Note: Descriptions are shown in the official language in which they were submitted.
~1~53~
It is known that enzymes, which are proteinaceous
in nature and which are commonly water soluble, comprise
biological catalysts which serve to regulate many and
varied chemical reactions which occur in living organisms.
The enzymes may al50 be isolated and used in analytical,
medical and industrial applications. For example, they find
use in industrial applications in the preparation of food
products such as cheese or bread as well as bein~ used
in the preparation of alcoholic beverages. ~Some specific
uses in industry may be found in the use of enzymes such
as in the resolution of amino acids; in the ~odification
; of penicillin to form various substrates thereof; the use
of various proteases in cheese making, meat tenderizing,
detergentformulations, leather manuEacture and as digestive
aids; the use of carbonhydrases in starch hydrolysLs, sucrose
inversion, gIucose isomerization, etc.; the use of nucleases
in flavor control; or the use of oxidases in oxidation pre-
vention and in the color control of food products. These
uses as well as many others have been well delineated in
the literature.
As hereinbefore set forth, inasmuch as enzymes are
commonly water soluble as well as being generally unstable
and readily deactivated, they are also difficult either to
remove from the solutions in which they are utilized for sub-
sequent reuse or it is difficult to maintain their catalytic
, . . .
activity for a relatively extended period of time. The a-
forementioned difficulties will, of course, lead to an in-
creased cost in the use of enzymes for commerical purposes
due to the necessity for frequent replacement of the enzyme,
`:
r ~ --2--
~- .
05853~
this replacement being Isu~l~y necessary with eac1- application.
To counteract the high cost of replacement, i~ has been
suggested to immobilize or insolubilize the enzymes prior to use
thereof. By immobilizing the enzymes through various systems
hereinafter set forth in greater detail, it is possible to
stablizied the enzymes in a relative manner and, thereofre, to
permit the reuse of the enzyme which may otherwise undergo de-
activation or be lost in the reaction medium. Such immobilized
or insolubilized enzymes may be employed in various reactor
systems such as in packed columns, stirred tank reactors, etc.,
depending upon the nature of the substrate which is utilized
therein. In general, the immobilization o~ the enzymes provides
a more favorable or broader environmental stability, a minimum
of effluent problems and materials handling as well as the
possibility of upgrading the activity of the enzyme itself.
As hereinbefore set forth, several genera] methods, as well
as many modiEications thereof, have been described by which the
immobilization of enzymes may be effected. One general method
is to adsorb the enzyme at a solid surface as, for example, when
an enzyme such as amino acid acylase is adsorbed on a cellulosic
derivative such as DE~E--cellulose; papain (3.4.4.10) or ribonuc-
lease (2.7.7.16/2.7.7.17) is adsorbed on porous glass; catalase
(1.11.1.6) is adsorbed on charcoal; trypsin t3.4.4.4) is adsorbed
on quartz glass or cellulose; chymotrypsin t3.4.4.5) is adsorbed
on kaolin:Lte, etc. ~nother general method is to trap an enzyme
in a gell lattice such as glucose oxidase (1.1.3.4), urea
(3.5.1.5) papain (3.4.4.10), etc., being entrapped in a polyacryl-
amide gel; acetyl cholinesterase (3.1.1.7) being entrapped in a
starch gel or a silicone polymer; glutamic-pyruvic transamînase
(2.6.1.2) being
jl/ ~~~ ~ -3-
5~S3~
entrapped in a polyamide or cel]ulvse acetate gel, etc. A
~urther general method is a cross-linking by means of bifunc-
tional reagents and may be effected in combination with either
of the aforementioned general methods of immobilization. When
utilizing this method, bifunctional or polyfunctional reagents
which may induce intermolecular cross-linking will covalently
bind the enzymes to each other as well as to a solid support.
This method may be exemplified by the use of glutardialdehyde or
bisdiazobenzidine-2,21-disulfonic acid to bind an enzyme such
as papin (3.4.4.10) to a soild support etc. A still further
method of immobilizing an enzyme comprises the method of a co-
valent binding in which enzymes such as glucoamylase ~3.2.1.3),
trypsin (3.4.4.4), papain ~3.4.4.10), pronase ~3.4.21.4/3.4.24.4)
amylase (3.2.1.1/3.2.1.2), glucose oxidase (1.1.3.4), pepsin
(3.4.4.1) rennin (3.~ .3) fungal protease, lactase (3.2.1.23),
etc., are immobilized by covalent attachment to a polymeric
material which is attached to an organic or inorganic solid
porous support. This method may also be combined with the afore-
said immobilization procedures.
The above enumerated methods of immobilizing enzymes all
possess some drawbacks which detract from their use in industrial
processes. For example, when an enzyme is directly adsorbed on
the surface of a support, the binding forces which result be-
tween the enzyme and the carrier support are often quite weak,
although some prior art has indicated that relatively stable
conjugates of this type have been obtained when the pore size of
the support and the spin diameter of the enzyme are correlated.
~lowever, the pore size of the support cannot exceed a diameter of
about 1000 Angstroms. In view of this weak bond~ the en-
jl/ J C ~,
3~358538
zyme is often readily desorbed in the presence of solution~ of
the substrate being processed. In addition to this, the enæyme
may ~e partially or extensively deactivated due to its lack of
mobility or due to interaction between the support and the ac-
.~ ~ .
tive site of the enzyme. Another process which may be employedis the entrapment o enzymes in gel lattices which can be ef~ec-
ted by polymerizing an aqueous solution or emulsion containing
the ~onomeric form of the polymer and the enzyme or by in~orpox-
ating the enzyme into the preformed polymer by various techniques,
often in the presence of a cross-linking agent. ~hile this meth-
od of immobilizing enzymes has an advantage in that the reaction
conditions utilized to effect the entrapment are usually mild so
~ ` that often there is little alteration or deactivation of the en-
zyme, it also has disadvantages in that the conjugate has poor
mechanical strength, which results in compacting when used in
~olumns in continuous flow systems, with a concomitant plugging
of the column. Such s~stems also have rather ~tide variations
in pore size thus leading to some pore sizes which are large
enough to permit the loss of enzyme. In addition, some pore
sizes may be sufficiently small so that large diffusional bar-
riers to the transport of the substrate and product will lead
to reaction retardation, this being especially true when using
a high molecular weight. substrate. The disadvantages which are
present when lmmobilizing an enzyme b-y intermolecular cross-
linkage, as already noted, are due to the lack of mobility withresulting deactivation because o~ inability of the enzyme to as-
- sume the natural configuration necessary for maximum activity,
particularly when the active site is involved in the ~inding
process.
.
~5~3538
Covalent hindin~ methods have ~ound wide a~Plications
and may be used either a~ the sole immohilization techni~ue
or as an integral part of many of the methods already described
in which cros~-lin~ingreactions are em~loved. This method is
often used to bind the enzyme as well as the sup~ort throu~h
a bifunctional intermediary molecule in ~hich the functional
qroups of the molecule, such as, for example, q3mma-aminoproPvl-
triethoxysilane, are capable of reactinq with functional moieties
present in ooth the en~yme and either an organic or inorqanic
porous support. A wide variety of reaqents and su~ports has
been employed in this manner and the method has the advantaqe
of provlding strong covalent bonds throuqhout the conju~ate
product as well as qreat activity in many cases. The covalent
linka~e of the enzyme to the carrier must be accomPllshed
throughfunctional groups on the enzyme which are non-es~sential
for its catalytic activity such as free amino arouPs, carboxvl
groups, hvflroxyl ~roups, phenolic ~rouPs, su]fhYdryl qrouDs,
etc. These functional qrou~s will also react with a wide
variety of other functional groups such as an a~dehyae, i~ocyanato,
acyl, diazo, azido, anhydro, activated ester, etc., to produce
covalent bonds. Nevertheless, this method also often has many
disadvantages involving costly reactants and solvents, as well
as specialized and costly ~orous suP~orts and cumhersome multi-
step procedures, which render the method of preparation un-
economical for commercial a~plication.
The prior art is thexefore replete with variou~ methodsfor immobilizing enzymes which, however, in various ways fail
to meet the reqairements of inAustria] use. ~lowever, as will
'
_fi _
~L~5~ii38
hexeinafter be discu~seæ in ~reater detail, none of the Prior
art compositions eomprise the composition of matter of the
present invention which constitutes an inorqanic porous sbpport
containing a polymeric material fon~ea in situ from a monomer
or preformed polymer, of natural or svnthetic'oriclin, which is
entrapped and also adsorhed in Part within the pores of said
sunport ana wHich contains f~nctionalized, pendent grou~s ex- : ~
tending therefrom; the enzyme bein~ ~artially adsorbe'~ to the ''
~atrix and also covalently boun~ to the active moieties at or
adjacent to terminal portions of the pendent qrouPs, thus Per- '
.itting the freedom of ~.ovement which ~ill enab}e the enzy~e
to e~ercise maximum activity. For examDle, Il .S~ Patent No.
3,556,945 relates to enzyme compo.sites in which the enzvme is ..
adsorbed directly to an inorganic carrier such as qlass. U.~.
Patent ~o. 3,519,538 is concerned with enzyme composites in Yhich
.
the enzymes are chemically couPled bY ~eans o.f an interme~iary
silane coupling a~ent to an inorqanic carrier. ~n similar ' ~
fashion, U. S. Patent ~o. 3,7~3,101'also utilizes an orqano~
silane composite as a b'inding aqent, the enzYme being covalently
counled to a ~lass carrier hy ~ean.s of an intermediate sil~ne
couplinq aqent, the silicon portion of the cou~linq aqent bein~
attached to the carrier wh.ile the orqanic oortion of the counlinq ' . '
a~ent is coupleA to the enæyme, the com~osition containing ~ .
metal oxide on the surface of the carrier disposéd bet~een the ' .
carrier and the silicon portion of the couPling ac.~ent. In ll. S.
Patent No. 3,821,083 the inert carrier is coated ~7ith a preformed
polymer such as polyacrolein which has bonded thereto an enzy7~e.
~lowever, according to most of ~he examples set ~orth in the patentr
it is necessary to first acicl hydrolvze the comPosite prior to
-7-
'
~05~3S38
the deposition oE the enzyme on the polymer. Another prior
art patent, namely, U.S, Patent No. 3,705,084 discloses
a macroporous enzyme reactor in which an enzyme is adsorbed
on the polymeric surface of a macroporous reactor core and
thereafter is cross-linked in place. By cross-linking the
enzymes on the polymeric surface after adsorption thereof,
the enzyme is further immobilized in part and cannot act
freely as in its native state as a catalyst. The cross-link-
age of enzymes in effect links them together, thereby pre~
venting a free movement of the enzyme and decreases the
mobility of the enzyme which is a necessary prereguisite for
maximum activity.
This invention rela-tes to novel compositions of
matter comprising immobilized enzyme conjugates. More spe-
cifically the invention is concerned with novel compositions
of matter which comprise an immobilized enzyme conjugate
which consists of an organic-inorganic matrix constituting
an inorganic porous support material containing an organic
polymeric material which has been formed in situ from a
monomer, hydrolyzed polymer, or preformed polymer of synthetic
ox natural origin by reaction with a bifunctional monomer
containing suitable reactive moieties. Said polymer material
is both entrapped and adsorbed in t~e pores of the aforesaid
support material, and is further provided with functionalized
pendent groups extending therefrom, the functional mo.ieties
being located at or adjacent to the terminal portions thereof,
and an enzyme which is both covalently bound to said function-
alized pendent groups as well as being adsorbed in part on
the organic-inorganic ~atrix.
8-
.
.
_~ ~8
.
~05~538
As hereinbefore set forth, the use of enzymes in
analytical, medical or industrial applications may be greatly
enhanced if said enzymes are in an immobilized condition,
that is, said enzymes, by being in combination with other
5 solid materials, are themselves in such a ~ondition where-
by they are not water soluble and therefore they may be sub-
jected to repeated use while maintaining the catalytic
activity of said enzyme. In order to be present in an im-
mobilized state, the enzymes must be bound in some manner
to a water insoluble carrier, thereby being commerically
- usable in a non-water soluble statq.
It is therefore an object of this invention to pro-
vide novel compositions of matter in which enzymes are present
in an immobilized state.
A further object of this invention is to provide
compositions of matter in which an enzyme is both adsorbed on
an organic-inorganic matrix and covalently bound to functional-
ized pendent groups, attached to said matrix, which is, in
turn, aiso both adsorbed and ~ntrapped in the pores of the
- 20 inorganic porous support material.
- In one aspect an embodiment of this invention re~
sides in an immobilized enzyme conjugate comprising an com-
bined organic-inorganic matrix consisting of an inorganic
porous support containiny an organic polymeric material ad-
sorbed and entrapped in the pores of said support, said
polymeric material containing functionalized pendent groups,
and an enzyme adsorbed to said matrix and covalently bound
to the functional moieties of said pendent groups of said
organic polymeric material at or adjacent to the terminal
portions thereof.
_g_
.,. , ~ - '' '''''I
~OS853B
A specific embodiment of thi9 invention is found
in an immobilized enzyme conjugate comprising an organic-
inorganic matrix consisting of a low bulk density, porous
silica-alumin support of relatively high surface area which
may also contain inorganic additives and an in situ~prepared
tetraethylenepentamine-glutaralaehyde polymeric material
which is adsorbed as well as e~ntrapped in the pores of sai~
h3 silica-alumina, and an enzyme comprising glucoamy~ase~bein~
covalently bound to the glutaraldehyde pendent groups of
the polymeric material at or adjacent to the terminal por-
tion of said groups as welI as being adsorbed in part on
the matrix.
Other objects and embodiments will be found in
the following further detailed description of the present
invention.
As hereinbefore set forth the present invention is
concerned with immobilized enzyme conjugates comprising a
combined organic-inorganic matrix consisting of an inor-
ganic porous support material containing an organic polymeric
material adsorbed and entrapped in the pores of said inor-
ganic porous support. In addition, the polymeric material
will contain pendent groups, said pendent groups having an
enzyme being covalently bound to said groups at or adjacent
to the terminal portions thereof and, in addition, said
enzyme is also partially adsorbed on said matrix. In con-
tradistinction to the compositions of matter containing
immobilzied enzymes as set forth in the prior art the com-
positions of matter of this invention may be prepared by
utilizing relatively inexpensive reactants as well as
--10--
~ . ~.
~058538
utilizing more simple steps in the procedure for preparing
said compositions. In addition, the mechanical strength
and stability of the enzyme conjugates of the present in-
vention will be greater than that wnich is possessed by the
immobilized enzymes of the prior art. There~ore, it is
readily apparent that the compositions of matter of the
present invention possess economical advantages which are
useful for industrial applications.
The compositions of matter of the present invention
may be prepared in a relatively simple manner. In the pre-
ferred-method of preparation, the inorganic porous support
material will be treated witn a solution, preferably aqueous
in nature, of a bi- or polyfunctional monomer, a polymer
hydrolysate or a preformed polymer, following which the
unadsorbed solution is removed by any means known in the art
as draining, etc. It is also contemplated that other in-
expensive organic solvents such as acetone, tetrahyarofuran,
etc., may also be used as the carrier for the aforementioned
monomers or polymers. Following the removal of the unad-
sorbed solution, the wet porous support is then contacted
with a relatively large excess of from 5 to 20 mole percent
of a second bifunctional monomer in which the reactive groups
- are preferably separated by a chain containing from ~ to 10
carbon atoms, this second bifunctional monomer also being
added in an aqueous solution, whereby a polymeric matrix
which is both adsorbed and entrapped in the pores of the
support will be formed and from which pendent groups of the
second monomer will extend. These pendent groups will con-
tain unreacted functional moieties due to the fact that an
excess amount of the second bifunctional monomer was employed
.
-11- , '
,
~LIt)5~353~ `
in treating the support. The unreacted functional moieties
are then available for covalent binding to the enzyme, which
is added to the resulting organic-inorganic matrix, again
usually in an aqueous solution. After removal of the un-
reacted materials such as by treating, wahsing, etc., theenzyme, besides being covalently bound to the pendent fun-
ctionalised groups at or adjacent to the terminal por~ions
- thereof, will also, in par~ be adsorbed to the matrix. It
is therefore readily apparent that the entire immobili-
sation procedure can be conducted in a simple and inex-
pensive manner utilising an aqueous or inexpensive solvent
media, the procedure being conducted over a temperature
differential which may range from subambient (about 5C.)
up ~o elevated temperatures of about 60C., and preferably
at ambient ~about 20-25C.)temperature, said procedure
be_ng effect by utilising a minimum of operating steps
and, in addition, permitting a ready recovery of the excess
reactants and finished composition of matter.
Many of the inorganic supports reported in the prior
art specify "controlled pore" materials such as glass, alumina,
etc., having a pore diameter of from 500 to 700 Angstroms for
abou 96 ~ of the material and a maximum pore diamter of lO00
Angstroms,a surface area of 40-70 m2~gm and 40-80 mesh size
particles. In addition, these supports may be coated with
metallic oxides such as zirconium oxide and titaniu~ oxide
for greater stability. In contradistinction to these sup-
ports, it is contemplated within the scope of this inven~
tion that the inorganic porous supports which are utilised
hcrein, will constitute materials which possess pore dia-
metres ranging from lO0 Angstrom up ~o SS,oO0 Angstroms
-12-
~ 58538
with as much as 25-60% of the porous support material
possessing pores having diametres above 20,000 Angstroms
and surface areas ranging from 150 to 200 m2/gm. The
particle size may also vary over a wide range of from 10-20
mesh to a fine powder, said particle size depending up~n
the particular system in which the~ are to be used. It
is also contemplated that the porous support materials
may be coated with various oxides of the type hereinbe- -
fore set forth or may have incorporated therein various
other inorganic materials such as boron phos~hate, etc.;
- these inorganic materials imparting special properties to
the support material. A particularly useful ~orm of sup-
port will constitute a ceramic body which may have the
type of porosity herein described for materials of the
present invention or it may be honeycombed with connecting
macro size channels throughout, such materials being com~
monly known as monoliths, and which may be coated with
various types of porous alumina, zirconia, etc. The use of
such a type of support has the par-ticular advantage of per-
mitting the free flow of even highly viscous substrateswhich are o~ten encountered in commercial enzyme catalyzed
reactions.
The inorganic porous support materials which are
utilised as one component of the combined organic-inorganic
matrix will include certain metal oxides such as alumina,
and particularly gamma-alumina, silica, ~irconia or mix-
tures of the metal oxides such as silica-alumina, silica-
zirconia, silica-magnesia, silica-zirconia-alumina, etc.,
or gamma-alumina containinq other inorqanic compounds such
as boron phosphate, etc., ceramic bodies, etc., as well as
combinations of the aforementioned material, one of said
materials which may serve as a coating for another material
comprising the support.
-13-
~05~538
The polymeric materials which are formed in situ
in such a manner so that the E,olymeric material is both
partially adsorbed and partially entrapped in the pores of
the inorganic support of the type hereinbefore set forth
may be produced according to the general methods herein- -
before described, that is, by first adsorbing a solution
containing from 2 to 25~ of a bi- or polyfunctional monomer,
polymeric hydrolysate, or a preformed polymer, the monomer
or polymer being synthetic or naturally occurring in origin,
and which are pre~erably soluble in water or other solvents
which are inert to the reactions subsequently employed. As
hereinbefore set forth, it is contemplated within the scope
of this invention that a second bifunctional monomer is
then added in similar manner to form an organic-inorganic
matrix by reaction with the original additive adsorbed on the in-
organic support. The func-tional groups which are present on the
bifunctional monomer wil~ comprise well known reactive moieties
such as amino, hydro~yl, carboxy, thiolr carbonyl, etc. moi-
eties. As was also hereinbefore set forth, the reactive groups
of the bifunctional compounds are preferably, but not neces-
sarily, separated by chains containing from 4 to 10 carbons
atoms. The reactive moieties are capable of covalently
bonding with both the initial additives and subsequently,
after washing out unreacted materials, with the enzyme
which is to be added in a subsequent step, said enzyme
being then covalently bound to the functional group at or
adjacent to the terminal portion of the functional chain
' ' " . ' ' .
-14-
lQS853~
a~ well as concomitantly adsorbed on the matrix. After
addition of the enzyme to this composition, a relatively
stable enzyme conjugate will be produced which possesses
high activity and high stability. In addition, the com-
position of matter of the present invention also possessesappreciable versatility in addition to the other advantages
hereinbefore enumerated in that it can be applied to pre-
pare conjugates in the absence of an inorganic support
which are soluble in either acidic or alkaline media. As
will hereinafter be set forth in greater detail, depen-
ding upon the reactants employed, the conjugates will re-
tain their stability in such media when prepared in com-
bination with the inorganic support according to the pro-
cesses set forth in the prior art. sy possessing these
properties, it .is possible to widen the uses to which these
conjugates may be applied.
Specific examples of bi- or polyfunctional monomers,
polymer hydrolysates or preformed polymers which may be
initially adsorbed on the inorganic support will include
water soluble polyamines such as ethylenediamine, diethyl-
enetriamine, triethylenetetramine, tetraethylenepentamine,
pentaethylenehexamine, hexamethylenediamine, polyethylene-
- imine, etc.; water insolu~le polyamines such as methylene-
dicyclohexylamine, methylenedianiline, etc.; natural and syn-
thetic, partially hydrolyzed polymers and preformed polymers
SUC}I as nylon, collagen, polyacrolein, polymaleic anhydride,
alginic acid, casein hydrolysate, gelatin, etc. Some
specific examples of intermediate bifunctional materials
which may be added to tne above enumerated products to pro-
-15-
l~S8~38
duce an organic-inorganic matrix and which possess the
necessary characteristics hereinbefore set forth include
compounds such as glutardialdehyde, adipoyl chloride,
sebacoyl chloride, toluenediisocyanate,hexamethylenedi-
isocyanate, etc. It will be noted that when a polyeth~l-
eneamine of the type hereinbefore set forth is reacted with
glutardialdehyde in-the absence o~ an inoxganic porous
support, an aqueous acid soluble material is obtained,
whereas when a polyethyleneamine is reacted with a di-
isocyanate or acyl halide, a water insoluble product is ob-
tained. Conversely, if a reaction com~lex without the in-
organic support coneains free carboxyl groups, an alka-
line soluble complex can be obtained. Due to the large
excess of intermediary, or spacer, bifunctional molecules
which are used, the polymexic matrix which is formed wi}l
contain pendent groups comprising the spacer molecules,
said molecules extending from the matrix and having reac-
tive moieties available at or ad~acent to the terminal pox- ~ -
tions thereof which are capable of reacting with and bind-
ing the enzyme to the spacer molecules via covalent bonas.
In addition, the enzyme, when applied after the unreacted
reagents have been removed ~rom the organic-inorganic matrix
by washing, will also concomitantly undergo adsorption in
part with said matrix. Binding the enzyme solely to the
organic matrix will not usually affect the de~endency of
the solubility of the aggregate on the pll of the solution but
when the inorganic support is included as heretoeore des-
cribed, the total conjugate exhibits high stability over ~
relatively wide pl~ range from 3 to ~, tne stability of course,
-16-
.
.
l~S~53~
also being a function of the optimal pH characteristics
of the particular enzyme employed as well as the inorganic
support used. Therefore, it is readily apparent that a
suitable organic-inorganic matrix which is applicable in
many situations will be formed with the support material
by adsorbing any of the type of materials hereinbefore
described which are known to the art and then treated with
any bifunctional molecule which is also known to the art and
; is suitably unctionalised to react with the original ad-
ditive, provided that a large enough excess of the bi-
functional molecule is used to provide pendent groups which
are capable of subsequently reacting with ~he enzyme which
is desired to be immobilised. By utilising these functional
pendent groups as a binding site for the enzymes, it will
permit the enzymes to have a greater mobility and thus per-
mit the catalytic activity of the enzyme to remain at a
high level for a relatively longer period of time than will
' be attained when the enzyme has heen immobllised by any of
the other methods such as entrapment in a gel lattice, ad-
sorption on a solid surface or cross-linkage of the enzyme
by means of bifunctional reagents, etc. Not all formulations,
however, will produce equivalent results in terms of stabili-
ty or activity.
Examples of enzymes which may be immobilised by a
covalent bonding reaction and which contain an amino group
capable of reacting with an aldehydic or isocyana-to moiety
of the pendent group which is attached to a polymeric ma-
terial entrapped and adsorbed in t,he pores of a porou,s sup- ~
~ port material will include trypsin~p:paln)~ ~exo~l~nase~J(~ ta- J
:
~058S38
"
` galactosidase (3.2.1.23), ficin (3.~.4.12), bromela:in (3.~.4.2~), lactic
dehydrogenase ~1.1.1.27), glucoamylase (3.2.1.3)~ chymotxypsin (3.~ .5),
pronase, acylase, invertase (3.2.1.26), amylase (3.2.1.1/3.2.1.2) glucose
oxidase (1.1.3.4), pepsin (3.4.4.1) rennin (3.4.4.3) f~mgal protease, etc.
In general any enzyme whose active site is not involved in the covalent
bonding can be used. While the aforementioned discuss:io~ was centred about
pendent groups which contain as a functional moiety thereon an aldehydic or
isoc~anato group, it is also contemplated within the scope of this invention
that the pendent group can contain other functional moieties capable of
reaction with carboxy, sulfhydryl or other moieties usually present in
enzymes. However, the covalent bonding of enzymes containing these other
I moieties with equivalent results and may also involve apprrciably greater
costs in preparing intermediates. It is to be understood that the afore-
mentioned listing of porous solid supports, monomers, hydrolysates, polymers
and enzymes are only representative of the various classes of compounds which
may be used, and that the present invention is not necessarily limited
thereto.
The preparation of the compositions o matter of the present invention
is preferably effected in a batch type operation as heretofore already
described in detail, al-though it is also contemplated within the scope of
this invention that the formation of the finished composition of matter
may also be effected in a continuous manner of operation. When a continuous
type operation is used, a quantity of the porous solid support material is
placed in an appropriate apparatus, usually constituting a column. The
porous solid support
jl/ ~ ~ -18-
; ` ` ~
3Ll)5~538
material may be in any form desired such as powder, pellets,
monoliths, etc., and i9 charged to the column, after which
a preferably aqueous solution of, for example, a poly-
functional amine is contacted ~ith the porous support un-
til the latter is saturated with the amine solution and thee~cess is then drained. A spacer or intermediary bifun-
ctional molecule such as glutardialdehyde is then contacted
with the saturated support. The formation of the polymeric
; matrix is thus effected in an aqueous system, said reaction
being effected during a period of time which may range from
1 to 10 hours in duration, but is usually of short duration.
After removing the ~xcess glutard;aldehyde by draining and
washing out any water soluble and unreacted materials, which
in the case of a polyamine is preferably done with a buffer
solution possessing a p~ of about 4, an aqueous solution of
the enzyme is contacted or recycled through the column,
this step effecting a covalent bonding of said enzyme to
the terminal aldehydic groups of the functionalised pendent
molecules which extend from the matrix. This occurs until
there is no further physical adsorption andfor covalent
binding of the enzyme to the organic-inorganic matrix and
pendent molecules. The excess enzyme is recovered in the
effluent after draining and washinq the column. The column
is thus ready for use in chemical reactions in which the
catalytic effect of the enzyme is to take place. The pro-
cedures are, for the most part, conducted within the time,
temperature and concentration parametres hereinbefore des-
cribed in the batch type procedure and will result in
- comparable immobilised enzyme complexes. It is also con-
' .
,
.. ~
~5~53J!3
templated within the scope of this invention that with
suitable modifications of pH and temperature parametres
w~ich will be obvious to those sXilled in the art, the
process may be applied to a wide vaxiety of inorganic
porous supports, polymer forming reactants and enzymes.
The following examples are given for purposes of
illustration of the novel compositions of matter of the
present invention and to methods for preparing the same. -
~lowever, these examples are given merely for purposes of
illustration and it is to be understood that the present
invention is not necessarily limited thereto.
EX~MPLE ~
In tbis example 2 grams of a porous silica-alumina
composite which contained boron phosp;late incorporatedthere-
in having a particle size of ~0-80 mesh, a pore diametre
ranging from about 100 to about 55,000 Angstroms and a sur-
face area of about 150-200 m2/gm was utilised as the in-
organic support for the novel composition of matter of the
present invention. This support was calcined at a temper-
ature of about 500F. to remove any adsorbed moisture con-
tained tnerein. Thereafter the support was treated with 25
- ml of a ~ aqueous solution of tetraethylenepentamine at
ambient temperature for a period of 1 hour in vacuo to
facilitate the penetration of the solution into the pores of
the support. The excess unadsorbed solution was then decanted,
about 25~ of the tetraethylenepentamine having been adsorbed
into the pores of the support. Following this, the wet sup-
port was then treated with 25 ml of a 5% aqueous solution
of glutardialdehyde at ambient temperature and an almost
~ -20-
~LOS~538
immediate reaction took place with the formation of an in-
soluble reaction product both on the surface and within
the pores of the support. The excess glutardialdehyde sol-
ution was then decanted and the organic-inorganic complex
was washed to remove unreacted and unadsorbed reagent~,
said washing being accomplished first with water followed
by washing with a 0.02 molar acetate buffer solution which
possessed a pH of 4.2, the washing operation being effected
at a temperature of 45C. Therea~er an enzyme solution
containing about 200 mg of glucoamylase~per 25 ~1 of water
was added and allowed to react with the composite at am-
bient temperature for a period of 1 hour. At the e~d of
this l-hour period, the excess glucoamylas~As~o~u~l~o~ was
decanted and the enzyme conjugate was washed with water to
remove any unbound and/or unadsorbed enzyme. The composi-
tior was then leached for a period of 24 hours with an ace-
tate buffer solution similar to that hereinbefore desoribed.
The amount of adsorbed and/or covalently bonded enzyme was
determined by micro Dumas gas chromatography analyses both
before and after addition of the enzyme. The activity of
the en2yme conjugate was then determined by the amount of
glucose produced using 30~ thinned starch solution as sub-
strate at a pH of 4.2 and 60C., and employing Worthington's
glucostat procedure for analysing glucose, the latter being
considered the more reliable procedure for determining the
utility of the conjugate. An activity of 28 units per gram
of support with an enzyme loading of 29 mg/gm of support was
obtained by this procedure (one unit representing the pro-
duction of l gram glucose per hour at 60 C. according to the
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\
~058S38
assay specifications~. It will be noted that despi~e the
known solubility at p~l of 4.2 of the enzyme conjugate when
prepared in the abse~ce of an inorganic support, negligible
loss of enz~me from the combined inorganic-organic complex
oacurred during leaching with the 4.2 pH buffer solution.
This was demonstrated by assaying the effluent from this
treatment.
EXAMPLE II
In th'is example the procedure of Example I was
followed with the exception that the inorganic porous sup-
port had a particle size of 10-30 mesh. This silica-alumina
composite containing boron phosphate incorporated therein
was treated with tetraethylenepentamine, glutardialdehyae
~ and glucoamylasel ln ~a ma L er similar to that set forth a-
bove. An active immobilised enzyme complex was obtained
although of decreased activity probably because a diffusion
problem is produced by the larger particle size of the com-
posite.
EXAMPLE III
In a manner similar to that set forth in Example
I above, 2 grams of a silica-alumina composite possessing
the same physical characteristics of particle size, pore
diameter and surface area as that set forth in Example I
was treated with an acetone solution of tetraethylenepen-
tamine and followed by a tolunediisocyanate solutionalso in
acetone instead of aqueous glutardialdehyde. After de-
canting the excess diisocyanate solution and washing with
water, the organic-inorganic complex was further treated
with an aqueous glucoamylase solution. As in Example I,
the finished product comprised an active completely in~
soluble enzyme complex.
,
'1
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8538
EXAMPLE IV
To illustrate the point that various concentra-
tions of solutions can be used to prepare the desired pro-
duct, the procedure set forth in ExampleI above was re-
S peated with the exception that more highly concentratedsolutions of the various reagents were used. For example,
2 grams of a 10-30 mesh silica-alumina composite was treated
with 25 ml of a 20% tetraethylenepentamine solution and
` after decanting 50 ml of a 25~ glutardialdehyde solution
was added thereto. This complex, after washing, was then
treated with aqueous glucoamylas~ to prepare an immobilised
enzyme conjugate which showed an activity of akout 12 units
per gram based on the glucostat test.
EXAMPLE V
- 15 To a silica-alumina composite comprising 2 grams
of 10-30 mesh particles was added 25 ml of a 5% aqueous,
partially hydrolyzed collagen solution which was in place of
the tetraethylenepentamine. After decanting and treating
with glutardialdehyde, the organic-inorganlc matr ~ was
washed and then treated with a glucoamyla ~solution. The
finished composition of ma-tter was treated in a manner similar
to that set forth in Example I above by decanting, washing
and leaching with a buffered (pH of 4.2) solution to give an
immobilised enzyme conjugate which had an activity of about
10 units per gram.
EX~PLE VI
` In this example a silica-alumina composite having
a particle size of 10-30 mesh, a pore diametre ranging from
about 100 to about 55,000 Angstroms and a surface area of
-23-
,
~ . ''' '"' ' ' .
~IS1353~
- ~ from about 150-200 m2/gm was treated by adding tetraethylenepentamine in
a 1% partially hydrolyzed aqueous collagen solution, the collagen being
utilized as an additional bonding agen~. Afte~ draining and reacting with
glutartialdehyde, the organic-inorganic matrix was then treated with a
glycoamylase (3.2.1.3) solution according to the general procedure of
Example I to prepare an active enzyme conjugate.
EX~MPL~ VII
To illustrate that various enzymes can be used in preparing the
desired compositions of matter, a silica-alumina composite containing boron
phosphate incorporated therein was treated with a tetraethylenepentamine
solution, decanted, washed, followed by addition of a glutartialdehyde
solution and the resulting composite was then treated with an aqueous lactase
(3.2.1.23) solution. This produced an active enzyme conjugate. Similar
procedures can be used to bind enzymes such as proteases, glucose isomerase
(5.3.1.18), and glucose oxidase ~1.1.3.4) to produce active conjugates.
EXAMPLE VIII
In this example a column possessing an inside diameter of 20 mm
contained 1402 grams of an active enzyme conjugate prepared from glucoamylase
(3.2.1.3) which was bonded to a 10-30 mesh silica-alumina porous support
containing boron phosphate incorporated therein, the conjugate having been
prepared in a manner similar to that set forth in Example I above. The
column was used continuously for a period of 30 days at a temperature of
45C to hydrolyze an aqueous 30% thinned starch solution which had been
buffered to a pH of 4.2. The eEfluent was monitored for the glucose
production using the
~ -2~-
~L~5853~!3
Worthington glucostat procedure. ~t was found that there
was no apparent loss of enzyme activity during this perioa
of time and that the percentage of conversion of qtarch
to glucose at this temp2rature and at a flow rate of a-
5 bout 150 ml per haur was 62%.EXAMPLE IX
To illustrate the fact that various substrates or
supports may be utilised to prepare the desired composi-
tions of matter, an alumina coated ~onolith which consisted
of a Ceramic body honeycombed with connecting macro size
channels was treated in a manner similar to that set forth
- . in Example I aboYe, that is, the monolith was treated with
olutions of tetrae~hylenepentamine, giutar~ialdehyde and a
glucoamylase(~en~zyme) the treatment being carried out in a
sequential operation which included decanting~ washing, and
leaching ~rocedureshereinbefore described. The original
ceramic monolith possessed a dry weight of 256 grams, of
whlch 13% consisted of an alumina coating. The finished
immobilised enzyme conjugate was elaborated into a column
within a glass tube having an inside diametre of 70 mm in
order that it could be operated continuously by means of a
suitable pUmping apparatus within a temperature controlled
container, said container being maintained at a temperature
of 45C. Over a 40-day period of continuols usage for the
hydrolysis of a 10% buffered thinned starch solution, it
was found that only about 3~ of the original activity of
the enzyme conjugate was lost while maintaining a flow rate
of about 85 ml per hour. In addition, it was found that
during the 40-day pe~iod th;re was an approximate 80%
L
` ` ` ...
~qD5~31S3~3
co~e~sion of the starch to glucose. In order to further
study the properties of the system, subsequent variations
in f low rate were made during which it was found that at a
flow rate of about 38 ml per hour it was possible to obtain
a conversion in the range of from 92~93% of starch to glu-
cose. The relatively long period of timeiduring ~hich this
enzyme was used to convert starch to glucose without a
significant loss of enzyme activity eith~r by desorption or
deactivation indicated a long half life of the catalyst.
EXAMPLE X
In this example a monolith type of conjugate and
column similar to that described in Example IX above was pre-
pared, the exception being that the enzyme which )was used
~ to prepare the~ omplex comprised lactas(e~n p~ace oE gluco-
amylase~ ~ ~ é conjugate was tested for stability under acontinuous flow while maintaining the temperature at 3/C.
for a period of 29 days. It was again found that there was
no apparent loss of activity of the immobilised enzyme con-
jugate. This immobilised enzyme was used in the treatment
of a 5% lactose solution which had been buffered to a pH
o 4.2, said lactose solution being charged to the column
at a rate of 54 ml per hour. It was found during the 29-
day period that there was about 35~ conversion of lactose to
glucose and galactose.
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