CA1257555A - Immobilised cholinesterases - Google Patents
Immobilised cholinesterasesInfo
- Publication number
- CA1257555A CA1257555A CA000205671A CA205671A CA1257555A CA 1257555 A CA1257555 A CA 1257555A CA 000205671 A CA000205671 A CA 000205671A CA 205671 A CA205671 A CA 205671A CA 1257555 A CA1257555 A CA 1257555A
- Authority
- CA
- Canada
- Prior art keywords
- cholinesterase
- polymer
- acrylic acid
- homopolymer
- methacrylic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/087—Acrylic polymers
Landscapes
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Materials For Medical Uses (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
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 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.
, An immobilized cholinesterase composition is described, which comprises a polymer containing anilided acrylic acid or substituted acrylic acid units of the type 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.
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-~2S7555 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.
~Z575SS
Particular examples of the production of immobilised cholinesterases in accordance with the present invention will now be described by way of example.
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.
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.
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).
~A Trademark for an ion exchange resin.
"~ - 5 -l~
~257555 Table 2 Cholinesterase conc Activity bound (mg/ml) (IU/g) O O
0.5 4 0.8 8 1.0 12
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-~2S7555 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.
~Z575SS
Particular examples of the production of immobilised cholinesterases in accordance with the present invention will now be described by way of example.
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.
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.
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).
~A Trademark for an ion exchange resin.
"~ - 5 -l~
~257555 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.
~ r ~J .,
~ r ~J .,
Claims (6)
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 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.
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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB36188/73 | 1973-07-30 | ||
| GB36188/73A GB1605289A (en) | 1973-07-30 | 1973-07-30 | Immobilised cholinesterase composition |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1257555A true CA1257555A (en) | 1989-07-18 |
Family
ID=10385786
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000205671A Expired CA1257555A (en) | 1973-07-30 | 1974-07-25 | Immobilised cholinesterases |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA1257555A (en) |
| GB (1) | GB1605289A (en) |
-
1973
- 1973-07-30 GB GB36188/73A patent/GB1605289A/en not_active Expired
-
1974
- 1974-07-25 CA CA000205671A patent/CA1257555A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| GB1605289A (en) | 1988-03-02 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |