Note: Descriptions are shown in the official language in which they were submitted.
3~
This invention relates to the immobilization of enzymes on a
carrier to which enzymes had not previously beem immobilized.
It is very well known to immobilize enzymes on a vast number of
insoluble carriers. Immobilization of enzymes facilitates their recovery
and reuse. Furthermore immobilized enzymes are often more stable than
soluble enzymes. Enzyme immobilization is especially important if relatively
costly enzymes are applied. However, these advantages have often been
seriously handicapped by the high cost of materials and processing employed
for enzyme immobilization.
The advantages of using an immobilized enzyme in comparison with
a soluble one is that the immobilized enzyme is reusable and does not
contaminate the reaction products and therefore is ~min~ntly suitable for
continuous or repeated use. There are, however, some disadvantages of the
known immobilized enzymes, a ngst them being the following: some of them
have to be prepared by rather complicated methods and, accordingly, are
relatively expensive; and others require a rather large proportion of a
carrier, which means that these known immobilized enzymes are not very
compact. Some of the known immobilized enzymes suffer from the disadvantage
that, when packed in a column, they exhibit flow properties which are not
satisfactory.
The interest in carrier-bound enzymes, is continuously increasing
and numerous carrier materials and fixing methods have already been described.
~'
-- 1 --
'~2~3~.t7
However, only a few of the previously known methods and carrier materials
give really satisfactory products with a high activity and in good yield
and of sufficient stability. Therefore, only a Eew carrier-bound enzymes
have hitherto been commercially available. This is particularly due to the
fact that the more sensitive enzymes or enzyme complexes either completely
lose their activity when fixed by the previously known methods or are so
unstable that they cannot be used for technical purposes.
,
Methods employed for the immobilization of enzymes by
attachment to or on a matrix fall into four principal methods: (a) covalent
chemical linkage via functional groups of the enzyme that are not essential
to enzyme activity; (b) entrapment or inclusion of the enzyme within a
hydrophilic gel lattice which retains the enzyme but allows
substrate and product to pass through; (c) ionic binding (physical adsorp-
tion) on hydrophilic ion exchangers, or on charcoal or glass beads; and (d)
cross-linking enzymes into large aggregates by reaction with bifunctional
compounds.
The covalent linkage of enzymes to insoluble carriers offers a
method of preparing water-insoluble derivatives which will not be solubilized
when used or when the composition of the medium is charged provided that the
covalent bonds formed are such as will not be broken under the conditions
of biochemical use. In general, the binding of a biologically active
protein to an insoluble carrier by covalent bonds must be carried out via
functional groups on the enzyme which are non-essential for its biological
activity. The binding reaction should obviously be performed under con-
ditions which do not cause denaturation.
Various physical attributes of the carrier e.g. solubility,
-- 2 --
~2C~3~
mechanical stability, swelling characteristics and porosity, as well as
its electric charge and hydrophilic or hydrophobic nature, play a major
role in detPrm;n;ng the maximal amount of enzyme which can be covalently
bound, and the stability and biological activity of the insoluble product.
Minimal solubility, high -ch~n; cal stability and adequate particle size
are essential for the preparation of biologically-active, bound enzymes
which can be readily and completely removed from reaction mixtures by
filtration or centrifugation. Similar requirements must be met in the
preparation of stable, biologically-active columns with well defined flow
rates.
m e hitherto described preparations of this type have not proved
to be useful from an economic-technical point of view, as the coupling
reagents, carrier substances or production processes required are too
expensive. Thus, in the coupling processes presently known, it is neces-
sary to work with a substantial excess of enzymes, as a large proportion
of the enzymes is inactivated during the coupling procedure. Most of the
processes provide only for a small proportion of enzymes to be attached
to the carrier, as the carrier can only be covered with enzymes on their
surface. This problem can be partially solved by pulverization of the
finished products, but, due to their fineness, the resulting preparations
significantly restrict the flow of liquids therethrough and their use
in a p~acked-bed reactor is limited.
The i~mobilization of en~ymes by inclusion techniques is not com-
pletely satisfactory since small amounts of enzymes leak out from such
prepared gels.
Enzymes immobilized by adsorption to an insoluble carrier, e.g.
~203~
active charcoal or a polysaccharide have the disadvantage that, as a result
of the relatively weak adsorptive attachment, desorption easily occurs.
Thus, if changes in the concentration of ions and changes of temperature
occur, a detachment of the enzyme from the carrier may easily happen result-
ing in the so-called "bleeding-out" of the enzymes.
Ionic binding is not a reliable technique when a totally insoluble
preparation is desired since partial or total desorption of enzyme may
result from a change in ionic strength, pH or temperature, or addition of
substrate. In the case of ionic bonding of enzymes to a polyanionic or
polycationic carrier, (such as, for example, ion-exchange resins), the
further disadvantage exists of a relatively weak bonding between the poly-
ionic carrier and the enzymes. This is due to the enzymes generally having
only weakly ionic groups. E`urthermore, an ion-exchange effect occurs with
enzymes thus attached, leading to the unintended removal of certain ions
from a liquor in the course of the treatment thereof, which may be a
decisive disadvantage disturbing many reactions.
Enzymes encapsulated into polymeric substances (for example, cross-
linked polyacrylamides) have the disadvantage that substrate and products
suffer diffusional resistance though the molecular structure of the encap-
sulating material. The apparent Michaelis' constant of an encapsulated
enzyme is therefore increased. In addition, there is the possibility that
the encapsulated enzymes, due to their elasticity escape the interstices
of the encapsulating material and thus "bleed-out". The cross-linking of
enzymes with bi- or polyfunctional reagents is now known. It is also known
to bring the enzyme with a gelling protein into a particulate form and to
cross-link the mixture in particulate form with a bi- or polyfunctional
reagent.
12C~3~L~7
In many cases, however, it is desirable to use soluble enzyme con-
jugates. Textiles will not be desized to any extent by an insoluble ~ -
amylase because, on a solid carrier, the starch cannot react with the in-
soluble enzyme or only in a slight degree. Moreover enzymes affixed to an
in~soluble carrier have a relatively short conversion rate as compared with
enzymes in solution. The soluble, stable form will often be preferred to
the insoluble form for therapeutic and cosmetic purposes. Further, it is
known to obtain soluble enzyme conjugates by cross-linking, e.g. to form
water-soluble conjugates of chymotrypsin with poly-acrylic acid, poly-
glutamic acid and carkoxymethylcellulose.
Most of the above-mentioned processes for i D bilization of enzymes
have the disadvantage that the carriers may be restricted to certain
spatial forms and it may be impossible to give them a desired shape, or to
use them as coatings for other materials. Some of the carriers and coupling
reagents which have been proposed may also be undesirable due to their
toxicity. Some of the above-mentioned disadvantages of known immobilized
enzymes to collagen, are said to be overcome by the use, for example, of
glutaraldehyde. The preparations thus produced, however, are easily
attacked by microbes and their specific activity is low. A similar problem
arises with preparations obtained by cross-linking enzymes with gel-forming
proteins. In such preparations a homogeneous mixture is obtained as the
enzymes and the gel-forming protein are cross-linked at random. While
these preparations are more stable towards microbes, their activity is poor
as the
-- 5 --
~2~3~7
enzymes are homogeneously distributed over the whole cross-section of the
product. It is possible to obtain a high activity only by cross-linking
relatively weakly. This leads however to soft preparations, which when
used in a packed-bed reactor cause blocking and bleeding out of the enzymes.
On the other hand, stronger cross-linking causes inactivation due to too
strong a bonding between the enzyme and the carrier and due to the inclusion
of enzyme molecules, as indicated above. A considerable disadvantage of
the last mentioned immobilization processes is the fact that they cannot
be used for proteolytic enzymes.
More specifically, moreover during recent years, efforts have been
devoted particularly to the development of immobilization technology in the
starch processing industry in connection with the manufacture of such pro-
ducts as high dextrose and high fructose syrups. Lately, the relative im
portance of the latter has been increasing in food and related industries,
high fructose syrup being an advantageous substitute for sucrose and for
(the less sweet) high dextrose syrup. Two additional types of sugar syrups
based on starch hydrolysis, with both syrups having a substantial content
of maltose, namely high maltose and high conversion syrup, are being used
to an increasing extent, particularly in the confectionary (hard candy) and
canning industries, respectively.
The over-all industrial process of converting starch via high
dextrose syrup in-to high fructose syrup comprises three consecutive steps,
namely the starch thinning or liquefaction process to dextrins, catalyzed
by acid and/or by a bacterial alpha-amylase, followed by saccharification
to a high dextrose syrup and then the conversion of the latter into high
fructose syrup, each process being catalyzed by its specific enzyme, viz.
amyloglucosidase and glucose isomerase, respectively. However,
-- 6 --
1;2~31~7
industrial saccharification is still pred~m;n~ntly a batch-type process.
The starting material of a high maltose type of syrup is also liquified starch
produced by either one of the methods just deseribed. However, in this case
the subsequent saceharifieation step resulting in a syrup of high maltose
eontent (and usually eontaining relatively little glueose) is effected by
means of a maltogenic amylase, preferably a fungal alpha-amylase. Like
glucogenic saccharification, the corresponding maltogenic process is usually
condueted batch-wise using the soluble enzyme.
The phenomenon of increased reversion eneountered with an immobilized
enzyme produet of this type appears at least partly to be an inherent dis-
advantage of using porous enzyme carrier materials. Clearly the porous
strueture eontributes to a very substantial degree to the total surfaee area
lending itself to enzyme bonding and, eonsequently, to the maximal enzyme
aetivity obtainable of the immobilized enzyme produet.
However, the high eoncentration of amyloglueosidase attaehed to
the earrier pore surfaee eombined with a reduced diffusion rate within the
pores inevitably results in locally high glucose concentrations, thus eon-
s-tituting favourable eonditions for the promotion of enzyme processes having
high K -values. This is the ease with the undesirable reversion reaetions,
and partieularly those in whieh isomaltose and isomaltriose are formed.
Many patents have been proposed ~o overcome the problems discussed
above. Thus Canadian Patent 900,391 issued May 16, 1972 to N.V. Organon
provided a process for the preparation of stable, water-soluble enzyme con-
jugates by forming covalent bonds with compounds containing several reactive
groups. In that patent an enzyme and a protein hydrolysate are reacted
together with these reactive compounds to obtain soluble conjugates with a
much higher yield of activity as compared with the known methods. The con-
-- 7 --
121333L~'7
jugate is then c~oss-linked with a water soluble reactive polymer e.g.
glutaraldehyde. The product is used as a laundering agent.
~ AnA~;an Patent 965,716 issued April 8, 1975 to Beecham Group Limited
provided water insoluble enzyme preparations in the form of an enzyme,
absorbed on a water insoluble material, e.g. a finely divided polymeric
material, e.g. a cellulose powder, and thereafter rendered water insoluble
by means of a water-soluble dialdehyde, e.g. glutaraldehyde.
Canadian Patent 970,305 issued July 1, 1975 to MonsantoCompany
provided immobilized enzymes which were adsorbed to a polymeric material.
The act~ve enzyme adsorbed on the polymeric surface was further immobilized
by cross-linking in place with a crosslinking agent, e.g. dialdehyde, mono-
meric polyisocyanat~, an imidoester, disulfonyl halide, and the like. The
macroporous reactor core was said to be made of acrylic-type or polyamide-
type macromolecules, polyurethane, and the like, thereby providing an ad-
sorption-promoting surface, or to be made of inert material, e.g. a foamed,
open cell, polyolefin or the like, and then the surfaces thereof coated so
as to provide the desired polymeric surface thereon.
Canadian Patent 993,387 issued July 20, 1976 to Beecham Group Limited
provided an immobilized enzyme in the form of an enzyme associated with a
water insoluble absorbent substrate, e.g. a cellulose derivative or an ion
exchange resin, and then treated with both a water-soluble dialdehyde, e.g.
glutaraldehyde and then with an aliphatic diamine, e.g. l,3-diaminopropane
or l,6-diaminohexane.
Canadian Patent 1,011,672 issued June 7,1977 to Nova Terapeutisk
Laboratorium provided an enzyme preparation in the form of a shaped body of
a water insoluble structure, e.g. polyamine fibres or microspheres, and a
non-proteinaceous binder, e.g. carbohydrates or polyamines, which had been
cross-linked with a cross-linking agent, e.g. glutaraldehyde.
-- 8 --
~203~
Canadian Patent 1,020,894 issued Nov.15, 1977 to Gist-Brocades
N.V. provided a water-insoluble enzyme preparation in semi-solid or solid
particulate form, e.g. a gelling protein, i.e. gelatin and an enzyme, treated
with a polyfunctional protein cross-linking agent, e.g. glutaraldehyde
or epichlorohydrin.
Canadian Patent 1,023,287 issued Dec. 27, 1977 to Boehringer ~nnhPi~
G.m.b.H. provided an enzyme preparation in the form of an enzyme first
reacted with an acylating or alkylating coupling compound, e.g. one containing
ethylene imine groups, and then bound on a carrier substance, e.g. a hydro-
philic easily swellable water insoluble material, e.g. films or filaments of a
cellulose material.
Canadian Patent 1,025,382 issued Jan 31, 1978 to Pfizer Inc. provided
an immobilized enzyme in the form of the reaction product of the enzyme
with a polymerizable, ethylenically unsaturated monomer, e.g. acrylic or
methacrylic acid, which was then polymerized in the presence of a cross-
linking agent, e.g. N,N'-methylene-bisacylamide, and an initiator, e.g. a
Redox initiator, i.e. dimethyIaminoproprionitrile and ammonium persulfate.
Canadian Patent 1,034,523 issued July 11, 1978 to Beecham Group
~imited provided an immobilized enzyme preparation in the form of an enzyme
cross-linked onto a macroporous polymer of methacrylic acid, e.g. AMBERLITE.
The cross-linking agent may be glutaraldehyde.
Canadian Patent 1,072,029 issued Feb. 19, 1980 to Novo Industri
A/S provided an insolubilized enzyme in the form of water insoluble carrier
particles, e.g. casein or albumin, coated with a proteinaceous layer, in
which the enzyme is immobilized by cross-linking with glutaraldehyde.
Canadian Patent 1,088,009 issued Oct. 21, 1980 to Boehringer
Ingelheim G.m.b.H. provided immobilized enzymes in the form of a porous water-
insoluble protein polymer e.g. gelatin or albumin having the enzyme
_ g _
~2~31~7
covalently bonded thereto by glutaraldehyde.
U.S. Patent No. 3,705,084 describes the adsorption of enzymes onto
polymeric surfaces and their subsequent cross-linking into place. Polymers
and copolymers of methacrylic acid are stated to be suitable for making such
polymeric surfaces. The patent however specifies that before polymers and
copolymers of methacrylic acid can be used according to this patent, carboxyl
groups on the polymer and copolymer must be converted to one of the specifically
taught adsorption promoting groups.
U.S. Patent No. 4,011,137 (Thompson et al.) teaches an insolubilized
maltogenic alpha-amylase product, prepared by bonding the soluble enzyme to
aminoethylcellulose by means of glutaraldehyde.
It has been proposed in British Patent Specification No. 1,193,918
to overcome these difficulties by bonding the enzyme to a polymer substrate
and thereby rendering it water-insoluble. However, such polymer-enzyme
complexes have been found to have a poor mechanical stability when the poly-
mers employed as substrate are those generally available on a commercial
scale for other purposes. These polymers have to be specially designed and
prepared for this purpose and this is unduly expensive.
The concept of adsorbing an enzyme on a substrate and cross-linking
it in situ, is described in British Patent Specification No. 1,257,Z63. It
teaches the use of polyfunctional reagents other -than water-soluble dialde-
hydes, for example by the use of biz-diazo-o-dianisidine.
It has also been proposed in the specification of German Offen-
legungsschrift No. 2,143,062 to prepare a water-insoluble penicillin acylase
preparation by adsorbing the acylase on a substrate and cross-linking it
there by the action of a water-soluble dialdehyde with or without the forma-
tion of links between the polymer substrate and the enzyme.
- 10 -
~L2(~3~37
Immobilized enzymes and their preparation are also described
in the following patents:
Vnited States 3,536,587
United States 3,574,062
United States 3,607,653
United States 3,616,229
United States 3,619,371
United States 3,645,852
United States 3,650,900
United States 3,650,901
British 1,224,947
British 1,274,869
German 1,935,711
German 2,012,089
one commercially useful enzyme, namely yeast invertase is used
commercially in free form for the production of invert sugar. Although
the enzyme has been immobllized, such immobilized invertase has not been
used commercially, even though the following are typical patents teaching
that immobilized enzymes may be used isomerizing glucose to fructose:
3:18~
1) 1,004,616 issued Feb. 1, 1977 to Standard Brands Inc. which teaches the
passage of the solution through a bed of microorganisms containing intra-
cellular xylose isomerase;
2) 1,020,476 issued Nov. 8, 1977 to Standard Brands Inc. which teaches the
passage of the solution through a bed of cells of microorganisms containing
intra-celluloar xylose isomerase which is naturally fixed or stabilized;
3) 1,036,089 issued Aug. 8, 1978 to CPC International Inc., which teaches the
passage of the solution through a column packed with dextrose isomerase
immobilized by being absorbed on macroreticular-type or porous-type basic
anion exchange resins;
4) 1,060,824 issued Aug. 21, 1979 to CPC International Inc., which teaches the
passage of the solution through a bed containing a dextrose isomerase enzyme
preparation and a soluble chelating agent;
5) 1,060,825 issued Aug. 21, 1979 to CPC International Inc., which teaches the
passage of the solution first through a bed containing a chelating resin and
then through a bed containing a dextrose isomerase en~yme;
6) 1,106,306 issued Aug. 4, 1981 to Pro~ektierung Chemische Verfahrenstecknik
Bm.b.~. which teaches the passage of a xylan containing solution through a column
filled with a carrier having the enzymes bonded thereto;
and
7) 1,133,410 issued Oct. 12, 1982 to National Research Development Corporation,
which teaches the passage of the solution through a reactor containing
a bed of permeable whole cell preparation immobili~ed on a gel matrix or on
an ion exchange support material.
- 12 -
~12~3~L~7
In spite of the many proposals made to immobilize enzymes, and in
spite of the many uses to which such immobilized enzymes may be put there
still is a need to provide a less costly, simpler and safer me-thod for
enzyme immobilization.
An object of an ~aspect of this invention is to provide new,
stable, immobilized enzyme preparations with high enzyme incorporation and
high enzyme activity, which stable preparations can be used repeatedly
without significant loss of enzyme activity.
An object of another aspect of the present invention is to provide an
especially mild process for binding enzymes to insoluble carriers, which gives
products which not only have a high activity and activity yield but which are
also, above all, so stable that they can be used for technical purposes.
An object of yet another aspect of this invention is to provide insoluble
long-acting and reusable forms of enzymes which do not suffer from the dis-
advantage of high cost.
An object of still another aspect of this invention is to provide
such enzymatically-active materials in forms adapted for ready contact of a
substrate with the insoluoilized enzyme.
An object of yet another aspect of this invention is to provide an
insolubilized product having process characteristics essentially similar to
those of the soluble enzyme, irrespective of whether the immobilized product
is used in a batch process or in a continuous mode of operation.
Z03~
An object of another aspect of the present invention is the
provision of economically favourable, insolublized products, by the utili-
zation of comparatively inexpensive, commercially readily available carriers
and other auxiliary materials and, furthermore, the achievements of sub-
stantial recoveries of enzyme activities in the immobilization process and
that all materials used should be acceptable for food processing purposes.
By a broad aspect of this invention a process is provided for
immobilizing an enzyme on the surface of a cotton cloth, which comprises:
treating the cloth with a chemical agent to provide an ionic surface thereon;
adsorbing the enzyme on the surface by ionic interactioni and finally
immobilizing the adsorbed enzyme on the cloth by cross-linking with a cross-
linking agent.
By one variant thereof, the chemical agent is selected to provide
an anionic surface on the cloth.
By variants thereof, the chemical agent is selected from the
group consisting of polyalkylene polyamines and quaternized polyalkylene
polyamines.
By a variation thereof, the chemical agent is polyethyleni-
mine.
By a further variant thereof, the cross-linking agent is a bi-
functional compound or a multifunctional compound, each containing groups
selected from the group consisting of aldehydes, isocyanates, sulfonyl
halides, imidoesters, succinimides, diazonium compounds, azides, anhydrides,
chlorotriazines, and polysaccharides activated by cyanogen bromide.
By a variation thereof, the cross-linking agent is glutaraldehyde.
- 14 -
~LZC~3~7
By a variation thereof, the polyethylenimine is in the form of
0.1% by weight aqueous solution having a pH of 7Ø
By a further variation thereof, the glutaraldehyde is in the
form of a 0.2 - 2% by weight aqueous solution.
By yet a further variation thereof, the polyethylenimine is in
the form of a 0.1% by weight aqueous solution having a pH of 7.0 and
the glutaraldehyde is in the form of a 0.2 - 2~ by weight
aqueous solution.
By another variant thereof, the enzyme is selected from the
0
group consisting of: inv~rtase; glucoamylase (amyloglucosidase)
(3.2.1.3); lactase (3.2.1.23); lipase (3.1.1.3); esterase (3.1.1.1);
glucose-isomerase (5.3.1.18); glucose-oxidase (1.1.3.4)i alcohol
dehydrogenase (1.1.1.1); penicillinase (3.5.2.6); penicillin amidase;
catalase; amino-acylase; phenol oxidase; inulase; carboxy-peptidase;
ribonuclease; trypsin; ficin; subtilisin; pepsin; rennin; and chymo-
trypsin.
By a variation thereof, the enzyme is invertase.
By another aspect of this invention, a cotton cloth is pro-
vided to which an enzyme is immobilized by cross-linking after having
been adsorbed into an ionic surface thereof by ionic interaction.
By one variant, the cotton cloth is one to which an enzyme
is immobilized by cross-linking after having been adsorbed onto a sur-
face which has been made ionic by a chemical agent selected Erom the
group consisting of polyalkylene polyamines and quaternized polyalkylene
polyamines.
By a variation thereof, the surface has been made ionic by
polyethylenimine.
- 15 -
~LZ03~L~37
By another variant, the cloth is one to which an enzyme is
immobilized by cross-linking with a bifunctional compound or a multi-
functional compound each containing groups selected from the group
consisting of aldehydes, isocyanates, sulfonyl halides, imidoesters,
succinimides, dlazonium compounds, azides, anhydrides, chlorotriazines,
and polysaccharides activated by cyanogen bromide,after having been
adsorbed onto a surface which has been made ionic by a chemical agent
selected from the group consisting of polyalkylene polyamines and
quaternized polyalkylene polyamines.
By another varia~ion thereof, the cross-linking is with glu-
taraldehyde after having been adsorbed onto a surface which has been
made ionic by polyethylenimine.
By yet another variant the cotton cloth is one to which
an enzyme selected from the group consisting of:invertase; glucoamylase
(amyloglucosidase) (3.2.1.3); lactase (3.2.1.23); maltase (3.2.1.20);
amylase (3.2.1.1); urease (3.9.1.5); lipase (3.1.1.3); esterase
(3.1.1.1); glucose-isomerase (5.3.1.18); glucose -oxidase (1.1.3.4);
alcohol dehydrogenase (1.1.1.1); penicillinase (3.5.2.6); penicillin
amidase; catalase; amino-acylase; phenol oxidase; inulase; carboxy-
peptidase; ribonuclease; trypsin; ficin; subtilisin; pepsin; papain;rennin; and chymotrypsin,is immobilized by cross-linking with a
bifunctional compound or a multifunctional compound each containing
groups selected from the group consisting of aldehydes, isocyanates,
sulonyl halides, imidoesters, succinimides, diazonium compounds,
azides, anhydrides, chlorotriazines, and polysaccharidos activated
by cyanogen bromide,after having been adsorbed onto a surface which
has been rnade ionic by a chemical agent selected from the group con-
sisting of polyalkylene polyamines and ~uaternized polyalkylene
- 16 -
~;2C33~7
polyamines.
By yet another variation thereof, the enzyme is invertase which
is immobilized by cross-linking with glutaraldehyde after having been
adsorbed onto a surface which has been made ionic by polyethylenimine.
By yet another aspect of this invention an apparatus is provided
for carrying out an enzymatic reaction comprising a cylindrical column
packed with pieces of cotton cloth to which an enzyme is i D bilized by
cross-linking after having been adsorbed into an ionic surface thereof.
By a further aspect of this invention an apparatus is provided
for carrying out an enzymatic reaction comprising a cylindrical column
packed with a cylindrically wound core of cotton cloth to which an enzyme
is i D bilized by cross-linking after having been adsorbed into an ionic
surface thereof.
By yet another aspect of this invention, an apparatus is provided
for carrying out an enzymatic reaction comprising a cylindrical column
packed with pieces of cotton cloth to which invertase is immobilized by
cross-link'ng with glutaraldehyde after having been adsorbed onto a surface
whlch has been made ionic by polyethylenimine.
By still another aspect of this invention, an apparatus is provided
for carrying out an enzymatic reaction comprising a cylindrical column
packed with a cylindrically wound core of cotton cloth to which invertase
is immobilized by cross-linking with glutaraldehyde after having been
adsorbed oLto a surface which has been made ionic by polyethylenimine.
By variants thereof, the cotton cloth is one of the variants or
variations described above.
- 17 -
:~L2~3~7
By a still further aspect of this invention, a process is pro-
vided for the inversion of sucrose comprising passing a solution of
sucrose at a space velocity of up to 50 through a cylindrical colu~n
packed with a cylindrically wound core of cotton cloth to which invertase
is immobilized by cross-linking with glutaraldehyde after having been
adsorbed onto a surface which has been made ionic by polyethylenimine.
Cotton flannel cloth has an open and compaet structure devoid
of fines. Columns packed with staeks of such a fabric exhibit excellent
flow. Since yeast invertase is an acidic protein, it can adsorb to an
,10 anion exchanger. A simple way according to the present invention to
convert cot-ton cloth to an anion exchanger is by coating with polyethyleni-
mine.
In one embodiment of the present invention, a simple, inexpensive
and safe process for immobilizing yeast invertase has been provided. Cot-
ton flannel is first coated with polyethylenimine. Invertase is then
adsorbed to this cloth and is fixed thereon by glutaraldehyde treatment.
A column packed with cloth segments exhibited good flow and efficient
sucrose hydrolysis over 3 months.
One procedure for carrying out the process of an embodiment of
this invention is as follows:
A solution of polyethylenimine (M.W. 40,000 - 60,000) was pre-
pared by dilution with H2O and titration with HCl to obtain a 0.1% by
weight solution of pH 7Ø Polyethylenimine-cloth was prepared by soaking
cloth squares of 0.5 or 2 em in the so-prepared polyethylenimine solution
for 30 minutes at room temperature. The cloth was then washed with H2O,
air-dried, and stored at 4C until use. m e resulting polyethylenimine-
cloth was soaked in a 0.1% by weight solution of invertase in H2O for 16
hours at room temperature. Glutaraldehyde was then added to the solution
-- 1?3 --
~031~'7
to a final concentration of 2%. After 2 hours at room temperature, the
invertase cloth was washed wlth H2O, blotted dry and stored at 4C until
use. When recycling of unadsorbed invertase was desired, the invertase
solution was separated from the cloth aftar enzyme adsorption and the
cloth was then treated with glutaraldehyde.
T -hilized invertase was assayed by shaking invertase cloth (2cm
squares) at 320 rpm in 2.0 ml of 1.0 M sucrose (pH 5.0) for 10 min. at
30C. me activity was calculated by measuring the production of reducing
sugars (glucose and fructose) from non-reducing sucrose. me reducing
sugars were assayed by 3,5-dinitrosalicylic acid reagent.
Free invertase was assayed by measuring reducing sugars
produced during 10 min. incubation in 1.0 M sucrose at 30C.
Cotton cloth coated with 1~ polyethylenimine for 1 h was found
to absorb an aqueous solution very slowly. Therefore, 0.1~ polyethyleni-
mine was used instead. The effect of coating time on adsorption of E.
coli proteins was first studied. A 30 min. coating of the cloth with 0.1%
polyethylenimine allowed ~; adsorption of E, coli proteins to poly-
ethylemimine-cloth.
Table 1 shows that when polyethylenimine-cloth was soaked in 0.1
invertase in H20 (pH 5.3), invertase adsorbed to the polyethylenimine
cloth in an active form. Soaking for 16 h was found to m~;m; 7,e adsorption.
Table 1: Adsorption of yeast invertase to polyethylenimine-cloth
cloth (soaking hoursl specific activity
untreated cloth (16 h) ` 180
polyethylenimine-cloth (2 h) 400
polyethylenimine-cloth ~16 h) 1670
The adsorption of yeast invertase to polyethylenimine cloth was
carried out as follows:
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,
~2~3~7
Squares of PEI-cloth and untreated cloth (controi) were soaked
in 0.1% invertase (pH 5,3) for 2 h and 16 h at room temperature. The
squares were washed with H20 and assayed for invertase activity.
The specific activity is given in micromoles of reducing sugars.
produced per min. per g of cloth.
In the accompanying drawings,
Figure 1 is a graph showing the effects of pH and concentration
of invertase solutions on invertase adsorpt'lon onto polyethylenimine-cloth.
Figure 2 is a graph showing the effect of pH on immobilized
invertase; and
Figure 3 is a graph showing the hydrolysis of sucrose in an
immobilized invertase column.
The pH effect (0), was studied by adjusting 0.1% invertase
solutions to various pH by addition of tris(hydroxymethyl) aminomethane
powder. The concentration effect (~), was studied by using invertase
solutions in H20 at different concentrations without pH adjustment- Two
cm squares of PEI-cloth were soaked in these invertase solutions for 16 h
at room temperature. The squares were washed with H20 and assayed for
invertase activity. The specific activity on the cloth treated with 0.1~
invertase in H20 was taken as 100. The relative invertase activity was plotted
against pH and % (w/v) of the invertase solutions.
Fig. 1 shows the effects of the pH and concentration of the invertase
solution on enzyme adsorption. Adsorption was maximal between pH 5.3 and 6.3.
Use of invertase solutions lower than 0.1% resulted in a sharp reduction in
adsorption while higher concentrations resulted in only a gradual increase.
Therefore, use of 0.1~ invertase solution in H20 which exhibited pH 5.3 was
continued.
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~;203~
The adsorbed invertase was found to be easily eluted from the
polyethylenimine cloth with a 0.2 M NaCl solution. However, when the
adsorbed invertase was treated with 2% glutaraldehyde for 2 h at room
temperature it was no longer elutable with NaCl solutions (as high as 1M).
Glutaraldehyde treatment for 2 h did not reduce the specific activity of
the invertase cloth but a longer treatment caused a gradual decrease (40%
decrease after 8 h).
The invertase activity was assayed at 30C using l M sucrose in
0.01 M sodium acetate buffers of various pH. The specific activities of
the i ~hi-;7ed ( 0 ) and free ( ) invertase were expressed as millimoles
of reducing sugars produced per minute per g of the invertase cloth or
lO mg of free invertase.
Fig. 2 shows that immobilization shifted the optimal pH range of
the invertase from 4.8 - 6.0 ~free enzyme) to 4.4 - SØ As the activity
of i hili zed invertase sharply declined above pH 5.2, sucrose solutions
adjusted to pH 5.0 were subsequently used as substrates.
The invertase cloth was kept at 30C in a l M sucrose (pH 5.0)
solution which was changed every 3 days. The activity of the invertase
cloth was assayed every 3 days. No significant change in activity was
observed for at least 3 months.
To evaluate the performance of the invertase cloth in a column
operation, 0.5 cm squares of the invertase cloth were tightly packed into
a lO ml bed in a jacketed column of 10 mm diameter~ 0.5 M and 1 M sucrose
solutions (pH 5.0) were pumped into the column at various space velocities
(hourly flow rates divided by the bed volume). As shown in Fig. 3, 0.5 M
(~ ) and l.0 M ( O ) sucrose solutions (pH 5.0) were pumped into the column
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~za3l~
(equilibrated at 30C) at various space velocities by means of a peristallic
pump. Figure 3 also shows that 100% hydrolysis of 0.5 M sucrose occurred
at a space velocity of 10, while maximum hydrolysis (! 90%) of 1 M
sucrose was achieved at a space velocity of 5. Higher space velocities
resulted in approximately linear reduction in the degree of hydrolysis.
The degree of hydrolysis was calculated from the concentration of reducing
sugars in the effluent which was assayed after pumping through 5 to 10 bed
volumes of the sucrose solutions. The degrees of sucrose hydrolysis ob-
served at these velocities did not change for at least 2 months.
Since sucrose supports microbial growth, the invertase column
requires periodic cleaning. Quaternary ammonium chlorides, e.g. benzalkonium
chloride may be used as a sanitizing agent. Treatment of the invertase cloth
with benzalkonium chloride (as high as 200 ppm) did not affect the invertase
activity.
While polyethylenimine has been used as the agent to provide an
anionic surface on cloth, other agents may be used, e.g. polyalkylene poly-
amines and quaternized polyalkylene polyamines. However, since polyethyleni-
mine has been cleared by both the Environmental Protection Agency and the
Food and Drug Administration of the United States for use in potable water, it
is the agent of clloice.
The reagent with which the enzyme is cross-linked should pre-
ferably be a bifunctional compound or a multifunctional compound, each con-
taining groups selected from the group consisting of aldehydes, isocyanates,
sulfonyl halides, imidoesters, succinimides, diazonium compounds, azides,
anhydrides, chlorotriazines, and soluble polysaccharides activated by cyanogen
bromide. However, the cross-linking agent which should be selected, must be
- 22 -
~2~3~7
one which is safe and free from possible toxicity side effects. Since
glutaraldehyde as a cross-linking agent is generally rcgar~ as safe, whereas
most other cross-linking agents may not be acceptable because of possible
toxicity,glutaraldehyde is the agent of choice.
While the enzyme used in the specific examples in the present inven-
tion was invertase, other enzymes may be used. Examples include the following
enzymes, identified by numbers according to the Florkin and Stotz system
(Florkin M. Stotz E.H., "Comprehensive Biochemistryl~ Vol. 13, 3rd Edition,
Elsevier Publishing Company, ~ew York,1973), are as follows: glucoamylase
(amyloglucosidase) (3.2.1.3); lactase (3.2.1.23); maltase (3.2.1.20); amylase
(3.2.1.1); urease (3.9.1.5); lipase (3.1.1.3); esterase (3.1.1.1); glucose-
isomerase (5.3.1.18); glucose-oxidase (1.1.3.4); alcohol dehydrogenase
(1.1.1.1); penicillinase (3.5.2.6); penicillin; amidase; catalase; amino-
acylase; phenol oxidase; inulase; carboxy-peptidase; ribonuclease; trypsin;
ficin; subtilisin; pepsin; papain; rennin; and chymotrypsin.
The present invention emphasizes practical aspects of enzyme immobili-
zation: cost, operational ease, and safety. Both polyethylenimine and cotton
cloth are relatively inexpensive. In carrying out a practical aspect of this
invention the enzyme was first adsorbed to polyethylenimine-coated cotton
cloth by ionic interaction and then was permanently immobilized by cross-
linking with glutaraldehyde. Yeast invertase immobilized by this method
exhibited good activity and stability. Unlike a column made of cellulose
fibers, a column made of cloth segments can produce a space velocity as high
as 30 in a bed height of 50 cm. The process of aspects of the present inven-
tion does not impose safety problems.
- 23 _
~C)3.1L~3 7
In summary, the immobilization process of aspects of this inven-
tion has potential to be used in food modifying processes. The process
is applicable to immobilization of other industrial enzymes, if they
adsorb to polyethylenimine-cloth and are not inactivated by glutaraldehyde.
The pH of enzyme solutions may need to be adjusted so as to maximize ionic
attraction between enzymes and polyethylenimine during adsorption.
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