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WO1992015674A2 - Enzymes modifiant l'adn ou l'arn immobilise sur un support - Google Patents

Enzymes modifiant l'adn ou l'arn immobilise sur un support Download PDF

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Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
enzyme
support
groups
aldehyde
reacting
Prior art date
Application number
PCT/GB1992/000370
Other languages
English (en)
Other versions
WO1992015674A3 (fr
Inventor
Steven Minter
Terence Colley
Original Assignee
Tepnel Medical Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Tepnel Medical Limited filed Critical Tepnel Medical Limited
Publication of WO1992015674A2 publication Critical patent/WO1992015674A2/fr
Publication of WO1992015674A3 publication Critical patent/WO1992015674A3/fr

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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
    • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • 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

On décrit un système réactif enzymatique comprenant une enzyme modifiant l'ADN ou l'ARN qui est immobilisée sur un support tel qu'un support en plastique ou en verre, ainsi que des procédés de production d'un tel système.
PCT/GB1992/000370 1991-03-02 1992-03-02 Enzymes modifiant l'adn ou l'arn immobilise sur un support WO1992015674A2 (fr)

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 (fr) 1992-09-17
WO1992015674A3 WO1992015674A3 (fr) 1992-12-10

Family

ID=10690905

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1992/000370 WO1992015674A2 (fr) 1991-03-02 1992-03-02 Enzymes modifiant l'adn ou l'arn immobilise sur un support

Country Status (3)

Country Link
AU (1) AU1347392A (fr)
GB (1) GB9104453D0 (fr)
WO (1) WO1992015674A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996042017A1 (fr) * 1995-06-08 1996-12-27 Tepnel Medical Limited Determinations immunologiques
US5625055A (en) * 1990-09-22 1997-04-29 University Of Strathclyde Rapid isolation of polynucleotides
WO2001046213A3 (fr) * 1999-12-21 2002-05-10 Lion Bioscience Ag Compose comprenant un groupe fonctionnel peptidique et un groupe fonctionnel silane organique
WO2003080862A1 (fr) * 2002-03-25 2003-10-02 Epigenomics Ag Procede et dispositifs pour analyser la methylation de l'adn

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4342833A (en) * 1977-05-31 1982-08-03 Bethesda Research Laboratory Immobilized restriction endonucleases
BE889858A (fr) * 1981-08-05 1982-02-05 Mta Kozponti Kemiai Ki Procede de synthese de polydesoxynucleotides
JPH01148186A (ja) * 1987-12-03 1989-06-09 Tosoh Corp Ｄｎａ制限酵素固定化担体

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5625055A (en) * 1990-09-22 1997-04-29 University Of Strathclyde Rapid isolation of polynucleotides
WO1996042017A1 (fr) * 1995-06-08 1996-12-27 Tepnel Medical Limited Determinations immunologiques
WO2001046213A3 (fr) * 1999-12-21 2002-05-10 Lion Bioscience Ag Compose comprenant un groupe fonctionnel peptidique et un groupe fonctionnel silane organique
WO2003080862A1 (fr) * 2002-03-25 2003-10-02 Epigenomics Ag Procede et dispositifs pour analyser la methylation de l'adn

Also Published As

Publication number Publication date
WO1992015674A3 (fr) 1992-12-10
GB9104453D0 (en) 1991-04-17
AU1347392A (en) 1992-10-06

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