
filtered cake (1,000 grams, 128.0 .times. 10.sup.6 DV units) and
the mixture agitated at room temperature for one hour.
Al.sub.2 (SO.sub.4).sub.3.sup.. 18H.sub.2 O (25 grams, as a 20
percent (w/v) solution) was then added and the pH maintained at 6.4
by the simultaneous addition of Na.sub.2 SO.sub.3 (30 grams). After
the addition of the salts, Chem-Flo 325 CK filteraid (350 grams)
was added and the mixture filtered on a Buchner funnel, followed by
a tap water wash. The combined filtrate and wash (7,850 ml., pH
6.65) was evaporated in vacuo to a concentrate (33.6 percent dry
solids, pH 6.3) containing 121.1 .times. 10.sup.6 DV units for an
amylase enzyme recovery of 94.6 percent. The resultant concentrate
was a visually tan turbid solution, appearing to contain colloidal
protein solid in suspension. Aliquots of this concentrate were
tested at 4.4.degree.C., 28.degree.C. and 42.degree.C. for storage
stability. The results obtained are shown below:
Amylase Activity (DV/Gm.) After 36 Days at Indicated % Dry Solids
Temperature In Concentrate 4.4.degree.C. 28.degree.C. 42.degree.C.
__________________________________________________________________________
33.6 87,200 86,300 83,700
__________________________________________________________________________
EXAMPLE 8
a. Using the procedures described in Example 3, a portion of the
aqueous concentrate (200 grams) from Example 7 was added to
glycerol (100 grams, U.S.P. grade) and the mixture evaporated in
vacuo to 200 grams final weight. The resultant product was a clear
solution. Aliquots of this solution were tested at 4.4.degree.C.,
28.degree.C., 42.degree.C. and 55.degree.C. for storage stability.
The results obtained are shown below:
Amylase Activity (DV/Gm.) After % non-glycerol 30 Days at Indicated
% glycerol dry Temperature in concentrate solids 4.4.degree.C.
28.degree.C. 42.degree.C. 55.degree.C.
__________________________________________________________________________
50 33.6 87,600 90,000 92,900 89,200
b. Using the procedures described in Example 3, a portion of the
aqueous concentrate (450 grams) from Example 7 was added to
glycerol (225 grams, U.S.P. grade) and the mixture evaporated in
vacuo to 450 grams final weight. The resultant product was a clear
solution. Aliquots of this solution were tested at 4.4.degree.C.
and 55.degree.C. for storage stability. The results obtained are
shown below:
Amylase activity (DV/Gm.) After 12 Days % Non-Glycerol at Indicated
% Amylase % Glycerol in Dry Temperature Activity Concentrate Solids
4.4.degree.C. 55.degree.C. remaining
__________________________________________________________________________
None 33.6 84,990 44,410 52.3 50 33.6 90,070 85,720 95.2
EXAMPLE 9
An enzyme concentrate was prepared by mixing equal parts by weight
of a bacterial protease-glycerol concentrate containing 50 percent
glycerol by weight and having an activity of 464,000 PV units per
gram with a bacterial amylase concentrate containing 50 percent
glycerol by weight and having an activity of 90,680 DV units per
gram and 20,000 PV units per gram.
The final product contained a protease concentration of 242,000 PV
units per gram and 45,340 DV units per gram.
EXAMPLE 10
The procedure was the same as in Example 9 except that additional
glycerol was added and the relative proportions of bacterial
protease-glycerol concentrate, bacterial amylase-glycerol
concentrate and glycerol were, respectively, 12.42 percent, 65.70
percent and 21.88 percent by weight. The final product had a
protease concentration of 70,760 PV units per gram and 59,580 DV
units per gram.
EXAMPLE 11
The procedure was the same as in Example 9 except that additional
glycerol was added and the relative proportions of bacterial
protease-glycerol concentrate, bacterial amylase-glycerol
concentrate and glycerol were 46.25 percent, 37.05 percent and
16.70 percent by weight, respectively. The final product contained
a protease concentration of 222,200 PV units per gram and 33,600 DV
units per gram.
The following examples illustrate the preparation of enzyme
compositions of the type described containing surfactants.
EXAMPLE 12
The bacterial protease-glycerol concentrate of Example 9 containing
50 percent by weight glycerol and having a protease concentration
of 464,000 PV units per gram was mixed in a weight ratio of 4:1
with Alrospere DC, to produce a composition having a protease
concentration of 371,200 PV units per gram.
EXAMPLE 13
The procedure was the same as in Example 12 except that 80 parts of
the protease-glycerol concentrate was mixed with 5 parts Alrosperse
DC and 15 parts of additional glycerol. The protease concentration
was the same as in Example 12.
EXAMPLE 14
The procedure was the same as in Example 12 except that the
surfactant Triton QS-15 was used.
EXAMPLE 15
The procedure was the same as in Example 13 except that the
surfactant Triton QS-15 was used.
EXAMPLE 16
The procedure was the same as in Example 12 except that the
surfactant Triton BG-5 was used.
EXAMPLE 17
The procedure was the same as in Example 13 except that the
surfactant Triton BG-5 was used.
EXAMPLE 18
The procedure was the same as in Example 12 except that a bacterial
amylase-glycerol concentrate containing 50 percent by weight
glycerol was used instead of the bacterial protease-glycerol
concentrate. Since the bacterial amylase contained some protease,
the final product had an amylase concentration of 72,540 DV units
per gram and a protease concentration of 16,000 PV units per
gram.
EXAMPLE 19
The procedure was the same as in Example 13 except that a bacterial
amylase-glycerol concentrate of the type employed in Example 18 was
used. The final product had an amylase concentration of 72,540 DV
units per gram and a protease concentration of 16,000 PV units per
gram.
EXAMPLE 20
The procedure was the same as in Example 18 except that Triton
QS-15 was used instead of Alrosperse DC.
EXAMPLE 21
The procedure was the same as in Example 19 except that Triton
QS-15 was used instead of Alrosperse DC.
EXAMPLE 22
The procedure was the same as in Example 18 except that Triton BG-5
was used instead of Alrosperse DC.
EXAMPLE 23
The procedure was the same as in Example 19 except that Triton BG-5
was used instead of Alrosperse DC.
EXAMPLE 24
An enzyme-surfactant concentrate was prepared by mixing 40 parts of
bacterial protease-glycerol concentrate containing 50 percent
glycerol and having a protease concentration of 464,000 PV units
per gram, 40 parts of a bacterial amylase-glycerol concentrate
containing 50 percent glycerol and having an amylase activity of
90,680 DV units per gram, and a protease activity of 20,000 PV
units per gram, 5 parts of Alrosperse DC and 15 parts glycerol. The
final product had a protease concentration of 193,600 PV units per
gram and an amylase concentration of 36,270 DV units per gram.
EXAMPLE 25
The procedure was the same as in Example 24 except that Triton BG-5
was substituted for the Alrosperse DC.
EXAMPLE 26
The procedure was the same as in Example 24 except that Triton
QS-15 was substituted for the Alrosperse DC.
It should be noted that the bacterial amylase enzyme normally
contains some bacterial protease enzyme. In Example 7 the protease
enzyme activity is not given.
In Examples 12 to 26 all of the surfaces were found to be
compatible with the enzyme concentrates. The Alrosperse DC is an
oil soluble surfactant which is non-ionic plus a cationic mixture
of fatty alkoamides. Triton BG-5 is a non-ionic surfactant. Triton
QS-15 is a liquid anhydrous amphoteric surfactant in the form of an
oxyethylated sodium salt containing both anionic and cationic
centers. All of these surfactants are identified in McCutcheon's
1969 Annual, supra.
In the foregoing examples the recovery procedures used in preparing
bacterial protease and/or the bacterial amylase, as illustrated
specifically in Example 1, do not constitute a part of the present
invention but are disclosed and claimed in U.S. application Ser.
No. 849,148 filed Aug. 11, 1969.
It may be mentioned that the clarity of the enzyme solution depends
upon the particular enzyme and the concentration thereof. The
addition of the polyhydric alcohol serves to reduce the turbidity
and thereby increase the clarity of the solution. This does not
necessarily mean, however, that one can see through a given sample
of solution after it has been clarified. Thus, in the case of
enzyme compositions which are concentrated to the point that they
are of a syrupy consistency, the composition prior to treatment
with the polyhydric alcohol will contain suspended particles which
cause turbidity and will usually have a tan appearance whereas
after the treatment with a polyhydric alcohol the turbidity will be
reduced due to the dissolution of the particles and the resultant
clarified composition will have a darker brown appearance.
* * * * *