Note: Descriptions are shown in the official language in which they were submitted.
ifA~3~
The present invention is concerned with the fixing or insolu-
bilising of proteins.
An object of the Canadian Patent Appllcation No. 192,733 is
the manufackure of the novel condensation product obtained by reacting
1,3-phenylenediamine with glutardialdehyde, which is distinguished by
having a large bonding capacity for proteins.
It has now been found in accordance with the present invention
that a series of further structurally related amines may be reacted
with the same or with structurally similar dialdehydes, or also with
acrolein, in the same manner to give condensation products which are
likewise suitable in an outstanding manner for the same purpose~j namely
for the fixing or insolubilising of proteins.
Accordingly, the present invention provides a process for the
fixing or insolubilising of proteins, which process comprises bonding
the protein to a polymeric condensation product obtained by reacting
a carbocyclic, aromatic polyamine with an aliphatic dialdehyde or acro-
~ lein, other than the 1,3-phenylenediamine glutardialdehyde condensation
; product.
The polyamine-aldehyde resins can be made reacting a carbocyclic
aromatic polymaine with an aliphatic dialdehyde or acrolein to give a
condensation product suitable for the fixing or insolubilising of proteins
,:
~s~s
~the manufacture of the 1,3-phenylenediamine glutardialdehyde condensation
product being excluded).
Carbocyclic, aromatic polyamines which can be used in the for-
mation of the condensation product include diamines or triamines, that is
mononuclear compounds such as phenylenediamines (e.g. p-phenylenediamine)
and phenylenetriamines ~e.g. 2,4,6-triaminophenol, 2,~,6-triaminotoluene),
polynuclear compounds such as biphenyldiamines (e.g. ben~idine) or poly-
nuclear compounds with aliphatic bridges, such as diaminodiphenylalkanes
~e.g. diaminodiphenylmethanes).
Apart from amino groups, the aromatic nuclei can also carry
other substituents such as halogen atoms ~e.g. chlorine), lower alkyl
groups ~e.g. methyl, ethyl), lower alkoxy groups ~e.g. methoxy, ethoxy)
or hydroxy, carboxy, mercapto, lower alkylthio or sulphonyl groups.
Finally, mixtures of different polyamines can also be employed
as reaction components in the manufacture of the condensation products
in accordance with the invention.
Preferred polyamine reaction components are banzidine and 2,4,
6-triaminotoluene.
~xamples of aliphatic dialdehydes which can be used in the for-
mation of the condensation product include glutardialdehyde, succinic acid
dialdehyde, glyoxal, etc. Glutardialdehyde is preferably used.
-- 3 --
. . , ~
~/~S'~3~5
~:`
~hether or not the condensation products manufactured
according to the process of the present invention are suitable
for the fixing or insolubilising of enzymes can be experimentally
determined in a simple manner by treating the condensation product
with an enzyme, preferably amyloglucosidase~ and measuring the
activity of the fixed ~nzyme. The suitabili~y is verified if
this activity comes to at least 50 to 100 units per gram of
; condensation product carrier material.
The following test procedure can be used for this purpose:
A chromatography column (1.5 x 15 cm~ is filled with 1 g
of the condensation product carrier materialO A solution of 1 g
of amyloglucosidase (50 units/mg) in 100 ml of 16 mmol acetate
buffer (pH 4.8) is passed through the column at rdom temperature
and with a flow rate of 20 ml per hour. As already mentioned,
; the fixe~ enzyme should have an activity of at least 50 to 100
units ~ of carrier material.
The activity is measured according to Zulkoski by way of
the glucose liberated from soluble starch. The liberated
glucose is determined enzymatically by means of the glucose-
oxidase/peroxidase test [Bergmeyer~ Methoden der enzymatischen
Analyse, Volume I, (19~0) page 41~]. [1 ~lit = that amoun~ of
enzyme which liberates 1 ~mol of glucose/min from soluble starch
at a reaction temperature of 60 C and at pH 4.8].
Alternatively, the suitability is verified if the
condensation product carrier material fixes at least 10 mg of a
protein (e.g. beef serum albumin) per gram of carrier. The
--'1--
~z~os
amount of fixed protein can be determined by hydrolysing the carrier-fixed
protein with 6-N hydrochloric acid and subsequently determining the amount of
free amino acids with the aid of any practicable analysis method for amino
acids.
Proteins which can be bonded to the condessation products in ac-
cordance with the present invention include polypeptides, antigens, anti-
bodies, protein inhibitors and~ especially~ enzymes~ The enzymes can be of
vegetahle, animal or microbial origin. The enzymes may be hydrolases
(peptidases, proteinases, desaminases, carbohydrases, esterases~ nucleases),
lyases or desmolases (hydrolyases, decarboxylases, aldolases), transferases,
iso~erases, oxidoreductases and ligases. Examples of enzymes from which
insoluble enzyme preparations provided by this invention can be n~nufactured
are alcoholdehydrogenase, naringinase, hesperidinase~-glucosidase, ~-amyl-
la~ invertase, amyloglucosidase, urease, trypsin, ficin, papain, bromelin,
subt;lopeptidase, rennin, glucoseisomerase, glucoseoxidase, peroxidase,
catalase, acylase, cytochrome, ribonuclease, phosphodiesterase and
adenyldeaminase.
According to ~Comprehensive Biochemistryl~ by Florkin ~. and Stotz
E. H. Volume 13, third edition, Elsevier Publishing Company, the enzymes have
the following enzyme numbers:
alcoholdehydrogenase 1 ol~
~-glucosidase 3.~.1.21
-amylase 3.2.1.2
invertase 3.2.1~26
amyloglucosidase 3.2.1.33
urease 3.5.1.5.
t~ypsin 3.4.4.4
ficin 3.4.4.12
papain 3.4~4.10
~, ', ' ! ~
~05'~3~5
bromelin 3.4.4.24
subtilopeptidase 3.4.4~16
rennin 3.4.4 3
glucoseisomerase 5.3~1 5
glucoseoxidase 1.1.3 4
peroxidase 1.11.l.7
catalase 1.11.1.6
acylase 3~1.1.6
cytochrome 1.9.3.1
ribonuclease 2.7.7.16
phosphodiesterase 3.1.4.1
adenyldeaminase 3.5.4.2
Naringinase and hesperidinase ha~e no numbers because they are
enzyme mixtures of ~-glucosidase and rhamnosidase~
The molar ratio of the polyamine and aldehyde reaction partners
expediently lies at between l:l and l:10, a ratiolof about 1:3 being pre-
ferred.
The reaction can be carried out in a manner known per se; for
example~ in aqueous solution, preferably with the addition of an acid such
2~ as a mineral acid (e.g. hydrochloric acid). In a particular embodiment,
the reaction is carried out in the presence of an inert, fine-grained,
preferably inorganic,
- 5a-
~5'~3~5
especially silicate-contain~g~ juuant.~ p~e~fo~-u~ch
adjuvants are silica gel, pumice-stone, diatomaceous earth
(kieselguhr)~ bentonite, wollastonite, porous glass and also
metal oxides such as aluminium oxide or hydroxylapatite. It
is preferred to use silica gel ~e.gO with a particle si7~e of
0.05-0.2 mm, 70-325 mesh) or pumice-stone (e.g. with a particle
siæe of 0.05-10 mm). The presence of such an adjuvant gives
rise to a homogeneous particle formation in the reaction andg
as a result~ an improved sedimentation is achieved. The
reaction in the presence of an adjuvant is expediently carr~d
out by initially bringing the particles into contact with one
of th~ two reaction compo~ènts and then adding the second
component with simultaneous or subsequent slight acidification.
The reaction can be carried out in a homogeneous phase or,
preferably~ in a two-phase system with the addition of an acid
such as a mineral acid ~e.g. sulphuric acid, phosphoric acid
or, preferably, hydrochlori~ acid) or an organic acid (e.g. a
carboxylic acid such as acetic acid) with vigorous stirring or
shaking. The use of a two-phase system promotes the formation
~0 of spherical~ substantially homogeneous particles which are
easy to filter and which sediment well, these particles being
especially well suited as a carrier materialO
Inert, water-immiscible organic solvents (e.g. di~hcloro-
methane, chloroform, carbon tetrachloride~ benzene~ toluene,
ethyl acetate~ dioxane, carbon disulphide3 are suitable as the
second phaseO Chlaroform is preferably used as the inertg
water-immiscible solvent, but acetone is also suitable for this
purpose.
~SZ3~
The reaction temperature is not critical, it can lie,
for example, at between 0C and 50C, preferably at room
temperature (i.eO at about 18-22 C.). In accordance with the
process provided by this invention there is obtained a conden-
sation product carrier material which, although already very
active, can be still further activated by diazotisation. The
diazotisation can be carried out in a manner known per se by
treatment with nitrite and acid.
In order to fix the protein to the carrier material, the
latter is treated with an aqueous s~-lution, preferably a
buffered solution~ of the protein (1-50 mg/ml) at a temperature
of 0 -30 C~ preferably 4 C to room temperature. This treatment
can be carried out while stirring or shaking. Because of the
high activity of the carrier material, the fixing of the proteins
can also be advantageously carried out by simply filtering the
protein solution through a carrier layer, preferably a column
filled with the carrier material~ as is common, for example,
; in column chromatography. Thus, for example, culture f~ltrates
of microorganisms which contain proteins or enzymes can be
allowed to run directly through a column of carrier material,
the proteins or enzymes being selectively fixed to the carrier.
The column is expediently rinsed with a buffer solution and
1-M potassium chloride solution in order to remove the non-fixed
proteins.
The proteins fixed to the carrier material are generally
very stable and very high specific activities (unit/g) are
achieved in the case of in enzymesD Loadings of, for example,
1:3-10 parts by weight of protein/carrier material are achieved.
3~
The carrier-bonded proteins provided by this inven~ion,
especially enzymes, can be used in a manner known per se; for
example, for analytical or preparative purposes or in food
technology such as in the manufacture of glucose from starch
~see3 or example., Scientific American 224~ (No. 3) 26-33
( 1971); Angew. Chemie 849 (8) 319-268 ( 1972); Chemiker
Zeitung 96, (11), 595-602 ~1972); ~ & EN, ~15.2.71), 86-87].
In this important large-scale process, amyloglucosidase
fixed to the condensation product provided by the present
invention can be utilised for the manufacture of glucose from
the "glucose syrup" (prehydrolysed starch). The condensation
products can then be transferred~ for example, onto columns
(fixed-bed process). A further application results in the
enzymatic degradation of lactose in milk products by means of
lactases (e.g. I'sweets" from whey).
The following Examples illustrate the present invenkion.
The quantitative data of the carrier material obtained or used
is given with respect to dry weight.
xample 1
To a solution or suspension of 5 g of the polyamine in
200 ml of chloroform, there are added portionwise with vigorous
s~irring, firstly 20 g of aldehyde (80 ml of a 25% solution ~
water) and, after a further 5 minutes, 20 ml of 7-N hydrochloric
acid. The reaction mixture solidifies. It is treated with
300 ml of water and shaken for 5 minutes until it again becomes
liquid. The polymeric particles are left to stand for 1 hour
with occasional stirring. After vacuum filtration of the
mixture over a Bùchner funnel, the polymeric part:icles are
30~i
washed with three 200 ml portions of acetone and then with
0.1-~ sodium hydroxide solution. The residual chloroform is
removed by washing with acetone. The thus obtained carrier
particles are spherical, homogeneous and capable of good
filtration; they are stored in water or in d~llute sodium
hydroxide. They can be employed for enzyme fixing~9 optionally
after diazotisation.
Example 2
A chromatography column (1.5 x 15 cm) is filled with 1 g
of carrier material and washed with the buffer solution used
in the following enzyme fixing. The buffered enzyme solution
(1-100 mg of protein per ml of buffer) is passed through the
column with a flow rate of 20 ml/hour.
The coupling capacity is exhausted ~eh protein can be
detected in the eluate, which can be detected, for exa~ple by
measurement of the absorption spectrum at 280 m,u. The non-
fixed enzyme is removed by washing the column with 1-M
potassium chloride and the buffer solution. There then follows
an activity determination of an aliquot of the enzyme-fixed
material with the respective substrate under given reaction
conditions. Then an aliquot of the enzyme solution is washsd
with waterg dried and the dried material determined analogously.
The number of units of enzyme activity/g of carrier material
can be determined in this manner.
The condensation products manufactured according to
Example 1 and their corresponding activities determined
according to Example 2 are compiled in the following Table:
~ ~5~3~5
_ ~ _ _ . .
~ O
h ~ ~ >
~ ' _ _ _ __ . _ _ ~ .
~9'~ '7
_10 -
