WO1992015674A2 - Dna -or rna- modifying enzymes immobilized to supports - Google Patents
Dna -or rna- modifying enzymes immobilized to supports Download PDFInfo
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- WO1992015674A2 WO1992015674A2 PCT/GB1992/000370 GB9200370W WO9215674A2 WO 1992015674 A2 WO1992015674 A2 WO 1992015674A2 GB 9200370 W GB9200370 W GB 9200370W WO 9215674 A2 WO9215674 A2 WO 9215674A2
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- enzyme
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- groups
- aldehyde
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- 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
-
- 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
- C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
-
- 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
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
Definitions
- the present invention relates to enzymes and more particularly to restriction endonucleases.
- the invention comprises an enzymatic reagent system comprising a DNA or RNA modifying enzyme immobilised to a support.
- the enzyme may be a digestion or a ligation ' enzyme.
- the enzyme may for example be a DNA restriction enzyme or an RNA restriction enzyme, or a DNA ligase, DNA polymerase or DNA inase, or an RNA ligase,
- the enzyme may be covalently immobilised on a support. Any such support will require surface groups to which either the enzyme to be immobilised may be bonded, or to which may be bonded a "coupling" compound which will provide a "link" between the enzyme and the support.
- the support may, for example, have free hydroxyl groups and/or may be of plastics, silica, acrylamide or sephadex.
- the support may have different functionalities on its surface (for example COOH, OH, NH-) to maximise the efficiency of the immobilised enzyme.
- An alternative to the covalent bonding of the enzyme to the support it is possible to "entrap" the enzyme in or on the support with the intention that the enzyme will be released into a medium in which a reaction is being conducted.
- the enzyme may be entrapped within a polymer which gives a stable rigid film.
- a particularly suitable material for this purpose is a mixture of hydrolysed polyvinyl alcohol and polyethylene • glycol (PVA/PEG) .
- PVA polyvinyl alcohol and polyethylene • glycol
- the PVA has an AMW of 5,000 to 40,000 and a degree of hydrolysis in the range 75-95%.
- the PEG preferably has a molecular weight of 100 to 400.
- PEG 200 and PVA 88% hydrolysed AMW 10,000 has been found to be particularly suitable when used in a PVA:PEG ratio of 90%:10%. Such a mixture gives a firm rigid gel on drying.
- the gel is mixed with the enzyme and applied to a support (e.g. a plastic film) which is then placed in a desiccator and allowed to dry (preferably in vacuo).
- a support e.g. a plastic film
- Enzymes which are suitable for this entrapment procedure include EcoRl, Hind III, Pst 1, and Bam HI.
- Figure 2 illustrates the production of free amino groups on a surface for the method of Figure 1;
- Figure 3 illustrates a support with an epoxy group reacting with an enzyme amino group
- Figure 4 illustrates a carbodiimide coupling
- Figure 5 illustrates the use of a spacer molecule.
- the enzyme is immobilised onto the support by means of glutaldehyde as a "coupling agent".
- the support has surface groups which can be derivatised to amino groups.
- Suitable supports include control pore glass, plastics with surface hydroxyl groups, Eupegit (a synthetic polymer having a controlled particle size), plasticard, polycarbonate, glass fibre, and glass wool.
- the basic requirement for the use of glutaldehyde is that the support has free amino groups on its surface.
- the glutaldehyde is bonded to the free amino groups on the support surface and subsequently the enzyme is bonded by means of its free amino groups to the remaining free aldehyde group of glutaldehyde.
- the support does not already have free surface amino groups, then such groups can be introduced by means of reagents such as aminopropyl trithoxysilane. These reagents react with surface hydroxyl groups so as to provide free amino groups is shown in Figure 2 the resultant support, now including free amino groups, may now be treated with glutaldehyde for the subsequent immobilisation of an enzyme thereon (following the procedure of Figure 1) .
- reagents such as aminopropyl trithoxysilane.
- FIG. 3 A further scheme for the immobilisation of an enzyme is shown in Figure 3 in which an epoxy group provided on the support reacts with an amino group of the enzyme to effect immobilisation thereof.
- the epoxy groups may be introduced onto the support by the use of reagents such as 3-glycidoxypropyl trimethoxysilane. These reagents add an epoxy group to free hydroxy groups.
- Support including the epoxy group are commercially available, for example Eupegit. These are acrylic beads with the oxiraine group in the environment,
- FIG. 4 A further scheme is shown in Figure 4 and uses soluble carbodiimide coupling.
- a soluble carbodiimide such as l-ethyl-3-(3-dimethyl- aminopropyl) carbodiimide hydrochloride (EDC) is used to activate the carboxyl groups of restriction endonucleases such as Sau 3A and EcoRl.
- EDC l-ethyl-3-(3-dimethyl- aminopropyl) carbodiimide hydrochloride
- a "spacer" molecule is provided for increasing the distance between the support matrix and the functional centre of the enzyme.
- the reaction scheme uses amino butyl aldehyde as the spacer molecule.
- This reagent reacts with amino groups on the support to form an imine which may subsequently be reacted with a hydride (e.g. sodium cyanoborohydride) .
- a hydride e.g. sodium cyanoborohydride
- the use of this reaction chemistry increases the arm length and thus the distance of the reactive group (i.e. the a ine) from the support. This in turn increases the space available for the enzyme to maintain a functional, 3-dimensional structure. This results in an apparent increase in reactivity of the enzyme.
- the pro_ision of an enzyme on a support has several advantages over current methods of using enzymes. These advantages include :-
- each enzyme may be presented immobilised on a suitable support (e.g. a strip or tube);
- each enzyme may be pre-measured in terms of a defined number of units per pack;
- the enzyme can be stored dry
- the enzyme may be covalently immobilised (to be used in situations where more than one reaction is required to be performed) or immobilised such that the enzyme can be dissolved into a buffer in which the support is located;
- a 2.5% aqueous glutaldehyde solution was mixed with amino propyl controlled pore glass (NH CPG at pH 7.0).
- the support was washed with water and a restriction endonuclease added.
- An addition of bovine serum albumin (a similar carrier protein could be used instead) was then made and the support subsequently washed with water.
- the support with immobilised enzyme could then be dried or stored moist at 4°C for greater than two months without loss of activity.
- Enzymes that have been immobilised in this manner include Bam HI, Pst 1, and Hind 111.
- a support including oxiraine groups (e.g. Eupegit) was washed in a buffer at pH 7.5. The enzyme was then added in the same buffer and the support then washed in the buffer. The support bound enzyme was then stored moist at 4°C.
- oxiraine groups e.g. Eupegit
- a support was washed with water at pH 5.5 and the enzyme added. EDC was added at a molar ratio of 2:1 (enzyme:EDC) and the reaction medium was maintained at room temperature with agitation.
- the support was washed with 0.5M sodium chloride.
- the support (with immobilised enzyme) was stored in 0.5M NaCl at pH 7.5 and 4°C.
- restriction endonucleases on to a support as above described does not interfere with their activity.
- the enzymes remain active and usable after extensive washing whether in a solumn or by centrifu- gation.
- the loading of the enzyme on the support can be determined by a dilution series experiment. A standard amount (1 mg) of resin is added to 1, 2, 3, 5, and 10 ug amounts of lambda DNA and, after one hour the resulting digested DNA is run on an agarose gel, which then gives a direct measurement of enzyme activity per mg of resin.
- Immobilisation as described above increases the stability of the enzyme. Storage temperature may be no longer critical. It is possible to re-use the enzyme and to wash the resin in different buffers which allows the activity (in terms of specificity) of the enzyme to be changed.
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Inorganic Chemistry (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
An enzymatic reagent system is disclosed comprising a DNA- or RNA-modifying enzyme immobilised to a support such as a plastic or glass support, and methods for producing such a system.
Description
DNA-OR RNA MODIFYING ENZYMES IMMOBILIZED TO SUPPORTS
The present invention relates to enzymes and more particularly to restriction endonucleases.
The invention comprises an enzymatic reagent system comprising a DNA or RNA modifying enzyme immobilised to a support.
The enzyme may be a digestion or a ligation ' enzyme. The enzyme may for example be a DNA restriction enzyme or an RNA restriction enzyme, or a DNA ligase, DNA polymerase or DNA inase, or an RNA ligase,
%_, polymerase or kinase.
The enzyme may be covalently immobilised on a support. Any such support will require surface groups to which either the enzyme to be immobilised may be bonded, or to which may be bonded a "coupling" compound which will provide a "link" between the enzyme and the support. The support may, for example, have free hydroxyl groups and/or may be of plastics, silica, acrylamide or sephadex. The support may have different functionalities on its surface (for example COOH, OH, NH-) to maximise the efficiency of the immobilised enzyme.
An alternative to the covalent bonding of the enzyme to the support, it is possible to "entrap" the enzyme in or on the support with the intention that the enzyme will be released into a medium in which a reaction is being conducted.
In this embodiment, the enzyme may be entrapped within a polymer which gives a stable rigid film. A particularly suitable material for this purpose is a mixture of hydrolysed polyvinyl alcohol and polyethylene • glycol (PVA/PEG) . Preferably the PVA has an AMW of 5,000 to 40,000 and a degree of hydrolysis in the range 75-95%. The PEG preferably has a molecular weight of 100 to 400. For example, PEG 200 and PVA 88% hydrolysed AMW 10,000 has been found to be particularly suitable when used in a PVA:PEG ratio of 90%:10%. Such a mixture gives a firm rigid gel on drying.
The gel is mixed with the enzyme and applied to a support (e.g. a plastic film) which is then placed in a desiccator and allowed to dry (preferably in vacuo).
Enzymes which are suitable for this entrapment procedure include EcoRl, Hind III, Pst 1, and Bam HI.
Embodiments of enzymatic reagent systems according to the invention will now be described with reference to .the accompanying diagrams, of which :-
Figure 1 illustrates a method producing immobilisation of an enzyme on to a support with free amino surface groups using glutaldehyde;
Figure 2 illustrates the production of free amino groups on a surface for the method of Figure 1;
Figure 3 illustrates a support with an epoxy group reacting with an enzyme amino group;
Figure 4 illustrates a carbodiimide coupling;
and Figure 5 illustrates the use of a spacer molecule.
In the embodiment illustrated in Figure 1, the enzyme is immobilised onto the support by means of glutaldehyde as a "coupling agent". In this embodiment, the support has surface groups which can be derivatised to amino groups. Suitable supports include control pore glass, plastics with surface hydroxyl groups, Eupegit (a synthetic polymer having a controlled particle size), plasticard, polycarbonate, glass fibre, and glass wool.
The basic requirement for the use of glutaldehyde is that the support has free amino groups on its surface.
Initially, the glutaldehyde is bonded to the free amino groups on the support surface and subsequently the enzyme is bonded by means of its free amino groups to the remaining free aldehyde group of glutaldehyde.
If the support does not already have free surface amino groups, then such groups can be introduced by means of reagents such as aminopropyl trithoxysilane. These reagents react with surface hydroxyl groups so as to provide free amino groups is shown in Figure 2 the resultant support, now including free amino groups, may now be treated with glutaldehyde for the subsequent immobilisation of an enzyme thereon (following the procedure of Figure 1) .
A further scheme for the immobilisation of an enzyme is shown in Figure 3 in which an epoxy group provided on the support reacts with an amino group of the enzyme to effect immobilisation thereof. The epoxy groups may be introduced onto the support by the use of reagents such as 3-glycidoxypropyl trimethoxysilane. These reagents add an epoxy group to free hydroxy groups.
Support including the epoxy group are commercially available, for example Eupegit. These are acrylic beads with the oxiraine group in the environment,
A further scheme is shown in Figure 4 and uses soluble carbodiimide coupling. In this method, a soluble carbodiimide such as l-ethyl-3-(3-dimethyl- aminopropyl) carbodiimide hydrochloride (EDC) is used to activate the carboxyl groups of restriction endonucleases such as Sau 3A and EcoRl.
In the further embodiment of the invention illustrated in Figure 5, a "spacer" molecule is provided for increasing the distance between the support matrix and the functional centre of the enzyme. The reaction scheme uses amino butyl aldehyde as the spacer molecule. This reagent, as do others in the class, reacts with amino groups on the support to form an imine which may subsequently be reacted with a hydride (e.g. sodium cyanoborohydride) . The use of this reaction chemistry increases the arm length and thus the distance of the reactive group (i.e. the a ine) from the support. This in turn increases the space available for the enzyme to maintain a functional, 3-dimensional structure. This results in an apparent increase in reactivity of the enzyme.
The pro_ision of an enzyme on a support has several advantages over current methods of using enzymes. These advantages include :-
(a) each enzyme may be presented immobilised on a suitable support (e.g. a strip or tube);
(b) each enzyme may be pre-measured in terms of a defined number of units per pack;
(c) there will be no problems of cross-contamination, as is possible at present;
(d) the enzyme can be stored dry;
(e) the enzyme may be covalently immobilised (to be used in situations where more than one reaction is required to be performed) or immobilised such that the enzyme can be dissolved into a buffer in which the support is located; and
(f) the system will be more economical since the enzyme may be pre-measured.
The invention will be further illustrated by reference to the following examples.
Example 1
The process of this example follows the reaction scheme shown in Figure 1.
A 2.5% aqueous glutaldehyde solution was mixed with amino propyl controlled pore glass (NH CPG at pH 7.0). The support was washed with water and a restriction endonuclease added. An addition of bovine serum albumin (a similar carrier protein could be used instead) was then made and the support subsequently washed with water.
The support with immobilised enzyme could then be dried or stored moist at 4°C for greater than two months without loss of activity.
Enzymes that have been immobilised in this manner include Bam HI, Pst 1, and Hind 111.
Example 2
This example uses the scheme of Figure 3.
A support including oxiraine groups (e.g. Eupegit) was washed in a buffer at pH 7.5. The enzyme was then added in the same buffer and the support then washed in the buffer.
The support bound enzyme was then stored moist at 4°C.
This method has been used for Hind 111, Sau 3A, and XhO 1.
Example 3
This example uses the procedure of Figure 4.
A support was washed with water at pH 5.5 and the enzyme added. EDC was added at a molar ratio of 2:1 (enzyme:EDC) and the reaction medium was maintained at room temperature with agitation.
The support was washed with 0.5M sodium chloride.
The support (with immobilised enzyme) was stored in 0.5M NaCl at pH 7.5 and 4°C.
It has been demonstrated experimentally that immobilising restriction endonucleases on to a support as above described does not interfere with their activity. The enzymes remain active and usable after extensive washing whether in a solumn or by centrifu- gation.
The loading of the enzyme on the support can be determined by a dilution series experiment. A standard amount (1 mg) of resin is added to 1, 2, 3, 5, and 10 ug amounts of lambda DNA and, after one hour the resulting digested DNA is run on an agarose gel, which then gives a direct measurement of enzyme activity per mg of resin.
Immobilisation as described above increases the stability of the enzyme. Storage temperature may be no longer critical. It is possible to re-use the enzyme and to wash the resin in different buffers which allows the activity (in terms of specificity) of the enzyme to be changed.
Claims
1. An enzymatic reagent system comprising a DNA modifying enzyme or an RNA modifying enzyme immobilised to a support.
2. A system as claimed in claim 1, wherein the enzyme is a digestion enzyme or a ligation enzyme.
3. A system as claimed in claim 1, wherein the enzyme is a DNA restriction enzyme or an RNA restriction enzyme.
4. A system as claimed in claim 1, wherein the enzyme is a DNA ligase, a DNA polymerase, a DNA kinase, and RNA ligase, an RNA polymerase or an RNA kinase.
5. A system as claimed in any one of claims 1 to 4, wherein the enzyme is covalently bonded to the support.
6. A system as claimed in claim 5, wherein the support comprises a plastics material, a polycarbonate, silica, glass fibre or glass wool.
7. A system as claimed in any one of claims 1 to 4, wherein the enzyme is entrapped.
8. A system as claimed in claim 7, wherein the enzyme is entrapped in a rigid film.
9. A method of producing the enzymatic reagent system of claim 5, comprising providing a support having free aldehyde groups and reacting said aldehyde groups with amino groups of the enzyme.
10. A method as claimed in claim 9, wherein the support having, free aldehyde groups is obtained by reacting a support material having free amino groups on its surface with a di- or higher functionality aldehyde.
11. A method as claimed in claim 10, wherein the aldehyde is glutaldehyde.
12. A method as claimed in claim 10 or claim 11, wherein the support material having free amino groups is obtained by reacting a support material having free hydroxyl groups with an aminoalkyl trialkoxy silane.
13. A method as claimed in claim 12, wherein the hydroxyl groups are bonded to silicon atoms.
14. A method as claimed in claim 12 or claim 13, wherein the silane is aminopropyltriethoxysilane.
15. A method of producing the enzymatic reagent system of claim 5, comprising proving a support having epoxy groups, and reacting said epoxy groups with amino groups of the enzyme.
16. A method as claimed in claim 15, wherein the support is obtained by reacting a support material having hydroxy groups with a glycidoxyalkyl trialkoxy- silane.
17. A method as claimed in claim 16, wherein the glycidoxyalkyl trialkoxysilane is 3-glycidoxypropyl trimethoxy silane.
18. A method as claimed in claim 15, wherein the support comprises acrylic beads.
19. A method of producing the enzymatic reagent system of claim 5, comprising providing a support having free amino groups and using carbodiimide coupling to form amide linkages derived from said amino groups and carboxylic acid groups of the enzymes.
20. A method as claimed in claim 19, wherein the carbodiimide is l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.
21. A method of producing the enzymatic reagent system of claim 5, comprising providing a support having free amino groups, reacting said amino groups with a spacer compound having one aldehyde group and an amino group separated by at least one carbon atom to form an imine linkage, reducing said imine linkages to a secondary amino linkage, and reacting a carboxylic acid group of the enzyme.
22. A method as claimed in claim 21, wherein the amino group and the aldehyde group of the spacer compound are separated by an alkylene chain.
23. A method as claimed in claim 22, wherein the spacer compound is amino butyl aldehyde.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB919104453A GB9104453D0 (en) | 1991-03-02 | 1991-03-02 | Improvements relating to enzymes |
| GB9104453.7 | 1991-03-02 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1992015674A2 true WO1992015674A2 (en) | 1992-09-17 |
| WO1992015674A3 WO1992015674A3 (en) | 1992-12-10 |
Family
ID=10690905
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/GB1992/000370 WO1992015674A2 (en) | 1991-03-02 | 1992-03-02 | Dna -or rna- modifying enzymes immobilized to supports |
Country Status (3)
| Country | Link |
|---|---|
| AU (1) | AU1347392A (en) |
| GB (1) | GB9104453D0 (en) |
| WO (1) | WO1992015674A2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996042017A1 (en) * | 1995-06-08 | 1996-12-27 | Tepnel Medical Limited | Immunological determinations |
| US5625055A (en) * | 1990-09-22 | 1997-04-29 | University Of Strathclyde | Rapid isolation of polynucleotides |
| WO2001046213A3 (en) * | 1999-12-21 | 2002-05-10 | Lion Bioscience Ag | Compound comprising a peptide moiety and an organo-silane moiety. |
| WO2003080862A1 (en) * | 2002-03-25 | 2003-10-02 | Epigenomics Ag | Method and devices for dna methylation analysis |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4342833A (en) * | 1977-05-31 | 1982-08-03 | Bethesda Research Laboratory | Immobilized restriction endonucleases |
| BE889858A (en) * | 1981-08-05 | 1982-02-05 | Mta Kozponti Kemiai Ki | PROCESS FOR THE SYNTHESIS OF POLYDESOXYNUCLEOTIDES |
| JPH01148186A (en) * | 1987-12-03 | 1989-06-09 | Tosoh Corp | Carrier supporting immobilized dna restriction enzyme |
-
1991
- 1991-03-02 GB GB919104453A patent/GB9104453D0/en active Pending
-
1992
- 1992-03-02 AU AU13473/92A patent/AU1347392A/en not_active Abandoned
- 1992-03-02 WO PCT/GB1992/000370 patent/WO1992015674A2/en active Application Filing
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5625055A (en) * | 1990-09-22 | 1997-04-29 | University Of Strathclyde | Rapid isolation of polynucleotides |
| WO1996042017A1 (en) * | 1995-06-08 | 1996-12-27 | Tepnel Medical Limited | Immunological determinations |
| WO2001046213A3 (en) * | 1999-12-21 | 2002-05-10 | Lion Bioscience Ag | Compound comprising a peptide moiety and an organo-silane moiety. |
| WO2003080862A1 (en) * | 2002-03-25 | 2003-10-02 | Epigenomics Ag | Method and devices for dna methylation analysis |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1992015674A3 (en) | 1992-12-10 |
| GB9104453D0 (en) | 1991-04-17 |
| AU1347392A (en) | 1992-10-06 |
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