CO2 Solutions Inc.
`Exhibit 2010
`Akermin, Inc. v. CO2 Solutions Inc.
`IPR2015-00880
`Page 1 of 7
`
`

`
`COPYRIGHT © 1975, BY ACADEMIC PREss, INC.
`ALL RIGHTS RESERVED.
`NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR
`TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC
`OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY
`INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT
`PERMISSION IN WRITING FROM THE PUBLISHER.
`
`ACADEMIC PRESS, INC.
`111 Fifth Avenue, New York, New York 10003
`
`United Kingdom Edition published by
`ACADEMIC PRESS, INC. (LONDON) LTD.
`24/28 Oval Road, London NW1
`
`Librmy of Congress Cataloging in Publication Data
`Main entry under title:
`
`Immobilized enzymes for industrial reactors.
`
`Includes bibliographical references and index.
`1.
`Immobilized enzymes—Industrial applications.
`Biochemical engineering.
`I.
`Messing, Ralph A., ed.
`2.
`[DNLMI
`1.
`Enzymes.
`2.
`Technology.
`TP248.E5 M5 85i]
`TP248.E5146
`660’.63
`74-27521
`ISBN O—12—492350—9
`
`if
`
`PRINTED IN THE UNITED STATES OF AMERICA
`
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`

`
`Chapter‘7
`
`CHARACTERISTICS OF FREE VS.
`
`IMMOBILIZED ENZYMES
`
`Donald J. Lartigue
`
`The main difference between a free and an imo=
`bilized enzyme is that, once immobilized,
`the en=
`zyme is no longer completely surrounded by an aque=
`ous environment. One can suspend the immobilized
`enzyme in a solution of substrate, activators. or
`other components at a particular pH and ionic
`strength; but one does not have the assurance that
`the conditions in the medium immediately surround=
`ing the enzyme are the same as those in the exter=
`nal solution.
`Indeed they may be quite different.
`This phenomenum can be the result of the charges
`or the physico—chemical properties of the support=
`.ing matrix or may result from diffusional limitae
`tions. This latter aspect will be discussed later.
`Consider the simple case of proteins adsorbed’
`onto glass which has a negative surface charge.
`This system will usually exhibit an apparent pH
`optimum higher than that observed with the free en=
`zyme.
`In other words,
`the negative charges in the
`immediate neighborhood of the enzyme must be neuu
`tralized before the pH in this area is raised to
`that of the solution. Similarly, any charged lo=
`cations can affect the apparent pH optimum shifting,
`it up or down as the case may be. Goldstein and
`his group at the Weizmann Institute purposely prea
`pared immobilized enzyme systems containing large
`numbers of charged groups. They copolymerized chy=
`motrypsin with polyornithine and the resultant co=
`polymer, containing between 34 and 820 positive I
`groups per molecule shifted the apparent pH optimum
`from 8.3 to as low as 7.5. Similarly,
`the ethylene
`maleic anhydride copolymer of chymotrypsin contain=
`ing 290 carboxyl groups per molecule raised the
`_
`apparent pH to 9.5 (1).
`g
`In addition to charge. other properties of the
`carrier may influence the observed reactivity of
`the immobilized enzyme. An excellent case in point
`is the study by Brockman,et.al(2).‘They adsorbed;mm=
`creatic lipase to solid glass beads which had been
`siliconized creating a surface with a strong hydro=
`phobic character.
`They found that the catalytic
`
`125
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`

`
`DONALD J. LARTIGUE
`
`efficiency of the surfacewbound enzyme upon the
`substrate tripropionin was about
`three orders of
`magnitude greater than that for the homogeneous
`reaction. Yet, no change was observed in the inw
`trinsic reactivity of the enzyme when thus bound.
`They concluded that the apparent activation was due
`to the ability of the hydrophobic interface to in—
`crease the local concentration of the substrate.
`These types of effects are major ones and can
`be summed up in the term ”microenvironmental ef—
`fect”, a term coined by Katchalski (3). This means
`that, with the exception of hollow fiber, dialysis,
`gel entrapped, or ultra=filtration systems, one can
`never say with certainty just what actual condi=
`tions exist in the neighborhood of the enzyme re=
`gardless of the conditions set in the external so»
`lution.
`In most of these cases,
`the changes in the
`properties observed are a arent changes and not
`Other
`'changes in the actual enzymatic properties.
`apparent changes which can result from the micro=
`environmental conditions are changes in the ob»
`served Michaelis constant and the effect of product,
`substrate, inhibitor, or activator concentrations.
`In addition,
`these would result in changes in the
`observed rate of the reaction.
`The immobilization process, particularly if the
`enzyme is entrapped in a gel, copolymerized, or ad=
`sorbed or covalently coupled within pores of a ma=
`trix, can impart diffusional problems which must be
`considered.
`In order for the substrate to be acted
`upon, it must diffuse from the external solution
`into the rather static liquid layer that surrounds
`the particle and then into the pore where the solumn
`tion is almost stagnant and where the enzyme is lo=
`cated.
`The product must diffuse in the reverse die
`rection. These mass transfer effects can create
`problems in assay and in the use of the immobilized
`enzyme system.
`In many cases,
`the kinetic expres=
`sions are considerably altered. This matter, priw
`marily with respect to industrial applications,
`will be treated in detail in a subsequent chapter
`in this text. Certain representative laboratory
`studies or theoretical approaches to describing the
`kinetics of particular systems are included in the
`references (lO=24)
`to this chapter.
`Diffusional effects also result in apparent
`changes in the measured enzymatic properties.
`
`Im=
`
`126
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`

`
`HVIMOBILIZED ENZYMES FOR INDUSTRIAL REACTORS
`
`mobilization techniques may result in changes in
`the actual enzymatic properties.
`The most obvious change that can occur is the
`inactivation of the enzyme.
`It is not unusual to
`read that attempts to immobilize enzyme X by tech=
`nique Y resulted in total inactivation. An obvious
`explanation is that the immobilization occurred
`through side chains required for catalytic activity
`Even under the best conditions of immobilization,
`it is unusual to immobilize more than 80% of the
`available enzyme in an active form.
`A good example
`to illustrate this point is the data of Bernfeld,
`et.al.
`(4) which is listed in Table 1.
`Since a
`crystalline, uniformly labeled protein was used,
`radioactivity measurements are indicative of total
`protein. These data show that 55% of the radioac=
`- tivity but only 10% of the enzymatic activity was
`recovered in the immobilized enzyme preparation.
`
`_I__MMOBILIZATI_Q_1§I OF 1-4C==.LABELED ALDOLASE ON POL §_gR3_z_:
`LAMIDE
`A
`
`TABLE 1
`
`Source
`
`Insoluble enzyme
`Aqueous phase after
`polymerization
`Liquid phase after
`first wash
`’
`Liquid phase after
`second wash‘
`Liquid phase after
`third wash.
`~
`TOTAL RECOVERY
`
`% Recovery
`Activity Radioactivity
`
`10.4
`
`33.1
`
`1.0
`
`.
`
`0
`
`0
`44.5
`
`55.0
`
`44.2
`
`0.7
`
`0.3
`
`0
`100.2
`
` .
`It is dangerous to assume that all unrecovered
`enzyme is immobilized and active.
`For this reason,
`many of the reported effects of immobilization on
`V and the value of the catalytic constant (k') are
`invalid since any change in the effective enzyme
`concentration will affect these values. Unless an
`independent method of determining the_active enzyme
`concentration, other than simple assay is employed,
`these reports should be minimized.
`One of the primary causes of thermal deactiva=
`tion of the enzyme is the disruption of the relaa
`
`127
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`

`
`DESIGN AND OPERATION OF IMMOBILIZED ENZYME EEACTORS
`
`Chapter 9
`
`Wayne H. Pitcher, Jr,
`
`1,
`
`
`INTRODUCTION
`Although certainly any industrial process utie
`lizing immobilized enzymes requires engineering
`throughout,
`the major concern is the unique part of
`the process,
`the reactor,
`Even though unique,
`the
`immobilized enzyme reactor has much in common with
`other reactors utilizing heterogeneous catalysts.
`Since with immobilized enzymes this is an area of
`limited experience, especially in industrial applim
`cations, generalizations are hazardous at best, At
`'least while immobilized enzyme engineering is still
`in its infancy, it is necessary to consider each
`system as an individual case,
`Thus this chapter.
`consists of an examination of the various aspects
`of immobilized enzyme reactor design and operation
`illustrated with practical examples. Specific to=
`pics, such as reactor types, reactor behavior, and
`mass transfer are treated individually with conclu=
`ding sections placing in perspective a wide range
`.of parameters and their effect on overall processw
`ing costs,
`
`II, REACTOR TYPES
`ImmobiIized enzyme (IME) reactors fall into
`several general categories including batch reactors
`(such as an agitated tank), continuous stirred tank
`reactors,
`fixedwbed reactors, and fluidized=bed re=
`actors as shown in Figure l.
`A large body of apw
`plicable information concerning reactor design al=
`ready exists in the heterogeneous catalysis litera=
`ture (1,2). More specific discussions of reactors
`for immobilized enzymes are also available <35495)o
`Other variations of the basic reactor types in=
`elude a stirred tank reactor in which the immobili=
`zed enzyme (IME)
`is enclosed in mesh containers at=
`tached to a stirrer (Figure 2)
`to give adequate
`agitation with minimal
`IME attrition (6),
`Emery
`(7) has reported the use of alternate layers of
`paper to which enzyme has been attached and nylon
`mesh, rolled into a cylinder,
`in a tubular flow rem
`actor.
`Closset, Shah and Cobb (8,9) have analyzed
`
`151
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`

`
`WAYNE H. PITCHER, JR.
`
`
`
`' PRODUCT
`
`CONTTNUOUS STTRRED TANK
`REACTOR
`
`
`
`BATCH
`REACTOR
`
`PRODUCT
`
`
`
` PRODUCT
`
`FUJMZED BED
`
`PACKED BED
`
`REACTOR
`
`REACTOR
`
`Fig, 1. Reactor types,
`
`and presented data for a tubular membrane reactor
`for the hydrolysis of starri1bY @=3mY13S€%Enzyme
`and,
`starch were contained by the membrane, which was
`permeable to the product, maltose, but not to the
`starch. Venkatasubramanian and Vieth (10) have
`used an arrangement consisting of alternate colla=
`gen=enzyme membrane and backing layers wound around
`a feed distributor,
`Enzymes have been immobilized on the inner sur=
`faces of tubes (ll,l2) contained in hollow fiber
`devices (13), and spun into synthetic fibers (14,
`-
`l5,l6),
`Robinson, Dunnill and Lilly (l7) have immobili=
`zed enzymes on magnetizable particles, which can be
`recovered magnetically using existing technology,
`This approach has been carried a step further by
`Gelf and Boudrant
`(18) who used a fluidizedebed re=
`actor containing papain bound to a magnetic support
`The particulate IME was retained in the column by
`
`152
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