CA1229808A - Preparation of hydrophobic cotton cloth - Google Patents
Preparation of hydrophobic cotton clothInfo
- Publication number
- CA1229808A CA1229808A CA000462923A CA462923A CA1229808A CA 1229808 A CA1229808 A CA 1229808A CA 000462923 A CA000462923 A CA 000462923A CA 462923 A CA462923 A CA 462923A CA 1229808 A CA1229808 A CA 1229808A
- Authority
- CA
- Canada
- Prior art keywords
- enzyme
- aryl
- beta
- hydrophobic alkyl
- phenol
- 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
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M13/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment
- D06M13/10—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with non-macromolecular organic compounds; Such treatment combined with mechanical treatment with compounds containing oxygen
- D06M13/11—Compounds containing epoxy groups or precursors thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
- C12N11/12—Cellulose or derivatives thereof
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Textile Engineering (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
Novel hydrophobic cotton cloths of alkyl or aryl polyhydroxy com-pounds, e.g. butyl, hexyl, octyl, decyl, dodecyl, phenyl, naphthyl or anthranyl on which enzymes can be immobilized, and their process of prepara-tion and method of use are provided herein. The process involves reacting the polyhydroxy compound with a bifunctional compound and with an alcohol, or with a phenol, in a single step in an aqueous basic solution. An enzyme is then immobilized on the hydrophobic cloth by absorption of a solution of the enzyme thereon. The enzyme which is so immobilized is stabilized thereon by means of a cross-linking agent. Preferably the bifunctional compound is epichlorohydrin and the cross-linking agent is glutaraldehyde.
Novel hydrophobic cotton cloths of alkyl or aryl polyhydroxy com-pounds, e.g. butyl, hexyl, octyl, decyl, dodecyl, phenyl, naphthyl or anthranyl on which enzymes can be immobilized, and their process of prepara-tion and method of use are provided herein. The process involves reacting the polyhydroxy compound with a bifunctional compound and with an alcohol, or with a phenol, in a single step in an aqueous basic solution. An enzyme is then immobilized on the hydrophobic cloth by absorption of a solution of the enzyme thereon. The enzyme which is so immobilized is stabilized thereon by means of a cross-linking agent. Preferably the bifunctional compound is epichlorohydrin and the cross-linking agent is glutaraldehyde.
Description
This invention relates to a process for -the immobilization of en~ymes on an inert insoluble support, and to the support containing such i~nohilized en~ymes so produced.
En7ymes are ideal catalysts for the transformation of biological ,ubstances. Since transfonnation can be carried out under moderate con-ditions of pH and temperature and with high speci~icity, the process will generate products in higher yields with less use of energy and other chemi-cals, and is generally safer and less polluting than conventional chemical processes.
At the present time, enzymes ~ind industrial use in the produc-tion of glucose and dextrin from starch, wort modification in brewing, the clarification of beverages, and similar applications in the pro-essin~ of foods and bevetages. Other industrial uses of en~ymes include the textile and leather industries, the manufacture of paper and adhesives~ sewage disposal, garment cleaning, animal feedincJ, and chemical and pharmace~ltical applications. Most of these processes involve the production of relatively cheap products. Since enzymes are hi~hly efficient catalysts, their adaptation to such operations as the production of phar~ceutieals and petrochemicals and the treatment of domeskic and industrial effluents might well introduce a new technology with a per~ormance level superior to those now in use, both cost wise and in product quality.
The annual world pr~duc~ion of the en~y~es which ~re employed for the catalysis of che~ical reactions in the pharn~lceutical ~.nd nutrition inc'ustry is well over thousands o ~etric tons. Their more extensive use is above all hindered ~)y the act that they are di~ficult to preparc and their cost is high.
Besi~es the hiyh cost, the low stability and the un~on~!nical i- ~
method of application prevent the enzymes Erom being more extensively used on an industrial scale for the catalysis of chemical reac-tions. In a normal enzyme-catalyzed reaction, the enzyme is added to a solution as one of the reaction components. After the reaction has been completed, the enzyme is either re ved from the resulting mixture of products by a pro-cess which usually destroys its activity, or the en~yme remains in the mix-ture, usually together with inactivators which are added to stop the enzyme-catalyzed process. Methods which could be usecl for the recovery of the active enzymes are generally unsuitable from a technological point of view.
Therefore, the enzyme is used only once for the catalysis of the reaction and is thereafter discarded.
In order to attempt to recover such enzymes, it has become well known in the art to bond the enzyme chemically to an insoluble support member so that the enzyme is not lost during the process. Various support members for bondin~ enæymes thereto are well known in the art. In fact, the art is replete with various techniques Eor bonding a given enzyme to a support member. In general, some of such techniques simply re~uire that the desired enzyme be brought into contact with an active support member.
While such prior art immobilizing techniques result in a more efficient utilization of the enzyme they are generally characterized by certain inherent inefficiencies. For example, the so-bonded enzyme often exhibits a significantly reduced degree of activity. In addition, the enzyme may be substantially damaged during the bonding procedure which, obviously resuits in either an ineffective enzyme or one which exhibits a significantly re- -duced degree of activity.
It is there~ore known to attach the enzyme to an insoluole solid support either by adsorption or by covalent honds eit~ler directly or i indirectly via bridging groups. ~lowever, such insoll~le prep,arations suffer from a number of disadvantages. Firstly, being solids, they are subject to mechanical decay and eventually break ~p. Secondly, the incor-poration of enzymes onto the surface of the solid support, and thus the specific ~ctivity of the preparation, is often low. Thirdly, access of the substrate to the active site of the enzyme is often hindered. Attempts to improve the specific activity of such insoluble enzymes/polymer preparations by increasing the external surface area of the solid, re~uires a decrease in particle size of the solid preparation, rendering handiing and in par-ticular separation by filtration more difficult, and increasiny the internal surface area by making the particles more highly porous, produces particles with less mechanical strength.
In addition certain water-soluble enzyme-polymer complexes hav~
been disclosed, wherein the enzyme is bound either directly or indirectly via a bridging group to a water soluble polymeric support. These enzymes~
polymer comple~es are recoverable from the aqueous reaction medium by ultrafiltration. Moreover, ultrafiltration is a difficult and ex~ensive techni~ue, especially on a large scale and there~ore undesirable for industrial applications.
Depending upon the method oF immobilization employed, the immo-bilized en~ymes may be divided into the classes o~ bound, included, andcross-linked enzymes. 80und enzymes can be obtained by covalent bonding to active carriers, heteropolar bonding and/or van de Wa~l's exchange action on ion exchangers, as well as on absorben~s. Included enzymes are enzymes which are mechanically immobilized in cross-linked polymers and microcap-sules, as well as in regenera~ed cellulose derivatives. ~inally, enzymes can also be cross-linked with bifunctional low molecular wPight reagents and tnus made insoluble.
~ 3 -It is clready known superficially to hydrolyze tubular bodies made of nylon and chen to Eix enzymes on to the surface ~hereof by means ; of glutaraldehyde. However, this process is relatively laborious and cannot be applied to other materials.
Furthermore, it is known to bind enzymes onto the surfaces of formed bodies, for example, onto glass spheroids, by cross-llnking with bifunctional reagents, e.g., glutdraldehyde. A disadvantage of this method is that the "enzyme film" thus produced is very sensitive and the enzymatic activity is easily lost in the case of mechanical stressing due to denaturing of the enzyme. Furthermore, this method can only be applied with difficulty to carriers having hydrophobic surfaces.
In addition, it is known to coat a formed body with ~ material which is suitable as an enzyme carrier and suhsequently to carry out, adsorptively or co valently, the binding of the enzyme to the carrier in known manner.
Moreoverl since the prior ~ixing procedures must take place under precisely controlled conditions, especially with regard to pH and temperature, the production is made very difficult and the yields obtained are extremely low. Since an activated carrier must always be present, ther~ is the furtller problem that charged substrates or reactants are ad~
sorbed, the pH optimum of the enzyme is displaced and the .~ichealis con-stants are changed and, in ~he case of cross-linked enzymes, high losses of acti~ity due to irre~ersible denaturing. Furthermore, in some cases, fixing only takes place by adsorption so that the activity rapidly "bleeds away".
~ method by which the enzyme may be mechanically enclosed into a three-dimensional network of a highly cross-linked gel is frequently used for its fixation. The diffusion of ~he enzyme is then cletermined by the
En7ymes are ideal catalysts for the transformation of biological ,ubstances. Since transfonnation can be carried out under moderate con-ditions of pH and temperature and with high speci~icity, the process will generate products in higher yields with less use of energy and other chemi-cals, and is generally safer and less polluting than conventional chemical processes.
At the present time, enzymes ~ind industrial use in the produc-tion of glucose and dextrin from starch, wort modification in brewing, the clarification of beverages, and similar applications in the pro-essin~ of foods and bevetages. Other industrial uses of en~ymes include the textile and leather industries, the manufacture of paper and adhesives~ sewage disposal, garment cleaning, animal feedincJ, and chemical and pharmace~ltical applications. Most of these processes involve the production of relatively cheap products. Since enzymes are hi~hly efficient catalysts, their adaptation to such operations as the production of phar~ceutieals and petrochemicals and the treatment of domeskic and industrial effluents might well introduce a new technology with a per~ormance level superior to those now in use, both cost wise and in product quality.
The annual world pr~duc~ion of the en~y~es which ~re employed for the catalysis of che~ical reactions in the pharn~lceutical ~.nd nutrition inc'ustry is well over thousands o ~etric tons. Their more extensive use is above all hindered ~)y the act that they are di~ficult to preparc and their cost is high.
Besi~es the hiyh cost, the low stability and the un~on~!nical i- ~
method of application prevent the enzymes Erom being more extensively used on an industrial scale for the catalysis of chemical reac-tions. In a normal enzyme-catalyzed reaction, the enzyme is added to a solution as one of the reaction components. After the reaction has been completed, the enzyme is either re ved from the resulting mixture of products by a pro-cess which usually destroys its activity, or the en~yme remains in the mix-ture, usually together with inactivators which are added to stop the enzyme-catalyzed process. Methods which could be usecl for the recovery of the active enzymes are generally unsuitable from a technological point of view.
Therefore, the enzyme is used only once for the catalysis of the reaction and is thereafter discarded.
In order to attempt to recover such enzymes, it has become well known in the art to bond the enzyme chemically to an insoluble support member so that the enzyme is not lost during the process. Various support members for bondin~ enæymes thereto are well known in the art. In fact, the art is replete with various techniques Eor bonding a given enzyme to a support member. In general, some of such techniques simply re~uire that the desired enzyme be brought into contact with an active support member.
While such prior art immobilizing techniques result in a more efficient utilization of the enzyme they are generally characterized by certain inherent inefficiencies. For example, the so-bonded enzyme often exhibits a significantly reduced degree of activity. In addition, the enzyme may be substantially damaged during the bonding procedure which, obviously resuits in either an ineffective enzyme or one which exhibits a significantly re- -duced degree of activity.
It is there~ore known to attach the enzyme to an insoluole solid support either by adsorption or by covalent honds eit~ler directly or i indirectly via bridging groups. ~lowever, such insoll~le prep,arations suffer from a number of disadvantages. Firstly, being solids, they are subject to mechanical decay and eventually break ~p. Secondly, the incor-poration of enzymes onto the surface of the solid support, and thus the specific ~ctivity of the preparation, is often low. Thirdly, access of the substrate to the active site of the enzyme is often hindered. Attempts to improve the specific activity of such insoluble enzymes/polymer preparations by increasing the external surface area of the solid, re~uires a decrease in particle size of the solid preparation, rendering handiing and in par-ticular separation by filtration more difficult, and increasiny the internal surface area by making the particles more highly porous, produces particles with less mechanical strength.
In addition certain water-soluble enzyme-polymer complexes hav~
been disclosed, wherein the enzyme is bound either directly or indirectly via a bridging group to a water soluble polymeric support. These enzymes~
polymer comple~es are recoverable from the aqueous reaction medium by ultrafiltration. Moreover, ultrafiltration is a difficult and ex~ensive techni~ue, especially on a large scale and there~ore undesirable for industrial applications.
Depending upon the method oF immobilization employed, the immo-bilized en~ymes may be divided into the classes o~ bound, included, andcross-linked enzymes. 80und enzymes can be obtained by covalent bonding to active carriers, heteropolar bonding and/or van de Wa~l's exchange action on ion exchangers, as well as on absorben~s. Included enzymes are enzymes which are mechanically immobilized in cross-linked polymers and microcap-sules, as well as in regenera~ed cellulose derivatives. ~inally, enzymes can also be cross-linked with bifunctional low molecular wPight reagents and tnus made insoluble.
~ 3 -It is clready known superficially to hydrolyze tubular bodies made of nylon and chen to Eix enzymes on to the surface ~hereof by means ; of glutaraldehyde. However, this process is relatively laborious and cannot be applied to other materials.
Furthermore, it is known to bind enzymes onto the surfaces of formed bodies, for example, onto glass spheroids, by cross-llnking with bifunctional reagents, e.g., glutdraldehyde. A disadvantage of this method is that the "enzyme film" thus produced is very sensitive and the enzymatic activity is easily lost in the case of mechanical stressing due to denaturing of the enzyme. Furthermore, this method can only be applied with difficulty to carriers having hydrophobic surfaces.
In addition, it is known to coat a formed body with ~ material which is suitable as an enzyme carrier and suhsequently to carry out, adsorptively or co valently, the binding of the enzyme to the carrier in known manner.
Moreoverl since the prior ~ixing procedures must take place under precisely controlled conditions, especially with regard to pH and temperature, the production is made very difficult and the yields obtained are extremely low. Since an activated carrier must always be present, ther~ is the furtller problem that charged substrates or reactants are ad~
sorbed, the pH optimum of the enzyme is displaced and the .~ichealis con-stants are changed and, in ~he case of cross-linked enzymes, high losses of acti~ity due to irre~ersible denaturing. Furthermore, in some cases, fixing only takes place by adsorption so that the activity rapidly "bleeds away".
~ method by which the enzyme may be mechanically enclosed into a three-dimensional network of a highly cross-linked gel is frequently used for its fixation. The diffusion of ~he enzyme is then cletermined by the
2~
density of the net~ork of the surrounding gel. Such ma-terials do not possess very satisfactory mechanical properties and their biological activity is strongly influenced by the type and cross-linking of the sur-roundinq gel. Cellulose gels and dextran gels have been used for such purpose. Agarose gel has also been utilized as a similar carrier. The cellulosic material, however, tends to pass into the reaction solution by solubilization during continuous use and, hence, lacks in durability; it is also difficult to immobilize a large amount of enzyme on a unit weight of the cellulosic carrier. Although a cross-linked dextran (dextran gel) and an agarose gel can immobilize a relatively large amount of enzyme per unit weight, yet it is difficult directly to obtain these materials having desired degree of polymerization. For this reason, the dextran gel and agarose gel are very expensive, which discourages the use of these ~terials as enzyme~immobilizing carriers on a commercial scale.
A disadvantage of the immobilized enzymes is that carriers suitable for fixing frequently have inadequate mechanical properties and either cannot be worked up to glve formed bodies with enzymatically-active surfaces or can only be so worked up wlth great difficulty. In principle, only those carriers based on synthetic resins, biopolymers or inorganic substances can be used in which reactive groups are present or can be pro-duced and which, if possible, contain hydrophilic groups or centres. On the other hand, however, for many fields of use for immobilized enzymes, mechanically stable formed bodies are necessary. Thus, for example, membranes, films, tubes and other formed bodies with enzymatically-actlve surfaces are much more suitable for many purposes than the conventional granula~es and permit, for example, a much higher flowthrough rate of the substrate solu-tion, a reduced diffusion and the use of solid bed reactors. In the field of chemical analysis, too, as well as in therapeutic medicine, enzymatically-active formed bodies of this type would be very desirable.
The velocity of the enzymatically cataly2ed reaction during the passage of a solution of substrate through a column packed with the gel chemically bound with an en~yme, is not only influenced by the type and activity of the enzyme, but also by the character of the en~yme, such as carrier linkage, physico-chemical properties of the carrier (porosity, surface area), hydrodynamic parameters of the e~uipment, and flow-rate of the substrate solution. A simultaneous or subsequent ac-tion of several enzymes may be achieved either by mixing or stratifying pacXinq beds or by connecting in series columns containing different insoluble enzymes.
The following materials have also been used as the carriers of enzymes: porous glass, cellulose and cellulose derivativest starch, derivatives of cross-linked polystyrene, copolymers o~ ethylene and maleic anhydride, polyacrylamide and polysaccharides. ~ecause of their relatively high porosity in a swollen state, the latter compounds are often used as carrier. Except for porous glass and polystyrene~ most of the gels men-tioned are noted for their low mechanical stability and the operation under pressure re~uired for higher flow rates through the gel column is ~xcluded.
A low hydrolytic stability of the materials recently used is often an obstacle and such cases are Xnown when the reaction of the substrate ~ith a biologically active material bound to the carrier is complicated by an insufficieht hy~lrophilic character of the ~eJ surface of by non-specific sorptions at the gel matrix~ In addition, highly cross-linked polysacchar-ides or polyacrylamides cannot be prepared in the form o~ discrete particles in a dry state.
The immobilization of enæymes on such inert insoluble supports is the subject of many patent. For example, Canadian Patent 881,483 issued September 21, 1971 to S~ ~lopps provides untreated tissue paper merely im-pregnated with a small amount of an enzyme. For example, a convenient paper according to that patent may consist of a toilet tissue which has been impregnated with 1% by weight of a mixture of enzymes, an adhesive agent, a deordorant, and a neutralizing agent.
Canadian Patent 999,820 issued November 16, 1976 to Ceskoslovanska akademie ved provides insoluble enzymes in the form of activated gels, e.g. methacrylates activated by diisocyanates treated with the enzyme, and chemically bound thereto. Thus the patentee provides a process for the preparation of insoluble enzymes on polymeric homogeneous or heterogeneous macroporous gels, in which the surface of the ~els, which were prepared by coopolymerization of hydrophilic nomers, with divinyl or polyvinyl mono-mers are activated by compounds which react with th~ hydroxyl or amino func~
tional group. The gels activated in this way are treated with the soluble enzymes, which react with the hydroxyl OI amino groups o~ the gel. The enzymes are thus chemically bound to the polymeric material and are said to retain their chemical and biological reactivity.
Canadian Patent 1,~32,882 issued June 13, 1979 to the Carborund~un Company provided a porous cartridge, fonned o~ a fibrous or bonded particular material, e.g. formed by winding a permeable fibrous material around a central core with an enzyme deposi~ed thereon from an en~yme-con~ainin~
fluid. Thus~ the patentee provided a mechanically self-sustaining and fixed enzyme supportin~, depth-type cartrid~e comprising an annular and ~A>~
porous body of permeable fibrous filter material wo~md around a center core and having pores throughout with average pore diameters ranging from about 0.1 micrometer to about 100 micrometers and at least one enzymatic substance fixedly associated with the body by attachment to the permeable fibrous filter material, for enzymatic treatment of fluid substrates ~low-ing through the cartridge. The patentee taught furthermore that the fibres or particulate materials within the permeable cartridge body may be treated with appropriate coupling agents, selected to allow a covalent attachment with the desired en~ymatic substance.
Canadian Patent 1,036,965 issued August 22, 1~78 to The Ohio State University Research Foundation provided an immobilized enzyme by first ~ix-ing an enzyme with a substrate to form an enzyme with a substrate to form an enzyme-substrate complex and then contacting the en7yme-substrate complex with an active support men~er to ~ransfer the enzyme to the support member!
and then removing the substrate.
Canadian Patent 1,053,5~5 issued ~ay 1, 1979 to Beecham Group Limited provided an immobilized enzyme, preferable supported on a cellulose derivative, attached to a non-polar group, in contact with an inert water-i~iscible liquid. The patentee discovered that certain en7~es could be attached to a non polar group to render the preparation separable from aqueous media by virtue of the affinity for water-imiscible liquids.
The enzyme could be attached to a polymeric support, for example, cellulose powder and cellulose derivatives such as carboxymethyl-cellulose powder and cellulose derivatives such as carboxymethyl-cellulose ion exchange resins, nylon, high ~olecular polysaccharides such as agarose and cross-linked dex-trans; polysaccharides modified ~ith modi~ying agents sucn as epichlorhy-drin or modified to incorporate carboxymethyl or aminoethyl groups; poly-acrylates and polymethacrylates.
Canadian Patent 1,054,079 issuecl May 8, 1979 to Boehringer ~nnheim GmbH provided an im~obilized enzyme on the fonn of a formed body, e.g. of glass or synthetic polymers coated with an adhesive, e.g. a rubber adhesive, to which is bonded an enzyme which was immobilized on a solid carrier, e.g. a synthetic resin. Such formed bodies can consist of any desired organic or inorganic material and can be of any desired shape.
Canadian Patent 1,060,366 issued August 1~, 1979 to Exxon Research ~ Engineering Company provided immobilized en~ymes prepared by ~irst con-tacting the enzyme with an oxidizing agent, and then contacting the product with an amino~containing material.
Canadian Patent 1,093,991 issued January 20, 1981 to Sumitomo Chemical Company provided an enzynle immobili~ed on a pullulan gel in bead form by means oE cross-linking with epichlorohydrin. The patent was based on the discovery that an immobili~.ed enzyme could be obtained by using, as the carrier, a hydrophilic gel prepared by cross-linking pullulan or by using ionic pullulan gel as the carrier.
As described hereinabove, neu-tral hydrophobic derivatives of agarose ha~e been prepared by coupling alcohols or phenols via stable ether linkages. Since s~ch pxoced~lres required the a~arose to be swollen 2~ in nonaqueous solvents, the agarose had to be washed with an extensive series oE solvents of decreasing polarity. The synthesis rt~g~ired a series of steps and a variety of organic reagents. After these reactions, the agarose had to ~é washed with a series of solvents of increasing polarity. To increase the physical and chemical stability of the gel, cross-linked agarose WaS used in the co~mercial preparation of hydrophobic media. The cost o~ the cross-linked agarose and preparation of these hydrophobic media limits their application to co~nercial biotechnologicaa processes .
density of the net~ork of the surrounding gel. Such ma-terials do not possess very satisfactory mechanical properties and their biological activity is strongly influenced by the type and cross-linking of the sur-roundinq gel. Cellulose gels and dextran gels have been used for such purpose. Agarose gel has also been utilized as a similar carrier. The cellulosic material, however, tends to pass into the reaction solution by solubilization during continuous use and, hence, lacks in durability; it is also difficult to immobilize a large amount of enzyme on a unit weight of the cellulosic carrier. Although a cross-linked dextran (dextran gel) and an agarose gel can immobilize a relatively large amount of enzyme per unit weight, yet it is difficult directly to obtain these materials having desired degree of polymerization. For this reason, the dextran gel and agarose gel are very expensive, which discourages the use of these ~terials as enzyme~immobilizing carriers on a commercial scale.
A disadvantage of the immobilized enzymes is that carriers suitable for fixing frequently have inadequate mechanical properties and either cannot be worked up to glve formed bodies with enzymatically-active surfaces or can only be so worked up wlth great difficulty. In principle, only those carriers based on synthetic resins, biopolymers or inorganic substances can be used in which reactive groups are present or can be pro-duced and which, if possible, contain hydrophilic groups or centres. On the other hand, however, for many fields of use for immobilized enzymes, mechanically stable formed bodies are necessary. Thus, for example, membranes, films, tubes and other formed bodies with enzymatically-actlve surfaces are much more suitable for many purposes than the conventional granula~es and permit, for example, a much higher flowthrough rate of the substrate solu-tion, a reduced diffusion and the use of solid bed reactors. In the field of chemical analysis, too, as well as in therapeutic medicine, enzymatically-active formed bodies of this type would be very desirable.
The velocity of the enzymatically cataly2ed reaction during the passage of a solution of substrate through a column packed with the gel chemically bound with an en~yme, is not only influenced by the type and activity of the enzyme, but also by the character of the en~yme, such as carrier linkage, physico-chemical properties of the carrier (porosity, surface area), hydrodynamic parameters of the e~uipment, and flow-rate of the substrate solution. A simultaneous or subsequent ac-tion of several enzymes may be achieved either by mixing or stratifying pacXinq beds or by connecting in series columns containing different insoluble enzymes.
The following materials have also been used as the carriers of enzymes: porous glass, cellulose and cellulose derivativest starch, derivatives of cross-linked polystyrene, copolymers o~ ethylene and maleic anhydride, polyacrylamide and polysaccharides. ~ecause of their relatively high porosity in a swollen state, the latter compounds are often used as carrier. Except for porous glass and polystyrene~ most of the gels men-tioned are noted for their low mechanical stability and the operation under pressure re~uired for higher flow rates through the gel column is ~xcluded.
A low hydrolytic stability of the materials recently used is often an obstacle and such cases are Xnown when the reaction of the substrate ~ith a biologically active material bound to the carrier is complicated by an insufficieht hy~lrophilic character of the ~eJ surface of by non-specific sorptions at the gel matrix~ In addition, highly cross-linked polysacchar-ides or polyacrylamides cannot be prepared in the form o~ discrete particles in a dry state.
The immobilization of enæymes on such inert insoluble supports is the subject of many patent. For example, Canadian Patent 881,483 issued September 21, 1971 to S~ ~lopps provides untreated tissue paper merely im-pregnated with a small amount of an enzyme. For example, a convenient paper according to that patent may consist of a toilet tissue which has been impregnated with 1% by weight of a mixture of enzymes, an adhesive agent, a deordorant, and a neutralizing agent.
Canadian Patent 999,820 issued November 16, 1976 to Ceskoslovanska akademie ved provides insoluble enzymes in the form of activated gels, e.g. methacrylates activated by diisocyanates treated with the enzyme, and chemically bound thereto. Thus the patentee provides a process for the preparation of insoluble enzymes on polymeric homogeneous or heterogeneous macroporous gels, in which the surface of the ~els, which were prepared by coopolymerization of hydrophilic nomers, with divinyl or polyvinyl mono-mers are activated by compounds which react with th~ hydroxyl or amino func~
tional group. The gels activated in this way are treated with the soluble enzymes, which react with the hydroxyl OI amino groups o~ the gel. The enzymes are thus chemically bound to the polymeric material and are said to retain their chemical and biological reactivity.
Canadian Patent 1,~32,882 issued June 13, 1979 to the Carborund~un Company provided a porous cartridge, fonned o~ a fibrous or bonded particular material, e.g. formed by winding a permeable fibrous material around a central core with an enzyme deposi~ed thereon from an en~yme-con~ainin~
fluid. Thus~ the patentee provided a mechanically self-sustaining and fixed enzyme supportin~, depth-type cartrid~e comprising an annular and ~A>~
porous body of permeable fibrous filter material wo~md around a center core and having pores throughout with average pore diameters ranging from about 0.1 micrometer to about 100 micrometers and at least one enzymatic substance fixedly associated with the body by attachment to the permeable fibrous filter material, for enzymatic treatment of fluid substrates ~low-ing through the cartridge. The patentee taught furthermore that the fibres or particulate materials within the permeable cartridge body may be treated with appropriate coupling agents, selected to allow a covalent attachment with the desired en~ymatic substance.
Canadian Patent 1,036,965 issued August 22, 1~78 to The Ohio State University Research Foundation provided an immobilized enzyme by first ~ix-ing an enzyme with a substrate to form an enzyme with a substrate to form an enzyme-substrate complex and then contacting the en7yme-substrate complex with an active support men~er to ~ransfer the enzyme to the support member!
and then removing the substrate.
Canadian Patent 1,053,5~5 issued ~ay 1, 1979 to Beecham Group Limited provided an immobilized enzyme, preferable supported on a cellulose derivative, attached to a non-polar group, in contact with an inert water-i~iscible liquid. The patentee discovered that certain en7~es could be attached to a non polar group to render the preparation separable from aqueous media by virtue of the affinity for water-imiscible liquids.
The enzyme could be attached to a polymeric support, for example, cellulose powder and cellulose derivatives such as carboxymethyl-cellulose powder and cellulose derivatives such as carboxymethyl-cellulose ion exchange resins, nylon, high ~olecular polysaccharides such as agarose and cross-linked dex-trans; polysaccharides modified ~ith modi~ying agents sucn as epichlorhy-drin or modified to incorporate carboxymethyl or aminoethyl groups; poly-acrylates and polymethacrylates.
Canadian Patent 1,054,079 issuecl May 8, 1979 to Boehringer ~nnheim GmbH provided an im~obilized enzyme on the fonn of a formed body, e.g. of glass or synthetic polymers coated with an adhesive, e.g. a rubber adhesive, to which is bonded an enzyme which was immobilized on a solid carrier, e.g. a synthetic resin. Such formed bodies can consist of any desired organic or inorganic material and can be of any desired shape.
Canadian Patent 1,060,366 issued August 1~, 1979 to Exxon Research ~ Engineering Company provided immobilized en~ymes prepared by ~irst con-tacting the enzyme with an oxidizing agent, and then contacting the product with an amino~containing material.
Canadian Patent 1,093,991 issued January 20, 1981 to Sumitomo Chemical Company provided an enzynle immobili~ed on a pullulan gel in bead form by means oE cross-linking with epichlorohydrin. The patent was based on the discovery that an immobili~.ed enzyme could be obtained by using, as the carrier, a hydrophilic gel prepared by cross-linking pullulan or by using ionic pullulan gel as the carrier.
As described hereinabove, neu-tral hydrophobic derivatives of agarose ha~e been prepared by coupling alcohols or phenols via stable ether linkages. Since s~ch pxoced~lres required the a~arose to be swollen 2~ in nonaqueous solvents, the agarose had to be washed with an extensive series oE solvents of decreasing polarity. The synthesis rt~g~ired a series of steps and a variety of organic reagents. After these reactions, the agarose had to ~é washed with a series of solvents of increasing polarity. To increase the physical and chemical stability of the gel, cross-linked agarose WaS used in the co~mercial preparation of hydrophobic media. The cost o~ the cross-linked agarose and preparation of these hydrophobic media limits their application to co~nercial biotechnologicaa processes .
3~8 While cellulose is inexpensive, and chemically and physically stable, it has not heretofore been used as a carrier for immobilizing enzymes. Perhaps this was because a column packed with the fibrous form of its derivatives tended to exhibit high hydrodynamic resistance due to compaction and clogging by fine fibres.
For successful industrial applications, improved methods and apparatus for supporting the enzyme-bearing matrix and assuring adequate contact with the substrate materials are desirable. This is especially so in applications where the substrate solution or suspension is passed over or through the enzyme containing matrix. Moreover, it would be desirable to pr~vide:enzymes which could be easily separated from products and be reused; immobilized enzymes which would exhibit greater stability; and immobilized enzymes packed in columns (a "bioreactor") whereby continuous, controlled and sequential operations would be possible.
An object of one aspect of the presen-t invention is to provide a simple process for preparing an immobilized enzyme which is stable and of high enzymatic activity.
~ n object of another aspect of the present invention is to pro-vide a process for preparing an immobilized enzyme which can be utilized in such an efficient way that it permits of an enzymatically catalyzed continuous reaction of the substrate.
An object of yet another aspect of the present invention is to provide a method for immobilizing an enzyme by bonding it to ~ suitable sup-port member in such a manner that the bonded enzyme exhibits a suitable high degree of activity.
B
An ob~ec~ of an additional aspect of the present invention is to provide a method of immobilizing an enzyme by bondiny it to a support member without significantly damaging the enzyme during the bonding or immobili~ing procedure.
An object of yet another aspect of the present invention is to provide a support with an enzymatically-active surface which can be used as widely as possible in analytical and preparative chemistry and, on the other hand, can be produced in a simple manner.
By a br~d aspec~ or ~x~ent of this inventiont a process ispro~ ~d for preparing a hydrophobic alkyl or aryl polyhydroxy compound suitable for use as a carrier to im~obili~e enzymes thereon, the process comprisin~ reactiny the polyhydroxy compound with a bifunctional con~ound and with a phenol or with an alcohol in a single step in an aqueous, basic solution.
By~mother broad as~ct or H~x~ ent of this invention, a processis pro~ided for preparing hydrophobic alkyl or aryl cellulose suitable for use as a carrier to immobili~e enzymes thereon, the process comprising reacting the cellulose with a bifunctional compound and with a phenol or with an alcohol in a single step in an aqueous basic solution.
By y~t al~t~r broad as~ct or e~di~ent o~this invention, a pr~ess is provided for preparing hydrophobic alkyl or aryl cotton cloth suitable for use as a carrier to immobilize enzymes thereon, the process co~prisiny reacting the cotton cloth with a bi~unctional compound and with a phenol or with ~n alcohol in a single step in an ~queous basic solution.
The bi-~unctional co~pound is selecred from the group consisting of epichlorohydrin, epibromohydrin, dichlorohy-drin, dibromohydrin, e~hylene glycol diglycidyl ether, triethylene glycol diqlycidyl ether, diqlycidyl ether~ and 1,6-hexanediol diglycidyl et~er, but preferably is epichlorohydrin.
The aqueous basic solution may be provided by a solution of sodium hydroxide or of potassium hydroxide. The concentration of such basic compound is generally 4 to 5 M.
By one embodiment of the process of the invention, the reaction is carried out at an eleva~ed temperature of up to 100C for a period oEc I to 24 hours.
In the reactiorl, the phenol may be phenol, ~ -naphthol or anthranol. The alcohol may be butyl, hexyl, octyl, decyl, or dodecyl alcohols.
By another aspect or embodiment of this invention, a hydrophobic alkyl or aryl polyhydroxy compound is providtd. Preferably, this takes the form of a hydrophobic alkyl or aryl cellulose. Most preferably, a hylrophobic alkyl or aryl cotton cloth is prcvidecl.
In such product, the alkyl group may be butyl, hexyl, octyl, decyl or dodecyl, and the aryl group may be phenyl, naphthyl or anchranyl.
By yet another aspect or embodilllent oc this inventiorl, a pro-cess is provided for immobilizing an enzyme on a polyhydroxy compound which comprises: reacting the polyhydroxy compouncl with a biEunctional compound and with an alcohol, or with a phenol in a single step in an aqueous basic solution, immobilizing an enzyme thereon by absorption of a solution of the enzyme thereon; and stabilizing the immobili2ed enzyme thereon by reaction with a cross-linking agent.
By still another aspect or embodiment:, a process is provided for immobilizinc3 an en2yme or cellulose which comp~ises reacting ~he cellulose with a bifunctional compound and wi-th a phenol or with ~n alcohol in a single step in an aqueous basic solution; immobili~in~ an enzyme thereon by absorption of a sclution of the enzyme thereonj and stabilizing the immobilized enzyme thereon by reaction with a cross-li~cing agent.
~ y yet ~nother aspect of this invention, a process is provided for imnobilizing an enzyme on cotton cloth which comprises reacting the cotton cloth with a bi~unction~l compound and with a phenol or with an alcohol in a single step in an ac~ueous basic solution; i~obilizing ~n enzyme thereon by absorption of a solution of the unzyme rhereon; on stabili~ing the immobilized en~yme thereon by reaction~lith a cross-linking agent. In the above eMbodiments, the cross-linking agent is preEerably glutaralde-hyde.
The enzyme selected Eor these embodiments oE this invention may be any of the major industrial en~ymes, e.g. alcohol dehyciro~enases, c~ -amylase (E.C. 3.2.1.1), ~ -amylase (E.C. 3.2.1.1), ~ -amy1ase or glusoamylase (E.C. 3.2.1.3), asparaginase (E.C. 3.5.1.1), aspartase (E.C.
For successful industrial applications, improved methods and apparatus for supporting the enzyme-bearing matrix and assuring adequate contact with the substrate materials are desirable. This is especially so in applications where the substrate solution or suspension is passed over or through the enzyme containing matrix. Moreover, it would be desirable to pr~vide:enzymes which could be easily separated from products and be reused; immobilized enzymes which would exhibit greater stability; and immobilized enzymes packed in columns (a "bioreactor") whereby continuous, controlled and sequential operations would be possible.
An object of one aspect of the presen-t invention is to provide a simple process for preparing an immobilized enzyme which is stable and of high enzymatic activity.
~ n object of another aspect of the present invention is to pro-vide a process for preparing an immobilized enzyme which can be utilized in such an efficient way that it permits of an enzymatically catalyzed continuous reaction of the substrate.
An object of yet another aspect of the present invention is to provide a method for immobilizing an enzyme by bonding it to ~ suitable sup-port member in such a manner that the bonded enzyme exhibits a suitable high degree of activity.
B
An ob~ec~ of an additional aspect of the present invention is to provide a method of immobilizing an enzyme by bondiny it to a support member without significantly damaging the enzyme during the bonding or immobili~ing procedure.
An object of yet another aspect of the present invention is to provide a support with an enzymatically-active surface which can be used as widely as possible in analytical and preparative chemistry and, on the other hand, can be produced in a simple manner.
By a br~d aspec~ or ~x~ent of this inventiont a process ispro~ ~d for preparing a hydrophobic alkyl or aryl polyhydroxy compound suitable for use as a carrier to im~obili~e enzymes thereon, the process comprisin~ reactiny the polyhydroxy compound with a bifunctional con~ound and with a phenol or with an alcohol in a single step in an aqueous, basic solution.
By~mother broad as~ct or H~x~ ent of this invention, a processis pro~ided for preparing hydrophobic alkyl or aryl cellulose suitable for use as a carrier to immobili~e enzymes thereon, the process comprising reacting the cellulose with a bifunctional compound and with a phenol or with an alcohol in a single step in an aqueous basic solution.
By y~t al~t~r broad as~ct or e~di~ent o~this invention, a pr~ess is provided for preparing hydrophobic alkyl or aryl cotton cloth suitable for use as a carrier to immobilize enzymes thereon, the process co~prisiny reacting the cotton cloth with a bi~unctional compound and with a phenol or with ~n alcohol in a single step in an ~queous basic solution.
The bi-~unctional co~pound is selecred from the group consisting of epichlorohydrin, epibromohydrin, dichlorohy-drin, dibromohydrin, e~hylene glycol diglycidyl ether, triethylene glycol diqlycidyl ether, diqlycidyl ether~ and 1,6-hexanediol diglycidyl et~er, but preferably is epichlorohydrin.
The aqueous basic solution may be provided by a solution of sodium hydroxide or of potassium hydroxide. The concentration of such basic compound is generally 4 to 5 M.
By one embodiment of the process of the invention, the reaction is carried out at an eleva~ed temperature of up to 100C for a period oEc I to 24 hours.
In the reactiorl, the phenol may be phenol, ~ -naphthol or anthranol. The alcohol may be butyl, hexyl, octyl, decyl, or dodecyl alcohols.
By another aspect or embodiment of this invention, a hydrophobic alkyl or aryl polyhydroxy compound is providtd. Preferably, this takes the form of a hydrophobic alkyl or aryl cellulose. Most preferably, a hylrophobic alkyl or aryl cotton cloth is prcvidecl.
In such product, the alkyl group may be butyl, hexyl, octyl, decyl or dodecyl, and the aryl group may be phenyl, naphthyl or anchranyl.
By yet another aspect or embodilllent oc this inventiorl, a pro-cess is provided for immobilizing an enzyme on a polyhydroxy compound which comprises: reacting the polyhydroxy compouncl with a biEunctional compound and with an alcohol, or with a phenol in a single step in an aqueous basic solution, immobilizing an enzyme thereon by absorption of a solution of the enzyme thereon; and stabilizing the immobili2ed enzyme thereon by reaction with a cross-linking agent.
By still another aspect or embodiment:, a process is provided for immobilizinc3 an en2yme or cellulose which comp~ises reacting ~he cellulose with a bifunctional compound and wi-th a phenol or with ~n alcohol in a single step in an aqueous basic solution; immobili~in~ an enzyme thereon by absorption of a sclution of the enzyme thereonj and stabilizing the immobilized enzyme thereon by reaction with a cross-li~cing agent.
~ y yet ~nother aspect of this invention, a process is provided for imnobilizing an enzyme on cotton cloth which comprises reacting the cotton cloth with a bi~unction~l compound and with a phenol or with an alcohol in a single step in an ac~ueous basic solution; i~obilizing ~n enzyme thereon by absorption of a solution of the unzyme rhereon; on stabili~ing the immobilized en~yme thereon by reaction~lith a cross-linking agent. In the above eMbodiments, the cross-linking agent is preEerably glutaralde-hyde.
The enzyme selected Eor these embodiments oE this invention may be any of the major industrial en~ymes, e.g. alcohol dehyciro~enases, c~ -amylase (E.C. 3.2.1.1), ~ -amylase (E.C. 3.2.1.1), ~ -amy1ase or glusoamylase (E.C. 3.2.1.3), asparaginase (E.C. 3.5.1.1), aspartase (E.C.
4.3.1.1), catalase (E.C. l.ll.l.6), cellobiase (E.C. 3.2~1.21)~ cellu~se (E.C. 3.2.1.4), chloride peroxidase (E.C. 1.11.1.10), dextranase (E.C.
3.2.1.11), ~ -galactosidase (E.C. 3.2.1.22), ~ -galactosidase or lac-tase (E.C. 3.2.1.23), ~ -glucanase (E.C. 3.2.1.6), glucose or xylose isomerase (E.C. 5.3.1.5), glucose cxidase (E.C. 1.1.3.4), hemicellulase (E.G.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), sLeapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.2l.7), pectinase (E.C. 3.2.1.15j, phenol oxidases, ribon~lc1eases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline pro~eases, papain ~E.C. 3.4.22.2), Eicin (E.C. 3.4.22.3), bromelain (E.~. 3.4.22.4), pepsin (E.C. 3.4.23.1), _ 13 -chymosin (E.C. 3.4.23.4), microbial proteases (E.C. 3.4.23.6), micro-bial metallo proteases (E.C. 3.4.24.4)1 pullunase (E.C. 3.2.1.41j, ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tannase ~E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.7.303), and xylanase (E.C. 3.2.1.32).
By preferred embodiments, the enzyme is ~ -galactosidase or -glucosidase.
In variations of these embodiments of the invention, where the cross-linking agent is glutaraldehyde and~or the enzyme is ~ -galactosi-dase or ~ -glucosidaset the following may be pro~ided: the bifunctional compound is epichlorohydri~; the a~ueous basic solution is provided by a solution of sodium hydroxide or of potassium hydroxide, having a concen-tration of 4 to 5 M; the reaction is carried out at an elevated tempera-ture of up to 100 C Eor a period of I to 24 hours; the alcohol is one oE
butyl, hexyl, octyl, decyl, or dodecyl alcohols; and che phenol is phenol~
-naphi~hol or anthranol.
By another aspect or embodiment of this invention, a novel enzyme is pro~ided, na~ely one which is immobilized on a hydrophobic aikyl or aryl polyhydroxy compound, preferably one which is immobilized on a hydrophobic alkyl or aryl cellulose, and still more desirably, one which is immobilized on a hydrophobic alkyl or aryl cotton cloth.
In such novel enzyme, the alkyl group may be butyl, hexyl, octyl, decyl or dodecyl, and the aryl group may be phenyl, naphthyl or anthranyl.
The enzyme selected for these embodiments o~ ~his invention m~y be any of the m~or industrial enzymes, e.g. alcohol dehydrogenases, c~ -amylase ~E.C. 3.2.1.1), ~ -amylase (E.C. 3.2.1.1~ amylase or gluc~amylase (E.C. 3.2.1.3), asparaginase ~E.C. 3.5.1.1), aspartase ~E.C.
4.3.1.1), catalase (E.C. 1.11.1.6), cellobiase (E.C~ 3.2.1.21), celluL~se (E.C. 3.2.1.4), chloride peroxldase ~E.C. 1.11.1.10), dextran,ise ~E.C.
_ 1 4 -12~ P~3 3.2.1.11), ~ -galactosidase (E.C. 3.2.1.22), ~ -galactosldase or lac-tase (E.C. 3.2.1.23), ~ -glucanase (E.C. 3.2.1.6), glucose ~r xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E.C. 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papair! (E.C. 3.4.22.2), ~icin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), 1~) chymosln (E.C. 3.4.~3.4), mlcroblal proteases (E.C. 3.4.23.6), micro-bial me~allo proteases (E.C. 3.4.24.4), pullunase (E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6)9 tannase (E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.7.3O3),and xylanase (E.C. 3.2.1.32).
Preferably, the enzytne may be g -galactosidase or ~ -glucosidase.
By yet another aspect or embodiment of this invention, a method is provided for carrying out an enzyrne-catalyzed reaction compris-ing: packing a column with an enzyme which has been immobilized on a hydro-phobic alkyl or aryl polyhydroxy compound, namely with an enzyme which has been immobilized on a hydrophobic alkyl or aryl celllulose, prefera-bly with an enzyme which has been immobilized on a hydrophobic alkyl or aryl cotton cloth; and passing a solution of the material on which that enzyme catalyzed reaction is to take place through the column.
By one embodiment of such compound on which the enzyme has been immobilized, the alkyl group may be butyl, hexyl, octyl, decyl, or dodecyl, and the aryl group is phenyl, naphthyl or anthranyl.
The enzyme selected for these embodiments of this Invention may be any of the major industrial enzymes, e.g. alcohol dehydrogenases.
c~ -amylase (E.C. 3.2.1.1), ~ -amylase (E.C. 3.2.1.1), ~ -amylase or gluc~amylase (E.C. 3.2.1.3), asparaginase (E.C. 3.5.1.1), aspartase (E.C.
4.3.1.1), catalase (E.C. I.ll.l.o), cellobiase (E.C. 3.2.1.21), cellu~se (E.C. 3.2.1.4), chloride peroxidase (E~C. 1.11.1.10), dextranase (E.C.
3.2.1.11), ~ -galactosidase (E.C. 3.2.1.22), ~ -galactosidase or lac-tase (E.C. 3.2.1.23), ~ -glucanase (E.C. 3.2.1.6), glucose or xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.2~), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E.C. 3.2.1.15), phenol oxidases, ribc.nucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papain (E.C. 3.4.22.2), Eicin (E.C. 3.4.22.3)~ bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), chymosin ~E.C. 3.4.23.4)7 m~croblal proteases (E.C. 3.4.23.6), micro-bial metallo proteases (E.C. 3.4.24.4), pullunase ~E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.~), tannase (E.C. 3.1.1.20), urease ~E.C.
3.5.1.5), urlcase (E.C. 1.7.3.3),~ and xylanase (E.C. 3.2.1.32).
PreEerably, the enzyme is ~ -galactosidase or ~ -glucosidase.
By d presently preferred embodiment of this invention, the cot- -ton cloth is glucosamylase naphthyl cloth, and the material being sub-jected to the en~yme-catalyzed reaction is liquified starch.
The hydrophobic alkyl or aryl polyhydroxyl con-pounds may be regenerated aEter use according to another aspect or embodiment o~ this invention. Thus, a method is provided Eor carrying our an en~yme-ca~alyzed 3~
reaction comprising: packing a column with an enzyme immobilized on a hydro-phobic alkyl or aryl polyhydroxy compound e.g. a hyclrophobic ~1lkyl or i3ryl cellulose, preferably alkyl or aryl cotton cloth; passing a solution of the material on which the enzyme catalyzed reaction is to take place through the column; removing that hydrophobic alkyl or aryl compound ~rorn the column; heat-ing that removed hydrophobic alkyl or aryl compound in a basic solution (i.e.
2 M ~aO~1)for d time and at a temperature sufficient to regenerate the compound (e.g. 100 C for 1 hr.); immobilizing the same enzyme on that regenerated hydrophobic alkyl or aryl polyhydroxy compound by the steps of reacting the lo selected polyhydroxy compound e.g. cellulose, preferably cotton cloth~ with a bifunctional compound and with an alcohol, or with a phenol, in a single step in an aqueous basic solution, immobilizing the enzyme therecn by absorption of a solution of the enzyme thereon, and stabilizing the immobilized enzyme thereon by reaction with a cross-linking agent; and carrying out the enzyme-catalyzeci reaction with the immobili2ed enzyme by again packing a column with an en~yme immobilized on d hydrophobic alkyl or aryl polyhydroxy compound e.g.
a hydrophobic alkyl or aryl cellulose, preterably alkyl or ~1ryl cotton cloth; and passing a solution of the material on which the en~yme catalyzed reaction is to take place through the column.
In one embodiment of such a hydrophobic polyhydroxy compound, the alkyl group may be butyl, hexyl, octyl, decyl or dodecyl anci the aryl group may be phenyl, naphthyl or anthranyl.
The enzyme selected may be one of alcohol dehydrogenases, c~ -amylase (E.C. 3.2.1.1~, ~ -amylase (E.C. 3.2.1.1~, ~ -amylase or gluc~amylase (E.C. 3.2.1.3), asparaginase ~E.C. 3.~ asparcase (E.C.
.3.1.1), catali3se (E.C. 1.11.1.6~, cellobiase ~E.C. 3.~.1.21), cellu~se (E.C. 3.2.1.4~, chloride peroxidclse (E.C. 1.11.1.10), dextranast (E.C.
3.2.1.11), ~ -galactosidase (E.C. 3.2.1.22~ galac~osid~3se or lac-tase (E.C. 3.2.i.23~ glucanase (E.C. 3.2.1.o~, ~lucose or xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E~C. 3.2.1.15), phenol oxidases, rihonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subcilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papdin (E.C. 3.4.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), chymosin (E.C. 3.4.23.4), microbia proteases (E.C. 3.4.23.6), micro-bial metallo proteases (E.C. 3.4.24.4), pullunase (E.C.3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tannase (E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.7.3.3), and xylanase (E.C. 3.2.1.32), preferably -galactosidase or /~-glucosidase.
In a especially preferred embodiment, the cotton cloth is gluco-amylase naphthyl cloth, and the material on which the reaction is to cake pla^e is liquified starch.
Thus, the present invention preferably provides hydrophobic cotton cloths, e.g. phenyl cloth, naphthyl cloth, anthranyl cloth, butyl cloth, hexyl cloth, octyl cloth, decyl cloth or dodecyl cloth, preferably by ~he reaction of cotton flannel cloth with epichlorohydrin and the selected reactive alcohol or phenol in an aqueous NaOil solution. The hydrophobic cloths may then be con-verted into immobilized enzyme clochs by the immobilization of an enzyme there-on by absorption oE a solution of the enzyme on the cloth, and then stabili-zation oE the enzyme thereto by means of reaction with a cross-linking agent, e.g. gll1tardldehyde.
3~
The present process is a dramatic improvement over the prior art derivatization procedure. That procedure consisted of two steps namely: i) preparation of glycidyl ethers from alcohols and epichlorohy-drin; and ii) coupling of the glycidyl ethers to the hydroxyl groups of a support (agarose). Those reactions were carried out in nos~aqueous sol-vents which dissol~e alcohols, epichlorohydrin and ~oron trifluoride etherate (a moisture-sensitive ca~alyst~. Thus, the procedure requires agarose swollen in the nonaqueous sol~ents.
By the present inventive process, however, it has been found that these reactions can readily be perforrned in aqueous NaOH solution at elevated temperatures, e.g. up to 100C in a single step. This greatly simplifies and economi~es the preparation of hydrophobic media from cellulose, as well as from other polyhydroxy compounds. Hydropho~ic media were therefore prepared from cotton flannel (cloth) which is much less expensive than agarose derivatives. Moreover, it ~las been found according to the ~nethod of an a~pect of this invention that fabric forms (cloth) of fibers, when stacked in a column, provide good flow rates.
Enzymes which may be immobili~ed on the hydrophobic cloch to provide an embodimellt of this i~ention include alcohol clehyclrogenases c~ -arnyla~e (E.C. 3.2.1.1), ~ -amylase (E~C. 3.2.1.1), ~ -amylase or ~luc~amylase tE.C. 3.2.1.3), asparaginase (E.C. 3.5.1.1`), aspartase (E.C.
4.3.1.1), catalase (É.C. 1.11.1.6)t cellobiase (E.C. 3.2.1.21), cellu~se (E.C. 3.2.1.4), chloride peroxidase (E.C. 1.11.1.10~, dextranase (E.C.
3.2.1.11), ~ -galactosidase (E.C. 3.2.1.22), ~ -galactosidase or lac-tase (E.C. 3.2.1.23), ~ -glucai-ase (E.C. 3.2.1.6), ~IUCOSe or xylose isomerase ~E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), 1ip3se ~E.(. 3.1.1,3), steapsin, _ 19 _ nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidas2 (E.C.
3.S.I.II), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectir~se (E.C. 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteascs, papain (E.C. 3.4.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase(E~c~ 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papain (E.C. 3.6.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4~, pepsin (E.C. 3.4.23.1), chymosln (E.C. 3.4.23.4), mlcroblal proteases ~E.C. 3.4.23.6), micro-blal n~tallo proteases (.C. 3.4.24.4), pullunase (E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tr~nnase (E.C. 3.1.1.20), urease ~E.C.
3.5.1.5), uricase (E.C. 1.7.3.3), and xylanase (E.C. 3.2.1.32).
Preferably the enzyme isi~ -g~ ctosidase or ~ -gluosidase.
The hydrophobic cloth of one embodimént of this invention may be prepared accorcling to Lhe following process within the scope of an aspect of this invention.
Examples h 2 Cill square of cotton flannel cloth (0.05 g) was soaked in I ml of 4 M NaOii containing 2 mg/ml Nai31i4 for 15 min. at room cernpera-ture. Five rnmoles (0.4 ml) of epichlorohydrin were mixed with 5 nunoles of the desired alcohol (e.g. outyl, hexyl, octyl, decyl or dodecyl) or the desired phenol (phenol, ~ -n.lphthol o: alltnrone, whit-h is converttd in _ru to arlthrallcl in alkali.) This W~tS Idde(l ro tne so.lking clcth, mix~a and heated at 100 C for 2 h for the alc-ilol mixCure oi~ I h fc~r the phenol mixture. A~ter cooling to room tempirature~ rht clorh was washed with I N ilCL, hot ethallol. arld distilled water and blotreti dry.
- 2(~ -3~3 Thus, for example, a phenyl derivative of the eellulose ean be obtained aeeording to the following reaetions:
OH
C _ r ~--C ~ ~ C ~ -- ~ L ~ C ~ ~ C ~ C~
phenol epichlDrohydrin . __ +NaOH cellulose-OH
N~C~ U;L--CH CH~ ~o{~ cH2-o-cc~ osc ~ phenyl cellulose Sinee 1 ml of eoneen~rated NaOH solution ean dissolve 5 nmoles of epiehlorohydrin at 100C, the derivi~ation mixture eontained, per ml of NaOH
solution, 5 mmoles of epiehlorohydrin and 5 r~moles of ROH ~aleohols or phenols).
Exeess RO~ was found to reduee derivati~ationr possibly beeause it reaeted with the glyeidyl ether and thus lowered the level of this eoupling r~aetant.
The optimal derivati~ation eonditions were examined in terms of the eapaeity of hydrophobie cloth to absorb bovine serum albumin (BSA)-e adsorption of BSA on eloth ~as earried out as follows:
A eloth was soaked in 1 ml of 10~ BSA (Sigma NO. A 7906~ for 16 h at 25C. The eloth was thoroughly washed with water, blotted dry, and then heated in 2 ml of 1~ sodium dodeeyl sulfate at 100C for 10 min. The extraeted BSA was eolorimetrioally assayed (aeeording to the proeedure of Lowry et al, 1951).
~ -Galactosidase and ~ -glucosidase were immobilized on the resepctive al~yl or aryl cloth, namely on hexyl cloth, octyl cloth, decyl cloth, dodecyl cloth, phenyl cloth, or naphthyl cloth (accordin~ to dif-Eerent selected aspects oE this invention) and - 21 _ on oc-tyl SEPHAROSE and phenyl SEP2~.ROSE (as comparisons) according to the following techniques;
~ -Galactosidase (Sigma G 6008 ~rade VI) was dissolved in 0 05"~
sodium phosphate buffer (pH 7.0) ~o a final concentration of 20Jug~2~
Glucosidase (Sigma G 8625) was dissolved in 0.05 M sodium acetate buffer (p.-. 5.6) to a final concentration of 200 ~g/ml. A 2 cm square of hydro-phobic cloth was covered with lOO~ul of the enzyme solutions and left for 16 h at 25C. The cloth was then soaked in 3 ml of the buffer for 30 min at 25C (the steep liquid was kept for assaying the unbound en~yme). The l~ cloth was washed with the buffer and assayed for the immobilized enzyme.
For en~yme adsorption to a hydrophobic agarose gel, lOOful of an en~yme solution was mixed with l g of wet gel ~0.3 g dry weight). After 16 h at 25C, the gel was washed with ~he buffer and assayed for the im~wbilized enzyme.
~2 ~ lactosidase and ~ -glucosidase were assayed according to the following proceclure :
A cloth containin bound enzyme was shaken at 320 rpm and 30C in 3 ml of 2 mM o-nitrophenyl ~ -galactoside in phosphate buffer (Eor ~ -galac-tosidase) or l mM p-nitrophenyl ~ -glucoside in acetate buffer (for~ -glucosidase). ~fter 5 min. 2 ml of the reaction mixture was mixed with l ml of l M Na2C03 and absorbance was determined at ~120 nm (for ~ -galac-tosidase) or 400 nm (for b -glucosidase).
In the accom~nying drawings, Figure l A is a graph of BSA adsorption capacity of a hydrophobic cloth showing the eEfect of NaOH concentration, while ~igure lB is a similar graph showing the efEect of reaction time; ar,d Figure 2 is a graph showing the hy-'2rcl1ysis oE sol~2b1e starch in a column packed with ,y uc~s~lmylas~ naphthyl cloth.
- 2~ -The adsorption capacity of octyl ~C~) and phenyl ( ~ ~ cloths are shown in Figures lA and lB. For the results in Figure lA derivatiza-tion mixtures consisting of various concentrations of NaOH were heated for 1 h. For the results in Figure lB derivatization mixtures were heated for various lengths of time. The resulting hydrophobic cloths were assayed for BSA adsorption ~mg BSA/g cloth). The data are the average of the triplicate samples.
Figure lA shows that the use of 4 to 5 M NaOH in the derivati-zation mixture produced octyl and phenyl cloths with the highest BSA
adsorption. Thus, 4 M NaOH was used in the derivatization mixture.
Figure 1~ shows that the optimal reaction time was 1 h for phenyl cloth and 2 h for octyl cloth. Thus 1 h and 2 h were used in the derivatizations involving phenols and alcohols, respectively. Longer reaction times reduced the ~SA adsorption capacity. It is conceiva~le that excess derivatization encourages interaction between the introduced hydrophobic groups rathar than these groups and proteins.
Table 1 (below) shows that the resulting hydrophobic cloths exhibit ~SA adsorption capacities comparable to those obtained by commercial octyl and phenol agarose (Sepharose). m ese hydrophobic cloths also adsorbed ~ ~galactosidase and ~ -glucosidase efficiently and immobilized enzymes were nearly 50% as active as free enzymes. On the other hand, commercial hydrophobic agarose did not adsorb ~ -galactosidase in an active form, and the activity of adsorbed ~ -glucosidase was less than that observed on the hydrophobic cloth.
l.Z ~ ;?~3 Table 1: AdsorPtion of ~S~ gal~ctosidase and ~-glucosid~se Hydrophobic Inedia BSA ~-~alactosidase ~-Glucosidase He~yl cloth 24 19 (86) 37 (76) Octyl cloth 46 22 (88) 43 (84) Decyl cloth 51 22 ~88) 51 (92) Dodecyl cloth 35 26 (88) 48 (97~
Phenyl cloth 51 28 ~95) 56 (96) Naphthyl cloth 51 28 (96) 49 (96) Octyl SEPHAROSE 60 ND 21 (55) Phenyl SEPHAROSE 50 ND 22 (60) 1 o In the aL~ove Table BSA adso.rption is expressed as mg BSA per g dry cloth or Sepharose. Enzyme adsorption is expressed as activity (nanomoles of substrate hydrolyzed per min) of enzymes immobilized on a cloth square The enzyme activities added to a square were 54 nanomoles~
min of ~ -galaotosidase and 105 nanomol.es/min of ~ -glucosidase (activities of free enzymes).
~ In the columns for ~ -galactosidase and ~ -glucosidase, the numbers in the parentheses indicate the percentage aE enzyme absorption which was calculated from the activitLes of applied enzme and unadsorbed enzymes. No activity o~ ~ -gdlactosiddse was detected on the hydrophobic octyl SEPiiAROSE or phenyl SEP~IAROSE, nor in the steep I iquid .
A glucoamylase column to evaluate the adsorption o~ Rhi70pus glucoamylclse r.o naphthyl cloth was prepared dS ~ollows:
.
- ~4 -Naphthyl cloth segments (O.S cm square) were soaked in a 5~
suspension of crude Rhizopus glueo~nylase (Sign~ A 72553 in 0.02 M sodium acetate (pH 4.8~ for 16 h at 25C. The segments were washed with ~2~ soaked in 1~ glutaraldehyde for 30 min, washed with H20 and packad into a jacketed eolumn (10 mm diameter) to a bed volume of 10 ml. A 5~ soluble starch suspension in H20 was pumped into the eolumn (equilibrated at 50C) at various space velocities. The degree of hydrolysis was calculated from the coneentration of redueing sugars in the effluent obtained after pump-ing through about 10 volumes of the stareh solution. The redueing sugar was assayed ~y 3,5-dinitrosalieylate reagent,(aeeording to the procedure of Miller, 1959).
Figure 2 shows that nedrly complete hydrolysis of soluble starch could be achieved in the Rhi~opus glucosidase column at a space velocity of 3 to ~. Higher space velocities resulted in a linear reduction of the degree of hydrolysis.
Nearly identical results were obtained with a column of Aspers~illus glucoamylase which was similarly prepared.
~ ration of Used hYdroohobic Cloth _ Used hydrophobic cloth can be regenerated by heating it in 2 M NaOH at lOO C for I h. The regenerated cloth may be loaded with fresh enzyme, and the enzyme immobilized thereon. The enzyme loading capacity did not change after 6 cycles of immobili~ation, use, and regenerat iOIl.
This regeneration urther economizes the application of rtle hydrophobic cloth.
Other enzyme~ which may be immobili~ed according to the process of an aspect o~ this invention incl~lde gl~cose oxidase and peroxidase. Glucose oxidase is a flavoenzyme which catalyzes the following reactions.
-D-glueose + Enzyme-FAD c Enzyme-FADH2 + ~ D-glueonolactone Enzyme-FADH2 ~ 2 - ~ Enzyme-FAD -~ H202 Peroxidase catalyzes the oxidation of a number of oxidizable substrates by H202. If a chromogenic substrate (e.g. o-dianisidine) is oxidized, a eolor will develope aecording to the following reaetion.
2H202 + ehromogenic substrate -~2H20 + oxidized color product Thus, glucose can be assayed by a mixture of glucose oxidase and peroxidase in the presence of a chro~ogenic substrate. such glucose assays have been extensively used in food industries, and in diagnosis of patients (particularly diabetics).
Unless heavily glycosylated, pro~eins exhibit ilydrophobic inter-action with hydrophobic materials due to the presence of hydrophobic groups on their surface. Thus, proteins can be fractionated on the basis of the differing hydrophobieity. The sum of hydrophobic interaction is often so large to cause the binding (immobilization) of many proteins to a hydro-phobic material. This type of in~obilization is very mild and nardlyaffects the aetivity of the proteins (e,g. enzymes and antibody).
Thus, according to aspects of the present invention, depending 8~`~
on the enzyme immobilized on the hydrophobic cloth, the followlng pro-cess may be carried out: removal of 2 and glucose from solutions (~lucose oxidase); hydrolysis of starch (~ -amylase, glucoamylase); hydrolysis o~
sucrose (invertase); hydrolysis of lactose ( ~-~alactosidase); hydrolysis of peptides, amides and esters (brom~lain); synthesis of carbon-halogen bonds (chloroperoxidase); oxidation of phenols, aminophenols, diamines and amino acids in the presence of H202 (peroxidase); and hydrolysis of proteins (proteases).
While epichlorohydrin is the preferred compound for this reaction, 0 other suitable bifunctional compounds include epibromohydrin, dichloro-hydrin, dibromohydrin, ethylene glycol diglycidyl ether, triethylene glycol diglycidyl ether, diglycidyl ether, and 1,6-hexanediol diglycidyl ether.
The crosslinking reaction for producin~ the hydrophobic cotton cloth is carried out in an aqueous solution in the presence of an alkaline substance, e.g. sodium hydroxide, an portassium hydroxide, usually at an elevated temperature up to 100 C for I to 24 hoursl preferably 2 to l0 hours.
Thus, according to aspects of the present invention, hydropho-bic cotton cloths may be prepared by heating cotton flannel in a mixture of alcohols or phenols, epiehlorohydrin and 4 M NaOH. These cloths adsorbed as much bo~ine serum albumin as did a commercial preparation of phenyl agarose. ~ -Galactosidase and ~ -glucosidase adsorbed on the eloths were 50~ as active as free enzymes. Glucoamylase immobilized on naphthyl cloth in a packed bed column efficiently hydrolyzed soluble starch to glucose. These inexpensive media would be useful for commercial-scale hydrophobic chromatography and enzyme immobilization.
.
Alkyl and aryl derivatives of cotton cloth can thus be easily prepared by aqueous reactions in a single batch operation. This derivatization method is simpler and less costly than the previous method.
Hydrophobic cloth is much less expensive than hydrophobic agarose and yet adsorbs as much protein. Inexpensive hydrophobic cloths which exhibit high protein adsorption can be easily prepared by heating cotton cloth in a mixture of alcohols or phenols epichlorophydrin and ~ M NaOH.
Therefore, hydrophobic cloth is useful for commercial-scale hydrophobic chromatography and enzyme immobilization. The absence of charges on the cloth permits fractionation based solely on hydrophobic interactions.
Enzyme immobilization on hydrophobic cloth is particularly suited for transformation of large-sized substrates as an enzyme is e~posecl on the surface of cellulose.
The insoluble biologically active materials prepared in rhis way possess the properties of selectively acting heterogeneous cacalysts for chemical reactions,e.g., enzymes~ enzyme inhibi~ors, or compounds hav-ing a specific reactivity. The advantages of the such materials are obvious from the most frequently used biologically active compounds i.e.
insoluble enzymes, although the invention is not to be limited to this group oE compounds.
- 2~ -
3.2.1.11), ~ -galactosidase (E.C. 3.2.1.22), ~ -galactosidase or lac-tase (E.C. 3.2.1.23), ~ -glucanase (E.C. 3.2.1.6), glucose or xylose isomerase (E.C. 5.3.1.5), glucose cxidase (E.C. 1.1.3.4), hemicellulase (E.G.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), sLeapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.2l.7), pectinase (E.C. 3.2.1.15j, phenol oxidases, ribon~lc1eases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline pro~eases, papain ~E.C. 3.4.22.2), Eicin (E.C. 3.4.22.3), bromelain (E.~. 3.4.22.4), pepsin (E.C. 3.4.23.1), _ 13 -chymosin (E.C. 3.4.23.4), microbial proteases (E.C. 3.4.23.6), micro-bial metallo proteases (E.C. 3.4.24.4)1 pullunase (E.C. 3.2.1.41j, ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tannase ~E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.7.303), and xylanase (E.C. 3.2.1.32).
By preferred embodiments, the enzyme is ~ -galactosidase or -glucosidase.
In variations of these embodiments of the invention, where the cross-linking agent is glutaraldehyde and~or the enzyme is ~ -galactosi-dase or ~ -glucosidaset the following may be pro~ided: the bifunctional compound is epichlorohydri~; the a~ueous basic solution is provided by a solution of sodium hydroxide or of potassium hydroxide, having a concen-tration of 4 to 5 M; the reaction is carried out at an elevated tempera-ture of up to 100 C Eor a period of I to 24 hours; the alcohol is one oE
butyl, hexyl, octyl, decyl, or dodecyl alcohols; and che phenol is phenol~
-naphi~hol or anthranol.
By another aspect or embodiment of this invention, a novel enzyme is pro~ided, na~ely one which is immobilized on a hydrophobic aikyl or aryl polyhydroxy compound, preferably one which is immobilized on a hydrophobic alkyl or aryl cellulose, and still more desirably, one which is immobilized on a hydrophobic alkyl or aryl cotton cloth.
In such novel enzyme, the alkyl group may be butyl, hexyl, octyl, decyl or dodecyl, and the aryl group may be phenyl, naphthyl or anthranyl.
The enzyme selected for these embodiments o~ ~his invention m~y be any of the m~or industrial enzymes, e.g. alcohol dehydrogenases, c~ -amylase ~E.C. 3.2.1.1), ~ -amylase (E.C. 3.2.1.1~ amylase or gluc~amylase (E.C. 3.2.1.3), asparaginase ~E.C. 3.5.1.1), aspartase ~E.C.
4.3.1.1), catalase (E.C. 1.11.1.6), cellobiase (E.C~ 3.2.1.21), celluL~se (E.C. 3.2.1.4), chloride peroxldase ~E.C. 1.11.1.10), dextran,ise ~E.C.
_ 1 4 -12~ P~3 3.2.1.11), ~ -galactosidase (E.C. 3.2.1.22), ~ -galactosldase or lac-tase (E.C. 3.2.1.23), ~ -glucanase (E.C. 3.2.1.6), glucose ~r xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E.C. 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papair! (E.C. 3.4.22.2), ~icin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), 1~) chymosln (E.C. 3.4.~3.4), mlcroblal proteases (E.C. 3.4.23.6), micro-bial me~allo proteases (E.C. 3.4.24.4), pullunase (E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6)9 tannase (E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.7.3O3),and xylanase (E.C. 3.2.1.32).
Preferably, the enzytne may be g -galactosidase or ~ -glucosidase.
By yet another aspect or embodiment of this invention, a method is provided for carrying out an enzyrne-catalyzed reaction compris-ing: packing a column with an enzyme which has been immobilized on a hydro-phobic alkyl or aryl polyhydroxy compound, namely with an enzyme which has been immobilized on a hydrophobic alkyl or aryl celllulose, prefera-bly with an enzyme which has been immobilized on a hydrophobic alkyl or aryl cotton cloth; and passing a solution of the material on which that enzyme catalyzed reaction is to take place through the column.
By one embodiment of such compound on which the enzyme has been immobilized, the alkyl group may be butyl, hexyl, octyl, decyl, or dodecyl, and the aryl group is phenyl, naphthyl or anthranyl.
The enzyme selected for these embodiments of this Invention may be any of the major industrial enzymes, e.g. alcohol dehydrogenases.
c~ -amylase (E.C. 3.2.1.1), ~ -amylase (E.C. 3.2.1.1), ~ -amylase or gluc~amylase (E.C. 3.2.1.3), asparaginase (E.C. 3.5.1.1), aspartase (E.C.
4.3.1.1), catalase (E.C. I.ll.l.o), cellobiase (E.C. 3.2.1.21), cellu~se (E.C. 3.2.1.4), chloride peroxidase (E~C. 1.11.1.10), dextranase (E.C.
3.2.1.11), ~ -galactosidase (E.C. 3.2.1.22), ~ -galactosidase or lac-tase (E.C. 3.2.1.23), ~ -glucanase (E.C. 3.2.1.6), glucose or xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.2~), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E.C. 3.2.1.15), phenol oxidases, ribc.nucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papain (E.C. 3.4.22.2), Eicin (E.C. 3.4.22.3)~ bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), chymosin ~E.C. 3.4.23.4)7 m~croblal proteases (E.C. 3.4.23.6), micro-bial metallo proteases (E.C. 3.4.24.4), pullunase ~E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.~), tannase (E.C. 3.1.1.20), urease ~E.C.
3.5.1.5), urlcase (E.C. 1.7.3.3),~ and xylanase (E.C. 3.2.1.32).
PreEerably, the enzyme is ~ -galactosidase or ~ -glucosidase.
By d presently preferred embodiment of this invention, the cot- -ton cloth is glucosamylase naphthyl cloth, and the material being sub-jected to the en~yme-catalyzed reaction is liquified starch.
The hydrophobic alkyl or aryl polyhydroxyl con-pounds may be regenerated aEter use according to another aspect or embodiment o~ this invention. Thus, a method is provided Eor carrying our an en~yme-ca~alyzed 3~
reaction comprising: packing a column with an enzyme immobilized on a hydro-phobic alkyl or aryl polyhydroxy compound e.g. a hyclrophobic ~1lkyl or i3ryl cellulose, preferably alkyl or aryl cotton cloth; passing a solution of the material on which the enzyme catalyzed reaction is to take place through the column; removing that hydrophobic alkyl or aryl compound ~rorn the column; heat-ing that removed hydrophobic alkyl or aryl compound in a basic solution (i.e.
2 M ~aO~1)for d time and at a temperature sufficient to regenerate the compound (e.g. 100 C for 1 hr.); immobilizing the same enzyme on that regenerated hydrophobic alkyl or aryl polyhydroxy compound by the steps of reacting the lo selected polyhydroxy compound e.g. cellulose, preferably cotton cloth~ with a bifunctional compound and with an alcohol, or with a phenol, in a single step in an aqueous basic solution, immobilizing the enzyme therecn by absorption of a solution of the enzyme thereon, and stabilizing the immobilized enzyme thereon by reaction with a cross-linking agent; and carrying out the enzyme-catalyzeci reaction with the immobili2ed enzyme by again packing a column with an en~yme immobilized on d hydrophobic alkyl or aryl polyhydroxy compound e.g.
a hydrophobic alkyl or aryl cellulose, preterably alkyl or ~1ryl cotton cloth; and passing a solution of the material on which the en~yme catalyzed reaction is to take place through the column.
In one embodiment of such a hydrophobic polyhydroxy compound, the alkyl group may be butyl, hexyl, octyl, decyl or dodecyl anci the aryl group may be phenyl, naphthyl or anthranyl.
The enzyme selected may be one of alcohol dehydrogenases, c~ -amylase (E.C. 3.2.1.1~, ~ -amylase (E.C. 3.2.1.1~, ~ -amylase or gluc~amylase (E.C. 3.2.1.3), asparaginase ~E.C. 3.~ asparcase (E.C.
.3.1.1), catali3se (E.C. 1.11.1.6~, cellobiase ~E.C. 3.~.1.21), cellu~se (E.C. 3.2.1.4~, chloride peroxidclse (E.C. 1.11.1.10), dextranast (E.C.
3.2.1.11), ~ -galactosidase (E.C. 3.2.1.22~ galac~osid~3se or lac-tase (E.C. 3.2.i.23~ glucanase (E.C. 3.2.1.o~, ~lucose or xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E~C. 3.2.1.15), phenol oxidases, rihonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subcilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papdin (E.C. 3.4.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), chymosin (E.C. 3.4.23.4), microbia proteases (E.C. 3.4.23.6), micro-bial metallo proteases (E.C. 3.4.24.4), pullunase (E.C.3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tannase (E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.7.3.3), and xylanase (E.C. 3.2.1.32), preferably -galactosidase or /~-glucosidase.
In a especially preferred embodiment, the cotton cloth is gluco-amylase naphthyl cloth, and the material on which the reaction is to cake pla^e is liquified starch.
Thus, the present invention preferably provides hydrophobic cotton cloths, e.g. phenyl cloth, naphthyl cloth, anthranyl cloth, butyl cloth, hexyl cloth, octyl cloth, decyl cloth or dodecyl cloth, preferably by ~he reaction of cotton flannel cloth with epichlorohydrin and the selected reactive alcohol or phenol in an aqueous NaOil solution. The hydrophobic cloths may then be con-verted into immobilized enzyme clochs by the immobilization of an enzyme there-on by absorption oE a solution of the enzyme on the cloth, and then stabili-zation oE the enzyme thereto by means of reaction with a cross-linking agent, e.g. gll1tardldehyde.
3~
The present process is a dramatic improvement over the prior art derivatization procedure. That procedure consisted of two steps namely: i) preparation of glycidyl ethers from alcohols and epichlorohy-drin; and ii) coupling of the glycidyl ethers to the hydroxyl groups of a support (agarose). Those reactions were carried out in nos~aqueous sol-vents which dissol~e alcohols, epichlorohydrin and ~oron trifluoride etherate (a moisture-sensitive ca~alyst~. Thus, the procedure requires agarose swollen in the nonaqueous sol~ents.
By the present inventive process, however, it has been found that these reactions can readily be perforrned in aqueous NaOH solution at elevated temperatures, e.g. up to 100C in a single step. This greatly simplifies and economi~es the preparation of hydrophobic media from cellulose, as well as from other polyhydroxy compounds. Hydropho~ic media were therefore prepared from cotton flannel (cloth) which is much less expensive than agarose derivatives. Moreover, it ~las been found according to the ~nethod of an a~pect of this invention that fabric forms (cloth) of fibers, when stacked in a column, provide good flow rates.
Enzymes which may be immobili~ed on the hydrophobic cloch to provide an embodimellt of this i~ention include alcohol clehyclrogenases c~ -arnyla~e (E.C. 3.2.1.1), ~ -amylase (E~C. 3.2.1.1), ~ -amylase or ~luc~amylase tE.C. 3.2.1.3), asparaginase (E.C. 3.5.1.1`), aspartase (E.C.
4.3.1.1), catalase (É.C. 1.11.1.6)t cellobiase (E.C. 3.2.1.21), cellu~se (E.C. 3.2.1.4), chloride peroxidase (E.C. 1.11.1.10~, dextranase (E.C.
3.2.1.11), ~ -galactosidase (E.C. 3.2.1.22), ~ -galactosidase or lac-tase (E.C. 3.2.1.23), ~ -glucai-ase (E.C. 3.2.1.6), ~IUCOSe or xylose isomerase ~E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), 1ip3se ~E.(. 3.1.1,3), steapsin, _ 19 _ nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidas2 (E.C.
3.S.I.II), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectir~se (E.C. 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteascs, papain (E.C. 3.4.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase(E~c~ 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papain (E.C. 3.6.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4~, pepsin (E.C. 3.4.23.1), chymosln (E.C. 3.4.23.4), mlcroblal proteases ~E.C. 3.4.23.6), micro-blal n~tallo proteases (.C. 3.4.24.4), pullunase (E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tr~nnase (E.C. 3.1.1.20), urease ~E.C.
3.5.1.5), uricase (E.C. 1.7.3.3), and xylanase (E.C. 3.2.1.32).
Preferably the enzyme isi~ -g~ ctosidase or ~ -gluosidase.
The hydrophobic cloth of one embodimént of this invention may be prepared accorcling to Lhe following process within the scope of an aspect of this invention.
Examples h 2 Cill square of cotton flannel cloth (0.05 g) was soaked in I ml of 4 M NaOii containing 2 mg/ml Nai31i4 for 15 min. at room cernpera-ture. Five rnmoles (0.4 ml) of epichlorohydrin were mixed with 5 nunoles of the desired alcohol (e.g. outyl, hexyl, octyl, decyl or dodecyl) or the desired phenol (phenol, ~ -n.lphthol o: alltnrone, whit-h is converttd in _ru to arlthrallcl in alkali.) This W~tS Idde(l ro tne so.lking clcth, mix~a and heated at 100 C for 2 h for the alc-ilol mixCure oi~ I h fc~r the phenol mixture. A~ter cooling to room tempirature~ rht clorh was washed with I N ilCL, hot ethallol. arld distilled water and blotreti dry.
- 2(~ -3~3 Thus, for example, a phenyl derivative of the eellulose ean be obtained aeeording to the following reaetions:
OH
C _ r ~--C ~ ~ C ~ -- ~ L ~ C ~ ~ C ~ C~
phenol epichlDrohydrin . __ +NaOH cellulose-OH
N~C~ U;L--CH CH~ ~o{~ cH2-o-cc~ osc ~ phenyl cellulose Sinee 1 ml of eoneen~rated NaOH solution ean dissolve 5 nmoles of epiehlorohydrin at 100C, the derivi~ation mixture eontained, per ml of NaOH
solution, 5 mmoles of epiehlorohydrin and 5 r~moles of ROH ~aleohols or phenols).
Exeess RO~ was found to reduee derivati~ationr possibly beeause it reaeted with the glyeidyl ether and thus lowered the level of this eoupling r~aetant.
The optimal derivati~ation eonditions were examined in terms of the eapaeity of hydrophobie cloth to absorb bovine serum albumin (BSA)-e adsorption of BSA on eloth ~as earried out as follows:
A eloth was soaked in 1 ml of 10~ BSA (Sigma NO. A 7906~ for 16 h at 25C. The eloth was thoroughly washed with water, blotted dry, and then heated in 2 ml of 1~ sodium dodeeyl sulfate at 100C for 10 min. The extraeted BSA was eolorimetrioally assayed (aeeording to the proeedure of Lowry et al, 1951).
~ -Galactosidase and ~ -glucosidase were immobilized on the resepctive al~yl or aryl cloth, namely on hexyl cloth, octyl cloth, decyl cloth, dodecyl cloth, phenyl cloth, or naphthyl cloth (accordin~ to dif-Eerent selected aspects oE this invention) and - 21 _ on oc-tyl SEPHAROSE and phenyl SEP2~.ROSE (as comparisons) according to the following techniques;
~ -Galactosidase (Sigma G 6008 ~rade VI) was dissolved in 0 05"~
sodium phosphate buffer (pH 7.0) ~o a final concentration of 20Jug~2~
Glucosidase (Sigma G 8625) was dissolved in 0.05 M sodium acetate buffer (p.-. 5.6) to a final concentration of 200 ~g/ml. A 2 cm square of hydro-phobic cloth was covered with lOO~ul of the enzyme solutions and left for 16 h at 25C. The cloth was then soaked in 3 ml of the buffer for 30 min at 25C (the steep liquid was kept for assaying the unbound en~yme). The l~ cloth was washed with the buffer and assayed for the immobilized enzyme.
For en~yme adsorption to a hydrophobic agarose gel, lOOful of an en~yme solution was mixed with l g of wet gel ~0.3 g dry weight). After 16 h at 25C, the gel was washed with ~he buffer and assayed for the im~wbilized enzyme.
~2 ~ lactosidase and ~ -glucosidase were assayed according to the following proceclure :
A cloth containin bound enzyme was shaken at 320 rpm and 30C in 3 ml of 2 mM o-nitrophenyl ~ -galactoside in phosphate buffer (Eor ~ -galac-tosidase) or l mM p-nitrophenyl ~ -glucoside in acetate buffer (for~ -glucosidase). ~fter 5 min. 2 ml of the reaction mixture was mixed with l ml of l M Na2C03 and absorbance was determined at ~120 nm (for ~ -galac-tosidase) or 400 nm (for b -glucosidase).
In the accom~nying drawings, Figure l A is a graph of BSA adsorption capacity of a hydrophobic cloth showing the eEfect of NaOH concentration, while ~igure lB is a similar graph showing the efEect of reaction time; ar,d Figure 2 is a graph showing the hy-'2rcl1ysis oE sol~2b1e starch in a column packed with ,y uc~s~lmylas~ naphthyl cloth.
- 2~ -The adsorption capacity of octyl ~C~) and phenyl ( ~ ~ cloths are shown in Figures lA and lB. For the results in Figure lA derivatiza-tion mixtures consisting of various concentrations of NaOH were heated for 1 h. For the results in Figure lB derivatization mixtures were heated for various lengths of time. The resulting hydrophobic cloths were assayed for BSA adsorption ~mg BSA/g cloth). The data are the average of the triplicate samples.
Figure lA shows that the use of 4 to 5 M NaOH in the derivati-zation mixture produced octyl and phenyl cloths with the highest BSA
adsorption. Thus, 4 M NaOH was used in the derivatization mixture.
Figure 1~ shows that the optimal reaction time was 1 h for phenyl cloth and 2 h for octyl cloth. Thus 1 h and 2 h were used in the derivatizations involving phenols and alcohols, respectively. Longer reaction times reduced the ~SA adsorption capacity. It is conceiva~le that excess derivatization encourages interaction between the introduced hydrophobic groups rathar than these groups and proteins.
Table 1 (below) shows that the resulting hydrophobic cloths exhibit ~SA adsorption capacities comparable to those obtained by commercial octyl and phenol agarose (Sepharose). m ese hydrophobic cloths also adsorbed ~ ~galactosidase and ~ -glucosidase efficiently and immobilized enzymes were nearly 50% as active as free enzymes. On the other hand, commercial hydrophobic agarose did not adsorb ~ -galactosidase in an active form, and the activity of adsorbed ~ -glucosidase was less than that observed on the hydrophobic cloth.
l.Z ~ ;?~3 Table 1: AdsorPtion of ~S~ gal~ctosidase and ~-glucosid~se Hydrophobic Inedia BSA ~-~alactosidase ~-Glucosidase He~yl cloth 24 19 (86) 37 (76) Octyl cloth 46 22 (88) 43 (84) Decyl cloth 51 22 ~88) 51 (92) Dodecyl cloth 35 26 (88) 48 (97~
Phenyl cloth 51 28 ~95) 56 (96) Naphthyl cloth 51 28 (96) 49 (96) Octyl SEPHAROSE 60 ND 21 (55) Phenyl SEPHAROSE 50 ND 22 (60) 1 o In the aL~ove Table BSA adso.rption is expressed as mg BSA per g dry cloth or Sepharose. Enzyme adsorption is expressed as activity (nanomoles of substrate hydrolyzed per min) of enzymes immobilized on a cloth square The enzyme activities added to a square were 54 nanomoles~
min of ~ -galaotosidase and 105 nanomol.es/min of ~ -glucosidase (activities of free enzymes).
~ In the columns for ~ -galactosidase and ~ -glucosidase, the numbers in the parentheses indicate the percentage aE enzyme absorption which was calculated from the activitLes of applied enzme and unadsorbed enzymes. No activity o~ ~ -gdlactosiddse was detected on the hydrophobic octyl SEPiiAROSE or phenyl SEP~IAROSE, nor in the steep I iquid .
A glucoamylase column to evaluate the adsorption o~ Rhi70pus glucoamylclse r.o naphthyl cloth was prepared dS ~ollows:
.
- ~4 -Naphthyl cloth segments (O.S cm square) were soaked in a 5~
suspension of crude Rhizopus glueo~nylase (Sign~ A 72553 in 0.02 M sodium acetate (pH 4.8~ for 16 h at 25C. The segments were washed with ~2~ soaked in 1~ glutaraldehyde for 30 min, washed with H20 and packad into a jacketed eolumn (10 mm diameter) to a bed volume of 10 ml. A 5~ soluble starch suspension in H20 was pumped into the eolumn (equilibrated at 50C) at various space velocities. The degree of hydrolysis was calculated from the coneentration of redueing sugars in the effluent obtained after pump-ing through about 10 volumes of the stareh solution. The redueing sugar was assayed ~y 3,5-dinitrosalieylate reagent,(aeeording to the procedure of Miller, 1959).
Figure 2 shows that nedrly complete hydrolysis of soluble starch could be achieved in the Rhi~opus glucosidase column at a space velocity of 3 to ~. Higher space velocities resulted in a linear reduction of the degree of hydrolysis.
Nearly identical results were obtained with a column of Aspers~illus glucoamylase which was similarly prepared.
~ ration of Used hYdroohobic Cloth _ Used hydrophobic cloth can be regenerated by heating it in 2 M NaOH at lOO C for I h. The regenerated cloth may be loaded with fresh enzyme, and the enzyme immobilized thereon. The enzyme loading capacity did not change after 6 cycles of immobili~ation, use, and regenerat iOIl.
This regeneration urther economizes the application of rtle hydrophobic cloth.
Other enzyme~ which may be immobili~ed according to the process of an aspect o~ this invention incl~lde gl~cose oxidase and peroxidase. Glucose oxidase is a flavoenzyme which catalyzes the following reactions.
-D-glueose + Enzyme-FAD c Enzyme-FADH2 + ~ D-glueonolactone Enzyme-FADH2 ~ 2 - ~ Enzyme-FAD -~ H202 Peroxidase catalyzes the oxidation of a number of oxidizable substrates by H202. If a chromogenic substrate (e.g. o-dianisidine) is oxidized, a eolor will develope aecording to the following reaetion.
2H202 + ehromogenic substrate -~2H20 + oxidized color product Thus, glucose can be assayed by a mixture of glucose oxidase and peroxidase in the presence of a chro~ogenic substrate. such glucose assays have been extensively used in food industries, and in diagnosis of patients (particularly diabetics).
Unless heavily glycosylated, pro~eins exhibit ilydrophobic inter-action with hydrophobic materials due to the presence of hydrophobic groups on their surface. Thus, proteins can be fractionated on the basis of the differing hydrophobieity. The sum of hydrophobic interaction is often so large to cause the binding (immobilization) of many proteins to a hydro-phobic material. This type of in~obilization is very mild and nardlyaffects the aetivity of the proteins (e,g. enzymes and antibody).
Thus, according to aspects of the present invention, depending 8~`~
on the enzyme immobilized on the hydrophobic cloth, the followlng pro-cess may be carried out: removal of 2 and glucose from solutions (~lucose oxidase); hydrolysis of starch (~ -amylase, glucoamylase); hydrolysis o~
sucrose (invertase); hydrolysis of lactose ( ~-~alactosidase); hydrolysis of peptides, amides and esters (brom~lain); synthesis of carbon-halogen bonds (chloroperoxidase); oxidation of phenols, aminophenols, diamines and amino acids in the presence of H202 (peroxidase); and hydrolysis of proteins (proteases).
While epichlorohydrin is the preferred compound for this reaction, 0 other suitable bifunctional compounds include epibromohydrin, dichloro-hydrin, dibromohydrin, ethylene glycol diglycidyl ether, triethylene glycol diglycidyl ether, diglycidyl ether, and 1,6-hexanediol diglycidyl ether.
The crosslinking reaction for producin~ the hydrophobic cotton cloth is carried out in an aqueous solution in the presence of an alkaline substance, e.g. sodium hydroxide, an portassium hydroxide, usually at an elevated temperature up to 100 C for I to 24 hoursl preferably 2 to l0 hours.
Thus, according to aspects of the present invention, hydropho-bic cotton cloths may be prepared by heating cotton flannel in a mixture of alcohols or phenols, epiehlorohydrin and 4 M NaOH. These cloths adsorbed as much bo~ine serum albumin as did a commercial preparation of phenyl agarose. ~ -Galactosidase and ~ -glucosidase adsorbed on the eloths were 50~ as active as free enzymes. Glucoamylase immobilized on naphthyl cloth in a packed bed column efficiently hydrolyzed soluble starch to glucose. These inexpensive media would be useful for commercial-scale hydrophobic chromatography and enzyme immobilization.
.
Alkyl and aryl derivatives of cotton cloth can thus be easily prepared by aqueous reactions in a single batch operation. This derivatization method is simpler and less costly than the previous method.
Hydrophobic cloth is much less expensive than hydrophobic agarose and yet adsorbs as much protein. Inexpensive hydrophobic cloths which exhibit high protein adsorption can be easily prepared by heating cotton cloth in a mixture of alcohols or phenols epichlorophydrin and ~ M NaOH.
Therefore, hydrophobic cloth is useful for commercial-scale hydrophobic chromatography and enzyme immobilization. The absence of charges on the cloth permits fractionation based solely on hydrophobic interactions.
Enzyme immobilization on hydrophobic cloth is particularly suited for transformation of large-sized substrates as an enzyme is e~posecl on the surface of cellulose.
The insoluble biologically active materials prepared in rhis way possess the properties of selectively acting heterogeneous cacalysts for chemical reactions,e.g., enzymes~ enzyme inhibi~ors, or compounds hav-ing a specific reactivity. The advantages of the such materials are obvious from the most frequently used biologically active compounds i.e.
insoluble enzymes, although the invention is not to be limited to this group oE compounds.
- 2~ -
Claims (54)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for preparing a hydrophobic alkyl or aryl polyhydroxy compound suitable for use as a carrier to immobilize enzymes thereon, said process comprising reacting said polyhydroxy compound with a bifunctional com-pound and with an alcohol, or with a phenol, in a single step, in an aqueous basic solution.
2. The process of claim 1 wherein said hydrophobic alkyl or aryl polyhydroxide compound is hydrophobic alkyl or aryl cellulose, and the process comprises reacting cellulose with a bifunctional compound and with an alcohol, or with a phenol, in a single step, in an aqueous basic solution.
3. The process of claim 2 wherein said hydrophobic alkyl or aryl cellulose is hydrophobic alkyl or aryl cotton cloth, and the process comprises reacting cotton cloth with a bifunctional compound and with an alcohol, or with a phenol, in a single step, in an aqueous basic solution.
4. The process of claims 1, 2 or 3 wherein said bifunctional compound is selected from the group consisting of epichlorohydrin, epibromohydrin, dichlorohydrin, dibromohydrin, ethylene glycol diglycidyl ether, triethylene glycol diglycidyl ether, diglycidyl ether, and 1,6-hexanediol diglycidy1 ether.
5. The process of claims 1, 2 or 3 wherein said compound is epichlorohydrin.
6. The process of claims 1, 2 or 3 wherein said aqueous basic solution is provided by a solution of sodium hydroxide or of potassium hydroxide.
7. The process of claims 1, 2 or 3 wherein said aqueous basic solu-tion is provided by a solution of sodium hydroxide or of potassium hydroxide, having a concentration of 4-5 M.
8. The process of claims 1, 2 or 3 wherein said reaction is carried out at an elevated temperature of up to 100°C for a period of 1 to 24 hours.
9. The process of claims 1, 2 or 3 wherein said alcohol is one of butyl, hexyl, octyl, decyl, or dodecyl.
10. The process of claims 1, 2 or 3 wherein said phenol is phenol, .beta.-naphthol or anthranol.
11. A hydrophobic alkyl or aryl polyhydroxy compound.
12. A hydrophobic alkyl or aryl cellulose.
13. A hydrophobic alkyl or aryl cotton cloth.
14. The hydrophobic material of claims 11, 12 or 13 wherein said alkyl group is butyl, hexyl, octyl, decyl, or dodecyl.
15. The hydrophogic material of claims 11, 12 or 13 wherein said aryl group is phenyl, naphthyl or anthranyl.
16. A process for immobilizing an enzyme in a hydrophobic alkyl or aryl polyhydroxy compound which comprises: reacting said polyhydroxy compound with a bifunctional compound and with an alcohol, or with a phenol, in a single step, in an aqueous basic solution; immobilizing an enzyme thereon by adsorption of a solution of the enzyme thereon; and stabilizing said immobilized enzyme thereon by reaction with a cross-linking agent.
17. The process of claim 16 wherein said hydrophobic alkyl or aryl polyhydroxy compound is hydrophobic alkyl or aryl cellulose.
18. The process of claim 17 wherein said hydrophobic alkyl or aryl cellulose is hydrophobic alkyl or aryl cotton cloth.
19. The process of claims 16, 17 or 18 wherein said cross-linking agent is glutaraldehyde.
20. The process of claims 16, 17 or 18 wherein said enzyme is selected from the group consisting of alcohol dehydrogenases, .alpha.-amylase (E.C. 3.2.1.1), .beta.-amylase (E.C. 3.2.1.1), ?-amylase or glucoamylase (E-C. 3.2-1.3), asparaginase (E.C. 3.5.1.21), aspartase (E.C.
4.3.1.1), catalase (C.E. 1.11.1.6), cellobiase (E.C. 3,2,1.21), cellulase (E.C. 3.2.1.4), chloride peroxidase (E.C. 1.11.1.10), dextranase (E.C.
3.2.1.11), .alpha.-galacrosidase (E.C. 3.2.1.22), .beta.-galactosidase or lac-tase (E.C. 3.2.1.23), .beta.-glucanase (E.C. 3.2.1.6), glucose or xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E.C. 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papain (E.C. 3.4.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), chymosin (E.C. 3.4.23.4), microbial proteases (E.C. 3.4.23.6), micro-bial metallo proteases (E.C. 3.4.24.4), pullunase (E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tannase (E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.7.3.3), and xylanase (E.C. 3.2.1.32).
4.3.1.1), catalase (C.E. 1.11.1.6), cellobiase (E.C. 3,2,1.21), cellulase (E.C. 3.2.1.4), chloride peroxidase (E.C. 1.11.1.10), dextranase (E.C.
3.2.1.11), .alpha.-galacrosidase (E.C. 3.2.1.22), .beta.-galactosidase or lac-tase (E.C. 3.2.1.23), .beta.-glucanase (E.C. 3.2.1.6), glucose or xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E.C. 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papain (E.C. 3.4.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), chymosin (E.C. 3.4.23.4), microbial proteases (E.C. 3.4.23.6), micro-bial metallo proteases (E.C. 3.4.24.4), pullunase (E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tannase (E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.7.3.3), and xylanase (E.C. 3.2.1.32).
21. The process of claims 16, 17 or 18 wherein said enzyme is .beta.-galactosidase or .beta.-glucosidase.
22. The process of claims 16, 17 or 18 wherein said cross-linking agent is glutaraldehyde and wherein said compound is epichlorohy-drin.
23. The process of claims 16, 17 or 18 wherein said enzyme is .beta.-galactosidase or .beta.-glucosidase, wherein said cross-linking agent is glu-taraldehyde and wherein said compound is epichlorohydrin.
24. The process of claims 16, 17 or 18 wherein said cross-linking agent is glutaraldehyde and wherein said aqueous basic solution is provided by a solution of sodium hydroxide or potassium hydroxide, having a concentra-tion of 4-5 M.
25. The process of claims 16, 17 or 18 wherein said enzyme is .beta.-galactosidase or .beta.-glucosidase and wherein said aqueous basic solution is provided by a solution of sodium hydroxide, or potassium hydroxide, having a concentration of 4-5 M.
26. The process of claims 16, 17 or 18 wherein said cross-linking agent is glutaraldehyde and wherein said reaction is carried out at an elevated temperature of up to 100°C for a period of 1 to 24 hours.
27. The process of claims 16, 17 or 18 wherein said enzyme is .beta.-galactosidase or .beta.-glucosidase and wherein said reaction is carried out at an elevated temperature of up to 100°C for a period of 1 to 24 hours.
28. The process of claims 16, 17 or 18 wherein said cross-linking agent is glutaraldehyde and wherein said alcohol is one of butyl, hexyl, octyl, decyl, or dodecyl.
29. The process of claims 16, 17 or 18 wherein said enzyme is .beta.-galactosidse or .beta.-glucosidase and wherein said alcohol is one of butyl, hexyl, octyl, decyl, or dodecyl.
30. The process of claims 16, 17 or 18 wherein said cross-linking agent is glutaraldehyde and wherein said phenol is phenol, .beta.- naphthol or anthranol.
31. The process of claims 16, 17 or 18 wherein said enzyme is .beta.-galactosidase or .beta.-glucosidase and wherein said phenol is phenol, .beta.-naphthol or anthranol.
32. An enzyme immobilized on a hydrophobic alkyl or aryl poly-hydroxy compound.
33. An enzyme immobilized on a hydrophobic alkyl or aryl cellu-lose.
34. An enzyme immobilized on a hydrophobic alkyl or aryl cotton cloth.
35. The enzyme of claims 32, 33 or 34 wherein said alkyl group is butyl, hexyl, octyl, decyl, or dodecyl.
36. The enzyme of claims 32, 33, or 34 wherein said aryl group is phenyl, naphthyl or anthranyl.
37. The enzyme of claims 32, 33 or 34 wherein said enzyme is selected from the group consisting of alcohol dehydrogenases, .alpha.-amylase (E.C. 3.2.1.1), .beta.-amylase (E.C. 3.2.1.1), ?-amylase or glucoamylase (E.C. 3.2.1.3), asparaginase (E.C. 3.5.1.1), aspartase (E.C.
4.3.1.1), catalase (C.E. 1.11.1.6), cellobiase (E.C. 3,2,1.21), cellulase (E.C. 3.2.1.4), chloride peroxidase (E.C. 1.11.1.10), dextranase (E.C.
3.2.1.11), .alpha.-galactosidase (E.C. 3.2.1.22), .beta.-galactosidase or lac-tase (E.C. 3.2.1.23), .beta.-glucanase (E.C. 3.2.1.6), glucose or xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.304), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E.C. 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papain (E.C. 3.4.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), chymosln (E.C. 3.4.23.4), microbial proteases (E.C. 3.4.23.6), micro-blal metallo proteases (E.C. 3.4.24.4), pullunase (E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tannase (E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.1.3.3), and xylanase (E.C. 3.2.1.32).
4.3.1.1), catalase (C.E. 1.11.1.6), cellobiase (E.C. 3,2,1.21), cellulase (E.C. 3.2.1.4), chloride peroxidase (E.C. 1.11.1.10), dextranase (E.C.
3.2.1.11), .alpha.-galactosidase (E.C. 3.2.1.22), .beta.-galactosidase or lac-tase (E.C. 3.2.1.23), .beta.-glucanase (E.C. 3.2.1.6), glucose or xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.304), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E.C. 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papain (E.C. 3.4.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), chymosln (E.C. 3.4.23.4), microbial proteases (E.C. 3.4.23.6), micro-blal metallo proteases (E.C. 3.4.24.4), pullunase (E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tannase (E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.1.3.3), and xylanase (E.C. 3.2.1.32).
38. The enzyme of claims 32, 33 or 34 wherein said enzyme is .beta.-galactosidse or .beta.-glucosidase.
39. A method for carrying out an enzyme-catalyzed reaction compris-ing: packing a column with an enzyme immobilized on a hydrophobic alkyl or aryl polyhydroxy compound; and passing a solution of the material on which said enzyme catalyzed reaction is to take place through said column.
40. The method of claim 39 wherein said column is packed with an enzyme immobilized on a hydrophobic alkyl or aryl cellulose.
41. The method of claim 39 wherein said column is packed with an enzyme immobilized on a hydrophobic alkyl or aryl cotton cloth.
42. The method of claims 39, 40 or 41 wherein said alkyl group is butyl, hexyl, actyl, decyl, or dodecyl.
43. The method of claims 39, 40 or 41 wherein said aryl group is phenyl, naphthyl or anthranol.
44. The method of claims 39, 40 or 41 wherein said enzyme is .beta. -galactosidase or .beta.-glucosidase.
45. The method of claim 41 wherein said cotton cloth is glucoamylase naphthyl cloth, and said material upon which said enzyme-catalyzed reaction is to take place is liquified.
46. A method for carrying out an enzyme-catalyzed reaction compris-ing: packing a column with an enzyme immobilized on a hydrophobic alkyl or aryl polyhydroxy compound; passing a solution of the material on which said enzyme catalyzed reaction is to take place through said column; removing said hydro-phobic alkyl or aryl polyhydroxy compound from said column; heating said re-moved hydrophobic alkyl or aryl polyhydroxy compound in a basic solution for a time and at a temperature sufficient to regenerate said hydrophobic alkyl or aryl polyhydroxy compound; immobilizing the same enzyme on said regenerated hydrophobic alkyl or aryl polyhydroxy compound by the steps of reacting said regenerated polyhydroxy compound with a bifunctional compound and with an alcohol, or with a phenol, in 2 single step, in an aqueous basic solution, immobilizing said enzyme thereon by adsorption of a solution of said enzyme thereon and stabilizing said immobilized enzyme thereon by reaction with a cross-linking agent; and carrying out said enzyme catalyzed reaction with said immobilized enzyme by the steps of: packing a column with an enzyme immobilized said hydrophobic alkyl or aryl polyhydroxy compound; and passing a solution of the material on which said enzyme catalyzed reaction is to take place through said column.
47. The method of claim 46 wherein said basic solution is 2 M NaOH
and said heating is carried out at 100° C for 1 hr.
and said heating is carried out at 100° C for 1 hr.
48. The method of claim 47 wherein said hydrophobic alkyl or aryl polyhydroxy compound is hydrophobic alkyl or aryl cellulose.
49. The method of claim 48 wherein said hydrophobic alkyl or aryl cellulose is hydrophobic alkyl or aryl cotton cloth.
50. The method of claims 47, 48 or 49 wherein said alkyl group is butyl, hexyl, octyl, decyl or dodecyl.
51. The method of claims 47, 48 or 49 wherein said aryl group is phenyl, naphthyl or anthranyl.
52. The method of claims 47, 48 or 49 wherein the enzyme is selected from alcohol dehydrogenases, .alpha.-amylase (E.C. 3.2,1.1), .beta.-amylase (E.C. 3.2.1.1), ?-amylase or glucoamylase (E.C. 3.2.1.3), asparaginase (E.C. 3.5.1.1), aspartase (E.C.
4.3.1.1), catalase (C.E. 1.11.1.6), cellobiase (E.C. 3,2,1.21), cellulase (E.C. 3.2.1.4), chloride peroxidase (E.C. 1.11.1.10), dextranase (E.C.
3.2.1.11), .alpha.-galactosidase (E.C. 3.2.1.22), .beta.-galactosidase or lac-tase (E.C. 3.2.1.23), .beta.-glucanase (E.C. 3.2.1.6), glucose or xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E.C. 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papain (E.C. 3.4.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), chymosin (E.C. 3.4.23.4), microbial proteases (E.C. 3.4.23.6), micro-bial metallo proteases (E.C. 3.4.24.4), pullunase (E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tannase (E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.7.3.3), and xylanase (E.C. 3.2.1.32).
4.3.1.1), catalase (C.E. 1.11.1.6), cellobiase (E.C. 3,2,1.21), cellulase (E.C. 3.2.1.4), chloride peroxidase (E.C. 1.11.1.10), dextranase (E.C.
3.2.1.11), .alpha.-galactosidase (E.C. 3.2.1.22), .beta.-galactosidase or lac-tase (E.C. 3.2.1.23), .beta.-glucanase (E.C. 3.2.1.6), glucose or xylose isomerase (E.C. 5.3.1.5), glucose oxidase (E.C. 1.1.3.4), hemicellulase (E.C.3.2.1.78), invertase (E.C. 3.2.1.26), lipase (E.C. 3.1.1.3), steapsin, nitrate reductase (E.C. 1.7.99.4), penicillin acylase or amidase (E.C.
3.5.1.11), peroxidase (E.C. 1.11.1.7), lecithinase (E.C. 3.1.1.4), plasmin (E.C. 3.4.21.7), pectinase (E.C. 3.2.1.15), phenol oxidases, ribonucleases, chymotrypsin (E.C. 3.4.21.1), trypsin (E.C. 21.4), subtilisins (E.C.
3.4.21.14), Asparagillus alkaline proteases, papain (E.C. 3.4.22.2), ficin (E.C. 3.4.22.3), bromelain (E.C. 3.4.22.4), pepsin (E.C. 3.4.23.1), chymosin (E.C. 3.4.23.4), microbial proteases (E.C. 3.4.23.6), micro-bial metallo proteases (E.C. 3.4.24.4), pullunase (E.C. 3.2.1.41), ren-nets (E.C. 3.4.23.4 and 3.4.23.6), tannase (E.C. 3.1.1.20), urease (E.C.
3.5.1.5), uricase (E.C. 1.7.3.3), and xylanase (E.C. 3.2.1.32).
53. The method of claims 47, 48 or 49 wherein the enzyme is .beta.-galac-tosidase or .beta.-glucosidase.
54. The method of claim 49 wherein said cotton cloth is glucoamylase naphthyl cloth, and said material on which said reaction is to take place is liquified starch.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000462923A CA1229808A (en) | 1984-09-11 | 1984-09-11 | Preparation of hydrophobic cotton cloth |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000462923A CA1229808A (en) | 1984-09-11 | 1984-09-11 | Preparation of hydrophobic cotton cloth |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1229808A true CA1229808A (en) | 1987-12-01 |
Family
ID=4128695
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000462923A Expired CA1229808A (en) | 1984-09-11 | 1984-09-11 | Preparation of hydrophobic cotton cloth |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1229808A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6303119B1 (en) | 1999-09-22 | 2001-10-16 | The Procter & Gamble Company | Personal care compositions containing subtilisin enzymes bound to water insoluble substrates |
| US6410017B1 (en) | 1998-09-22 | 2002-06-25 | The Procter & Gamble Company | Personal care compositions containing active proteins tethered to a water insoluble substrate |
| US7544635B2 (en) | 2001-07-17 | 2009-06-09 | Zhi-Wei Liang | Process for changing hydrophilic/hydrophobic characteristics |
| EP2144681A1 (en) * | 2007-05-04 | 2010-01-20 | Kurt G. I. Nilsson | Material for separation of a biomolecule |
-
1984
- 1984-09-11 CA CA000462923A patent/CA1229808A/en not_active Expired
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6410017B1 (en) | 1998-09-22 | 2002-06-25 | The Procter & Gamble Company | Personal care compositions containing active proteins tethered to a water insoluble substrate |
| US6303119B1 (en) | 1999-09-22 | 2001-10-16 | The Procter & Gamble Company | Personal care compositions containing subtilisin enzymes bound to water insoluble substrates |
| US7544635B2 (en) | 2001-07-17 | 2009-06-09 | Zhi-Wei Liang | Process for changing hydrophilic/hydrophobic characteristics |
| EP2144681A1 (en) * | 2007-05-04 | 2010-01-20 | Kurt G. I. Nilsson | Material for separation of a biomolecule |
| EP2144681A4 (en) * | 2007-05-04 | 2011-10-05 | Kurt G I Nilsson | Material for separation of a biomolecule |
| US8691544B2 (en) | 2007-05-04 | 2014-04-08 | Glycorex Transplantation Ab | Material for separation of a biomolecule |
| US9561252B2 (en) | 2007-05-04 | 2017-02-07 | Glycorex Transplantation Ab | Material for separation of a biomolecule |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US3802997A (en) | Method of stabilizing enzymes | |
| Rosevear | Immobilised biocatalysts—a critical review | |
| Petro et al. | Immobilization of trypsin onto “molded” macroporous poly (glycidyl methacrylate‐co‐ethylene dimethacrylate) rods and use of the conjugates as bioreactors and for affinity chromatography | |
| Arica et al. | Covalent immobilization of α-amylase onto pHEMA microspheres: preparation and application to fixed bed reactor | |
| Arica et al. | Immobilization of glucoamylase onto activated pHEMA/EGDMA microspheres: properties and application to a packed-bed reactor | |
| RU2157407C2 (en) | Liquid coffee extract galactomannane hydrolysis method | |
| EP0490929B1 (en) | A process for the hydrolysis of hemicellulose by immobilized enzymes and a product comprising an immobilized hemicellulolytic enzyme | |
| US4839419A (en) | Method for immobilizing dissolved proteins | |
| US4033817A (en) | Pressure-driven enzyme-coupled membranes | |
| US4013514A (en) | Preparing a reactor containing enzymes attached to dialdehyde cellulose | |
| EP0641859A1 (en) | Method for preparing immobilized enzyme conjugates and immobilized enzyme conjugates prepared thereby | |
| Garcia III et al. | Cellulase immobilization on Fe3O4 and characterization | |
| Gemeiner et al. | Biochemical engineering of biocatalysts immobilized on cellulosic materials | |
| Campbell et al. | Enzymatic recycling of coenzymes by a multi-enzyme system immobilized within semipermeable collodion microcapsules | |
| US3730841A (en) | Encapsulated carrier bound enzymes | |
| Horvath | Pellicular immobilized enzymes | |
| Spasojević et al. | The enzyme immobilization: carriers and immobilization methods | |
| D'souza | Trends in immobilized enzyme and cell technology | |
| US3849253A (en) | Process of immobilizing enzymes | |
| US5916789A (en) | Immobilized enzyme | |
| CA1229808A (en) | Preparation of hydrophobic cotton cloth | |
| Pedersen et al. | Immobilized biocatalysts | |
| Powell | Developments in immobilized-enzyme technology | |
| Adlercreutz | Immobilized enzymes | |
| CA1203187A (en) | Immobilization of invertase on polyethylenimine- coated cotton cloth |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |