CO2 Solutions Inc.
`Exhibit 2011
` Akermin, Inc. v. CO2
`Solutions Inc. IPR2015-00880
`Page 1 of 22
`
`

`
`‘.
`
`
`
`«-- _.__:_.._.__.
`
`e‘-
`
`Jmmobilized
` Enzymes,
`
`Author:
`
`Oskar Zaborsky, Ph.D.
`Esso Research ::1”1‘T<—i'7'IE7f‘{éineering Company
`Lindén, New Jersey
`I
`
`published by:
`
`
`
`A DIVISION OF
`
`THE CIIEIVIICAL RUBBER co.
`
`18501 Cranwood Parkway - Cleveland, Ohio 44128
`
`Page 2 of 22
`
`Page 2 of 22
`
`

`
`This book represents information obtained from authentic and highly regarded sources. Reprinted material is quoted
`with ‘permission, and sources are indicated. A_ wide variety of references are listed. Every reasonable effort has been made
`to give reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all
`materials or for the consequences of their use.
`
`I
`
`All rights reserved. This book, or any parts thereof; may not be reproduced in any form without written consent from the
`publisher.
`
`© 1973 by THE CHEMICAL RUBBER CO.
`
`International Standard Book Number 0-87819-016-3
`
`Library of Congress Card Number 7295697
`
`Page 3 of 22
`
`Page 3 of 22
`
`

`
`Chapter 6
`
`ENTRAPMENT WITHIN CROSST INKE ) POLYMERS
`
`Enzymes can*be immobilized by entrapment
`within the interstitial space of crosslinked water-
`insoluble polymers. The method involves
`the
`formation of a highly crosslinked network of a
`polymer in the presence of an enzyme Enzyme
`molecules are physically entrapped within the
`polymer lattice and cannot permeate out. of the gel
`matrix, but appropriately sized substrate and
`product molecules can transfer across and within
`this network to insure apcontinuous transforma-
`tion (see Figure l7_). Other commonly used names
`for the method are inclusion, lattice entrapment,
`and occlusion.
`
`Lattice entrapment was first employed success-
`fully.
`for
`the
`immobilization of
`trypsin,
`a-
`. chymotrypsin, and other enzymes by Bernfeld and
`Wan355 in 1963. Since then, numerous enzymes
`
`have been immobilized in this manner, and several
`polymeric networks have been used (see Table 10).
`The most commonly employed crosslinked poly»
`mer for enzyme entrapment
`is the well-known
`polyacrylamide gel system, but silicone rubber
`A(Silastic®), starch, and silica gel have also been
`used. Dickey3 5 6 reported a partially successful en-
`trapment of urease and catalase in silica gel as early
`as 1955. Although immobilization of enzymes in
`
`these systems is visualized to occur by entrapment
`within the lattice structure of the polymer, part of
`the immobilization could be due to physical
`adsorption. This is especially‘ so with charged
`polymers such as‘ silica gel.
`
`7
`
`
`
`Nature of Crosslinked l50lymeric Matrices
`The polyacrylamide gel system is produced by
`the reaction of acrylamideland N,N'—methylene-
`bisacrylamide. The polymerization reaction can be
`initiated in several ways and that used most often
`for enzyme immobilization is shown in Equation
`52. The procedure for the formation of the cross-
`linked
`polyacrylamide-enzyme
`conjugate
`is
`identical
`to that used for
`the preparation of
`polyacrylamide
`gels
`employed commonly for
`separation and isolation of enzymes, except that in
`this case the protein is present during the poly-
`merization. A recent review of polyacrylamide gel
`electrophoresis by Chrambach and Rodbard357
`provides a quick survey of the more important
`parameters of the reaction and the structure of the
`produced gel. Several studies dealing with poly-
`acrylamide gel-immobilized enzymes have also
`described
`some pertinent
`details
`about
`this
`
`
`
`lattice-entrapped enzyme conjugate
`FIGURE 17. Cross~sectional view of
`showing polymer chains and occluded enzyme molecules.
`
`83
`
`Page 4 of 22
`
`Page 4 of 22
`
`

`
`em-CH, —(‘H~CH1 —(‘H um-
`
`I IN
`
`co
`H
`
`l (
`
`‘ONH2
`
`5
`
`dnl
`I
`l\[lH
`30
`(‘Hz a CH-—
`
`.
`
`(52)
`
`K2320”
`TEMED
`
`CH ‘CH + CH -CH
`7 ‘I
`2 “ I’
`CONH,
`$0NH
`
`1C
`
`H1
`H
`1.
`CO
`|
`CH; =. CH
`
`IN
`
`procedure. The total concentration and relative
`ratio of acrylamide and N,N'-methylenebisacryl—
`amide determine the pore size of the interstitial
`space within which the enzyme molecules are
`entrapped and the physical nature of the water-
`insoluble material produced.
`Hicks and Updike358 examined the character-
`istics
`and
`activity of
`lactate dehydrogenase
`immobilized in different compositions of acryl-
`amide
`and N,N'-methylenebisacrylamide
`and
`observed that ‘the best mechanical rigidity was
`obtained at higher gel
`concentrations
`(total
`acrylamide
`and N,N'-methylenebisacrylamide).
`However, at
`any one total concentration, an
`increase in the relative amounts of crosslinking
`reagent decreased the mechanical rigidity of the
`gel but gave a higher yield of immobilized enzyme
`activity per unit of soluble enzyme activity
`introduced before polymerization. For example, at
`a total concentration of 5% (acrylamide and
`N,N'~methylenebisacrylamide),
`a
`5% MN“
`methylenebisacrylamide
`concentration
`(95%
`acrylamide)
`produced
`a
`gel of
`“excellent”
`mechanical
`rigidity which showed a
`relative
`activity of 60%. The polyacrylamide gel composed
`of 10% A/',1V'—methylenebisacrylarnide exhibited
`only “fair” rigidity and showed a 66% relative
`activity. According to Hicks and Updike, the most
`suitable gel material requires both a relatively high
`concentration of the monomer (acrylamide) to
`give mechanical rigidity, and a high concentration
`of crosslinking reagent
`(N,N-methylenebisacryl-
`amide) to achieve the highest possible yield of
`immobilized enzyme activity. Immobilization of
`enzymes can be achieved by polymerization only
`of the crosslinking reagent as was done initially by
`Bernfeld and Wan,“ 5 but the gel so produced is
`very soft, sediments slowly, and is unsuitable for
`use in flow system applications. Moreover,
`the
`degree of concentration of the crosslinking reagent
`is severely limited by its solubility in water, which
`is approximately 3%. The appearance of poly-_
`
`84
`
`Immobilized Enzymes
`
`I
`
`to opaque,
`from clear
`acrylamide gels varies
`4
`depending on the exact composition.
`Hicks and Updike3 5 8 also examined the nature
`of the polymerization catalyst. They noted that
`mixtures with a high percentage of monomer
`polymerize more effectively with persulfate, and
`that‘ solutions with a higher percentage of cross-
`linking reagent polymerize better with riboflavin
`and a photocatalyst. Often, it is best to have both
`catalytic systems present. Other catalytic systems
`employed for polymerization during the immobili-
`zation of enzymes have been TEMED and persu1-
`fate,1 ° 7 33 5 5 ’3 5 9 ’3 6 ° B-dirnethylaminopro-
`pionitrile and persulfate,1°6’3“‘3‘4 and x-ray
`radiation.“ 5 The latter initiation method has, in
`principle, the inherent advantage of being non-
`chemical in nature, allowing better heat control
`and permitting quick termination and control of
`the
`initiation
`step. The
`polymerization of
`acrylamide and »MN’-methylenebisacrylamide is
`usually conducted at room temperature or some-
`what lowered temperatures and in the absence of
`atmospheric oxygen. The oxygen molecule, being
`a paramagnetic species, is a very potent polymeri-
`zation inhibitor.
`
`The trapping efficiency of polyacrylamide gels-
`of varying composition was examined by several
`investigators. Degani and Miron3.64 observed that
`the maximum. yield -of activity trapping (56%)
`occurred with cholinesterase at a crosslinking
`concentration of 5% and with a total and constant
`monomer concentrationof 15%. The activity of
`the Water-insoluble conjugate reached its highest
`value at the same 5% MN -‘methylenebisacrylamide
`concentration. Higher percentages of crosslinking
`reagent decreased both the activity of the con-
`jugate and the trapping efficiency. The effect of
`the total monomer concentration (at a constant
`N,N'—methylenebisacrylamide
`concentration of
`5%) on the activity of the conjugate. and the
`trapping efficiency was likewise investigated. With
`increasing concentrations of total monomer, the
`
`Page5 of 22.
`
`Page 5 of 22
`
`

`
`addition of the catalyst, stannous octoate, water-
`insoluble silicone rubber is produced. The water-
`insoluble enzyme conjugate is prepared by adding
`the enzyme to an‘ excess of the Silastic resin
`
`Silastic® resin
`
`ll
`
`CH3
`i — 0
`
`|S
`
`I
`CH3
`
`(100-fold w/w excess), stirring the mixture for sev-
`eral minutes, and then adding the stannous octoate
`in catalytic portions. Gel formation takes place
`within half an hour under normal circumstances,
`and the product is a rigid material that can be cut
`into any desired shape. Reasonable good entrap-
`ment efficiency seems to have been observed with
`trypsin and chymotrypsin.3“ However, Guilbault
`and Das3 7°
`have
`reported some unfavorable
`characteristics
`of Silastic-immobilized enzyme
`
`derivatives. They noted that the polymerization is
`“rigorous” and that up to 80% of the enzymic
`activity is lost in the immobilization. In addition,
`the silicone rubber pads cracked upon drying and
`. did not become uniform upon rehydration because
`of the hydrophobic nature of the Silastic resin.
`Starch gels haveubeen employed by Guilbault
`and colleagues3”‘374 for
`the preparation of
`urethane foam-supported,
`immobilized enzyme
`pads. These pads372 are prepared by pouring a
`slurry of Connaught-type starch into a boiling
`mixture of buffer and glycerine, heating the
`resulting mixture until a clear solution is obtained,
`and then cooling it
`to 47°. At
`this point, a
`solution of the enzyme is added to the clear starch
`solution with stirring for
`several seconds;
`the
`resulting mixture is
`immediately poured onto
`open-cell urethane foam. The enzyme-starch solu-
`tion is gently ‘worked into the urethane foam,
`excess liquid is removed by squeezing, and the pad
`» is cooled in a refrigerator for an hour in order to
`form the permanent gel. The large pad can be cut
`into smaller segments. This method of immobiliza-
`tion was reported only for cholinesterase. The
`addition of glycerine to the starch gel produces
`pads that are less subject to mechanical damage
`and which are able to rehydrate more quickly than
`pads without glycerine. Other additives (Triton®
`X-100 and Kraystay® K) are more superior storage
`additives than glycerine.3 74
`A
`Several enzymes, most notably trypsin, have
`also been immobilized by incorporation within a
`
`yield of proteirl trapping increased as expected by
`the decrease in gel porosity. However, the activity
`of the conjugate and the yield of activity trapping.
`reached a maximum at 15% total monomer con-
`
`centration. Higher total monomer concentrations
`decreased both the activity and the yield of
`activity trapping. Degani. and Miron attributed this
`decrease in activity to inactivation of the enzyme
`occurring in
`the
`polymerization mixture.
`Acrylamide apparently acts as a denaturing agent
`on cholinesterase in a manner similar
`to that
`observed with urea.
`
`the
`examined
`Smiley366
`and
`Strandberg
`entrapment of ‘glucose isomerase with varying
`N,N'-rnethylenebisacrylarnide
`concentrations
`(constant total monomer) and observed a similar
`trend of lower trapping with increasing concentra-
`tions of crosslinking reagent. The typical trapping
`efficiency of
`the polyacrylamide for glucose
`isomerase was roughly 40 to 50% of the enzyme
`added to the system. About 30% of the entrapped
`enzyme exhibited activity in the normal assay-
`Bernfeld et al.36°’367 examined the entrap—'
`ment and catalytic efficiency of a 14C-labeled
`aldolase-N,N'-methylenebisacrylamide water-
`insoluble
`conjugate prepared without
`added
`acrylamide. The water-insoluble conjugate pre-
`pared in this manner contained 55% of the
`radioactivity and exhibited 10.4% of the total
`enzymic activity. The aqueous solution contained
`44.2% of the radioactivity and exhibited 33.1% of
`
`the enzymic activity. The ratio of enzyme activity
`to radioactivity in the aqueous phase which
`remained after termination of the polymerization
`was reduced by only 25%. On the contrary, the
`
`ratio of activity to radioactivity for the insoluble
`enzyme-conjugate was
`reduced by about 80%.
`These
`results
`indicated that
`four
`times‘ more
`enzyme
`protein than enzymic
`activity was
`associated with the water-insoluble carrier and
`Bernfeld et al.3 6 ° reasoned that the most plausible
`explanation of the findings was that only a portion
`of the entrapped aldolase is enzymatically active.
`Only the entrapped aldolase near the polymer-
`water interface apparently exhibited activity.
`As mentioned,
`Silastic
`resin
`(silicone
`rubber),362’368’3 7° silica gel,356’3 7‘ and starch
`gel3 ""3 74 have been used for lattice entrapment
`of enzymes. Silastic resin (Dow Corning Co.,
`Midland, Mich.) has the general chemical structure
`shown below where n is in the order of 10,000.
`The resin also contains a silica filler, and on
`
`85
`
`Page 6 of 22
`
`Page 6 of 22
`
`

`
`polymerizing silicic acid sol.“ 61371 The use of
`this procedure for enhancing the storage stability
`of certain materials
`(including enzymes) was
`suggested by Dickey356 in 1955. Urease and
`V catalase immobilized in this manner exhibited
`some detectable activity, but muscle adenylic acid
`deaminase was completely inactivated by the
`condition used to form the gel. Only recently has
`this method been used successfully with an
`enzyme. Johnson and Whateley3” immobilized
`trypsin within the lattice structure ‘of a silica
`“xerogel.” The term'“xerogel” was used by them
`to mean a dried-out nonswelling structure such as
`commercial silica gel. The term “hydrogel” was
`used for a water-rich colloidal system with a finite,
`rather small yield stress. The procedure for pre-
`paring the entrapped trypsin was asfollows:
`the
`pH of the silicic acid sol was adjusted with sodium
`hydroxide to be between 6 and 7, a solution of the
`enzyme was added to the sol, and after adjusting
`the NaCl concentration, the sol was allowed to set
`
`to a hydrogel. Gel formation took place in about
`15 min. The hydrogel was allowed to age overnight
`and was then lyophilized to give the flaky White
`powder- xerogel. The enzyme-containing xerogel
`was washed with water and buffers to remove any
`
`physically adsorbed enzyme. The xerogel pro-
`duced in this manner seems to have a considerable
`
`degree of hydration.
`
`According to Johnson and Whateley, enzyme
`entrapment occurs via the condensation of silicic
`acid sols to hydrogels having the three-dimension-
`ally crosslinked networks composed of alternating
`silicon and oxygen atoms characteristic of silica.
`Initially, these networks result in only small but
`highly hydrated particles with little interparticle
`bonding. At
`a later stage,
`interparticle bonds
`become more important and these lead to gelation
`and formation of the hydrogel. The condensation
`reaction continues after the hydrogel has formed.
`
`A method for immobilizing enzymes by entrap-
`ment within the microspace of fibers was an-
`nounced recently.3 “"3 773 The enzyme molecules
`are entrapped in aqueous microdroplets within
`synthetic resin filaments which can be produced
`continuously by
`conventional wet-spinning
`
`techniques. A number of enzymes were im-
`mobilized and filaments of different chemical
`
`composition were described.
`
`86'
`
`Immobilized Enzymes
`
`Properties
`With this method of
`immobilization, no
`changes in the intrinsic properties of an enzyme
`are anticipated. Local microenvironmental effects
`such as those created by the nature of the carrier
`(e.g., its charge) or enzymatically generated ones
`created by the enzymic reaction itself can be
`expected. The charge of a carrier is an important
`consideration in trying to rationalize some of the
`effects observed. The charge of the water-insoluble
`matrix formed from polyacrylamide, starch, and
`Silastic resin is electrically neutral. On the other
`hand, the xerogel system formed from silicic acid
`sol is negatively charged at normal pH ranges (3
`and above) due to the relatively easy ionizable
`silanol hydroxyl groups.
`5
`'
`
`Activity
`The activity of lattice—entrapped enzymes (is
`critically dependent on the method of preparation.
`The relative activity of these water-insoluble con-
`jugates is usually low but can go up to approxi-
`mately 50 to 60% in the more ‘favorable cases.
`Some
`examples of immobilized enzymes
`ex-
`hibiting low- relative activities (with the percent
`activity given in parentheses) are trypsin (4 to
`5.5),355
`(2),358
`(11),378
`a-chymotrypsin
`(4.5),3'55
`papain
`(3.4
`to
`6),355
`0.-amylase
`(1.9),3 5 5
`B-amylase
`(6.6),35 5
`ribonuclease
`(4.6),355
`aldolase (4.2),355. alcohol dehydro-
`genase
`(5),3.78
`lactate dehydrogenase ((1)378
`steroid A1-dehydrogenase (7)379 citrate synthase
`(12 to l5),1°5 and glucose oxidase (l5),38° all
`immobilized in polyacrylamide
`gels. Relative
`activities in the range of 20 to 40% were reported
`for orsellinic acid decarboxylase (26 to 30)?“
`aldolase (ca. 2O),3 5° phosphoglycerate mutase (up
`to 64),38‘ and glucose isomerase (22 to 3_5),3“
`likewise entrapped in polyacrylamide gels. The
`highest
`relative activity (ca. 60%)
`for
`lattice-
`entrapped enzyme conjugates reported to date was
`observed for phosphoglycerate mutase38 1
`en-
`trapped in polyacrylamide and for trypsin and
`chymotrypsin362 entrapped within Silastic resin.
`The relative activity of these immobilized enzymes
`(as with other water-insoluble enzyme conjugates)
`is dependent on the particular substrate employed
`for enzymic activity. This
`can be illustrated
`dramatically with
`trypsin
`immobilized
`in
`xerogel.37‘ The relative esterase activity of this
`water-insoluble enzyme conjugate was 34% com-
`
`pared with soluble trypsin with BAEE as substrate.
`
`Page 27 of 22
`
`Page 7 of 22
`
`

`
`However, this same trypsin derivative exhibited no
`detectable proteolytic activity toward casein as the
`substrate. Although diffusion and steric repulsion
`of
`the macromolecular
`species are certain to
`diminish the relative activity of this immobilized
`enzyme conjugate, some activity was still expected
`(see Chapter 3). The complete lack of activity in
`this
`instance was attributed by Johnson and
`
`Whateley to the unfavorable charged character of
`the substrate and immobilized enzyme (both being
`
`negatively charged) and the steric hindrance.
`
`An interesting behavior of the relative activity
`with temperature of enolase immobilized in poly-
`acrylamide was
`observed
`by Bernfeld
`and
`Bieber.3 82 In their system, the relative activity of
`the insoluble form varied nonlinearly with temper-
`ature with a minimum occurring at 24°. At higher
`or lower temperatures,
`the ratio of the specific
`activity of the immobilized enzyme to the native
`enzyme increased.
`I
`
`pH—Actz'vz'ty Behavior
`Although very few studies have been conducted
`to date on the pH-activity behavior of lattice-
`entrapped enzymes,
`the same observations have
`been made with these immobilized enzymes as
`
`with other enzyme conjugates. The,pH-activity
`curves of the immobilized enzyme can be dis-
`
`placed toward either alkaline or acid pH values or
`need not be displaced at
`all. For example,
`trypsin371 immobilized in xerogel exhibited the
`expected shift of its pH-activity curve toward
`more alkaline pH’s. A shift of approximately 0.8
`pH units was observed at an ionic strength of 0.2
`with BAEE as substrate. No shift (or only a very
`negligible shift and well within experimental error)
`in the pH-activity curves was reported for several
`enzymes immobilized in polyacrylamide gel. For
`example, no shift was reported to occur with.
`355
`382
`ribonuclease,
`enolase,
`or other
`enzymes.” 5 This is the expected behavior due to
`the electrically neutral character of polyacryl-
`amide. However, an anomalous pH-activity be-
`havior has been observed for phosphoglycerate
`mutase381 immobilized in polyacrylamide. An
`unexpected and as yet unexplained shift toward
`more apid pH’s was observed for
`the lattice-
`entrapped enzyme. The pH-optimum of
`the
`water—insoluble, enzyme conjugate shifted to 6.5
`(pH optimum of soluble enzyme, pH 7.3).
`
`Michaelis Constant
`
`A slightly higher apparent Michaelis constant
`was found for glucose oxidase369 entrapped in
`Silastie resin (4 mM, compared with 2 mM for the
`soluble enzyme) and for urease3 7° immobilized in
`polyacrylamide gel
`(5 mM and 4 mM,
`respec-
`tively). Similar K'm values were observed for
`immobilized
`phosphoglycerate mutase,381
`enolase,382
`and lactate dehydrogenase358 im-
`mobilized in polyacrylamide. Decreased values of
`the apparent Michaelis constants wererobserved for
`cholinesterase37° in starch gel (K'm of 0.16 mM
`and Km of 0.25 mM for immobilized and soluble
`enzymes,
`respectively)
`and
`for
`acetylcholin-
`es'terase369 in silicone rubber (50 mM and'12O
`mM for immobilized and soluble enzyme, respec-
`tively). No explanation for the decreased K'm
`values was offered.
`
`Specificity
`Several publications have mentioned aspects of
`substrate and inhibitor specificity for entrapped
`enzymes. Brown et al.362 noted that the calcium
`ion activation optima for soluble and polyacryl-
`amide-immobilized apyrase were identical (1 mM)
`but that the extent of stimulation was different.
`High concentrations of Ca” inhibited the en-
`trapped enzyme. Pennington et al.368 observed
`that
`acetylcholinesterase entrapped in silicone
`rubber was less affected by inhibitors than the
`freely soluble enzyme. Bernfeld et al.38‘ observed
`similar substrate behavior for soluble and poly-
`
`acrylamide-immobilized phosphoglycerate mutase.
`Pronounced substrate inhibition started to become
`
`noticeable at higher substrate concentrations for
`both enzymes.
`P0lyacrylamide-immobilized
`enolase3“ exhibited different behavior
`toward
`magnesium ion inhibition. Both the soluble and
`immobilized enzymes required Mg” for maximum
`activity (0.68 mM) but the entrapped enolase, in
`contrast to the soluble enzyme, was not inhibited
`by an excess of magnesium. Zinc ions inhibited’
`both enzymes to about the same extent. At low
`magnesium ion concentrations,
`the polyacryl-
`amide-enolase conjugate was somewhat
`less af-
`fected by Zn” than was the soluble enolase.
`
`Stability
`Enhanced thermal stability (compared with the
`soluble enzyme) was observed for lactate dehydro-
`genase,3 5 8 apyrase,3 6 2 and trypsing‘ 6 5
`immobil-
`
`87
`
`Page 8 of 22
`
`Page 8 of 22
`
`

`
`ized in polyacrylamide gel and for acetylcholin—
`esterase358 entrapped in Silastic resin. A higher
`temperature optimum was noted for enolase382
`immobilized
`in
`polyacrylamide
`gel.
`Similar
`thermal stability was noted for glucose oxidase3 5 8
`immobilized in polyacrylainide gel. A similar
`temperature optimum was noted for phospho-
`glycerate mutase381 in polyacrylamide gel. The
`temperature optima for both the soluble and
`immobilized enzymes were between 40 to 45°.
`There was, however, a slight change in the shape of
`the activity—temperature curve of the immobilized
`enzyme compared with the curve of the soluble
`enzyme. Diminished stability has also been re-
`ported. Guilbault and Hrabankova383.
`reported
`that
`the polyacrylamide—entrapped L—amino acid
`oxidase was
`less
`stable
`than the cellophane-
`entrapped enzyme. In this example, it is not clear
`whether
`the
`diminished stability was
`solely
`thermal.
`
`Storage stabilities have been reported frequent-
`ly. Ribonuclease A355 immobilized in polyacryl—
`amide retained 99% of its original activity after
`one month at 0 to 4°, lactate dehydrogenase-.358
`lost 10% activity per month over a period of three
`months, glucose oxidase35 8 lost no activity during
`three months of storage at 0 to 4°, and orsellinic
`acid decarboxylase“ 1
`lost 3% of its activity after
`14 days
`at 20°. Storage stabilities of other
`enzymes immobilized in polyacrylamide gel have
`been
`reported for
`lactate dehydrogenase,373
`steroid, A1 -dehydrogenase,379
`citrate
`syn-
`thase,1°6
`and
`glucose—6-phosphate
`del1ydro—
`genase.1°7 Cholinesterase37°
`immobilized
`in
`starch gel lost 2.5% of its original activity after 5
`weeks of storage at ca. 4° and 32% after 20 weeks.
`At room temperature, 5% of its activity was lost in
`5 weeks and 41.5% in 20 weeks. Trypsin371
`immobilized in silica gel lost less than 10% of its
`activity in 75 days stored at 4°. At room temper-
`ature,
`the storage stability was considerably re-
`duced; after 70 days, only 27% of the original
`activity remained.
`A
`Stability of lattice—entrapped enzymes during
`continuous use was reported for cholinesterase3 72
`and for urease.384
`Latticeentrapped enzymes can be often lyo—
`
`philized without
`372,379
`
`serious
`
`inactivation.358=371’
`
`Advantages and Disadvantages
`Advantages of the lattice-entrapment method
`for immobilizing an enzyme include its overall
`experimental simplicity,
`the need for only small
`amounts of an enzyme in order to produce a
`water-insoluble enzyme conjugate, and the fact
`that it is a physical method. No chemical modifica-
`tion of the enzyme is expected and consequently
`
`no change_in an enzyme’s intrinsic properties is
`anticipated. The method also allows for a consider-
`.able choice of neutrally charged water-insoluble
`carriers. Perhaps most
`important,
`this method
`permits the preparation of water-insoluble enzyme
`derivatives of widely different physical forms. The
`often gelatinous nature of the enzyme derivative
`makes it easy to deposit an immobilized enzyme
`on either regular or highly irregular surfaces.
`Disadvantages of the method also exist. Oper-
`ationally, good immobilization (i.e., good mechan-
`ical prfoperties and activity) is dependent on a
`delicate balance of experimental factors. The exact
`physical nature of the crosslinked polymers is
`often quite important for obtaining high activity.
`Chemical and thermal
`inactivation of enzymes
`during the gel formation can also take place. The
`formed. lattice-entrapped enzyme often needs to
`be broken up or cut
`into suitable form. A
`considerable disadvantage of this method is leak-
`age of an enzyme from within the crosslinked
`polymeric network. During formation of
`the
`polymer network and entrapment of an enzyme,
`differently sized microspaces
`(micropores) are
`created,
`the size distribution of‘ which is deter-
`
`mined largely by the relative degree of cross-
`linking. The larger the micropores, the greater will
`be the leakage. Although leakage, in principle and
`practice, can be reduced by reducing the size of
`these micropores, some initial leakage of enzyme
`molecules is certain to occur.3 5 5 ’3‘8 5 Severe initial
`
`gel-
`starch
`especially with
`occurs
`leakage
`immobilized enzymes.37°’3” 573 Another major
`disadvantage of the method is the limitation to
`only small-sized
`substrates;
`lattice-entrapped
`enzymes show veryvlittle activity toward macro-
`molecular substrates.
`
`gs
`
`Immob ilizea’ Enzymes
`
`Page 9 of 22
`
`Page 9 of 22
`
`

`
`TAB LE 1 O
`
`Lattice-Entrapped Enzyme Conjugates
`
`Enzyme immobilized
`
`Entrapment matrixa
`
`Alcohol dehydrogenase
`(1.1.1.1)
`
`Lactate dehydrogenase
`(1.1.1.27)
`
`G1ucose—6-phosphate
`dehydrogenase
`(1.1 .1.49)
`
`Glucose oxidase
`(1.1.3.4)
`
`Polyacrylamide
`
`Polyacrylamide
`
`Polyacrylamide
`
`Polyacrylamide
`
`Starch
`
`Glucose oxidase ~
`
`Silastic resin
`
`peroxidase
`
`Steroid A‘ —dehydrogenase
`(1 .3.—.-)
`
`Polyacrylamide
`
`Glutamate dehydrogenase
`(1.4.1.2)
`
`L-Amino acid oxidase
`(1.4.3.2)
`
`—/
`
`D—Amino acid oxidase
`(1 .4.3.3)
`
`Amino acid oxidase
`(1.4-.3.?)
`
`Polyacrylamide
`
`Polyacrylamide
`
`Polyacrylamide
`
`Polyacrylamide
`
`Catalase
`(1.1 1.1.6)
`
`'
`
`Peroxidase
`(1.1 1.1.7)
`
`Hexokinase
`(2.7.1.1)
`
`,
`
`Polyacrylamide
`—
`Silica gel (hydrogel)
`
`Polyacrylamide
`
`Polyacrylamide
`
`Hexokinase — g1ucose—6-
`phosphate dehydrogenase
`
`.Polyacrylamide
`
`Phosphofructokinase
`(2.7 .1 .1 1)
`
`Polyacrylamide
`
`Phosphoglycerate mutase
`(2.7.5.3)
`
`Po1y(N,7N'-methylenebis
`acrylamide)
`
`Poly(N,N'—methy1enebis—
`acrylamide)
`
`Ref.
`
`378
`
`358, 378
`
`107
`
`358, 380,
`386, 387
`372
`
`369
`
`379
`
`358
`
`383
`
`388
`
`358
`
`358
`
`356
`
`389
`
`363
`
`107
`
`363
`
`381
`
`355
`
`Ribonuclease A
`(27.7.16)
`
`Acetylcholinesterase
`(3.1.1.7)
`
`Silastic resin
`
`368, 369
`
`89
`
`Page 10 of 22
`
`Page 10 of 22
`
`

`
`‘ TABLE 10 (Continued)
`
`Lattice-Entrapped Enzyme Conjugates
`
`Enzyme immobilized
`
`Entrapment matrixa
`
`Cholinesterase
`
`(3 .1 .1 .8)
`
`Alkaline phosphatase
`(3.1.3.1)
`
`K
`
`Polyacrylamide
`
`Silastic resin
`Starch
`
`. Polyacrylarnide
`
`<1-Amylase
`(32.1.1)
`
`I [3-Amylase
`(3.2.1.2)
`
`Trypsin
`(3 .4.4.4)
`
`a.-Chymotrypsin
`(3 .4.4 .5)
`
`Papain
`(3 .4 .4 .1 O)
`
`Asparaginase
`(3 .5 .1 .1 )
`
`Glutaminase
`(3 .5 .1 .2)
`
`3 Urease
`(3.5.1.5)
`
`AMP deaminase
`(3.5 .4 .6)
`
`Apyrase
`(3.6.1.5)
`
`Po1y(N,N'-1‘nethylenebis—
`acrylamide
`
`Po1y(N,N'—methylenebis-
`acrylamide)
`
`Polyacrylarnide
`
`Poly(N,N'-methy1enebis—
`acrylamide)
`—
`Silastic resin
`Silica gel (xerogel) .
`
`Po1y(N,N'-methy1enebis-
`acrylamide)
`Silasfic resin
`
`Poly (N,N' ~methy1er'1ebis-
`acrylamide)
`
`_ Polyacrylamide
`
`3 Polyacrylamide
`
`Polyacrylamide
`
`-
`Silastic resin
`Silica gel (hydrogel
`‘ Starch
`
`Silica gel (hydr0ge1)b ‘
`
`I Polyacrylarnide
`
`Silastic resin
`
`Oxsellinic acid decarboxy-
`ase
`
`Polyacrylamide
`
`
`
`Ref.
`
`364, 370
`
`370
`370, 372-374
`
`.
`
`3359
`
`355
`
`355
`
`‘ 361, 365,
`378
`355
`
`362
`371
`
`355
`
`362
`
`355
`
`388
`
`390
`
`370, 384, 385,
`391-393
`370
`356
`
`A370 '
`
`356 '
`
`362
`
`362
`
`361
`
`363
`
`(4.1.1.—)
`
`3 Aldolase
`(4.1.2.13)V
`
`90
`
`Immobilized Enzymes
`
`I Polyacrylamide
`
`Poly (N,N'-m‘ethy1enebis~
`acrylamide)
`
`355, 360, 367
`
`Page 11 of22'
`
`Page 11 of 22
`
`

`
`TABLE 10 (Continued)
`
`Lattice-Entrapped Enzyme Conjugates
`
`Enzyme immobilized
`
`Entrapment matrixa
`
`Citrate synthase
`(4 . 1 .3 .7)
`
`Enolase
`(4-.2.1.11)
`
`Glucose isomerase
`(5.3.1.—)
`
`Glucosephosphate
`isomerase
`
`(5.3 .1 .9)
`
`Polyacrylarnide
`
`Poly (N,N' -methy1enebis-
`acrylamide)
`
`Polyacrylamide »
`
`Polyaerylamide
`
`Ref.
`
`106 '
`
`382
`
`366
`
`363
`
`aPo1yacrylamide — mixture of acrylamide and N,N'—methy1enebisacry1amide.
`bNo activity observed.
`
`91
`
`Page 12 of 22
`
`Page 12 of 22
`
`

`
`Page 13 of 22
`
`

`
`
`
`Chapter 7
`
`MICROENCAPSULATEON
`
`of Chang’s method, however, depends on having a
`membrane
`as permeable
`as possible
`to the
`substrate and product but not to the enzyme.
`
`Preparation 0f Permanent Microcapsules
`Two general methods, based on coacervation
`and on interfacial polymerization, have been
`employed for the preparation" of semipermeable
`microcapsules 4 used
`for
`immobilizing
`en-
`394’39°’398’4°° Coacervation
`is
`the
`’ zymes.
`
`phenomenon of phase separation in polymer solu-
`tions, and the formationof a microcapsule is
`dependent on the lower solubility of the polymer
`at the interface of a microdroplet. Coacervation is
`a physical phenomenon. The interfacial polymeri-
`zation method for producing a microcapsule is
`based on a chemical process — the.synthesis of a
`water-insoluble copolymer at
`the interface of a
`microdroplet. One reactant is partially soluble in
`both the aqueous and organic phase and the other
`reactant (the second component of the copoly-
`mer) is soluble only in the organic phase. The
`
` P
`
`Schematic representation of a permanent
`FIGURE 18.
`microcapsule showing entrapped enzyme molecules (E).
`Continuous conversion is achieved by the diffusion of
`substrate (5) and product
`(P) molecules across
`the
`semipermeable membrane.
`
`Enzymes can be immobilized within micro-
`capsules that have either a permanent or non-
`permanent semipermeable membrane. Permanent
`membranes are formed by interfacial polymeriza-
`tion or by coacervation of preformed polymers.
`Nonpermanent membranes or “1iquid-surfactant
`membranesf are formed by the combination of
`.. appropriate surfactants, “additives,” and hydro-
`carbons. Both of these interesting and potentially
`highly useful microcapsules are discussed here.
`
`Immobilization with Permanent Microcapsules
`The immobilization of enzymes by entrapping
`the molecules within permanent semipermeable
`microcapsules was first reported by Chang in the
`mid-’6O’s.3 “'3 9 6 Since that time,various enzymes
`have been immobilized in microcapsules of differ-
`ent chemical composition (see Table 11). The
`principle of continuous operation using micro-
`encapsulated enzymes is based on the permselec-
`tivity of the membrane. The enzyme molecules,
`being larger than the mean pore diameter. of the
`spherical membrane within which they are en-
`trapped, cannot diffuse through the membrane
`into the external solution. On the other hand,
`substrate molecules whose size does riot exceed‘
`the diameter of the pore can readily diffuse
`through the membrane and be transformed to
`product by the entrapped enzyme molecules. The
`product(s) of the reaction then diffuse(s) through
`the membrane to the exterior phase (see Figure
`18). Only substrates and products of rather low
`molecular weights are applicable in this method.
`Typical microcapsules are shown in Figure 19.
`Prior to Chang’s first report, microencapsula-
`tion had been used for entrapping drugs, perfumes,
`detergents, dyes, adhesives, solvents, paints, chem-
`icals, etc. A good general review of such micro-
`encapsulation was
`published
`by Herbig.397
`Usually, a slightly permeable or even nonperme—
`able membrane was employed for the encapsula-
`tion of these materials and sudden release of the
`
`encapsulated substance was dependent on the
`rupture of the membrane by pressure or heat or by
`the dissolution of the membrane itself. The success
`
`93
`
`Page 14 of 22
`
`Page 14 of 22
`
`

`
`FIGURE 19.
`Photomicrograph of typical nylon—6,1O microcapsules containing entrapped
`soluble protein. Unpublished results, 0. R. Zaborsky and J. Ogletree. Photographed by
`R. Sherwood.
`
`parti