Note: Descriptions are shown in the official language in which they were submitted.
I
Title of the Invention:
PROCESS FOR PRODUCING CRUDE ELICITS
The present invention relates to a process
for obtaining crude elicits from an elicits-
containing solution obtained by activation treat-
Kent of the pancreas of porcine.
Elicits is present in the form of its prey
cursor in the mammalian pancreas. To obtain
elicits from, for example, the pancreas of porcine,
the elicits precursor is generally activated
to convert into elicits which is then extracted.
The activation is effected either by heating
minced fresh pancreas to effect atlases or by
the treatment by addition of mammalian duodenum
or an extract from the duodenum, trypsin or a
tripsin-containing substance such as pancreatic,
ethanol, acetone or isopropyl alcohol
Elicits thus activated is extracted from
the solution after the activation treatment
conventionally by adding an aqueous solution of,
for example, acetate buffer, filtering the mixture
to obtain a clear solution and salting out the
solution with ammonium sulfate. It is to be noted
that requisites for the conventional technique are
:
a salting-out operation for the precipitation of
elicits and preliminary filtration of the extract
to obtain a quite clear solution so as to make
the salting-out operation possible. In an exam-
pie of this technique, the elicits solution is
treated with an acetate buffer solution after
the activation then a large amount of a filter
aid is added, the solution is filtered to obtain
a quite clear solution, ammonium sulfate is added
thereto to attain 45 saturation and the mixture
.
is salted out. However, the operation per ye
for obtaining the clear solution is extremely
difficult, since the pancreas contains a large
amount of fat. This is quite unsuitable for the
mass production. Further, it is a fatal defect
that the recovery of elicits in the salting-out
is as low as below about 60 to 7Q % based on
the activated elicits. The large amount of the
balance is discharged as an unextractable matter
in the prior art. Thus the conventional process
has not been completed as an industrially sails-
factory process.
Under these circumstances, the inventors
made intensive investigations for the purpose of
developing a process for obtaining elicits with
I
a high recovery by a simple and easy chemical
operation without necessitating the difficult
clarification operation or complicated salting-
out operation. Consequently, the inventors have
found that the object can be attained by precipi~
toting elicits by the addition of a specific
amount ox a water-soluble organic solvent to the
elastase-containing solution after the activation
treatment. The present invention has been come
pleated on the basis ox this finding.
The present invention Jill now be described
in detail.
The term "the pancreas of porcine" includes not only the
pancreas so of porcine, but also a residue
obtained after extraction of insulin from the
pancreas of porcine, residue obtained after
extraction of kallikrein from the pancreas of
porcine and materials derived from the pancreas
of porcine such as defeated pancreas. From these
starting materials, an elastase-containing soul-
lion can be obtained by the activation treatment.
In the activation treatment, it is preferred
to mince the pancreas as finely as possible by
means of a meat grinder or by freeze pulverization.
It is also possible, however, to activate the pancreas
directly without mincing. As the activation
treatments, there have been known autolytic treat-
mint, enzymatic treatment and organic solvent
treatment. More particularly, there has been
known a process of addition of the mammalian
duodenum or an extract therefrom, trypsin, pan-
creating ethanol, acetone or isopropyl alcohol
as well as atlases my heating. In the active
lion treatment in the present invention, these
known processes may be employed directly.
The treatment can be effected advantageously by,
for example, a method disclosed in the specific
cation of Japanese Patent Laid-Open No. 61287/77
wherein the duodenum of porcine is added to the
fresh pancreas of porcine and the mixture is left
to stand at pi 7 to 7.5. In case the activation
is effected by the addition of an organic solvent,
it is preferred to use the same solvent as that
used in a subsequent precipitation operation.
In another method, a suitable quantity of
water or a dilute salt solution is added to the
minced starting material directly or after the
activation treatment to obtain a suspension, and
a filter aid is added to the suspension, if de-
sired, to remove rough fibrous substances previously.
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my this treatment, the subsequent operation of
preclpi~ating elicits is facilitated. Roy qua-
lily of water or the dilute salt solution may be
such that only rough fibrous materials are removed.
Namely, the quantity is about 0.5 to 2 parts by
volume per part by volume of the starting material
or treated material. The filter aid is used for
accelerating the coagulation of the fibrous ma-
trials. Diatomaceous en (icily) is most suitable.
However, this removing operation is not indispensa-
bye in the present invention. The invention is
not limited by this operation.
A suitable organic solvent may be added for
defeating the starting material immediately after
the activation treatment. However, also this
operation is not indispensable in the present
invention and it does not limit the invention.
The water-soluble organic solvents used in
the present invention are those miscible with
water at any desired ratio to form a transparent
solution. Concretely, the solvents are acetone,
ethanol, isopropyl alcohol, etc. Among them,
acetone is particularly preferred.
The amount of the water-soluble organic
solvent is desirably in the range of 30 to I V/V %
based on the total solution as will be shown in
experimental examples given below. In the present
invention, the water-soluble organic solvent
according to the invention is added to the anti-
voted, elastzse-containing solution to precipitate
elicits. If the concentration of the water-
soluble organic solvent is less than 30 V/V %
based on the solution including the solvent,
elicits is not precipitated. The amount required
for the precipitation is thus at least 30 TV %.
If the concentration is higher than 80 V/V %,
proteins and peptizes contained in the solution
are also precipitated together with elicits to
seriously lower the elicits purity. Thus, the
concentration of the water-soluble organic sol-
vent should be in the range of 30 to 80 V/V %,
particularly 50 to 70 V/V %
The precipitation method is not particularly
limited. or example, the water-soluble organic
solvent is added thereto to control the convent-
ration to a given value, and the mixture is then
stirred for, for example, 1 h to form a precipi-
late containing elicits, which is filtered out.
Alternatively, the precipitate may be again disk
solved in water, a filter aid such as Hyflo Super
.
Cot (diatomaceous earth us added lo the solution
the mixture is filtered to obtain an elicits-
containing solution, a water-soluble organic
solvent is added again thereto and the convent-
ration of the water-soluble organic solvent is
controlled in the range of from 30 to 80 V/V %
based on the resulting solution to reprecipitate
the intended product. The present invention,-
therefore, includes the repeated precipitation
treatment ox the elastase-containing solution.
The elicits precipitate obtained by the
process of the present invention is washed several
times with the water-soluble organic solvent and
vacuum-dried to isolate the same as dry product.
The crude elicits obtained my the process of
the present invention can be further purified by
a proper purification method. For the precipita-
lion, there may be employed for exam a method
disclosed in the specification of Japanese Patent
Publication No. 21557/75 wherein the dry product
obtained by the process of the present invention
is dissolved in an aqueous solution of pi 5 to
I the solution is left to stand at 5 to 50C
for longer than 1 h and then salted out and the
resulting precipitate is purified properly and
freeze-dried. This purification step is effected
when high-purity elicits is required particularly
and is unnecessary when crude elicits obtained
by the present invention satisfies the requirement.
Thus the purification step does not limit the
present invention.
The present invention will be illustrated
in more detail by the following effect examples
and experimental examples.
Effect Example 1
Samples
(1) Sample of the present invention:
A dry product obtained in Example 1 given
below was used as the sample.
(2) Control sample obtained by a conventional
process:
1 kg of the pancreas of porcine was minced.
2~0 ml of water and 7 g of pancreatic were added
thereto. Then, 5 ml of a 4Q % aqueous sodium
hydroxide solution was added thereto and the mixture
was stirred violently. It was left to stand at
17C for 2 h to effect the activation. There-
after, 3 1 of 0.1 M acetate buffer solution (pi
4.8) was added thereto and the mixture was stirred
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~66~3
for 4 h and then allowed to stand overnight.
300 g of Elite was added to the liquid and the
whole was stirred and then filtered. 2 1 of
the acetate buffer (pi 4.8) was added to the lit
traction residue to effect extraction. After
filtration, the filtrates were combined together
(4.5 1). Ammonium sulfate was added thereto to
attain 45 % saturation. After salting-out, the
resulting precipitate was filtered out and vacuum-
dried. The resulting dry product was used as
the control sample.
Method and results:
Elicits activities of the sample of the pro-
sent invention and control sample were determined
using succinyltrialanine p-nitroanilide as sub-
striate. The results are shown in Table 1.
Table
Sample Dry weight Elicits ' Total units
i (I activity obtained
_ _ (unit~mg)
Sample of Thea 1.11116,500
invention
Control 88 1.0088,000
_
_ g _
I
It is apparent from Table 1 that as compared
with the conventional process, a higher yield can
be obtained by the simple and easy chemical open
ration according to the present invention.
Effect Example 2
Samples:
(l) Sample of' the present invention:
Filtrate obtained by the rough filtration in
Example 1.
(2) Control sample obtained by a convention
net process:
A total filtrate obtained by combining the
filtrates obtained in the preparation of the count-
not sample in Effect Example 1.
Moth d and results:
Elicits activities were determined in the
same manner as in Effect Example 1. The results
are shown in Table 2.
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Table 2
.. _ I__ __
Sample Quantity of Elicits Total
filtrate activity units
(1) (unit/ml) obtained
- - _ _ __ __ ._
Sample of the 1.8 67.2120,960
invent ion
Control 4.5 20.893,600
__ _.
It is apparent prom Table 2 that the yield
obtained by the process of the present invention
was higher than that obtained my the conventional
process already in the extraction step.
experimental Example 1
2.4 1 of water and 17 g of pancreatic were
added to 2~4 kg of the need pancreas of porcine.
Then, 12 ml Or a 40 % aqueous sodium hydroxide
solution was added thereto and the mixture was
stirred and then left to stand at 25C for 2 h.
40 g of ~yflo So Cot ask was added to the mixture
and the mixture was subjected to rough filtration.
The resulting filtrate was divided into 400 ml
portions. Acetone was added to the respective
portions so as to control acetone concentration
to I, 20, 30, 40, 50~ 6Q, 70 and 80 V/V %, rest
pectively. After being left to stand at 25C for
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~18~9
1 h, the resulting precipitate was filtered out.
The respective precipitates were suspended in
aqueous acetone solutions having the same consent
rations as those in the precipitation step.
The resulting precipitates were again filtered
out. Elicits activities of the precipitates were
determined using succinyltrialanine p-nitroanilide
as substrate. The recoveries of elicits in
the respective precipitates were calculated from
the elicits activity thus obtained and the anti-
viny of elicits in the filtrate.
results:
The results are shown in Table 3. It is
apparent from this Table that in the precipitation
of elicits from the elastase-containing aqueous
solution obtained my the activation treatment of
the pancreas of porcine, the elicits precipitation
starts at an acetone concentration of 30 to 40
V/V and completed substantially at the convent-
ration of 60 V/V %.
The acetone concentration of higher than 80
V/V % is not preferred, since large amounts of
protein and peptizes contained therein are also
Precipitated.
::
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Table 3
Acetone concentration Recovery of elicits
TV % into the precipitate
(%)
lo 35
83
Lowe
. ` I
_ _
The following examples will further illustrate
the present invention.
Example l
200 ml of water and 7 g of pancreatic were
added Jo 1 kg of the minced pancreas of porcine.
Then, 5 ml of a 40 % aqueous sodium hydroxide
solution was added thereto and the mixture was
Stirred violently and then left to stand at 17C
for 2 h to effect the activation. Acetone was
added thereto to obtain an acetone concentration
of 70 V/V %. The mixture was stirred for l h.
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I
The acetone layer was removed. 1.2 1 of water
was added to the resulting precipitate and the
mixture was stirred for 2 h. Then, 10 g ox Hyflo
Super Cot was added to the mixture and the mixture
was subjected to rough filtration to obtain 1.8 1
of a rate. Acetone was added to the filtrate
to obtain an acetone concentration of 60 V/V %.
The resulting precipitate was filtered out.
The precipitate was further washed with 1.5 1 of
acetone three times and then dried in vacuum to
obtain a dry product.
Yield: 105 g. Activity: 1.11 units/mg.
Example 2
10 1 ox water and 70 g of pancreatîn were
added to 10 kg of the minced pancreas of porcine.
Then, 5Q ml of a 4Q % aqueous sodium hydroxide
solution was added thereto and the mixture was
stirred and then left to stand at 35C for 2 h
to effect the activation. Acetone was added
thereto to obtain a concentration of 30 V/V %.
100 g of Hyflo Super Cot was added to the mixture
and the mixture was subjected to rough filtration.
Acetone was added to the rough filtrate to a con-
cent ration of 60 V/V %. The resulting precipitate
_ 14 -
was filtered out. The precipitate was washed with
15 1 of acetone three times and vacuum-dried to
Obtain 1.2 kg of a dry product.
Activity: 0.93 unit/mg.
The dry product thus obtained was further
purified if necessary, as follows: 12 ] of a
0.1 M phosphate buffer solution (pi 7.4) was added
to 1.2 kg of the dry product. The mixture was
stirred for 1 h and then left to stand at 20C
for 2Q h. Then, 70Q g of Hoff Super Cell was
added to the mixture and the mixture was filtered.
Ammonium sulfate was added to the filtrate to
effect the salting-out at 40 % saturation.
The product salted out was filtered out. 370 g
of thus obtained product was dissolved in 3.7 1 of
0.1 M carbonate buffer solution (pi 7-Q2. The so-
lotion was filtered to obtain a clear solution,
which was stirred for 3 days to obtain elicits
crystals. The crystals were desalted and then
freeze-dried to obtain 35 g of a dry product.
Elastin-hydrolytic activity: 165 units/mg.
Example 3
4 1 of a 50 V/V aqueous acetone solution
was added to 10 kg of minced pancreas of porcine
and the mixture was left to stand at 10C for 16 h
to effect the activation. Then, acetone was added
thereto to obtain a concentration of 70 V/V %
and the mixture was stirred for 1 h. The acetone
layer was Ramada to obtain 12 kg of a precipitate.
13 1 of water was added to the precipitate and
the mixture was stirred for 2 h. Then 100 g of
Hyflo Super Cot was added thereto and the mixture
was subjected to rough filtration. Acetone was
added to the rough filtrate to obtain a 70 V/V %
concentration and the resulting precipitate was
filtered out. The precipitate was washed with
15 1 of acetone three times and vacuum-dried to
obtain 1.15 kg of a dry product. Activity: 0.97
unit/mg. If necessary, 1.2 kg of the obtained dry
product was purified in the same manner as in
the purification step in Example 2. 33 g of
freeze-dried elicits crystals were obtained.
Elastin-hydrolytic activity: 175 units/mg.
Example 4
4 1 of a 50 V/V % aqueous ethanol solution
was added to 10 kg of minced pancreas of porcine
and the mixture was left to stand at 10C for
2 days to effect the activation. Then, 8 1 of
water and 5 1 of ethanol were added thereto and
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subsequently 200 g of Hyflo Super Cell was added
thereto and the mixture was subjected to rough
filtration. Ethanol was added to the rough flit
rate to obtain a concentration of 60 TV I.
The formed precipitate was filtered out. The pro-
cipitate was further suspended in a 60 TV %
aqueous ethanol solution and the suspension was
filtered. The filtered precipitate was washed
with 15 1 of acetone three times and then vacuum-
dried to obtain 1 kg of a dry product. Activity:
1.12 units/mg. The resulting dry product was
further purified, if necessary, as follows: 10 1 of
a 0.1 M potassium dihydrogenphosphate solution
was added to 1 go of the dry product. The mixture
was stirred at 35C for 2 ho Then, 500 g of Hyflo
Super Cot was added to the mixture and the mixture
was filtered. Ammonium sulfate was added to the
filtrate to obtain I % saturation. After salting-
out, the product thus salted out was filtered out.
350 g of the obtained product was dissolved in
a Al M carbonate buffer solution (pi 7.0~.
The solution was filtered to obtain a clear soul-
lion, which was stirred for 3 days to obtain eras-
tease crystals. The crystals were filtered out,
desalted and freeze-dried to obtain 33 g of a dry
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product. Elastin-hydrolytic activity: 168 units/
my
Example 5
2 1 ox water and 70 g of pancreatic were added
to 10 kg of the pancreas of porcine. The mixture
was stirred violently and then left to stand at
35C for 4 h to effect the activation. Then,
isopropyl alcohol was added thereto to obtain a
concentration ox 80 V/V %. After stirring for 1 h,
the isopropyl alcohol layer was removed to obtain
12 kg of a precipitate. 13 1 of water was added
to the precipitate and the mixture was stirred for
2 h. Then, 100 g of Hyflo Super Cot was added
thereto and the mixture was subjected to rough
filtration. Isopropyl alcohol was added to the
filtrate to obtain a concentration of 70 V/V %
and the resulting precipitate was filtered out.
The precipitate was further washed with 15 1 of
isopropyl alcohol twice and vacuum-dried to obtain
1.2 kg of a dry product. Activity: 1.02 units/mg.
1.2 kg of the obtained dry product was further
purified, if necessary, according to the same
purification process as in Example 2 to obtain 34 g
ox freeze-dried ecstasy crystals. Elastin-hydrolytic
activity: 170 units/mg.
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~366~9
Example 6
2 1 of water and 70 g of pancreatic were
added to 10 kg of roughly divided pancreas of
porcine. The mixture was stirred and Lien let L to
Stand at 25C for 6 h to effect the activation.
The activated pancreas was minced and mixed with
10 1 of water and 200 g of Hyflo Super Cot. The
mixture was subjected to rough filtration. Acetone
was added to the filtrate to a concentration of
80 V/V %. The acetone layer was removed and the
precipitate was collected. The precipitate was
further suspended in 20 1 of a 70 V/V % aqueous
acetone solution. The suspension was filtered.
The weight of the precipitate (mixture) was 3.5 kg.
Activity Q 34 units/mg.
The resulting precipitate was further puff-
fled, if necessary, as follows: 14 1 of a 0.1 M
phosphate buffer solution (*H 7.4~ was added to
3.5 kg of the precipitate. After controlling
the pi to 7.0 with a 2 N aqueous sodium hydroxide
solution, the mixture was left to stand at 20C
for 20 h. Then, 700 g of Hyflo Super Cot was
added thereto and the mixture was filtered.
Ammonium sulfate was added to the filtrate to 40 %
saturation to effect salting-out. 340 g of a product
.
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:~86~
filtered out was dissolved in a 0.1 M carbonate
buffer solution (pi 7.0) and the solution was
filtered. The resulting clear solution was
Stirred for 3 days to obtain elicits crystals.
The Crystals were collected by centrifugation,
desalted and freeze-dried to obtain 34 g of a
dry product. Ela5tin-hydrolytic activity:
178 units/mg.
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