IMMOBILISED CHOLINESTERASES

CHOLINESTERASES IMMOBILISEES

English Abstract

ABSTRACT OF THE DISCLOSURE
,
An immobilized cholinesterase composition is described, which
comprises a polymer containing anilided acrylic acid or substituted acrylic
acid units of the type
<IMG>
wherein Y represents a cholinesterase molecule and R'R2R3 and R4 are the same
or different, and represent hydrogen atoms or alkyl, aryl, aralkyl, alkaryl,
alkenyl or alicyclic groups or R2 and R3 together form an alicyclic group.
The polymer may be a homopolymer or copolymer containing an acrylic acid or
a substituted acrylic acid. Preferably the polymer is a methacrylic acid
homopolymer or a copolymer of methacrylic acid with another alkene such as
styrene, and at least some of the methacrylic acid units may be anilided.

Claims

The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:
1. An immobilized cholinesterase composition, comprising a polymer
containing one of an anilided acrylic acid unit or a substituted acrylic
acid unit of the general type
<IMG>
wherein Y represents a cholinesterase molecule, and R1R2R3 and R4 represent
one of a hydrogen atom and an alkyl, aryl, aralkyl, alkaryl, alkenyl or
alicyclic groups.
2. The immobilized cholinesterase composition defined in Claim 1, wherein
R2 and R3 together form an alicyclic groups.
3. The immobilized cholinesterase composition defined in Claim 1, wherein
the polymer is one of a homopolymer or a copolymer containing an acrylic acid.
4. The immobilized cholinesterase composition defined in Claim 1, wherein
the polymer is one of a homopolymer or a copolymer containing a substituted
acrylic acid.
5. The immobilized cholinesterase composition defined in Claim 3 or 4,
wherein the acid is one of a methacrylic acid homopolymer or a copolymer of
methacrylic acid with another alkene.
6. A process for producing an immobilised cholinesterase composition
comprising the steps of
preparing an acrylyl chloride;
reacting the acrylyl chloride with a meta-halogenated aniline to
form an acrylic-3-halegeno anilide;
polymerising the anilide alone or with free or substituted acrylic
acid or with an olefinic compound;
nitrating the aromatic rings of the polymer; and
immobilising the cholinesterase on the nitrated polymer.

Description

~25755S
The invention relates to immobilised cholinesterases, that is
cholinesterase enzymes in the form of water insoluble derivatives which
retain their characteristic biological activity.
Such enzymes find various uses, notably in the catalysis of in vitro
reactions where their insolubility facilitates their removal at the end of
the reaction or permits their use in a bed through which reagents are
passed. One such use, for which immobilised cholinesterase in accordance
with the present invention are especially suitable, is in detectors for
nerve agents.
Various techniques have been proposed for the preparation of
immobilised enzymes, for example adsorption, physical occlusion within a
macromolecular lattice or covalent bonding of the enzyme either to an
insoluble substrate or via a cross-linking agent to other enzyme
molecules. Adsorption is sometimes useful although problems of elution
limit the useful life of the immobilised material and the range of
solutions with which it may be contacted. In addition the adsorption may
block active sites in the enzyme. Physical occlusion is generally less
susceptible to elution problems if the pore size of the macromolecule is
suitably controlled. However only enzyme molecules occluded within pores
near the surface and accessible to reagent molecules will be able to
display any of their enzymic activity. Much of the enzyme will be
inaccessible to reagent molecules and hence its activity lost. With
processes dependent on covalent bonding care must be taken that the
bonding does not affect the active sites on the enzyme.
According to the present invention, an immobilised cholinesterase
composition comprises a polymer containing anilided acrylic acid or
substituted acrylic acid units of the type

~2S75SS
_ , .
_ 1I C RZ~.3
L~
Wherein Y represents a cholinesterase molecule and Rl R2 R3
and R4 are the same or different and represent hydrogen atoms or alkyl,
aryl, aralkyl, alkaryl, alkenyl or alicyclic groups or R2 and R3
together form an alicyclic group.
The polymer may be a homopolymer or copolymer containing an acrylic
acid or a substituted acrylic acid but is preferably a methacrylic acid
homopolymer or a copolymer of methacrylic acid with another alkene such as
styrene and all or only some of the methacrylic acid units may be
anilided.
The polymers may be prepared by polymering a meta-halogeno
derivative of acrylic acid or a substituted acrylic acid alone or with
free acrylic, or substituted acrylic, acid or another alkene such as
styrene. Alternatively a preformed homo- or copolymer of acrylic acid or
a substituted acrylic acid may be reacted with a meta-halogeno aniline.
The meta substituent on the aniline nucleus is preferably a fluorine atom.
The cholinesterase is then attached to the polymer by reacting with the
meta-halogen atom, after nitrating the aromatic ring to increase
reactivity.
Thus a typical process for the production of a homo-polymer in
accordance with the invention may be represented by the following reaction
scheme. (It should be understood, however, that the invention is in no
way limited by the scheme given).
N l~ R
~R~ _ s~ce~ c,~2R~ R
~c~ ~;c\ ~c~ / R
~ , -2-

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t =C~R~ C- c~'R~ R R~ T
~rA~rlo~J
D~Hl- PRorFl~l) oD ~/
~! NHl- P~DTr ~rl
~0,,
Wherein X represents a halogen atom, m represents an average degree
of polymerisation of acrylate units, generally in the range 1 to 10, and
n represents the o~erall degree of polymerisation. Alternatively a
preformed acrylate polymer may be treated with thionyl chloride and a
halogeno-aniline to form the anilided polymer which may then be nitrated
and reacted with the enzyme.
Preferably polymer systems for forming polymers in accordance with
the present invention are methacrylic acid homopolymers and copolymers of
methacrylic acids with, for example, styrene.
The reaction of the enzyme with the nitrated polymer should
preferably be carried out by contacting the nitrated polymer with a
solution of the enzyme, preferably at a concentration of at least 1 IU/ml
(1 mg/ml) and a pH of 5 to 8, especially 7 to 7.5, for a period of
normally at least 10 minutes up to several days.

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Particular examples of the production of immobilised cholinesterases
in accordance with the present invention will now be described by way of
example.
EXAMPLE 1
r
Methacrylic acid was reacted with thienyl chloride to form
methacrylyl chloride which was then reacted with 3-fluoro-aniline to form
methacrylic-3-fluoranillde. This was then copolymerised with methacrylic
acid in the presence of 0.01 molar ratio (based on the total methacrylic
acid + methacrylic-3-fluoranilide) of divinylben~ene. The resulting
polymer had a structure of the type
//C\~
This polymer was then nitrated to a degree corresponding to 1 nitro
group per fluoranilide residue by reaction with 3:1 (v/v mixture of
concentrated sulphuric and concentrated nitric acids in order to activate
the fluorine atom towards nucleophilic attack.
Samples of the nitrated polymer were then added to a 60 IU/ml
solution of cholinesterase in 0.1M phosphate buffer at various levels of
pH. The cholinesterase activity bound to the polymer was estimated by
Ellman's method (A Ellman G L; Archs. Biophys 82, 70 (1959)). The results
are shown in Table 1.
, .~,

f~ ~7~;5S
Table 1
pH of medium Cholinesterase activity
bound (IU/g)
5.0 7.2
6.0 5'3
6.5 3.3
7,0 73.0
7.5 75.7
8.0 19505
Thus maximum binding of cholinesterase activity occurred at high pHo
However above pH 7.5 considerable hydrolysis of the polymer was observed.
EXAMPLE 2
Methacrylic-3-fluoranilide was copolymerised with styrene instead
of methacrylic acid as in example lo The copolymer was very hard and was
hydrophobic. Some chlorinesterase activity was bound, but considerably less
than with the methacrylic acid polymer.
EXAMPLE 3
A commercial cross-linked methacrylic acid polymer, Amberlite~
CG-50 type I (supplied by BDH Chemicals Ltd) having a particle size range of
75-150pm was fluoranilided with 3-fluoro-aniline and nitrated as in example
1. The resulting polymer contained a 1:6 molar ratio of fluoranilide to
methacrylic acid and an average 0.8 nitro group per fluoranilide. Samples
o~ the nitrated polymer were contacted with solutions of cholinesterase of
7 various concentrations at pH 7.4 for 2 hours at ambient temperature and the
amount of enzyme bound was measured. The results are shown in Table 2.
Similarly the amounts of enzyme bound after various contact times at ambient
- temperature and-pH 7.4 with a 2mglml solution were measured ~see Table 3).
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Table 2
Cholinesterase conc Activity bound
(mg/ml) (IU/g)
O O
0.5 4
0.8 8
1.0 12
2.0 33
4.0 72
5.0 70
Table 3
Contact Time Activity bound
(IU/g)
1 min 12
10 mins 14
2 hrs 34
18 hrs 53
2 days 66
7 days 24
The immobilised cholinesterases produced could be dried either by
freeze-drying or over P205 with 15% and 30% losses in activity
respectively. However, the P205 dried product was found to be more
stable.
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