CA1054079A - Particulate immobilized enzyme adhesively bound to formed body - Google Patents
Particulate immobilized enzyme adhesively bound to formed bodyInfo
- Publication number
- CA1054079A CA1054079A CA231,821A CA231821A CA1054079A CA 1054079 A CA1054079 A CA 1054079A CA 231821 A CA231821 A CA 231821A CA 1054079 A CA1054079 A CA 1054079A
- Authority
- CA
- Canada
- Prior art keywords
- adhesive
- formed body
- enzyme
- immobilised
- particulate
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
- B01J31/0234—Nitrogen-, phosphorus-, arsenic- or antimony-containing compounds
- B01J31/0235—Nitrogen containing compounds
- B01J31/0254—Nitrogen containing compounds on mineral substrates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/003—Catalysts comprising hydrides, coordination complexes or organic compounds containing enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
- B01J31/06—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides containing polymers
-
- 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Enzyme containing bodies are provided useful in re-search, industry and medicine; the bodies comprise formed bodies with an enzymatically-active surface, wherein the surface of a formed body is at least partially coated with adhesive and on the adhesive layer there are present particles which contain at least includes synthetic resin, metal, glass or a synthetic or natural polymer.
Enzyme containing bodies are provided useful in re-search, industry and medicine; the bodies comprise formed bodies with an enzymatically-active surface, wherein the surface of a formed body is at least partially coated with adhesive and on the adhesive layer there are present particles which contain at least includes synthetic resin, metal, glass or a synthetic or natural polymer.
Description
:~.05 4~
The present invention is concerned -with a formed body with an enzymatically-active surface ~Ld with the production thereof.
En~ymes f`ixed to a solid, insoluble carrier, i.e.
so-called i~mobil.ised enzymes, are of increasing importance in many different ~ields of use, such a~ research, industry and medicine. Depending upon the method of immobilisation employed, the immobilised enzymes are divided into the classes of bound, included and cross-linked enzymes. ~ound enzymes can be obtained by covalent bonding to active carriers, heteropolar bonding and/or van der Waal's exchange action on ion exchangers, as well as on adsorbents.
Included enzymes are enzymes which are mechanically immobilised in cross~ ed pol~mers and microcapsules, as well as in regenerated cellulQse derivatives. Finally, enzymes can also be cross-linked with bifunctional low mole-cular weight reagents and thus made insoluble. `~
A disadvantage of the immobilised enzymes is that thecarriers suitable for fixing frequently have inadequate mechanical properties and either cannot be worked up to give formed bodies with enzymatically-active surfaces or can only be so worked up with great difficulty. In principle~
o~ly those carriers based on synthetic resins, biopolymers or inorganic substances c~n be used in which reactive groups -are present or can be produced and which, if possible, con-tain hydrophilic groups or centres. On the other hand, however, for many fields of use for immobilised enzymes, mecha~Lically ~table formed bodies are necessary. Thus, for example, membranes, films, tubes and other formed bodies with enzymatically-active surfaces are much more suitable r~
~5~079 ABSl'RACT OF l~E DISCLOSURE MAY 8 1979 Enzyme containing bodies are provided useful in re-search, industry and medicine, the bodies comprise formed bodies with an enzymatically-active surface, wherein the surface of a formed body is at least partially coated with adhesive and on the adhe~ive layer there are present particles which contain at least one enzyme im~obilised on a solid carrier, suitable bodies include synthetic re~in, metal, glas~ or a synthatic ox natural polymer.
~ .
, , ~, V'`~
The pr~sent invention is concerned with a formed body with ~n enzymatically-active sur~ace and with the production thereof.
Enzymes fi~ed to a solid, insoluble carrier, i.e.
~o-oalled immobilised enzymes, are of increa~ing importance ln many different fields of use, such as research, industry and medicine. Depending upon the method of immobilisation employsd, the immobilised enzymes are divided into the classes of bound, included and cros~-linked enzymes. ~ound enzyme~ can be obtained by covalent bonding to active carriers, heteropolar bonding and/or van der Waal's exchange action on ion exchangers, as well as on adsorbents.
Included enzymes are enzymes which are mechanically immobilised in cross-linked polymers and microcapsules, as well as in regenerated cellulose derivat:ives. ~inally, enzymes oan also be cross~linked with bifunotional low mole-oular weight reagents and thus made insoluble.
A disadvantage of the immobilised enzymes is that the carriers suitable for fixing frequently have inadequate mech~nical properties and either cannot be worked up to ;~
give formed bodies with enzymatically-active surfaces or can only be so worked up with great difficulty. In p~inciple only those carriers based on synthetic resins, biopolymers or inorganic substances cQn be used in which reactive groups are present or can be produced and which, if possible, con-tain hydrophilic groups or centres. On the other hand, however, for many fields of use for i~mobilised enzymes, mechanically-~stable formed bodies are necessary. ~hus, for example, membranes, films, tubes and other formed bodies with enzymatically-active surfaces are ~uch more suitable ,;, ~ ' 40 r~ ~3 tubes, pipes, rods, foils, ~iller bodies or in any other desired for~ for which an enzymatically~active surface is desired, ~or example~ in the case of reaction vessels.
'rhe surEace of -the formed body according -to -the pre-sent invention is wholly or partially coated with an ad-hesive layer. IJsually, the adhesive layer is presen-t on those parts of the surface on which an enzymatic activit~
is desired. Any known and conventional -type o~ adhesive can be used ~hese include, for example~ water-insoluble vegetable adhe~ ves, such as adhesives made from rubber, natural rssins and cellulose derivatives which are soluble in organic solvents, as well as synthetic adhesives 7 such as cellulose esters, butadiene-styrene and butadiene-acrylonitrile co-polymers, pol~chlorobutadiene, phenoplasts~
such as phenol-~ormaldehyde condensation products and resorcinol-formaldehyd2 condensation products, aminoplasts 9 such as urea-formaldehyde and mela~i~e-~ormaldeb~ co-nden-sation products~ polyes-ters, polyisocyanates, epoxy resins, silicones, polyvinyl adhes~ves, for example, polyvinyl .~ . ,.
chloride, pol~isobutylene, po]yst~rene, polgvinyl alcoholS -polyvinyl acetate, polyvinyl ethers and polyacrylic and polymethacrglic acid esters, as well as mixtures thereof.
On the adhesive layer, there are present particles -which consist of a solid carrier on which are fixed the desired enzyme or enzymes, According to the present inven-tion, these include not only natural carriers, i.e~ micro~
organisms, cells and cell fragments, which carr~ or co~tain the enzyme, but also conventional enz~mes immobilised on solid carriers and especially enz~mes covalently fixed on to solid carriers. Immo~ilised enz~mes in which the carrier~
consists preponderantly of acrylamide ~nits are preferred, ~: 4 , .
': ' : , ,: , ~- , ;. . . .
,~
. - . : ,:. ~: :
,: . ' , -' ' - . ' ~
- , - .: - , . .. .
, . . . . .
- ~ i 1~5~
and the yields obtained are ex-tremely lot,r. ~'hese known processes also scarcely permit the simultaneous fixing of several enzymes. ~ince an acti~ated carrier must always be present, there is -the further feature that charged sub3tratcs or reactants are adsorbed, -the pH optimum of the enzyme is displaced and the Michealis constants are changed and, in the case of cross~ ked enzymes, high losses of a~tivity due to irreversible denaturing must be taken into account. Furthermore 9 in some cases, fixlng only takes place by adsorption so that the activity rapidly 'bleeds a~Jay".
It is, therefore, an object of the present invention to overcome these disadvantages and to provide a formed body with an enæymatically-active surface which can be used as widely as possible in analytical and preparative chemi~try and, on the other hand, can be produced in a simple manner. -~
Thus, according to the present invention, there is provided a ~ormed body with an enzymatically-active sur~ace, wherein the surface of the formed body is at least partially coated with an adhesive and on the adhesive layer there are present particles which contain at least one enzyrne immobilised on a solid carrier.
'~he formed bodies according to the present invention can consist of any desired organic or inorganic material ~ ; `;
and can be of any desired shape. Thus, the formed bodies --can be made of inorganic materials, such as glass, metal, corundum or the like, or of organic materials, such as ` ;~
, : .
natural or synthetic polymers, synthetic resins and the ~`~
.
like. They can be in the forrn of, for example~ spheroids7 :: '.
'j 3 ~ ~
~ ',': ~` .:
. : . : :
~, . , , , , . . ~ ..
S ~
tubes, pip~s, rods5 foils, filler bodies or in any other desired ~orm for wllich an enzyma-ticall~active surface is desired, for example, in the case of reaction vessels.
~rhe surface of the formed body according to -the pre-sent invention is wholl~ or partially coated with an ad-hesive la~er. IJsually~ the adhesive layer is present on ?
those parts of the surface on which an enzymatic activitg is desired. Any known and conventional -type of adhesive can be used. ~hese lnclude, for example~ wa-ter-insoluble vegetable adhesives, such as adhesives made from rubber, natural reSin~ and cellulose derivatives which are soluble in organic solvents, as well as sgnthetic adhesives, such as cellulose esters, butadiene-stgrene and butadiene-acrylonitrile co-pol~mers, pol~chlorobutadiene, phenoplasts~
such as phenol-formaldehyde condensation products and resorcinol-formaldehyde condensation products, aminoplasts, such as urea-formaldehyde and mela~ine-formaldeh~ conden-sation products~ polyesters, pol~isocganates, epoxy resins, silicones, polgvinyl adhesiYes, for example, pol~vinyl ~-chloride, polgisobutglene, pol~styrene, polyvingl alcohol, pol~vin~l acetate, polyvinyl ethers and pol~acr~lic and .
polgmethacr~lic acid esters, as well as mixtures thereof.
On the adhesive layer, there are present particles ~ -~
..
which consist o~ a solid carrier on which are fixed the desired enzyme or enzymes, According to the present inven~
tion9 these include not onlg natural carriers, i.e. micro- ;
organisms, cellæ and cell fragments, which carr~ or contain -the en~yme, but also convantional enz~mes immobilised on solid carriers and especiallg enzgmes cova~entlg fixed on to solid carriers. Immo~ilised enz~mes in which the carrier~
consists preponderantl~ of acrglamide ~nits are preferred, ,~
.- . . .... , .. - . , .... - - ... . - " .. ..
. - - : . . .
.. , . , " , ., ., .~: : , , :. - . , :,. . .
r~j ~
be used formed bodies, the surfaces of which can be con-verted n situ, by a chemical or physical treatment, into an adhesiveO This i~ possible, for example, in the case of cer-tain synthetic resins, such a~ polyvinyl chloride (PVC) by treatment with an appropriate organic solvent and/or pla~ticiser or modification a~ent.
~'he particles of the carrier-bound immobilised e"~f7/~yed enzyme ~k~e~ preferably have a size of from about 0.0001 to about 1 mm.~ the range of from 0.001 to 0.1 mm. being especially preferred. The particle size ~hich is best suited for a particular purpose depends upon the fixed enzyme, the carrier in question and ~lso upon the adhesive and especially upon the organic solvent used in the adhesive. In the case of especially sensitive enzymes, use is preferably made of a particle size in the upper half of the above-stated range of particle sizes. ~he more stable is the immobilised enzyme, the smaller can be ;
the particle size.
An important advantage of the present invention is ~hat enzymatically active formed bodies can be obtained, the physical properties of which are practically independent of the physical properties of the "primary carrier", i.e.
of the particulate material employed for the immobilisation of the enzyme. The actual formed body does nob need to be converted into an active form ~nd, in principle, carrier~
bound enzymes produced by all known methods of immobilis-ation can ~e employed for the enzymatic activation of the surfaces of the formed bodyO Apart from co-valently bound enzymes and preferably enzymes co-valently bound by protein co-polymerisation, there can ad~antageously also be employed, ~ ~ .
``~ ' .
10540 ~9 according to the present invention, enzyme~ immobilised by inclusion.
The formed bodies according to the present invention can be used, as already mentioned, for a large number of different purposes. For example, they can be used in the form of tubes, membranes and of other types of formed bodies in apparatus used for enzymatic analysis. ~hus, for example, it is possible, by the use of tubular material with enzymatically-active inner qurEaces, to carry out enzymatic analyses with 10 the use of conventional photometers which operate with flow-through cuvettes~ Another possibility of use is in automatic analysis devices which utilise dialysis membranes, us~ thereby being made of enzymatically-activated membranes covered by .
dialysis membranes. For simple detection reactions, enzymati- ;~
cally-active test tubes and object carriers can be used. In the case of preparative chemistry, formed bodies according to ~ ;
the present invention are especially useful which are in the form of reaction vessels, tubes, pipes and column fillings.
The invention is further described with reference 20 to the accompanying drawings in which:
Figure 1 illustrates graphically the enzyme ~ .
activity of a formed body of the invention according to Example 1, Figure 2 illustrates schematically an arrangement for analysis of the enzyme activity of a formed body of the invention according to Example 1, Figure 3 is a cross~section of one embodiment of a formed body of the invention, and : :.
Figure 4 is a cross-section of another formed body of the invention.
- 8 - :
~ ~ .
1 ()5~L~)r~
The following Examples are given for the purpose of illustrating the present invention:-Example 1 .
Enzy~el qlucose oxidase/catalase on tubesStarting materials:
tube made of polyvinyl acetate-polyethylene co-polymer, internal diameter 1.5 mm.
5 ml. adhesive based on synthetic rubber (Uhu- :
Kontakt 2000 ) 10 ml. methylene chloride 100 mg. of a glucose oxidase (GOD)-ca~alase tcab) '~ ''.
'~ ' ' . ~
., :
.
' '' ;,, trademark ~ ~
''~ `~ ' ' ' ` ~
;:
~40~g :~
mixed enzyme covalently bound to cross-linked polyacrylamide carrier ~Enzygel GOD/Cat), finely powdered ~ethod: , The adhesive ~olution, diluted with methylene chloride, is introduced with a syringe into the vertically suspended tube (length ~ 70 cm.). As soon as the solution hai~ run -through completely (after 1 - 2 minutes), the lower end of a spheroidially-shaped pipette tip, open at ;
both ends, is introduced into the tube. ~he pipette ,~ ~ . . . :;
synthetic resin sphere contains the enzyme powder. On the other part of the sphere~ with an opening of about 8 -12 mm., there is attached a pressure tube. Subsequently, `~
the enzyme powder is blown with about 1 - 3 ats. compressed air or nitrogen into the adhesive-coated tube. Exoess enzyme powder can be collected again at the lower end o~
the tube and used again. -The tube coated with the enzyme powder is, after ' ~ ;~
drying (about 1 hour), rinsed with a buffer solution until , . . .
no more enzyme particles come away. The ~inished enzyme ~
tube lS then used for the automatic analysis of glucose. ~ ;
A length of tube o~ about 48 cm~ shows 9 in the case of a si~mple ~requency of 24/hour and a ratio of sample to wash of 1:4, the activity indicated in Fig.1 of the accompanying drawing3. The sample frequency can be increased to about 40/hour.
~ he analysis can be carried out with the i~rrangement schematically illustrated in Fig.2 of the accompanying drawings~
The details of the apparatus iand measurement device ; ~ -are as follows~
lOS~O~
Photometer: Eppendor~ 1101 M
Flowthrough cuvette: ~ellma No. 178, lcm. layer depth Pump: Ismatic MP 13 G ~-10 :
Connector (air inlet): DO, H, Technicon No. 116-0203-00 Coil: 14 windings, Technicon , inner diameter 2 mm~
Air separator: C-5, Technicon No. 116-0202-05 Tubes: 1. air, inner diameter 1 mm.
The present invention is concerned -with a formed body with an enzymatically-active surface ~Ld with the production thereof.
En~ymes f`ixed to a solid, insoluble carrier, i.e.
so-called i~mobil.ised enzymes, are of increasing importance in many different ~ields of use, such a~ research, industry and medicine. Depending upon the method of immobilisation employed, the immobilised enzymes are divided into the classes of bound, included and cross-linked enzymes. ~ound enzymes can be obtained by covalent bonding to active carriers, heteropolar bonding and/or van der Waal's exchange action on ion exchangers, as well as on adsorbents.
Included enzymes are enzymes which are mechanically immobilised in cross~ ed pol~mers and microcapsules, as well as in regenerated cellulQse derivatives. Finally, enzymes can also be cross-linked with bifunctional low mole-cular weight reagents and thus made insoluble. `~
A disadvantage of the immobilised enzymes is that thecarriers suitable for fixing frequently have inadequate mechanical properties and either cannot be worked up to give formed bodies with enzymatically-active surfaces or can only be so worked up with great difficulty. In principle~
o~ly those carriers based on synthetic resins, biopolymers or inorganic substances c~n be used in which reactive groups -are present or can be produced and which, if possible, con-tain hydrophilic groups or centres. On the other hand, however, for many fields of use for immobilised enzymes, mecha~Lically ~table formed bodies are necessary. Thus, for example, membranes, films, tubes and other formed bodies with enzymatically-active surfaces are much more suitable r~
~5~079 ABSl'RACT OF l~E DISCLOSURE MAY 8 1979 Enzyme containing bodies are provided useful in re-search, industry and medicine, the bodies comprise formed bodies with an enzymatically-active surface, wherein the surface of a formed body is at least partially coated with adhesive and on the adhe~ive layer there are present particles which contain at least one enzyme im~obilised on a solid carrier, suitable bodies include synthetic re~in, metal, glas~ or a synthatic ox natural polymer.
~ .
, , ~, V'`~
The pr~sent invention is concerned with a formed body with ~n enzymatically-active sur~ace and with the production thereof.
Enzymes fi~ed to a solid, insoluble carrier, i.e.
~o-oalled immobilised enzymes, are of increa~ing importance ln many different fields of use, such as research, industry and medicine. Depending upon the method of immobilisation employsd, the immobilised enzymes are divided into the classes of bound, included and cros~-linked enzymes. ~ound enzyme~ can be obtained by covalent bonding to active carriers, heteropolar bonding and/or van der Waal's exchange action on ion exchangers, as well as on adsorbents.
Included enzymes are enzymes which are mechanically immobilised in cross-linked polymers and microcapsules, as well as in regenerated cellulose derivat:ives. ~inally, enzymes oan also be cross~linked with bifunotional low mole-oular weight reagents and thus made insoluble.
A disadvantage of the immobilised enzymes is that the carriers suitable for fixing frequently have inadequate mech~nical properties and either cannot be worked up to ;~
give formed bodies with enzymatically-active surfaces or can only be so worked up with great difficulty. In p~inciple only those carriers based on synthetic resins, biopolymers or inorganic substances cQn be used in which reactive groups are present or can be produced and which, if possible, con-tain hydrophilic groups or centres. On the other hand, however, for many fields of use for i~mobilised enzymes, mechanically-~stable formed bodies are necessary. ~hus, for example, membranes, films, tubes and other formed bodies with enzymatically-active surfaces are ~uch more suitable ,;, ~ ' 40 r~ ~3 tubes, pipes, rods, foils, ~iller bodies or in any other desired for~ for which an enzymatically~active surface is desired, ~or example~ in the case of reaction vessels.
'rhe surEace of -the formed body according -to -the pre-sent invention is wholly or partially coated with an ad-hesive layer. IJsually, the adhesive layer is presen-t on those parts of the surface on which an enzymatic activit~
is desired. Any known and conventional -type o~ adhesive can be used ~hese include, for example~ water-insoluble vegetable adhe~ ves, such as adhesives made from rubber, natural rssins and cellulose derivatives which are soluble in organic solvents, as well as synthetic adhesives 7 such as cellulose esters, butadiene-styrene and butadiene-acrylonitrile co-polymers, pol~chlorobutadiene, phenoplasts~
such as phenol-~ormaldehyde condensation products and resorcinol-formaldehyd2 condensation products, aminoplasts 9 such as urea-formaldehyde and mela~i~e-~ormaldeb~ co-nden-sation products~ polyes-ters, polyisocyanates, epoxy resins, silicones, polyvinyl adhes~ves, for example, polyvinyl .~ . ,.
chloride, pol~isobutylene, po]yst~rene, polgvinyl alcoholS -polyvinyl acetate, polyvinyl ethers and polyacrylic and polymethacrglic acid esters, as well as mixtures thereof.
On the adhesive layer, there are present particles -which consist of a solid carrier on which are fixed the desired enzyme or enzymes, According to the present inven-tion, these include not only natural carriers, i.e~ micro~
organisms, cells and cell fragments, which carr~ or co~tain the enzyme, but also conventional enz~mes immobilised on solid carriers and especially enz~mes covalently fixed on to solid carriers. Immo~ilised enz~mes in which the carrier~
consists preponderantly of acrylamide ~nits are preferred, ~: 4 , .
': ' : , ,: , ~- , ;. . . .
,~
. - . : ,:. ~: :
,: . ' , -' ' - . ' ~
- , - .: - , . .. .
, . . . . .
- ~ i 1~5~
and the yields obtained are ex-tremely lot,r. ~'hese known processes also scarcely permit the simultaneous fixing of several enzymes. ~ince an acti~ated carrier must always be present, there is -the further feature that charged sub3tratcs or reactants are adsorbed, -the pH optimum of the enzyme is displaced and the Michealis constants are changed and, in the case of cross~ ked enzymes, high losses of a~tivity due to irreversible denaturing must be taken into account. Furthermore 9 in some cases, fixlng only takes place by adsorption so that the activity rapidly 'bleeds a~Jay".
It is, therefore, an object of the present invention to overcome these disadvantages and to provide a formed body with an enæymatically-active surface which can be used as widely as possible in analytical and preparative chemi~try and, on the other hand, can be produced in a simple manner. -~
Thus, according to the present invention, there is provided a ~ormed body with an enzymatically-active sur~ace, wherein the surface of the formed body is at least partially coated with an adhesive and on the adhesive layer there are present particles which contain at least one enzyrne immobilised on a solid carrier.
'~he formed bodies according to the present invention can consist of any desired organic or inorganic material ~ ; `;
and can be of any desired shape. Thus, the formed bodies --can be made of inorganic materials, such as glass, metal, corundum or the like, or of organic materials, such as ` ;~
, : .
natural or synthetic polymers, synthetic resins and the ~`~
.
like. They can be in the forrn of, for example~ spheroids7 :: '.
'j 3 ~ ~
~ ',': ~` .:
. : . : :
~, . , , , , . . ~ ..
S ~
tubes, pip~s, rods5 foils, filler bodies or in any other desired ~orm for wllich an enzyma-ticall~active surface is desired, for example, in the case of reaction vessels.
~rhe surface of the formed body according to -the pre-sent invention is wholl~ or partially coated with an ad-hesive la~er. IJsually~ the adhesive layer is present on ?
those parts of the surface on which an enzymatic activitg is desired. Any known and conventional -type of adhesive can be used. ~hese lnclude, for example~ wa-ter-insoluble vegetable adhesives, such as adhesives made from rubber, natural reSin~ and cellulose derivatives which are soluble in organic solvents, as well as sgnthetic adhesives, such as cellulose esters, butadiene-stgrene and butadiene-acrylonitrile co-pol~mers, pol~chlorobutadiene, phenoplasts~
such as phenol-formaldehyde condensation products and resorcinol-formaldehyde condensation products, aminoplasts, such as urea-formaldehyde and mela~ine-formaldeh~ conden-sation products~ polyesters, pol~isocganates, epoxy resins, silicones, polgvinyl adhesiYes, for example, pol~vinyl ~-chloride, polgisobutglene, pol~styrene, polyvingl alcohol, pol~vin~l acetate, polyvinyl ethers and pol~acr~lic and .
polgmethacr~lic acid esters, as well as mixtures thereof.
On the adhesive layer, there are present particles ~ -~
..
which consist o~ a solid carrier on which are fixed the desired enzyme or enzymes, According to the present inven~
tion9 these include not onlg natural carriers, i.e. micro- ;
organisms, cellæ and cell fragments, which carr~ or contain -the en~yme, but also convantional enz~mes immobilised on solid carriers and especiallg enzgmes cova~entlg fixed on to solid carriers. Immo~ilised enz~mes in which the carrier~
consists preponderantl~ of acrglamide ~nits are preferred, ,~
.- . . .... , .. - . , .... - - ... . - " .. ..
. - - : . . .
.. , . , " , ., ., .~: : , , :. - . , :,. . .
r~j ~
be used formed bodies, the surfaces of which can be con-verted n situ, by a chemical or physical treatment, into an adhesiveO This i~ possible, for example, in the case of cer-tain synthetic resins, such a~ polyvinyl chloride (PVC) by treatment with an appropriate organic solvent and/or pla~ticiser or modification a~ent.
~'he particles of the carrier-bound immobilised e"~f7/~yed enzyme ~k~e~ preferably have a size of from about 0.0001 to about 1 mm.~ the range of from 0.001 to 0.1 mm. being especially preferred. The particle size ~hich is best suited for a particular purpose depends upon the fixed enzyme, the carrier in question and ~lso upon the adhesive and especially upon the organic solvent used in the adhesive. In the case of especially sensitive enzymes, use is preferably made of a particle size in the upper half of the above-stated range of particle sizes. ~he more stable is the immobilised enzyme, the smaller can be ;
the particle size.
An important advantage of the present invention is ~hat enzymatically active formed bodies can be obtained, the physical properties of which are practically independent of the physical properties of the "primary carrier", i.e.
of the particulate material employed for the immobilisation of the enzyme. The actual formed body does nob need to be converted into an active form ~nd, in principle, carrier~
bound enzymes produced by all known methods of immobilis-ation can ~e employed for the enzymatic activation of the surfaces of the formed bodyO Apart from co-valently bound enzymes and preferably enzymes co-valently bound by protein co-polymerisation, there can ad~antageously also be employed, ~ ~ .
``~ ' .
10540 ~9 according to the present invention, enzyme~ immobilised by inclusion.
The formed bodies according to the present invention can be used, as already mentioned, for a large number of different purposes. For example, they can be used in the form of tubes, membranes and of other types of formed bodies in apparatus used for enzymatic analysis. ~hus, for example, it is possible, by the use of tubular material with enzymatically-active inner qurEaces, to carry out enzymatic analyses with 10 the use of conventional photometers which operate with flow-through cuvettes~ Another possibility of use is in automatic analysis devices which utilise dialysis membranes, us~ thereby being made of enzymatically-activated membranes covered by .
dialysis membranes. For simple detection reactions, enzymati- ;~
cally-active test tubes and object carriers can be used. In the case of preparative chemistry, formed bodies according to ~ ;
the present invention are especially useful which are in the form of reaction vessels, tubes, pipes and column fillings.
The invention is further described with reference 20 to the accompanying drawings in which:
Figure 1 illustrates graphically the enzyme ~ .
activity of a formed body of the invention according to Example 1, Figure 2 illustrates schematically an arrangement for analysis of the enzyme activity of a formed body of the invention according to Example 1, Figure 3 is a cross~section of one embodiment of a formed body of the invention, and : :.
Figure 4 is a cross-section of another formed body of the invention.
- 8 - :
~ ~ .
1 ()5~L~)r~
The following Examples are given for the purpose of illustrating the present invention:-Example 1 .
Enzy~el qlucose oxidase/catalase on tubesStarting materials:
tube made of polyvinyl acetate-polyethylene co-polymer, internal diameter 1.5 mm.
5 ml. adhesive based on synthetic rubber (Uhu- :
Kontakt 2000 ) 10 ml. methylene chloride 100 mg. of a glucose oxidase (GOD)-ca~alase tcab) '~ ''.
'~ ' ' . ~
., :
.
' '' ;,, trademark ~ ~
''~ `~ ' ' ' ` ~
;:
~40~g :~
mixed enzyme covalently bound to cross-linked polyacrylamide carrier ~Enzygel GOD/Cat), finely powdered ~ethod: , The adhesive ~olution, diluted with methylene chloride, is introduced with a syringe into the vertically suspended tube (length ~ 70 cm.). As soon as the solution hai~ run -through completely (after 1 - 2 minutes), the lower end of a spheroidially-shaped pipette tip, open at ;
both ends, is introduced into the tube. ~he pipette ,~ ~ . . . :;
synthetic resin sphere contains the enzyme powder. On the other part of the sphere~ with an opening of about 8 -12 mm., there is attached a pressure tube. Subsequently, `~
the enzyme powder is blown with about 1 - 3 ats. compressed air or nitrogen into the adhesive-coated tube. Exoess enzyme powder can be collected again at the lower end o~
the tube and used again. -The tube coated with the enzyme powder is, after ' ~ ;~
drying (about 1 hour), rinsed with a buffer solution until , . . .
no more enzyme particles come away. The ~inished enzyme ~
tube lS then used for the automatic analysis of glucose. ~ ;
A length of tube o~ about 48 cm~ shows 9 in the case of a si~mple ~requency of 24/hour and a ratio of sample to wash of 1:4, the activity indicated in Fig.1 of the accompanying drawing3. The sample frequency can be increased to about 40/hour.
~ he analysis can be carried out with the i~rrangement schematically illustrated in Fig.2 of the accompanying drawings~
The details of the apparatus iand measurement device ; ~ -are as follows~
lOS~O~
Photometer: Eppendor~ 1101 M
Flowthrough cuvette: ~ellma No. 178, lcm. layer depth Pump: Ismatic MP 13 G ~-10 :
Connector (air inlet): DO, H, Technicon No. 116-0203-00 Coil: 14 windings, Technicon , inner diameter 2 mm~
Air separator: C-5, Technicon No. 116-0202-05 Tubes: 1. air, inner diameter 1 mm.
2. sample, inner diameter 2.5 mm.
3. reagent, inner diameter 2.5 mm. ~ ~
4. enzyme tube, inner diameter 1.5 mm., length 48 cm. - -Sample solution: glucose 0.25 mg./lOO ml. - 200 mg./100 ml.
in 0.LM phosphate buffer (pH 7.0) Reagent: 1.1 g. 2,2'-azino-di-(3-ethyl-benzthiazolin)-6-sulphonate (ABTS) ~ 0.6 ml. peroxidase (Boehringer, POD-l) in 1000 ml. 0.1 M phosphate buffer pH 7.0 Measurement radiation: 405 nm, temperature 25C.
Analogously as in the case of the Technicon Auto-Analyzer , not only~ the sample but also the reagent solution ~ ;
are segmented in the tube by air bubbles and the substrate concentration measured co~inuously as well as discontinuously by comparision with a standard solution. ;
As Fig. 2 of the accompanying drawings shows, the -sample solution (2) is segmented, before entry into the enzyme reactor, with air bubbles (1) and subsequently reacted in the enzyme tube (or by an enzyme foil present in a bifilar milled spiral). By the addition of reagent solution (3), a colour reaction takes place (or some other indicator reaction) which, after emergence of the air bubbles (immediately before .. . .
the flowthrough cuvette) can be measured photometrically. ~ -- li - . .
*trademark : " ',: ' '~ '": "
The readings registered with a recorder are directly proportional to the substrate concentration~
Example 2 Glucose oxidase/catalase on foil ', Starting materials: ' , polyamide-polyester random fibre fleece tViledon ' fleece material, H 3003)
in 0.LM phosphate buffer (pH 7.0) Reagent: 1.1 g. 2,2'-azino-di-(3-ethyl-benzthiazolin)-6-sulphonate (ABTS) ~ 0.6 ml. peroxidase (Boehringer, POD-l) in 1000 ml. 0.1 M phosphate buffer pH 7.0 Measurement radiation: 405 nm, temperature 25C.
Analogously as in the case of the Technicon Auto-Analyzer , not only~ the sample but also the reagent solution ~ ;
are segmented in the tube by air bubbles and the substrate concentration measured co~inuously as well as discontinuously by comparision with a standard solution. ;
As Fig. 2 of the accompanying drawings shows, the -sample solution (2) is segmented, before entry into the enzyme reactor, with air bubbles (1) and subsequently reacted in the enzyme tube (or by an enzyme foil present in a bifilar milled spiral). By the addition of reagent solution (3), a colour reaction takes place (or some other indicator reaction) which, after emergence of the air bubbles (immediately before .. . .
the flowthrough cuvette) can be measured photometrically. ~ -- li - . .
*trademark : " ',: ' '~ '": "
The readings registered with a recorder are directly proportional to the substrate concentration~
Example 2 Glucose oxidase/catalase on foil ', Starting materials: ' , polyamide-polyester random fibre fleece tViledon ' fleece material, H 3003)
5 mlO adhesive as in Example 1 7 ml. methylene chloride 500 mg. of the same enzyme particle preparation as in Example 1 Method:
Tpe adhesive is diluted with methylene chloride and thinly applied to the foil. After some time, the finely-divided enzyme powder is applied to the still moist and sticky foil, with the use of a sieve. After slow drying, the foil is slowly stirred in a O.lM phosphate buffer solution (pH 7.0) and, in this way, non-bound particles are removed. The foil is subsequently transferred into a ,!
bifilar milled spiral and employed'for substrate dete~mination in a similar way to the enzyme tube.
1 cm of the foil produced by the above-described ~ -process weighs, in a dry,state, 23.3 mg. and has an enzymatic activity of 16.1 U/g., corresponding to 0.375 U/cm .
Coatinq of a glass tip Starting materials:
1 ml. adhesive as in Example 1 1.5 ml. methylene chloride 50 mg. enzyme powder as in Example 1.
*trademark f"
. ~ ' ' ~ .
. ,: . . , ~ ~ , . . . ~ , ~. . , ~ .
.: : : . . . ~ . :
Metho~:
The ylass tip is dipped once or twice into the diluted adhesive solution and, after a few seconds, dipped into the enzyme powder. Af-ter slow drying, it is trans-ferred to a 0.1M buffer solution (pH 7.0) and tested after about 12 hours. In the case of a tip of 1 - 2 mm. diameter and with a length of 2 - 3 mm., there is obtained an average activity of 0.5 U/tip.
Example 4 A round polystyrenerod of 2.4 mm. diameter is coated -with an adhesive which was obtained from 6 g. of a PVC
adhesive (Tangit of the firm Henkel) and 2 ml. tetrahydro-furan. The rod is then dipped into a finely powdered enzyme preparation which consists of GOD (230 U/g.) and Cat (3600 U/g.~ fixed via acryloyl chloride by the process of enzyme co-polymerisation. The rod is then allowed to dry and the activity determined. It is found to be 0.49 U GOD/mm surface area. ;
Example 5 The process of Example 4 is repeated with the use of an alkaline phosphatase with an activlty of 200 U/g. ~ ;
bound to the same carrier. The polystyrene rod is, aft0r dipping into the adhesive,left to dry for 15 to 30 seconds in the air, whereafter the enzyme dust is sprinkled on, the adhesive is left to dry and the rod is washed with a buffer. The activity is found to be 0.23 U/mm surface area. --Example 6 The process of Example 5 is repeated w1th the use of an enzyme powder which contains 5 U/g. trypsin on a maleic ; *trademark ~ ` ~
- .: , .
~054~) ~9 anhydride-acrylamide carrier (according to German Patent Specification No. 1,908,290). The activity of the formed body is subsequently found to be 0.003 U/mm surface area.
Example 7 Exàmple 5 is repeated with the use of a two-component epoxy resin adhesive (Uhuplus ) and the GOD/Cat powder of Example 4. The measured activity of the formed body is found to be 0.5 U/mm2 surface area.
Example 8 The process of Example 5 is repeated with the use of a contact adhesive based on polychlorobutadiene, with the addition of resins and organic solvents (Pattex of the firm Henkel), as well as of the enzyme powder of Example 4. The measured activity of the formed body is found to be 0.45 U/mm2.
Example 9 The process of Example 8 is repeated with the use of the alkaline phosphatase enzyme powder according to Example 5. The measured activity of the formed body is found to be 0.021 U~mm .
ExamPle 10 Example 5 is repeated with the use of a commercially available adhesive based on polyvinyl resin with acetone ; and ethyl acetate as solvents (Uhu Alleskleber ) and of the enzyme powder of Example 4. The enzymatic activity measured on the formed body is 0.5 U/mm2.
Example 11 .
Example 10 is repeated with the use of the enzyme powder of Example 6. The measured activity is found to be ; 0.0026 U/mm . ' ;
.
*trademark :. ~ .'. ' .
~35~ ri~
Example 12 A round rod of PVC o~ 3 mm. diameter is superficially ;`
dissolved with cyclohexane until the surf`ace thereof becomes sticky. The enzyme powder of Example 4 is then ~prinkled on, the rod is left to dry and thereafter it is washed with a buffer solution. '~he enzymatic activity of the formed body is 0.38 U/mm2.
xample 1~.
Ex~mple 12 is repeated with the use of the alkaline phosphatase enzyme po~der according to Example 5. ~he ~-enzymatic acti~ity of the formed body is 0.117 U/mm2.
xample 14. `
The process of ~xample 12 is repeated with the use ~ ;
of the trypsin-containing enzyme powder of ~xample 6. ~The enzymatic activity of the formed body is 0.0042 U/mm2.
E_am~le 15.
A glass rod of 2.4 mm. diameter is dipped into the ;~
adhesive of Example 8, thereafter allowed to dry in the -air for 15 to 30 seconds and sprinkled with the enzyme p~wder of Example 4. After drying the rod9 it is washed with buffer. The enzymatic activity of the finished rod `
is 0.66 U/mm .
a ~ . ` ;~
A steel wire of 1 mm. diameter is dipped into the adhesive of ~xample 8, left to dry in the air for 15 to 30 seconds and then sprinkled with the enzyme powder of Example 4. The enzymatic activity of the finished formed body is found to be 0.80 U/mm2.
Example 17.
The process of Egample 16 is repeated, with the use ~ ~ .
~ -15-- j, . .. , -. ,. . . - : - : ~
.. .. , ... ~
... . . . - . . . . . .. -. ... ... .:,. :: .. .-. . .. : . . .
~ ~9 of the all~aline pho~ hatase enz~me powder accorcling to Exam~le 5. r~le enzymatic activit~ of the formed bod~ is found to be 0.003 U/mm2.
~æample 18~
Example 4 i9 repeat~d but, ins-tead of the poly-styrene rocl, there is used a '~eflon-coated magnetic s-tirrer rod with the dimensions 6.5 x 10.9 m~, ~he enz~matic a~-tivitg of the formea body is found to be 0.15 U/m~n2.
~ .
Example 5 is repeated but, instead of -the polg-st~rene rod, there was used a ~eflon-coated magnetic stirrer rod as described in Example 18. '~he enzymatic activitg of the formed body is 0.135 U/mm2, E ~
~ wo foils were coated with adhesive J 6610-21 (50~ polyacrylic ester) of the firm Henkel. The la~er thickness was 25 ~ and 50~, respectivel~ ~
cells were applied to 50 cm2 pieces of the foils.
~he foils were vigorously s-tirred overnight in a phosphate buffer (pX 7.0) in order -to remove non-~ixed cells. The amount of ~ niger stuck on was determined by ~ -weighing the dried foils. n the 25~ foill there was ascertained 0,18 mg, ~ ~ cells per cm and on the 50 ~ foil, 0;20 mg. ~ Q ~ ce~ls per cm .
~he activit~ o~ the micro-organisms on the ~oils was 0~027 U/cm2 in the case of the 25~ foil and 0~0~2 U/cm2 in the case of the 50~4 foil, ~he la~t runnings amounted to 15~, Since the ~ ~ ells used had an activity of 200 U/g., referred to GODI the calculated activit~ ~ield was 75~ in the case of the 25~ ~oil and 80~G in the case o~ the 50~ foll.
-16~
- .
.. : , :
i~540 ï9 ; ', ':
Nocardia ~ micro-organism cells were stuck to the same foils as were used in 3xample 20~ The Nocardia ~ cells used had a chole,sterol oxidase activitg of 16lJ/gD '~he 25~ foil was ~ound to ha~e 0.2 mg~
of ce]ls per cn2 with an activitvv of 0.002U/cm2, which corresponcls to an activity yield of 62.5'~, In the case o~
the 50~ foil, 0.~8 m~. o~ cells per cm2 were found with an activlty of 0.005 U/cm29 which corresponds to a gield o~ 64~ here were no last runnings~
3xample 22, On the same foîls as wers used in Example 20, there were stuck GOD/Cat commonly bound to a polymeric carrier and having an activitv of 300 U/g, ~he 25~ foil was found to have 0,2 mg. enzygel/cm2 with an activity of 0.047 U/
cm2, which corresponds -to an activitv yield of 78.3~ In the case of the 50t~ foil, 0.2 mg. enz~el/cm2 were found~
with an activi ty of 0.049 U/cm2, which corresponds -to an activit~ yield of 81.6~. ~here were no las~ runnings~
Figs. 3 and 4 of the accompan~ing drawings illust- -ra-te two embodiments of the present invention. Fig, 3 shows a cross section through a trlangular-shaped rod coated on the surface with an adhesive lavver and with enzvvme-con-taining parti&les and Figo 4 shows a cross-section through a tube, the inner sur~ace of which is coated with an adhesive to which has been applied enz~me-containing particles.
' '~
.
:
- . . . ... , ~ . . :~ .
- . , . . . ;,: :. . ~ .
~54~
The enzymes particularly referred to in this disclosure have the following enzyme numbers:
glucose oxidase 1.1.3.4 catalase 1.11.1.6 alkaline phosphatase 3.1.3.1 trypsin 3.4.21.4 ~
~ . .
.
:' ~:
. ~ .
Tpe adhesive is diluted with methylene chloride and thinly applied to the foil. After some time, the finely-divided enzyme powder is applied to the still moist and sticky foil, with the use of a sieve. After slow drying, the foil is slowly stirred in a O.lM phosphate buffer solution (pH 7.0) and, in this way, non-bound particles are removed. The foil is subsequently transferred into a ,!
bifilar milled spiral and employed'for substrate dete~mination in a similar way to the enzyme tube.
1 cm of the foil produced by the above-described ~ -process weighs, in a dry,state, 23.3 mg. and has an enzymatic activity of 16.1 U/g., corresponding to 0.375 U/cm .
Coatinq of a glass tip Starting materials:
1 ml. adhesive as in Example 1 1.5 ml. methylene chloride 50 mg. enzyme powder as in Example 1.
*trademark f"
. ~ ' ' ~ .
. ,: . . , ~ ~ , . . . ~ , ~. . , ~ .
.: : : . . . ~ . :
Metho~:
The ylass tip is dipped once or twice into the diluted adhesive solution and, after a few seconds, dipped into the enzyme powder. Af-ter slow drying, it is trans-ferred to a 0.1M buffer solution (pH 7.0) and tested after about 12 hours. In the case of a tip of 1 - 2 mm. diameter and with a length of 2 - 3 mm., there is obtained an average activity of 0.5 U/tip.
Example 4 A round polystyrenerod of 2.4 mm. diameter is coated -with an adhesive which was obtained from 6 g. of a PVC
adhesive (Tangit of the firm Henkel) and 2 ml. tetrahydro-furan. The rod is then dipped into a finely powdered enzyme preparation which consists of GOD (230 U/g.) and Cat (3600 U/g.~ fixed via acryloyl chloride by the process of enzyme co-polymerisation. The rod is then allowed to dry and the activity determined. It is found to be 0.49 U GOD/mm surface area. ;
Example 5 The process of Example 4 is repeated with the use of an alkaline phosphatase with an activlty of 200 U/g. ~ ;
bound to the same carrier. The polystyrene rod is, aft0r dipping into the adhesive,left to dry for 15 to 30 seconds in the air, whereafter the enzyme dust is sprinkled on, the adhesive is left to dry and the rod is washed with a buffer. The activity is found to be 0.23 U/mm surface area. --Example 6 The process of Example 5 is repeated w1th the use of an enzyme powder which contains 5 U/g. trypsin on a maleic ; *trademark ~ ` ~
- .: , .
~054~) ~9 anhydride-acrylamide carrier (according to German Patent Specification No. 1,908,290). The activity of the formed body is subsequently found to be 0.003 U/mm surface area.
Example 7 Exàmple 5 is repeated with the use of a two-component epoxy resin adhesive (Uhuplus ) and the GOD/Cat powder of Example 4. The measured activity of the formed body is found to be 0.5 U/mm2 surface area.
Example 8 The process of Example 5 is repeated with the use of a contact adhesive based on polychlorobutadiene, with the addition of resins and organic solvents (Pattex of the firm Henkel), as well as of the enzyme powder of Example 4. The measured activity of the formed body is found to be 0.45 U/mm2.
Example 9 The process of Example 8 is repeated with the use of the alkaline phosphatase enzyme powder according to Example 5. The measured activity of the formed body is found to be 0.021 U~mm .
ExamPle 10 Example 5 is repeated with the use of a commercially available adhesive based on polyvinyl resin with acetone ; and ethyl acetate as solvents (Uhu Alleskleber ) and of the enzyme powder of Example 4. The enzymatic activity measured on the formed body is 0.5 U/mm2.
Example 11 .
Example 10 is repeated with the use of the enzyme powder of Example 6. The measured activity is found to be ; 0.0026 U/mm . ' ;
.
*trademark :. ~ .'. ' .
~35~ ri~
Example 12 A round rod of PVC o~ 3 mm. diameter is superficially ;`
dissolved with cyclohexane until the surf`ace thereof becomes sticky. The enzyme powder of Example 4 is then ~prinkled on, the rod is left to dry and thereafter it is washed with a buffer solution. '~he enzymatic activity of the formed body is 0.38 U/mm2.
xample 1~.
Ex~mple 12 is repeated with the use of the alkaline phosphatase enzyme po~der according to Example 5. ~he ~-enzymatic acti~ity of the formed body is 0.117 U/mm2.
xample 14. `
The process of ~xample 12 is repeated with the use ~ ;
of the trypsin-containing enzyme powder of ~xample 6. ~The enzymatic activity of the formed body is 0.0042 U/mm2.
E_am~le 15.
A glass rod of 2.4 mm. diameter is dipped into the ;~
adhesive of Example 8, thereafter allowed to dry in the -air for 15 to 30 seconds and sprinkled with the enzyme p~wder of Example 4. After drying the rod9 it is washed with buffer. The enzymatic activity of the finished rod `
is 0.66 U/mm .
a ~ . ` ;~
A steel wire of 1 mm. diameter is dipped into the adhesive of ~xample 8, left to dry in the air for 15 to 30 seconds and then sprinkled with the enzyme powder of Example 4. The enzymatic activity of the finished formed body is found to be 0.80 U/mm2.
Example 17.
The process of Egample 16 is repeated, with the use ~ ~ .
~ -15-- j, . .. , -. ,. . . - : - : ~
.. .. , ... ~
... . . . - . . . . . .. -. ... ... .:,. :: .. .-. . .. : . . .
~ ~9 of the all~aline pho~ hatase enz~me powder accorcling to Exam~le 5. r~le enzymatic activit~ of the formed bod~ is found to be 0.003 U/mm2.
~æample 18~
Example 4 i9 repeat~d but, ins-tead of the poly-styrene rocl, there is used a '~eflon-coated magnetic s-tirrer rod with the dimensions 6.5 x 10.9 m~, ~he enz~matic a~-tivitg of the formea body is found to be 0.15 U/m~n2.
~ .
Example 5 is repeated but, instead of -the polg-st~rene rod, there was used a ~eflon-coated magnetic stirrer rod as described in Example 18. '~he enzymatic activitg of the formed body is 0.135 U/mm2, E ~
~ wo foils were coated with adhesive J 6610-21 (50~ polyacrylic ester) of the firm Henkel. The la~er thickness was 25 ~ and 50~, respectivel~ ~
cells were applied to 50 cm2 pieces of the foils.
~he foils were vigorously s-tirred overnight in a phosphate buffer (pX 7.0) in order -to remove non-~ixed cells. The amount of ~ niger stuck on was determined by ~ -weighing the dried foils. n the 25~ foill there was ascertained 0,18 mg, ~ ~ cells per cm and on the 50 ~ foil, 0;20 mg. ~ Q ~ ce~ls per cm .
~he activit~ o~ the micro-organisms on the ~oils was 0~027 U/cm2 in the case of the 25~ foil and 0~0~2 U/cm2 in the case of the 50~4 foil, ~he la~t runnings amounted to 15~, Since the ~ ~ ells used had an activity of 200 U/g., referred to GODI the calculated activit~ ~ield was 75~ in the case of the 25~ ~oil and 80~G in the case o~ the 50~ foll.
-16~
- .
.. : , :
i~540 ï9 ; ', ':
Nocardia ~ micro-organism cells were stuck to the same foils as were used in 3xample 20~ The Nocardia ~ cells used had a chole,sterol oxidase activitg of 16lJ/gD '~he 25~ foil was ~ound to ha~e 0.2 mg~
of ce]ls per cn2 with an activitvv of 0.002U/cm2, which corresponcls to an activity yield of 62.5'~, In the case o~
the 50~ foil, 0.~8 m~. o~ cells per cm2 were found with an activlty of 0.005 U/cm29 which corresponds to a gield o~ 64~ here were no last runnings~
3xample 22, On the same foîls as wers used in Example 20, there were stuck GOD/Cat commonly bound to a polymeric carrier and having an activitv of 300 U/g, ~he 25~ foil was found to have 0,2 mg. enzygel/cm2 with an activity of 0.047 U/
cm2, which corresponds -to an activitv yield of 78.3~ In the case of the 50t~ foil, 0.2 mg. enz~el/cm2 were found~
with an activi ty of 0.049 U/cm2, which corresponds -to an activit~ yield of 81.6~. ~here were no las~ runnings~
Figs. 3 and 4 of the accompan~ing drawings illust- -ra-te two embodiments of the present invention. Fig, 3 shows a cross section through a trlangular-shaped rod coated on the surface with an adhesive lavver and with enzvvme-con-taining parti&les and Figo 4 shows a cross-section through a tube, the inner sur~ace of which is coated with an adhesive to which has been applied enz~me-containing particles.
' '~
.
:
- . . . ... , ~ . . :~ .
- . , . . . ;,: :. . ~ .
~54~
The enzymes particularly referred to in this disclosure have the following enzyme numbers:
glucose oxidase 1.1.3.4 catalase 1.11.1.6 alkaline phosphatase 3.1.3.1 trypsin 3.4.21.4 ~
~ . .
.
:' ~:
. ~ .
Claims (12)
1. A formed body with an enzymatically-active surface, wherein the surface of a formed body is at least partially coated with adhesive and on the adhesive layer there are present particles which contain at least one enzyme immobilised on a solid carrier.
2. A formed body according to claim 1, wherein the formed body consists of synthetic resin, metal, glass or a synthetic or natural polymer.
3. A formed body according to claim 1 or 2, wherein the adhesive is a vegetable or synthetic adhesive.
4. A formed body according to claim 2, wherein the carrier of the immobilised-enzyme consists preponderantly of acrylamide units.
5. A formed body according to claim 2 or 4, wherein said formed body is in the form of a spheroid, tube, rod or foil.
6. A formed body according to claim 1, 2 or 4, wherein said surface is completely coated with said adhesive.
7. A formed body according to claim 1, 2 or 4, wherein said surface is partially coated with said adhesive.
8. A process for the production of a formed body as defined in claim 1, wherein a liquid adhesive is applied to at least part of the surface of a formed body or at least part of the surface of a formed body is rendered adhesive, the adhesive is then allowed to dry, whereafter a particulate, immobilised enzyme is applied thereto and the adhesive then allowed to harden.
9. A process according to claim 8, wherein the particulate immobilised enzyme has a particle size of from about 0.0001 to about 1 mm.
10. A process according to claim 9, wherein the particulate immobilised enzyme has a particle size of from 0.001 to 0.1 mm.
11. A process according to claim 8, 9 or 10, which comprises applying said liquid adhesive to said surface, allowing the adhesive to dry, applying thereto the particulate, immobilised enzyme and allowing the adhesive to harden.
12. A process according to claim 8, 9 or 10, which comprises rendering said surface adhesive, allowing the adhesive surface to dry, applying thereto the particulate, immobilised enzyme and allowing the adhesive surface to harden.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE2438436A DE2438436B2 (en) | 1974-08-09 | 1974-08-09 | Shaped article with an enzymatically active surface and process for its production |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1054079A true CA1054079A (en) | 1979-05-08 |
Family
ID=5922876
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA231,821A Expired CA1054079A (en) | 1974-08-09 | 1975-07-18 | Particulate immobilized enzyme adhesively bound to formed body |
Country Status (13)
Country | Link |
---|---|
JP (1) | JPS5141495A (en) |
AT (1) | AT343584B (en) |
CA (1) | CA1054079A (en) |
CH (1) | CH619266A5 (en) |
DE (1) | DE2438436B2 (en) |
DK (1) | DK357675A (en) |
FI (1) | FI53705C (en) |
FR (1) | FR2281377A1 (en) |
GB (1) | GB1469191A (en) |
IL (1) | IL47685A (en) |
IT (1) | IT1039298B (en) |
NL (1) | NL7509345A (en) |
SE (1) | SE7508958L (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS52139777A (en) * | 1976-05-14 | 1977-11-21 | Omron Tateisi Electronics Co | Fixed enzyme membrane |
FI54842C (en) * | 1977-01-14 | 1979-03-12 | Suovaniemi Finnpipette | FORMULATION OF THE IMMUNITATION FOR IMMUNITATION AND ENZYMATION |
GB1584129A (en) * | 1977-06-10 | 1981-02-04 | Nayak P | Method and apparatus for performing in vitro clinical diagnostic tests using a solid phase assay system |
EP0029411B1 (en) * | 1979-11-15 | 1984-05-30 | Battelle Memorial Institute | Adhesive composition for depositing an adhesive coating able to fix biofunctional molecules, coating obtained and process for its preparation, substrate covered with the coating and its use as a biocatalyst |
DE19830848A1 (en) * | 1998-07-10 | 2000-01-13 | Daimler Chrysler Ag | Methods and agents for treating the surfaces of articles of daily use |
AT500669B1 (en) * | 2001-09-24 | 2007-02-15 | Oesterr Forsch Seibersdorf | SOLID CARRIER FOR THE IMMOBILIZATION OF BIOMOLECULES |
-
1974
- 1974-08-09 DE DE2438436A patent/DE2438436B2/en not_active Withdrawn
-
1975
- 1975-06-30 AT AT500475A patent/AT343584B/en active
- 1975-07-10 IL IL47685A patent/IL47685A/en unknown
- 1975-07-16 IT IT7525475A patent/IT1039298B/en active
- 1975-07-18 CA CA231,821A patent/CA1054079A/en not_active Expired
- 1975-08-06 GB GB3285575A patent/GB1469191A/en not_active Expired
- 1975-08-06 NL NL7509345A patent/NL7509345A/en not_active Application Discontinuation
- 1975-08-06 DK DK357675A patent/DK357675A/en unknown
- 1975-08-06 CH CH1027975A patent/CH619266A5/en not_active IP Right Cessation
- 1975-08-08 SE SE7508958A patent/SE7508958L/en not_active Application Discontinuation
- 1975-08-08 JP JP50096585A patent/JPS5141495A/en active Pending
- 1975-08-08 FR FR7524892A patent/FR2281377A1/en active Granted
- 1975-08-08 FI FI752261A patent/FI53705C/en not_active IP Right Cessation
Also Published As
Publication number | Publication date |
---|---|
NL7509345A (en) | 1976-02-11 |
GB1469191A (en) | 1977-03-30 |
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DE2438436A1 (en) | 1976-02-26 |
IT1039298B (en) | 1979-12-10 |
FI53705C (en) | 1978-07-10 |
CH619266A5 (en) | 1980-09-15 |
DE2438436B2 (en) | 1979-11-29 |
SE7508958L (en) | 1976-02-10 |
IL47685A0 (en) | 1975-10-15 |
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