Note: Descriptions are shown in the official language in which they were submitted.
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
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 .
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.
-- 10 --
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.
-- 15 --
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.
- 18 -
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
- 26 -
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~ -
