CA1059936A - Immobilization of biologically active substances - Google Patents
Immobilization of biologically active substancesInfo
- Publication number
- CA1059936A CA1059936A CA230,056A CA230056A CA1059936A CA 1059936 A CA1059936 A CA 1059936A CA 230056 A CA230056 A CA 230056A CA 1059936 A CA1059936 A CA 1059936A
- Authority
- CA
- Canada
- Prior art keywords
- biologically active
- support material
- active substance
- pores
- cross
- 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
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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
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
Abstract
ABSTRACT
The invention relates to a support material having immobilized thereon a biologically active substance (e.g. an enzyme) and a process for producing such a support material. m e process comprises temporarily retaining a biologically active substance on a support material and treating the biologically active substance to cause cross-linking so as to immobilize biologically active substance on the support material.
The invention relates to a support material having immobilized thereon a biologically active substance (e.g. an enzyme) and a process for producing such a support material. m e process comprises temporarily retaining a biologically active substance on a support material and treating the biologically active substance to cause cross-linking so as to immobilize biologically active substance on the support material.
Description
~059936 The present invention relates to the immobilization of biologically active substances, for example, enzymes, on a support material.
~ Biologically active substances, such as enzymes, are useful as catalysts in certain processes ~e.g. amyloglucosidase in the production of glucose from starch). However, often in practice it is found that biologically active substances are available only in forms, such as enzyme preparations, which are water soluble. ~his means that such a substance cannot be economically isolated at the end of a process with th0 result that the substance is either lost in the spent liquor or con-taminates the product.
~ herefore it can be advantageous to immobilizé a biologically active substance on a support material so that it can be separated from reaction media by physical techniques such as filtrat~on or sedimentation. In addition biologically active substances immoS~lized on a support material are available for "localised" use, e.g. ac a bed in a column.
A biologically active substance is considered to be immobilized on a support material i~ it is fixed thereon in such a manner that permits at least a major proportion of the - substance to be retained by the support`material under t~e particular process conditions in which the support material and biologically active su~stance are used.
Varlous methods for ~fixing" or Rimmobilizing" (often referred to as n in~olubilising") biologically active substances on support materials have previously been proposed in the art.
However, these methods are unsatisfactory in that their products suffer frsm d~a~v~tages. For exampla, the biologically active substance i8 eit~er "leac~d~ from the 6upport material when ~n - 1 - ~
lOS9936 use and/or the physical, chemical or microbiological stabi-lity of the substance/support material combination is unsat~
isfactory with the result that the substance is not available for continued re-use over prolonged periods of time.
According to one aspect of the present invention there is provided a method for the preparation of a support material carrying an immobilised biologically active sub-stance which method comprises (a) introducing biologically active substance into the pores of a porous support material, (b) insolubilising the biologically active substance to hold it in the pores for cross-linking, and (c) cross-linking the biologically active substance in the pores of the porous support material so as to immobilise biologically active substance in the pores of the porous support material.
In another aspect the present invention provides a method for the preparation of a porous support material having a biologically active substance immobilised in the pores thereof comprising introducing biologically active substance into the pores of a porous support material, insolubilizing the biologically active substance to hold it in the pores of the porous support material for cross-linking, and cross-linking the biologically active sub-stance in the pores of the porous support material so as to immobilise biologically active substance in the pores of the porous support material, the introduction of the biologi-cally active substance and the insolubilising and cross-linking being such that a major proportion of the immo-bilised biologically active substance is present in ~31 ~.~
1~59936 the pores of, rather than on the external surface of, the porous support material. In a preferred e~bodiment the invention provides such a method wherein the insol~bilizat-on of biologically active substance in the pores of a porous support material is effected by precipitation from a solu-tion of a biologically active substance in the pores.
In a further embodiment the present invention provides such a method wherein a porous support material is soaked in a concentrated solution of biologically active substance to introduce solution into the pores of the porous support material, biologically active substance is freeze-dried in the pores of the porous support material, and then the porous support material and freeze-dried biologically active substance are treated with a cross-linking agent, under conditions such that substantially no freeze-dried substance goes into s~lution, to effect cross-linking of the biolo-gically active substance and thereby immobilize the substance in the pores of the support material.
The term "insolubilizing", as used in this specifica-tion does not mean "making ins~luble" in the sense of precipitation, but is meant to include any treatment of the biologically active substance in the support material which leads to the biologically active substance being held available in the pores of the support material for cross-linking. It implies treating so that mobility of the biologically active substance is inhibited during subsequent cross-linking treatment.
In a further aspect the present invention provides a porous support material carrying a biologically active substance immobilized by cross linking in the pores thereof, when made by the above processes.
~ -3-. , ~, ~.,i lOS9936 The term "biolo~ically active substance" as used inthis specification embraces- inter alia proteinaceous sub-stances (e.g. enzymes~, and includes substances which are biologically active per se and those which are not, but which can be activated after immobilization to make them biologically active.
In this specification certain enzymes are referred to.
For the convenience of the reader the following enzyme numbers are listed.
Enzyme Numbers (I.U.B.) ~ -a~ylase 3.2.1.1 lipoxidase 1.99.2.1 chymotrypsin 3.4.4.5 Trypsin 3.4.4.4 glucose oxidase 1.1.3.4 Urease 3.5.1.5 lactase 3.2.1.23 Amyloglucosidase 3.2.1.3 Furthermore, it is to be understood that the term "biologically active substance" embraces inher alia those substances capable of participating in specific interactions, such ~059936 substances including, for example, substances of biological origin and those which act on living organisms. Substances of synthetic origin which can participate in reactions involving specific interactions analogous to those which can occur with S ¦ naturally occurring substances are also embraced within the term "biologically active substance"~
In immobilising a biologically active substance in accordance with the present invention the substance is temporarily retained on the support material so that a substantial concentration of the substance is available to be immobilized on the ~upport by cross-linking. ~hus, the term "temporarily retaining" is not intended to relate particularly to the res~dence time of substance on the support, but to distinguish the nature of this retention step from the more permanent immobi~lization obtained by cross-~inkin~.
Whilst adsorption of biologically active su~stance by the support material can occur during temporary retention, it has been found that the use of adsorption alone to effect temporary retention can give a low yield of immobilized enzyme. Thus, temporary retention in accordance with the method of the present invention involves treating the substance so as to form more than mere adsorptive retention by the support material.
me temporary retention of ~iologically active substance on the support material can be effected, for example, by precipitation from a solution of a biologically active ., ~.
~059936 substance by use of a precipitating agent. Alternatively, freeze-drying can be used in con~unction with a precipitating agent to effect temporary retention.
Freeze-drying alone can be used to effect temporary retention although it has been found that this can be less effective than when a precipitating agent is used.
Although a portion of a biologically active substance can be immobilized on the outer surface of a support material, in most circumstances it is very desirable to utilise the pores of a porous support material, since in this way, the surface area/volume ratio of the support mater~al is greatly increased and thus a greater amount of biolo~ically active substance immobilized~ In these circumstances, the pore size and pore structure of the porous support mater~al should be such as to permit entry of the substance to be immobilized and eventual entry of species with which the immobilized substance is to i~teract.
Thus, by using a porous support material and a biologically active substance which can enter the pores thereof, the method of the present invention enables biologically active substance to be immobilized in the pores of a porous support material. m us, after temporary retention and cross-linking, biologically active substance immobilized on the porous support material will be present in the pores of the material as well as upon its outer surface. For example, in the case of a particulate porous support material a major proportion of the ~mmobilized substance would be expected to bè
present in the pores rather than on the external surface thereof.
I) According to another aspect of the present invention there is provided ~ support material having immobilized thereon, by temporary retention and cross-linking, a biologically active substance.
S In accordance with one particular embodiment of the invention the temporary retention is effected by precipitation and the precipitation and cross-linking processes are carried out sequentially by treating biologically active substance, previously introduced onto a porous support material, with a precipitating agent and then a cross-linking agent to cross-link the biologically act~ve substance.
It has been found that it is possible to treat some biologically active substances with a precipitating and a cross-linking agent, or agents, simultaneously. This is possible in the case of enzymes te.g. amyloglucosidase) where the time taken for cross-linking to occur far exceeds the time taken to precipitate the enzyme.
The temporary retention of some biologically active substances may be facilitated byp~etreating the substance to cause a degree of cross-linking; in such cases a certain degree of cross-linking can precede precipitation.
Preferably a porous support material is first soaked in a concentrated solution of biologically active ; substance (e.g. an enzyme) and then is subsequently treated with a precipitating agent, which does not denature the substance, temporarily to retain biolosically active substance on the porous support material and a cross-linking agent to cross-link and immobilize the substance on the porous support material.
In accordance with.another particular embodiment of the invention a porous support material is soaked in a concentrated solution of biologically active substance ; S .(e~g.~~an enzyme), biologically active substance is temporarily retained on the porous support material by freeze-drying and the porous support material and temporarily retained biologically active substance are treated with a cross-linking agent, under conditions such ~10 . that substantially no temporarily retained substance goes I into solution, to effect cross-linking and immobilization .
of the substance.on the support material.
Optionally, after temporarily retaining the substance on the porous support material by freeze-drying further ~ 15~ biologically active substance can be introduced onto the I porous support material, prior to cross-linking, by immersion in a concentrated solution of the substance and temporarily retained by treatment with a precipitating agent.
.
~ ~ . It will be appreciated that when carrying out the method :1 20 of the present invention with a porous support material , ~ , .
bioIogically active substance can be introduced, in solution, so .
as to fill the pore volume of the material and then treated, in such a way that significant amounts of the substance are.not displaced from the pores, to cause the substance - to come out of solution thereby to effect temporary retention of substance in the support material. The temporarily retained substance which is ~localised~' on the support material is subsequently cross-linked to . .
-7_ ,. . .
.
immohilize it in the pores.
The immo~ilization o~ hiologically active substances on a support material in accordance with the present invention involves sorption, entrapment and cross-linking.
Using the method of the present invention amyloglucosidase has been immobilized, using tannic acid as a precLpitating agent and glutaraldehyde as a cross-linking agent, on the following inorganic materials which are given by ~ay of example:
porous titania spheroids, porous calcium phosphate spheroids, porous alumina spheroids, porous zirconia spheroids, controlled pore glass (Corning Glass types CPG10-240; 10-370; 10-1250;
30-370 and 30-2000), crushed THERMALITE fly ash type block, Laporte Spent Catalyst, Crosfield Spent Catalyst (Laporte and Crosfield Spent Catalysts contain pODOUS alumino- silicate particles). It is to be understood that materials other than the foregoing examples are suitable for use as support materials.
*
For example, porous natural earths such as Celite, are suitable support materials and enzymes have been immobilized in accordance ~ith the present invention on porous spheroids fabricated from Celite. Furthermore, organic materials such as wood, Viscose, Sephadex (a cross-linked dextran), and Bio-gel (a polyacrylamide gel) can be used as support materials.
For example, amyloglucosidase has been immobilized on wood chips using tannic acid as a precipitating agent and glutaraldehyde as a cross-linking agent. Very desirably *Trade Marks 105g936 the support material should be substantially ~nsoluble under the procèss conditions and in the reaction media ~ ~ in which the support material and biologically active substance are used.
The following are examples of biologically active substances which have been immobilized on porous titania spheroids (having a particle size of 5~0~ diameter and 60%
of the pores in the range 2,700- lO,OO0 A diameter) using tannic acid as a precipitatinq agent and glutaraldehyde as a cross-linking agent: amyloglucosidase (four types), lactase (four types), ~-amalyse, chymotrypsin, trypsin urease, glucose oxidase, lipoxidase, glucose isomerase and papain.
More than one biologically active substance can be immobilized on the same support in accordance with the present ~nvention. Thus, amyloglucosidase and ~-amylase have been immobilized together.
A number of different reagents have been used as the precipitating agent in carrying out the method ~f the present invention. Thus, using glutaraldehyde as the cross-linking agent a commerclally available amyiogiucosidase (availàble under thename "Agidex") ~as been immob~lized on porous titania spheroids (particle size and pore size as here~nbefore stated) using the following examples of precipitating agents: tann~c acid in 70X ethanol, tannic acid in SOX acetone, tannic acid in 2:1 water~sopropanol, synthetic polyphenols in SOX
* * *
acetone ~a~allable under the names Tannia, Fixoflex, F$xln, ~ysolad from Harshaw C~emicals Ltd.),~acetone, LT 24 Floccular . ~ . ~
*Trade Mhr~
_ g _ ~, i S
_.__,_ __._, _ _ __ _ _,, .,,_ ~, _ , . ., . ... _ _ .. . , .. , . . ~r ~;
.
(a flocculating agent available from Allied Colloids Ltd.), aqueous solutions of tannic acid, salmine sulphate, alginic acid, protamine sulphate, pectin, gallic ad d, pyrogallol, polyethylene glycol, DEAE dextran, dextran sulphate, j polygalacturonic acid and polyethyleneimine.
Amyloglucosidase (Agidex) has also been immo~ilized in accordance with the present invention on porous titania spheroids (particle and pore size as hereinbefore stated) using formaldehyde as the cross-linking agent and tann~c acid as the precipitating agent and, additionally, using diethylpyrocarbcnate as a cross-linking agent and each of the following examples of precipitating agents: aqueo~s tannic acid, salmine sulphate, alginic acid, protamine sulphate, pectin, and tannic ac~d ~n 70~ ethanol. In addition, amyloglycosidase (Agidex) has been ~5 ~mmobilized on porous titania spheroids (particle size and pore size as hereinbefore stated) using tannic acid as the precip-itating agent and alkaline oxidation to cause cross-linking.
Other cross-linking agents include glyoxal and bis-~iaZniUm salts.
~lso in accordance with the present invention glucose oxidase has been immobilized on titania spheroids using tannic acid and diethyl pyrocarbonate and papain has been immobilized on titania spheroids using tannic acid and formaldehyde.
~he optimum conditions for immobilizing ~iologically active substances in accordance with the present invention are, at least in part, dependent upon the biochemical properties of the substance. Thus, the choice of, for example, precipitating agent, cross-liking agent and cross-lin~ing time is optimised . .
by experiment.
The ti~e for immobilization can vary hetween say ~ hour to 24 hours depending on the biologically active substance.
The method of the present invention is preferably carried out at about 4C. It has been noted that inactivation of certain biologically active substancescan occur at higher temperatures. However, this depends on the substance and -higher or lower temperatures could be appropriate.
It has been found that when immobilizing amylglucosidase (Agidex) on porous titania spheroids (particle size and pore size as hereinbefore stated) by precipitation and cross-linking, the best performance from the point of view of enzyme activity of the immobilized enzyme is obtained by using tannic acid in acetone as the precipitating agent and glutaraldehyde as the cross-linking agent. For example, it has been found that using this particular combination of precipitating agent and cross-linking agent the apparent enzyme acti~ity of amyloglucosidase (Agidex) immobilized on porous titania spheroids can be up to 20X o* the enzyme activity of the aqueous enzyme preparation used as the source of amyloglucosidase.
Using 5% tannic acid in 5:i acetone/water as a precipitating agent and gluteraldehyde as the cross-linking agent a batch of amyloglucosidase immobilized on porous titania spheroids (particle size and pore size as hereinbefore stated) was prepared from ~ 1109 titania spheroids and 30 ml Agidex, and packed into a column, 60 cm high and 100 mls in volume, to form a bed.
An acid thinned starch solution was passed through lOS9936 the column at a rate of between 52 and 230 ml/hr. The column was maintained at 60 for 8 days during which 9 litres of the solution were treated.
The Dextrose Equivalent (D.E.) of the product from the S column was as high as that expected when a soluble enzyme ls used for the same conversion process. ~he degree of hydrolysis of the product ~as constant during 7 days of steady use of the column.
A column of amyloglucosidase immobilized on porous t~tania spheroids was used continuously to hydrolyse a dextrin solution over a period of 7~ days. m e product from ; the column had a constant ~.E. value whlch indlcated almost total conversion of the starting material. Subsequently? the column was washed free of dextrin solution, was stored for 5 weeks at room temperature and was re-used for the same hydrolysis process. The D.E. of the product was found to be the same as that obtained previously. The column was stored for a further 11 weeks and it was found that there was no serious loss in the enzyme activity.
It has been found that ti~nia spheroids having amyloglucosidase immobilized thereon in accordance with the present invention may be used in either a packed bed, as described above t in a fluidised bed reactor or in a stirred tank reactor. It is to be expected that this wi11 be so for many particulate support materials having biologically active substances immobilized on them in accordance with the present invention.
Amyloglucosidase has been lmmobilized on porous t1tania `
spheroids (particle size and pore size as hereinbefore stated) using freeze drying and a preclpitating agent to effect temporary retention and gluteraldehyde to cause ; cross-lLnking.
Enzymes have been immobilized in situ on a column of porous titania spheroids.
During in situ immobilization excess biologically active substance not temporarily retained on the support material can be reclaimed for recycling by washing the ; 10 support material (e.g. with acetone/tannic acid) prior to cross-linking.
Spent immobilized enzyme has been removed from titania spheroids and the spheroids re-used for further immobilization.
Concentrated nitric acid, concentrated caustic soda or heating in a furnace were suitable for removing enzyme from the spheroids.
` A wide range of bioiogically active substances can be immobilized on a wide range of support materials in accordance with the present invention. Consequently, the invention ofers the advantage of flexibility over known "immobilization" methods in that a support material can be chosen from a wide range of materials, on the basis of its properties, to suit a particular application.
The present invention will now be illustrated by reference to the following examples.
Example 1 Agidex solution (1 ml) was added to porous titania ` ' 1059936 spheroids (3 ml; 500~ particle size and having 60% of the pores in the range 2700-lO000 ~ diameter), on an ice bath and was allowed to distribute throughout the spheroids. A
5% solution of tannic acid in 5:1 acetone : water (1.5 ml), previously cooled on an ice bath, was added followed by a 50% aqueous solution of glutaraldehyde (0.25 ml). The liquid level was then just above the surface of the porous titania spheroids. After 5 hours the spheroids were removed from the ice bath, washed and packed into a column so that the enzyme activity could be investigated. A solution of dextrin was used to investigate the properties of the column and, taking the activity of the original Agidex solution as 100%, the apparent activity of the immobilized enzyme in the column was found to be 20%.
Example 2 Agidex solution (4.5 ml) was distributed throughout porous titania spheroids (15 ml~ as in Example 1. A cooled
~ Biologically active substances, such as enzymes, are useful as catalysts in certain processes ~e.g. amyloglucosidase in the production of glucose from starch). However, often in practice it is found that biologically active substances are available only in forms, such as enzyme preparations, which are water soluble. ~his means that such a substance cannot be economically isolated at the end of a process with th0 result that the substance is either lost in the spent liquor or con-taminates the product.
~ herefore it can be advantageous to immobilizé a biologically active substance on a support material so that it can be separated from reaction media by physical techniques such as filtrat~on or sedimentation. In addition biologically active substances immoS~lized on a support material are available for "localised" use, e.g. ac a bed in a column.
A biologically active substance is considered to be immobilized on a support material i~ it is fixed thereon in such a manner that permits at least a major proportion of the - substance to be retained by the support`material under t~e particular process conditions in which the support material and biologically active su~stance are used.
Varlous methods for ~fixing" or Rimmobilizing" (often referred to as n in~olubilising") biologically active substances on support materials have previously been proposed in the art.
However, these methods are unsatisfactory in that their products suffer frsm d~a~v~tages. For exampla, the biologically active substance i8 eit~er "leac~d~ from the 6upport material when ~n - 1 - ~
lOS9936 use and/or the physical, chemical or microbiological stabi-lity of the substance/support material combination is unsat~
isfactory with the result that the substance is not available for continued re-use over prolonged periods of time.
According to one aspect of the present invention there is provided a method for the preparation of a support material carrying an immobilised biologically active sub-stance which method comprises (a) introducing biologically active substance into the pores of a porous support material, (b) insolubilising the biologically active substance to hold it in the pores for cross-linking, and (c) cross-linking the biologically active substance in the pores of the porous support material so as to immobilise biologically active substance in the pores of the porous support material.
In another aspect the present invention provides a method for the preparation of a porous support material having a biologically active substance immobilised in the pores thereof comprising introducing biologically active substance into the pores of a porous support material, insolubilizing the biologically active substance to hold it in the pores of the porous support material for cross-linking, and cross-linking the biologically active sub-stance in the pores of the porous support material so as to immobilise biologically active substance in the pores of the porous support material, the introduction of the biologi-cally active substance and the insolubilising and cross-linking being such that a major proportion of the immo-bilised biologically active substance is present in ~31 ~.~
1~59936 the pores of, rather than on the external surface of, the porous support material. In a preferred e~bodiment the invention provides such a method wherein the insol~bilizat-on of biologically active substance in the pores of a porous support material is effected by precipitation from a solu-tion of a biologically active substance in the pores.
In a further embodiment the present invention provides such a method wherein a porous support material is soaked in a concentrated solution of biologically active substance to introduce solution into the pores of the porous support material, biologically active substance is freeze-dried in the pores of the porous support material, and then the porous support material and freeze-dried biologically active substance are treated with a cross-linking agent, under conditions such that substantially no freeze-dried substance goes into s~lution, to effect cross-linking of the biolo-gically active substance and thereby immobilize the substance in the pores of the support material.
The term "insolubilizing", as used in this specifica-tion does not mean "making ins~luble" in the sense of precipitation, but is meant to include any treatment of the biologically active substance in the support material which leads to the biologically active substance being held available in the pores of the support material for cross-linking. It implies treating so that mobility of the biologically active substance is inhibited during subsequent cross-linking treatment.
In a further aspect the present invention provides a porous support material carrying a biologically active substance immobilized by cross linking in the pores thereof, when made by the above processes.
~ -3-. , ~, ~.,i lOS9936 The term "biolo~ically active substance" as used inthis specification embraces- inter alia proteinaceous sub-stances (e.g. enzymes~, and includes substances which are biologically active per se and those which are not, but which can be activated after immobilization to make them biologically active.
In this specification certain enzymes are referred to.
For the convenience of the reader the following enzyme numbers are listed.
Enzyme Numbers (I.U.B.) ~ -a~ylase 3.2.1.1 lipoxidase 1.99.2.1 chymotrypsin 3.4.4.5 Trypsin 3.4.4.4 glucose oxidase 1.1.3.4 Urease 3.5.1.5 lactase 3.2.1.23 Amyloglucosidase 3.2.1.3 Furthermore, it is to be understood that the term "biologically active substance" embraces inher alia those substances capable of participating in specific interactions, such ~059936 substances including, for example, substances of biological origin and those which act on living organisms. Substances of synthetic origin which can participate in reactions involving specific interactions analogous to those which can occur with S ¦ naturally occurring substances are also embraced within the term "biologically active substance"~
In immobilising a biologically active substance in accordance with the present invention the substance is temporarily retained on the support material so that a substantial concentration of the substance is available to be immobilized on the ~upport by cross-linking. ~hus, the term "temporarily retaining" is not intended to relate particularly to the res~dence time of substance on the support, but to distinguish the nature of this retention step from the more permanent immobi~lization obtained by cross-~inkin~.
Whilst adsorption of biologically active su~stance by the support material can occur during temporary retention, it has been found that the use of adsorption alone to effect temporary retention can give a low yield of immobilized enzyme. Thus, temporary retention in accordance with the method of the present invention involves treating the substance so as to form more than mere adsorptive retention by the support material.
me temporary retention of ~iologically active substance on the support material can be effected, for example, by precipitation from a solution of a biologically active ., ~.
~059936 substance by use of a precipitating agent. Alternatively, freeze-drying can be used in con~unction with a precipitating agent to effect temporary retention.
Freeze-drying alone can be used to effect temporary retention although it has been found that this can be less effective than when a precipitating agent is used.
Although a portion of a biologically active substance can be immobilized on the outer surface of a support material, in most circumstances it is very desirable to utilise the pores of a porous support material, since in this way, the surface area/volume ratio of the support mater~al is greatly increased and thus a greater amount of biolo~ically active substance immobilized~ In these circumstances, the pore size and pore structure of the porous support mater~al should be such as to permit entry of the substance to be immobilized and eventual entry of species with which the immobilized substance is to i~teract.
Thus, by using a porous support material and a biologically active substance which can enter the pores thereof, the method of the present invention enables biologically active substance to be immobilized in the pores of a porous support material. m us, after temporary retention and cross-linking, biologically active substance immobilized on the porous support material will be present in the pores of the material as well as upon its outer surface. For example, in the case of a particulate porous support material a major proportion of the ~mmobilized substance would be expected to bè
present in the pores rather than on the external surface thereof.
I) According to another aspect of the present invention there is provided ~ support material having immobilized thereon, by temporary retention and cross-linking, a biologically active substance.
S In accordance with one particular embodiment of the invention the temporary retention is effected by precipitation and the precipitation and cross-linking processes are carried out sequentially by treating biologically active substance, previously introduced onto a porous support material, with a precipitating agent and then a cross-linking agent to cross-link the biologically act~ve substance.
It has been found that it is possible to treat some biologically active substances with a precipitating and a cross-linking agent, or agents, simultaneously. This is possible in the case of enzymes te.g. amyloglucosidase) where the time taken for cross-linking to occur far exceeds the time taken to precipitate the enzyme.
The temporary retention of some biologically active substances may be facilitated byp~etreating the substance to cause a degree of cross-linking; in such cases a certain degree of cross-linking can precede precipitation.
Preferably a porous support material is first soaked in a concentrated solution of biologically active ; substance (e.g. an enzyme) and then is subsequently treated with a precipitating agent, which does not denature the substance, temporarily to retain biolosically active substance on the porous support material and a cross-linking agent to cross-link and immobilize the substance on the porous support material.
In accordance with.another particular embodiment of the invention a porous support material is soaked in a concentrated solution of biologically active substance ; S .(e~g.~~an enzyme), biologically active substance is temporarily retained on the porous support material by freeze-drying and the porous support material and temporarily retained biologically active substance are treated with a cross-linking agent, under conditions such ~10 . that substantially no temporarily retained substance goes I into solution, to effect cross-linking and immobilization .
of the substance.on the support material.
Optionally, after temporarily retaining the substance on the porous support material by freeze-drying further ~ 15~ biologically active substance can be introduced onto the I porous support material, prior to cross-linking, by immersion in a concentrated solution of the substance and temporarily retained by treatment with a precipitating agent.
.
~ ~ . It will be appreciated that when carrying out the method :1 20 of the present invention with a porous support material , ~ , .
bioIogically active substance can be introduced, in solution, so .
as to fill the pore volume of the material and then treated, in such a way that significant amounts of the substance are.not displaced from the pores, to cause the substance - to come out of solution thereby to effect temporary retention of substance in the support material. The temporarily retained substance which is ~localised~' on the support material is subsequently cross-linked to . .
-7_ ,. . .
.
immohilize it in the pores.
The immo~ilization o~ hiologically active substances on a support material in accordance with the present invention involves sorption, entrapment and cross-linking.
Using the method of the present invention amyloglucosidase has been immobilized, using tannic acid as a precLpitating agent and glutaraldehyde as a cross-linking agent, on the following inorganic materials which are given by ~ay of example:
porous titania spheroids, porous calcium phosphate spheroids, porous alumina spheroids, porous zirconia spheroids, controlled pore glass (Corning Glass types CPG10-240; 10-370; 10-1250;
30-370 and 30-2000), crushed THERMALITE fly ash type block, Laporte Spent Catalyst, Crosfield Spent Catalyst (Laporte and Crosfield Spent Catalysts contain pODOUS alumino- silicate particles). It is to be understood that materials other than the foregoing examples are suitable for use as support materials.
*
For example, porous natural earths such as Celite, are suitable support materials and enzymes have been immobilized in accordance ~ith the present invention on porous spheroids fabricated from Celite. Furthermore, organic materials such as wood, Viscose, Sephadex (a cross-linked dextran), and Bio-gel (a polyacrylamide gel) can be used as support materials.
For example, amyloglucosidase has been immobilized on wood chips using tannic acid as a precipitating agent and glutaraldehyde as a cross-linking agent. Very desirably *Trade Marks 105g936 the support material should be substantially ~nsoluble under the procèss conditions and in the reaction media ~ ~ in which the support material and biologically active substance are used.
The following are examples of biologically active substances which have been immobilized on porous titania spheroids (having a particle size of 5~0~ diameter and 60%
of the pores in the range 2,700- lO,OO0 A diameter) using tannic acid as a precipitatinq agent and glutaraldehyde as a cross-linking agent: amyloglucosidase (four types), lactase (four types), ~-amalyse, chymotrypsin, trypsin urease, glucose oxidase, lipoxidase, glucose isomerase and papain.
More than one biologically active substance can be immobilized on the same support in accordance with the present ~nvention. Thus, amyloglucosidase and ~-amylase have been immobilized together.
A number of different reagents have been used as the precipitating agent in carrying out the method ~f the present invention. Thus, using glutaraldehyde as the cross-linking agent a commerclally available amyiogiucosidase (availàble under thename "Agidex") ~as been immob~lized on porous titania spheroids (particle size and pore size as here~nbefore stated) using the following examples of precipitating agents: tann~c acid in 70X ethanol, tannic acid in SOX acetone, tannic acid in 2:1 water~sopropanol, synthetic polyphenols in SOX
* * *
acetone ~a~allable under the names Tannia, Fixoflex, F$xln, ~ysolad from Harshaw C~emicals Ltd.),~acetone, LT 24 Floccular . ~ . ~
*Trade Mhr~
_ g _ ~, i S
_.__,_ __._, _ _ __ _ _,, .,,_ ~, _ , . ., . ... _ _ .. . , .. , . . ~r ~;
.
(a flocculating agent available from Allied Colloids Ltd.), aqueous solutions of tannic acid, salmine sulphate, alginic acid, protamine sulphate, pectin, gallic ad d, pyrogallol, polyethylene glycol, DEAE dextran, dextran sulphate, j polygalacturonic acid and polyethyleneimine.
Amyloglucosidase (Agidex) has also been immo~ilized in accordance with the present invention on porous titania spheroids (particle and pore size as hereinbefore stated) using formaldehyde as the cross-linking agent and tann~c acid as the precipitating agent and, additionally, using diethylpyrocarbcnate as a cross-linking agent and each of the following examples of precipitating agents: aqueo~s tannic acid, salmine sulphate, alginic acid, protamine sulphate, pectin, and tannic ac~d ~n 70~ ethanol. In addition, amyloglycosidase (Agidex) has been ~5 ~mmobilized on porous titania spheroids (particle size and pore size as hereinbefore stated) using tannic acid as the precip-itating agent and alkaline oxidation to cause cross-linking.
Other cross-linking agents include glyoxal and bis-~iaZniUm salts.
~lso in accordance with the present invention glucose oxidase has been immobilized on titania spheroids using tannic acid and diethyl pyrocarbonate and papain has been immobilized on titania spheroids using tannic acid and formaldehyde.
~he optimum conditions for immobilizing ~iologically active substances in accordance with the present invention are, at least in part, dependent upon the biochemical properties of the substance. Thus, the choice of, for example, precipitating agent, cross-liking agent and cross-lin~ing time is optimised . .
by experiment.
The ti~e for immobilization can vary hetween say ~ hour to 24 hours depending on the biologically active substance.
The method of the present invention is preferably carried out at about 4C. It has been noted that inactivation of certain biologically active substancescan occur at higher temperatures. However, this depends on the substance and -higher or lower temperatures could be appropriate.
It has been found that when immobilizing amylglucosidase (Agidex) on porous titania spheroids (particle size and pore size as hereinbefore stated) by precipitation and cross-linking, the best performance from the point of view of enzyme activity of the immobilized enzyme is obtained by using tannic acid in acetone as the precipitating agent and glutaraldehyde as the cross-linking agent. For example, it has been found that using this particular combination of precipitating agent and cross-linking agent the apparent enzyme acti~ity of amyloglucosidase (Agidex) immobilized on porous titania spheroids can be up to 20X o* the enzyme activity of the aqueous enzyme preparation used as the source of amyloglucosidase.
Using 5% tannic acid in 5:i acetone/water as a precipitating agent and gluteraldehyde as the cross-linking agent a batch of amyloglucosidase immobilized on porous titania spheroids (particle size and pore size as hereinbefore stated) was prepared from ~ 1109 titania spheroids and 30 ml Agidex, and packed into a column, 60 cm high and 100 mls in volume, to form a bed.
An acid thinned starch solution was passed through lOS9936 the column at a rate of between 52 and 230 ml/hr. The column was maintained at 60 for 8 days during which 9 litres of the solution were treated.
The Dextrose Equivalent (D.E.) of the product from the S column was as high as that expected when a soluble enzyme ls used for the same conversion process. ~he degree of hydrolysis of the product ~as constant during 7 days of steady use of the column.
A column of amyloglucosidase immobilized on porous t~tania spheroids was used continuously to hydrolyse a dextrin solution over a period of 7~ days. m e product from ; the column had a constant ~.E. value whlch indlcated almost total conversion of the starting material. Subsequently? the column was washed free of dextrin solution, was stored for 5 weeks at room temperature and was re-used for the same hydrolysis process. The D.E. of the product was found to be the same as that obtained previously. The column was stored for a further 11 weeks and it was found that there was no serious loss in the enzyme activity.
It has been found that ti~nia spheroids having amyloglucosidase immobilized thereon in accordance with the present invention may be used in either a packed bed, as described above t in a fluidised bed reactor or in a stirred tank reactor. It is to be expected that this wi11 be so for many particulate support materials having biologically active substances immobilized on them in accordance with the present invention.
Amyloglucosidase has been lmmobilized on porous t1tania `
spheroids (particle size and pore size as hereinbefore stated) using freeze drying and a preclpitating agent to effect temporary retention and gluteraldehyde to cause ; cross-lLnking.
Enzymes have been immobilized in situ on a column of porous titania spheroids.
During in situ immobilization excess biologically active substance not temporarily retained on the support material can be reclaimed for recycling by washing the ; 10 support material (e.g. with acetone/tannic acid) prior to cross-linking.
Spent immobilized enzyme has been removed from titania spheroids and the spheroids re-used for further immobilization.
Concentrated nitric acid, concentrated caustic soda or heating in a furnace were suitable for removing enzyme from the spheroids.
` A wide range of bioiogically active substances can be immobilized on a wide range of support materials in accordance with the present invention. Consequently, the invention ofers the advantage of flexibility over known "immobilization" methods in that a support material can be chosen from a wide range of materials, on the basis of its properties, to suit a particular application.
The present invention will now be illustrated by reference to the following examples.
Example 1 Agidex solution (1 ml) was added to porous titania ` ' 1059936 spheroids (3 ml; 500~ particle size and having 60% of the pores in the range 2700-lO000 ~ diameter), on an ice bath and was allowed to distribute throughout the spheroids. A
5% solution of tannic acid in 5:1 acetone : water (1.5 ml), previously cooled on an ice bath, was added followed by a 50% aqueous solution of glutaraldehyde (0.25 ml). The liquid level was then just above the surface of the porous titania spheroids. After 5 hours the spheroids were removed from the ice bath, washed and packed into a column so that the enzyme activity could be investigated. A solution of dextrin was used to investigate the properties of the column and, taking the activity of the original Agidex solution as 100%, the apparent activity of the immobilized enzyme in the column was found to be 20%.
Example 2 Agidex solution (4.5 ml) was distributed throughout porous titania spheroids (15 ml~ as in Example 1. A cooled
2% solution of tannic acid in acetone (6.7S ml), into which a 50% aqueous solution of glutaraldehyde (1.05 ml) had rec~ntly béen mixed, was added and the resulting stiff slurry was thoroughIy mixed. After 5 hours the spheroids were washed and the`enzyme activity assayed as in Example l. The apparent enzyme activity of the immobilized enzyme was found to be similar to that obtained in Example 1.
ExamJ~le 3 Agidex solution, diluted l:l with water (8 ml) was distributed throughout porous titania spheroids (10 ml;
:.
"
.. ... .
.
- ~059936 particle size and pore size as in Example 1) and was freeze-dried (lyophilised). Agidex solution (1.~ ml) was distributed throughout 5 ml of these lyophilised spheroids and a ~ooled 2~ solution of tannic acid in acetone (2.25 ml) S ¦ and 50X glutaraldehyde (0.35 ml) were added,in the same way as Example 2. The apparent acti~ity of the immobilized enzyme was found to be lS~ more than the activity of immobilized enzyme prepared using porous titania spheroids, but no freeze-drying step.
ExamDle 4 Agidex solution t2 ml) was added to, and distributed throughout, S mls of porous glass particles tCorning CPG
~0-12S0, 36-75~ particle size) on an ice bath. A cooled 5 solution of tannic acid in 5:1 acetone : water (3 ml) was added followed by a S0~ aqueous solution of glutaraldehyde (O.S ml~. After S hours the glass particles were washed ~nd ~he enzyme acti~ity investigated in a small stirred batch reactor. The apparent activity of ~he immobilized enzyme was 93% of the acti~ity of enzyme immobilized on porous titania spheroids of the type used in Examples 1, 2 and 3.
~e~ .
A~idex solution (-l ml) was distributed throughout porous titania spheroids (3 ml) as in Example ~. l.S mls of a cooled lOq~ solution of ~Fixoblex~* (a synthetic polyphenol) in S:1 acetone : water was added followed ~y a 50% aqueous solu~ion of glutaraldehyde (0.25 ml). After 5 hours the spheroids were washed and when t;he immobilized enzyme was assayed as in Exa-ple 1 it ~:as found to have 82~ of the acti~ity of immobilized enzyme prepared with tannie acid in *Trademark ~ .
lOSg93~ .
acetone : water mixture as the precipitatin~ agent.
Example 6 ~ ml of an aqueous solution of lactase (Maxilact 7~ mg/ml) was added to and distributed throu~hout 3 mls of S porous titania spheroids. A cooled 0.25% solution of tannic ¦ acid in 5:1 acetone : water (~.5 ml) was added followed by a 20% aqueous solution of glutaraldehvde (0.1 ml). After 20 minutes the beads were washed and pac~ed into a column.
The apparent activity of the immobilized enzyme was ~lX Of ~0 the acti~ity of the soluble enzyme when o-nitro phenyl-~-D
galactoside was used as the substrate.
Example 7 Agidex solution (1.5 ml) was added to porous titan~a spheroids (500~; 5 ml) on an ice bath and distributed throughout the spheroids. A cooled 2X solution of tannic .
acid in acetone ~ 5 ml) was added followed by a 3~%
aqueous solution of formaldehyde (0.35 ml3. After 5 hours the spheroids were washed and the immobilized enzyme was -assayed as ~n Example 1. It was found to have an enzyme activity which was 56% of that of a similar immobilized - enzyme prepared ùsing glutaraldehyde as the cross-linkin~
agent.
Example 8 Agidex (1 ml) was distributed throughout a wad of soft wood chips, which occupied a volume of 3 mls, on ~n ice bath. A cooled 2% solution o~ tannic acid in acetone (1.5 ml) into which a 50~ aqueous solut~on of ~lutaraldehyde (0.2S ml) ~Trade Mark had recently been mixed, was added and the reagents were thoroughly mixed. After 5 hours the chips were washed .
and loosely packed into a small column to enable the enzyme acti~ity to be assayed as in Example 1. The chips had an enzymic activity which was 74X of the activity of the immobilized enzyme ~n Example 2.
Example 9 An aqueous solution of glucose isomerase enzyme (1.5 ml obtained by aqueous extraction of Max~zyme - GI
14,000 cells) was allowed to soak into 5 g of porous ~ titania spheroids (particle size of 500 ~ diameter and 60X of the pores in the range 2,700 - 10,000 2 diameter) on an ice bath.
m e enzyme was precipitated and cross-linked by i5 means of a solution of 3% tannic acid in acetone (2.25 ml~
and 50Xaqueous glutaraldehyde (0.05 ml).
After reacting for 1~ hours on an ice bath the spheroids were washed and the enzyme activity assayed.
It was ~ound that the sphero~ds contained 35X of the ,20 enzyme activity present in the starting solution of the enzyme.
*Trade Mark -~?-`
,~ ..
ExamJ~le 3 Agidex solution, diluted l:l with water (8 ml) was distributed throughout porous titania spheroids (10 ml;
:.
"
.. ... .
.
- ~059936 particle size and pore size as in Example 1) and was freeze-dried (lyophilised). Agidex solution (1.~ ml) was distributed throughout 5 ml of these lyophilised spheroids and a ~ooled 2~ solution of tannic acid in acetone (2.25 ml) S ¦ and 50X glutaraldehyde (0.35 ml) were added,in the same way as Example 2. The apparent acti~ity of the immobilized enzyme was found to be lS~ more than the activity of immobilized enzyme prepared using porous titania spheroids, but no freeze-drying step.
ExamDle 4 Agidex solution t2 ml) was added to, and distributed throughout, S mls of porous glass particles tCorning CPG
~0-12S0, 36-75~ particle size) on an ice bath. A cooled 5 solution of tannic acid in 5:1 acetone : water (3 ml) was added followed by a S0~ aqueous solution of glutaraldehyde (O.S ml~. After S hours the glass particles were washed ~nd ~he enzyme acti~ity investigated in a small stirred batch reactor. The apparent activity of ~he immobilized enzyme was 93% of the acti~ity of enzyme immobilized on porous titania spheroids of the type used in Examples 1, 2 and 3.
~e~ .
A~idex solution (-l ml) was distributed throughout porous titania spheroids (3 ml) as in Example ~. l.S mls of a cooled lOq~ solution of ~Fixoblex~* (a synthetic polyphenol) in S:1 acetone : water was added followed ~y a 50% aqueous solu~ion of glutaraldehyde (0.25 ml). After 5 hours the spheroids were washed and when t;he immobilized enzyme was assayed as in Exa-ple 1 it ~:as found to have 82~ of the acti~ity of immobilized enzyme prepared with tannie acid in *Trademark ~ .
lOSg93~ .
acetone : water mixture as the precipitatin~ agent.
Example 6 ~ ml of an aqueous solution of lactase (Maxilact 7~ mg/ml) was added to and distributed throu~hout 3 mls of S porous titania spheroids. A cooled 0.25% solution of tannic ¦ acid in 5:1 acetone : water (~.5 ml) was added followed by a 20% aqueous solution of glutaraldehvde (0.1 ml). After 20 minutes the beads were washed and pac~ed into a column.
The apparent activity of the immobilized enzyme was ~lX Of ~0 the acti~ity of the soluble enzyme when o-nitro phenyl-~-D
galactoside was used as the substrate.
Example 7 Agidex solution (1.5 ml) was added to porous titan~a spheroids (500~; 5 ml) on an ice bath and distributed throughout the spheroids. A cooled 2X solution of tannic .
acid in acetone ~ 5 ml) was added followed by a 3~%
aqueous solution of formaldehyde (0.35 ml3. After 5 hours the spheroids were washed and the immobilized enzyme was -assayed as ~n Example 1. It was found to have an enzyme activity which was 56% of that of a similar immobilized - enzyme prepared ùsing glutaraldehyde as the cross-linkin~
agent.
Example 8 Agidex (1 ml) was distributed throughout a wad of soft wood chips, which occupied a volume of 3 mls, on ~n ice bath. A cooled 2% solution o~ tannic acid in acetone (1.5 ml) into which a 50~ aqueous solut~on of ~lutaraldehyde (0.2S ml) ~Trade Mark had recently been mixed, was added and the reagents were thoroughly mixed. After 5 hours the chips were washed .
and loosely packed into a small column to enable the enzyme acti~ity to be assayed as in Example 1. The chips had an enzymic activity which was 74X of the activity of the immobilized enzyme ~n Example 2.
Example 9 An aqueous solution of glucose isomerase enzyme (1.5 ml obtained by aqueous extraction of Max~zyme - GI
14,000 cells) was allowed to soak into 5 g of porous ~ titania spheroids (particle size of 500 ~ diameter and 60X of the pores in the range 2,700 - 10,000 2 diameter) on an ice bath.
m e enzyme was precipitated and cross-linked by i5 means of a solution of 3% tannic acid in acetone (2.25 ml~
and 50Xaqueous glutaraldehyde (0.05 ml).
After reacting for 1~ hours on an ice bath the spheroids were washed and the enzyme activity assayed.
It was ~ound that the sphero~ds contained 35X of the ,20 enzyme activity present in the starting solution of the enzyme.
*Trade Mark -~?-`
,~ ..
Claims (25)
1. A method for the preparation of a support material carrying an immobilised biologically active substance which method comprises (a) introducing biologically active sub-stance into the pores of a porous support material, (b) insolubilising the biologically active substance to hold it in the pores for cross-linking, and (c) cross-linking the biologically active substance in the pores of the porous support material so as to immobilise biologically active substance in the pores of the porous support material.
2. A method for the preparation of a porous-support material having a biologically active substance immobilised in the pores thereof, comprising introducing biologically active substance into the pores of a porous support material, insolubilising the biologically active substance to hold it in the pores of the porous support material for cross-linking, and cross-linking the biologically active substance in the pores of the porous support material so as to immobilise biologically active substance in the pores of the porous support material, the introduction of the biologically active substance and the insolubilising and cross-linking being such that a major proportion of the immobilised biologically active substance is present in the pores of, rather than on the external surface of, the porous support material.
3. A method as claimed in claim 1, wherein the insolubilization of biologically active substance in the pores of the porous support material is effected by precipitation from a solution of a biologically active substance in the pores.
4. A method as claimed in claim 1, 2 or 3, wherein freeze-drying is used to provide the insolubilization.
5. A method as claimed in claim 3 wherein the precipi-tation and cross-linking processes are carried out sequen-tially by treating the solution of the biologically active substance in the pores with a precipitating agent and then treating the biologically active substance with a cross-linking agent.
6. A method as claimed in claim 5, wherein a porous support material is first soaked in a concentrated solution of biologically active substance to introduce solution into the pores of the porous support material, and then is subsequently treated with a precipitating agent, which does not denature the substance, to precipitate biologically active substance in the pores, and with a cross-linking agent to cross-link and immobilise the biologically active substance in the pores of the porous support material.
7. A method as claimed in claim 1, 2 or 3, wherein biologically active substance previously introduced into the pores of the porous support material is treated with a precipitating agent and a cross-linking agent, or agents, simultaneously.
8. A method as claimed in claim 1, 2 or 3 wherein the biologically active substance is pretreated to cause a degree of cross-linking prior to precipitation.
9. A method as claimed in claim 1, 2 or 3, wherein a porous support material is soaked in a concentrated solution of biologically active substance to introduce solution into the pores of the porous support material, biologically active substance is freeze-dried in the pores of the porous support material, and then the porous support material and freeze-dried biologically active substance are treated with a cross-linking agent, under conditions such that substantially no freeze-dried substance goes into solution, to effect cross-linking of the biologically active substance and thereby immobilise the substance in the pores of the support material.
10. A method as claimed in claim 1, 2 or 3, wherein a porous support material is soaked in a concentrated solution of biologically active substance to introduce solution into the pores of the porous support material, biologically active substance is freeze-dried in the pores of the porous support material, and then the porous support material and freeze-dried biologically active substance are treated with a cross-linking agent, under conditions such that substantially no freeze-dried sub-stance goes into solution, to effect cross-linking of the biologically active substance and thereby immobilize the substance in the pores of the support material, and wherein, after freeze-drying of the substance in the pores of the porous support material, but prior to cross-linking, further biologically active substance is introduced into the porous support material by immersion in a concentrated solution of the substance and is precipitated by treatment with a precipitating agent.
11. A method as claimed in claim 5 or 6, wherein the precipitating agent is tannic acid in ethanol, tannic acid in acetone, tannic acid in water/isopropanol, a synthetic polyphenol in acetone, acetone, or an aqueous solution of tannic acid, salmine sulphate, alginic acid, protamine sulphate, pectin, gallic acid, pyrogallol, polyethylene glycol, DEAE dextran, dextran sulphate, polygalacturonic acid, or polyethyleneimine.
12. A method as claimed in claim 5 or 6, wherein the precipitating agent is a flocculating agent.
13. A method as claimed in claim 1, 2 or 3, wherein the biologically active substance is immobilised by being treated with a cross-linking agent.
14. A method as claimed in claim 1, 2 or 3, wherein cross-linking is effected by a cross-linking agent which comprises glutaraldehyde, formaldehyde, diethylpyrocarbonate, glyoxal or a bis-diazonium salt.
15. A method as claimed in claim 1, 2 or 3, wherein cross-linking is effected by alkaline oxidation.
16. A method as claimed in claim 1, 2 or 3 wherein biologically active substance is introduced into the pores of a plurality of discrete particles of porous support material and the insolubilization and the cross-linking are effected to produce a plurality of discrete particles, each particle comprising a particle of porous support material having biologically active substance immobilised in the pores thereof.
17. A method as claimed in claim 1, 2 or 3, wherein the support material is porous titania spheroids, porous calcium phosphate spheroids, porous alumina spheroids, porous zirconia spheroids, controlled pore glass, crushed porous thermal insulation block, alumino-silicate catalyst particles, or a porous natural earth.
18. A method as claimed in claim 1, 2 or 3, wherein the support material is a porous natural diatomaceous earth.
19. A method as claimed in claim 1, 2 or 3, wherein the support material is wood, viscose, a cross-linked dextran or a polyacrylamide gel.
20. A method as claimed in claim 1, 2 or 3 wherein the support material is porous titania spheroids having a particle size of 500 µ diameter of 60% of the pores in the range of 2700 to 10,000 .ANG. diameter.
21. A method as claimed in claim 1, 2 or 3 wherein the biologically active substance is a proteinaceous substance.
22.A method as claimed in claim 1, 2 or 3 wherein the biologically active substance is an enzyme.
23. A method as claimed in claim 1, 2 or 3, wherein the biologically active substance is an enzyme chosen from amyloglucosidase, lactase; .alpha. -amylase, chymotrypsin, trypsin, urease, glucose oxidase, lipoxidase, glucose isomerase or papain.
24. A method as claimed in claim 1, 2 or 3, wherein the cross-linking is followed by a washing step.
25. A porous support material having a biologically active substance immobilised in the pores thereof, whenever prepared by the process of claim 1, 2 or 3 or by an obvious chemical equivalent thereof.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB28212/74A GB1514707A (en) | 1974-06-25 | 1974-06-25 | Immobilization of biologically active substances |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1059936A true CA1059936A (en) | 1979-08-07 |
Family
ID=10272097
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA230,056A Expired CA1059936A (en) | 1974-06-25 | 1975-06-24 | Immobilization of biologically active substances |
Country Status (14)
| Country | Link |
|---|---|
| US (2) | US4323650A (en) |
| JP (1) | JPS5912275B2 (en) |
| AU (1) | AU502784B2 (en) |
| BE (1) | BE830590A (en) |
| CA (1) | CA1059936A (en) |
| CH (1) | CH629251A5 (en) |
| DE (1) | DE2527884A1 (en) |
| DK (1) | DK148484C (en) |
| FR (1) | FR2276318A1 (en) |
| GB (1) | GB1514707A (en) |
| IE (1) | IE43057B1 (en) |
| IT (1) | IT1036359B (en) |
| NL (1) | NL7507575A (en) |
| ZA (1) | ZA753866B (en) |
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| ZA811104B (en) * | 1980-02-26 | 1982-03-31 | Tate & Lyle Ltd | Immobilized enzymes, a process for their preparation and their use in converting substrates to products |
| DE3021629A1 (en) * | 1980-06-09 | 1981-12-17 | C.H. Boehringer Sohn, 6507 Ingelheim | COIMMOBILISATES FROM GAERFAHFE YEARS WITH COUPLED ENZYMES AND THEIR PRODUCTION AND USE |
| EP0048109B1 (en) * | 1980-09-11 | 1985-11-06 | United Kingdom Atomic Energy Authority | Improvements in or relating to composite materials |
| DE3213074A1 (en) * | 1982-04-07 | 1983-10-20 | Linde Ag, 6200 Wiesbaden | METHOD AND DEVICE FOR BIOLOGICAL WASTE WATER TREATMENT |
| US4504582A (en) * | 1982-07-20 | 1985-03-12 | Genex Corporation | Vermiculite as a carrier support for immobilized biological materials |
| JPS5968344A (en) * | 1982-10-12 | 1984-04-18 | Agency Of Ind Science & Technol | Unsymmetrical functional membrane and production thereof |
| JPS602200A (en) * | 1983-06-20 | 1985-01-08 | Kunie Nakamura | Zymological diagnosticum for tumor |
| US4530905A (en) * | 1984-10-25 | 1985-07-23 | The Dow Chemical Company | Crosslinked gelatin foams |
| US4748121A (en) * | 1984-11-30 | 1988-05-31 | Ppg Industries, Inc. | Porous glass fibers with immobilized biochemically active material |
| US4861714A (en) * | 1985-04-04 | 1989-08-29 | Verax Corporation | Weighted collagen microsponge for immobilizing bioactive material |
| DE3515252C2 (en) * | 1985-04-27 | 1994-02-24 | Roehm Gmbh | Process for the immobilization of dissolved proteins |
| US5100783A (en) * | 1985-05-10 | 1992-03-31 | Verax Corporation | Weighted microsponge for immobilizing bioactive material |
| US4749653A (en) * | 1985-10-21 | 1988-06-07 | Owens-Corning Fiberglas Corporation | Enzyme immobilization on non-porous glass fibers |
| US4757008A (en) * | 1986-02-24 | 1988-07-12 | Miles Inc. | Enzyme immobilization in a macroporous non-ionic resin |
| JPH0418216Y2 (en) * | 1986-12-26 | 1992-04-23 | ||
| WO1989000446A1 (en) * | 1987-07-16 | 1989-01-26 | E.I. Du Pont De Nemours And Company | Affinity separating using immobilized flocculating reagents |
| GB2208649A (en) * | 1987-08-17 | 1989-04-12 | Vnii Biotekhnologii | Immobilization of fungal beta -galactosidase on an inorganic carrier |
| DK427987A (en) * | 1987-08-17 | 1989-02-18 | Vnii Biotekhnologii | PROCEDURE FOR IMMOBILIZATION OF FUNGAL BETA-GALACTOSIDASE |
| JPH01104661U (en) * | 1987-12-29 | 1989-07-14 | ||
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| US5614401A (en) * | 1993-06-23 | 1997-03-25 | Toyo Denka Kogyo Co., Ltd. | Enzyme immobilizing carrier containing kaolin |
| WO1997024177A1 (en) * | 1995-12-29 | 1997-07-10 | Allied Colloids Limited | Enzyme-containing particles and liquid detergent concentrate |
| US20020182184A1 (en) * | 1999-07-09 | 2002-12-05 | Pentagonal Holdings, Inc. | Composition for the safe removal of indoor allergens |
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| US9540631B1 (en) | 2004-09-14 | 2017-01-10 | Peter T. Pugliese | Immobilized glucose oxidase for use in oral hygiene |
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| US3705084A (en) * | 1970-03-18 | 1972-12-05 | Monsanto Co | Macroporous enzyme reactor |
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| US3736231A (en) * | 1971-11-01 | 1973-05-29 | Us Agriculture | Preparation of insolubilized enzymes |
| US3767531A (en) * | 1972-02-14 | 1973-10-23 | Us Agriculture | Preparation of insolubilized enzymes |
| GB1400468A (en) * | 1972-07-22 | 1975-07-16 | Beecham Group Ltd | Enzyme preparation and use thereof |
| US3804719A (en) * | 1972-08-07 | 1974-04-16 | Corning Glass Works | Adsorbing and crosslinking enzymes within the pores of porous glass |
| US3849253A (en) * | 1972-10-10 | 1974-11-19 | Penick & Ford Ltd | Process of immobilizing enzymes |
| US3850751A (en) * | 1973-02-16 | 1974-11-26 | Corning Glass Works | Enzymes immobilized on porous inorganic support materials |
-
1974
- 1974-06-25 GB GB28212/74A patent/GB1514707A/en not_active Expired
-
1975
- 1975-06-17 ZA ZA3866A patent/ZA753866B/en unknown
- 1975-06-19 AU AU82234/75A patent/AU502784B2/en not_active Expired
- 1975-06-23 DE DE19752527884 patent/DE2527884A1/en active Granted
- 1975-06-23 IT IT68620/75A patent/IT1036359B/en active
- 1975-06-24 JP JP50078541A patent/JPS5912275B2/en not_active Expired
- 1975-06-24 BE BE157639A patent/BE830590A/en not_active IP Right Cessation
- 1975-06-24 CA CA230,056A patent/CA1059936A/en not_active Expired
- 1975-06-24 IE IE1397/75A patent/IE43057B1/en unknown
- 1975-06-24 DK DK285875A patent/DK148484C/en active
- 1975-06-24 FR FR7519783A patent/FR2276318A1/en active Granted
- 1975-06-25 CH CH826075A patent/CH629251A5/en not_active IP Right Cessation
- 1975-06-25 NL NL7507575A patent/NL7507575A/en not_active Application Discontinuation
-
1979
- 1979-02-26 US US06/015,405 patent/US4323650A/en not_active Expired - Lifetime
-
1981
- 1981-08-11 US US06/291,958 patent/US4425434A/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| US4425434A (en) | 1984-01-10 |
| JPS5119185A (en) | 1976-02-16 |
| DK148484C (en) | 1985-12-16 |
| BE830590A (en) | 1975-12-24 |
| US4323650A (en) | 1982-04-06 |
| GB1514707A (en) | 1978-06-21 |
| IE43057L (en) | 1975-12-25 |
| ZA753866B (en) | 1977-01-26 |
| DE2527884C2 (en) | 1988-05-26 |
| AU502784B2 (en) | 1979-08-09 |
| IT1036359B (en) | 1979-10-30 |
| DK285875A (en) | 1975-12-26 |
| CH629251A5 (en) | 1982-04-15 |
| DK148484B (en) | 1985-07-15 |
| NL7507575A (en) | 1975-12-30 |
| IE43057B1 (en) | 1980-12-17 |
| FR2276318B1 (en) | 1979-06-22 |
| JPS5912275B2 (en) | 1984-03-22 |
| FR2276318A1 (en) | 1976-01-23 |
| AU8223475A (en) | 1976-12-23 |
| DE2527884A1 (en) | 1976-01-15 |
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