ATTACHMENT C
`
`Petition for Infer Parfes Review of U.S. Pat. No. 8,394,618 B2
`
`ATTACHMENT C:
`
`“Immobilization ofEnzymes: Techniques and Applications,”
`Chapter 13 in “Biocataiytic Production of Amino Acids and
`Derivatives: New Developments and Process Co11side1'ations,” Eds.
`David Rozzel and Fritz Wagner, Hanser Publishers, 1992.
`
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`

`
`Biocatalytic Production of
`Amino Acids and Derivatives
`
`7
`
`Edited by
`J. David Rozzell and Fritz Wagner
`
`With 115 Illustrations and 68 Tables
`
`Hanser Publishers, Munich Vienna New York Barcelona
`
`-1 - Page 126
`
`

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`

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`.nwLI.LSeCa..nUSe.NmaeR
`
`O101KEPLL
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`__
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`8216QaP
`
`

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`

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`13 Immobilized Enzymes: Techniques & Applications
`
`307
`
`13.2 GENERAL, IMMOBILIZATIOP} METHODS
`
`Five general techniques have been described for immobilizing enzymes:
`adsorption:
`covalent attachment;
`cross-linking;
`entrapment of an enzyme in a polymeric matrix: and
`encapsulation or confinement of an enzyme in a membrane [2}.
`
`Each will be discussed along with its advantages and disadvantages. Although each
`immobilization technique is conceptually distinct. it is important to realize that there
`is often a certain amount of overlap or even combining of techniques in actual
`practice.
`Furthennore,
`the results of over thirty years of work by numerous
`researchers suggest
`that
`there is no one universally applicable immobilization
`technique; rather, a range of methodologies is available and must be evaluated on a
`case by case basis. There is still some art mixed with the science of enzyme
`immobilization. Certain enzymes may be immobilized successfully with one method
`but not with others. Each immobilization method has both advantages and disadvan-
`tages which must be considered within the context of the enzyme to be immobilized
`and the overall process in which the enzyme is to be used.
`1
`
`13.2.1 Adsorption
`
`Adsorption is one of the most economical and operationally simple processes by which
`enzymes can be immobilized. and this simplicity is no doubt responsible for its
`attractiveness as an immobilization method. Adsorption was the technique used by
`Chibata and co—workers in their first commercial process involving an immobilized
`enzyme. However, even though this immobilization procedure may be straightforward
`to carry out. the interactions involved in adsorption are complex and not completely
`understood. A good definition of adsorption, given by Messing, is the adhesion of an
`enzyme to the surface of a support which has not been specifically modified for
`covalent attachment [3].
`-.
`One of the principal advantages of adsorption is its ease to perform; simply
`contacting an aqueous solution of the enzyme with the support is all that is required.
`Occasionally, with judicious selection of the support, the desired enzyme will have an
`affinity for the support relative to other proteinaceous material such that a partial
`
`iecLtd., LLP Ex. 1010 - Page 13
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`

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`.
`
`It has also been rep(mad that, generally
`may result from im
`'
`'
`mobmzation within a
`
`aminoacylase on DEAE-Sephadex [4]
`, and illustrates the '
`At room temperature (2
`()—25 °C), 100 g of DEAE-Scp
`head type) suspended in I
`'
`'
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`13 Immobilized Enzymes: Techniques & Applications
`
`309
`
`protein is detected in the washing solution. These washing procedures are carried out
`at room temperature. The resulting precipitate is ready for use in the enzyme-
`catalyzed reaction.
`In order to store the immobilized preparafionfor a long period or to measure its
`enzyme activity, it is suspended in 2.5 l of distilled water and lyophilized. Using this
`procedure, 106 g of immobilized DEAE-Sepha(lex—am'Lnoacylase is obtained. The
`activity of the preparation is reported to be about 700 pmollh-g of preparation, under
`standard assay Conditions.
`industrial process for the
`The above method formed the basis for the first
`production of an amino acid by Tanabe Seiyaku Company in Japan. N-Acetyl-
`DL—tnethionine was passed through the column, where the L—Esomcr was deacetylated
`stereoselectively. The L-methionine was separated from the unrcactcd N-acctyl-
`D-methlon inc, which itself was recovered, racemized and recycled through the process.
`This process was successfully cotnmercialized in 1969 and is considered to be the first
`commercial-scale immobilized-enzyme process.
`in this example, the useful lifetime of the immobilized enzyme was approximately
`30 days, and the support could be regenerated each month by adsorbing fresh enzyme
`on the column.
`It was reported that the same batch of support matrix could be used
`for a period of live years, with repetitive regeneration cycles.
`Other enzymes useful for amino acid production which have been immobilized by
`the adsorption method include aspartase on DEAE-cellulose and silica gel [5], leucine
`aminopcptitlase on hydroxylapalite [6]. glutamieaspanic transaminases on DEAE—
`Sepharosc [7], and oi-amino-s-caprolaetam racernase on DEAE-Sephadex [8]. There
`are numerous reports of the immobilization of otherenzymes such as amylase, glucose
`isomerase, glucose oxidase, and proteases on materials such as activated carbon,
`bentonite, and alumina. Given the data base, it is probably fair to sunnise that an
`appropriate material may be found that will allow the immobilization by adsorption of
`virtually any enzyme; however, the strength of the adsorptive forces binding the
`enzyme to the support will vary considerably from one enzyme to another. Adoption
`of such a catalyst in a process will likely depend on whether the mechanical properties
`and the operational lifetime of the catalyst are sufficient for commercial application.
`
`13.2.2 Covalent Attachtnent
`
`Covalent attachment of enzymes to surfaces is often employed when leaching of
`enzyme activity from the support is a concern. This method generally offers the
`advantage of an immobilized enzyme system that is more permanently anchored. and
`may also show greater stability and the ability to withstand a broader spectrum of pl-l
`conditions, ionic strengths, and temperatures. Enzymes immobilized by covalent
`attachment are also more resistant to attack by protcolysis. The main disadvantage
`
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`310
`
`J. D. Rozzell
`
`[Refs. on p 313]
`
`of this type of attachment is its somewhat greater complexity and higher cost to
`prepare. Cost notwithstanding, when covalent attachment results in a significantly
`more stable enzyme system or when the absence of enzyme in the product solution is
`of critical importance, it still may be the method of choice.
`There are three different techniques by which covalent attachment can be effected.
`The first is through exposure of the enzyme to a support which has been preactivated
`to accommodate covalent binding. Operationally, once the activated support has been
`prepared, the immobilization proceeds like adsorption, but the result is an enzyme
`covalently bonded to the support matrix. The second technique involves exposure of
`the enzyme to the support in the presence of an activating or cross-linking reagent.
`Inevitably, some chemical modification or cross-linking of enzyme molecules occurs
`during an immobilization of this type which can lead to a loss in catalytic activity.
`A third possibility—much less commonly used——is to preactivate the enzyme and
`expose it to a support functionalized for covalent binding. The risk of inactivation of
`the enzyme by chemical processes during the preactivation procedure is significantly
`higher, and the procedure is less reproducib1e.- Thus, virtually all practical systems for
`' immobilization fall into one of the first two types.
`Frequently, covalent coupling is preferred to other processes in cases where the
`enzyme is multimeric or contains prosthetic groups. There may be a reduced tendency
`to disrupt the complex nature of these enzymes since specific bonds can be formed
`with the functional group to bind the enzyme through multiple points of attachment
`to the support.»
`A list of the amino acid functional groups which are chemically reactive enough
`to participate in covalent binding reactions may be found in Table 13.1.
`Of those functional groups listed, -NH” _-COZH, and -SH are involved in most
`immobilization procedures due to their nucleophilicity. The phenolic ring of tyrosine
`is also extremely reactive in diazo-coupling reactions, and its hydroxyl group can be
`an excellent nucleophile at basic pH. The guanidino group of arginine can react with
`aldehydes. Histidine displays a lower nucleophilicity, but can sometimes react with
`supports activated with tosylates, tresylates, or other good leaving groups.
`Functional groups of a wide range of types have been used for the covalent
`attachment of enzymes. The variety of chemistries available for covalent attachment
`allows the conditions of immobilization to be tailored to each enzyme system. The
`microenvironment of the enzyme may also be tailored by appropriate modification of
`the support surface; hydrophobic residues or ionically charged groups may be used to L
`alter the support to affect in a desirable way the enzyme-catalyzed reaction of interest
`[9].
`The range of support materials that has been used for covalent attachment. includes
`porous glass, porous ceramics, sand, charcoal, modified cellulose, polymeric resins,
`and metallic oxides. A few examples are described here to illustrate typical covalent
`attachment methodology.
`
`'
`f
`
`_
`
`'
`
`.
`
`-
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`13 Immobilized Enzymes: Techniques & Applications
`
`311
`
`TABLE 13.] Useful Functional Groups for Covalent Attachment of Enzymes
`to :1 Support Matrix
`
`Functional Group
`
`Corresponding Amino Acid
`.._....___:.......____..
`I
`
`Lysine, N-terminus
`
`Gtutamic Acid. Asportic Acid, C-terminus
`
`Cysteine
`
`Tyrosine
`
`Arginine
`
`Hisiidine
`
`Serine
`
`—NH2
`
`—co2H
`
`— SH
`
`_©_0H
`/NH
`—' NH—C
`
`H N
`
`“"\<_.?
`
`13.2.2.1 Specific Examples of Covalent Coupling: Binding to Acti-
`vated Carbohydrate Supports
`'
`'
`'
`
`One of the most commonly used procedures for the covalent coupling of enzymes to
`carbohydrate support matrices is based on a preactivation of a support with cyanogen
`bromide E10, ll], The mechanism of this reaction has been studied extensively by
`Wilchck and Kohn £12. 13].
`i
`The activated carbohydrate will couple generally to an amino group of lysine on
`the protein or the free "N-terminus of the protein to yield a covalently bound product.
`Supports which have been preactivated with cyanogen bromide can be prepared in
`advance and stored for periods of up to one year at freezer temperatures. Prcactivatcd
`supports are also available commercially. Coupling of an enzyme to a CNBr-activated
`support requires no more than exposure of the enzyme to the support in aqueous
`solution for a few hours, followed by washing. This method, while extremely popular
`in lab-scale reactions, has not been widely used in Iarge—scalc systems due to the
`requirements to handle toxic reagents and the poorer mechanical stability of most
`carbohydrate gets and polymers compared to other support materials. The bond
`between the enzyme and the support is also potentially susceptible to hydrolylic
`cleavage.
`"
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`312
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`J. D. Rozzell
`
`IRefs. on p 318]
`
`through the formation of an
`Carbodiimide reagents activate carboxyl groups
`0-acylisourea intermediate, which reacts rapidly with nuclcophilic functional groups
`of proteins to form amide, tltioester, or ester linkages. Hydrolysis is a competing side
`reaction. This method has been described for the immobilization of transaminases
`useful for amino acid production [14]. The reaction typically forms a stable amide
`linkage between enzyme-bound carboxyl groups and support-bound primary amines.
`and the rate of the reaction is relatively rapid. One disadvantage of carbodiimide
`reagents is their relatively high cost. Chemical modification of the enzyme leading to
`a loss of catalytic activity is another potentially negative side reaction.
`
`Perhaps the most frequently used technique for the
`covalent attachment to inorganic surfaces is the preaclivation and functionalization
`with aminosilane reagents [15.
`I6, 17].
`This technique involves attaching an
`aminosilane to the inorganic surface,
`leaving the
`'
`attachment of the enzyme. The most frequently used '
`
`been preaetivated for enzyme
`attachment. Coupling of enzyme may also be achieved through the use of carbodi—
`imide reagents, in which case it is through enzymic carboxyl groups that attachment
`is achieved.
`Another process for immobilizing on amino-functionalizcd inorganic supports
`involves isocyanate bonding [18]. If the enzyme is attached under alkaline conditions.
`a substituted urea bond is fanned between an amine on the protein surface and the
`'
`nditions are employed, then the isocyanatc reacts
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`
`[Refs on p 313]
`
`Carbodiimide
`
`I the formation of an
`philic functional groups
`lyxis is a competing side
`nation of Iranszuninases
`b‘ forms a stable amide
`-bound primary amines,
`‘image Of Carborliimide
`E the enzyme leading to
`trcacluon.
`
`Amine—Bea1ing
`
`Itiliaed supports for the
`[Hi reagents has been
`Wing Pendent amines.
`for binding. Glmar.
`Ilk, reacts in complex
`331d Produces pendent
`5! which proteins may
`as and diisocyanates.
`1 widely used for the
`red technique for the
`and functionalization
`‘uvolves attaching an
`available for covalent
`Elllarized through the
`Hnysilane [16].
`‘I113
`Illiling agents such as
`activated for enzyme
`:31 the use of embodi-
`“NIPS that attachment
`
`‘
`
`inorganic supports
`ll
`ualkalinc conditions,
`niacin surface and the
`the isocyanate reacts
`
`13 Immobilized Enzymes: Techniques & Applications
`
`313
`
`with a hydroxyl group on the enzyme and a urethane bond is fonned. Isothiocyanates
`have also been used successfully [16].
`-
`in providing amino group
`Polyelhyleneiminc is a common polyarninc, useful
`functionality for attachment. It has the advantage that it is inexpensive and that a wide
`range of supports including those other man inorganic particles may be used.
`Examples are alumina [19], carbon [20]. diatomaceous earth E21], and poiy(vinyl
`chloride)-silica composites [22, 23].
`A typical procedure involving polyethylenimine activation, which has been used for
`immobilization
`of
`recombinant
`transaminase,
`aspartase,
`and flSpal1ale-
`the
`B-dccarboxylase in the industrial—scale production of L-amino acids, has been described
`by Rozzeli [24]. The activity retained after immobilization approached 90%, with the
`operational half-lives of the immobilized biocatalysts ranging from 2 to 6 months.
`Recently, Flaschel and co-workers have developed a method for the covalent
`attachment of enzymes involving a mineral or carbon particle coated with chilosan,
`providing a hydrophilic surface of attachment [25]. Chitosan, which is deacetylated
`chitin, contains available amino groups for chemical activation and is easily obtained
`at relatively low cost. Activation with a bifunctional reagent such as glutaraldehyde '
`provides a stable immobilized-enzyme preparation. The use of rigid, incompressible
`particles on which the chitosan is deposited allows this catalyst to be used in both
`fixed-bed and fluidized-bed reactors. Yields of activity after immobilization of up to
`90% have been reported.
`-
`
`13.2.2.4 Specific Examples of Covalent Coupling: Oxirane or Epoxy-
`Activated Polymers
`'
`
`Epoxy-activated polymers fall into the category of preaclivated supports for covalent
`attachment, and they have gained attention as commercially useful support matrices
`for immobilized enzymes [26]. One advantage is the irreversible reaction by which
`'cnzymcs may be attached to a support through epoxides; as the epoxide opens. in a
`displacement reaction involving a nucleophilic group on the enzyme." a nonhydrolyz-
`able linkage is formed. Another advantage is the ability to activate a wide range of
`different surfaces with epoxides. Yet an additional advantage is that binding through
`epoxides does not appreciably change the charge state of the enzyme once it is bound.
`For exampte, the pit’, of the secondary amine formed after binding of an enzyme
`through a lysine side chain is not too different from that of the lysine side chain prior
`to coupling.
`An epoxide-activated support has been developed by Rohm Pharma (Darmstadt.
`FRG), and is sold commercially under the trade name of Eupergir. The support is
`based on a methacrylic polymer bead bearing epoxidc functionality. Eupergit has been
`successfully applied to the immobilization of many enzymes including penicillin
`acyiase for use in the production of 6-aminopenicillanic acid {27].
`'
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`13 Immobilized Enzymes: Techniques & Applications
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`315
`
`co-workers reported the immobilization of cholincsterase in polymerized starch [33].
`Vieth and Venkatasubramanian investigated the entrapment of enzymes in collagen
`matrices [34]. Whitesides has published on the use of PAN (polyacrylonitrile) gels
`{S5, 36].
`One of the major concerns with respect to elftrapped enzymes is that of leaching.
`The enzyme tnay migrate out of the pore it the porn is too large.
`In many cases, this
`leaching may be overcome by simply cross-linlcing the enzyme after entrapment with
`a bifunctional reagent such as glutaraldehyde.
`that of pore diffusion
`The opposite effect is also a concern with entrapment:
`limitations.
`If the substrate is a rather large molecule, such as a protein, it may be
`restricted from entry into the pore and thus be inaccessible to the enzyme.
`Dead cells containing enzymes have been very successfully imrnobilizetl by
`entrapment. The size of the cells prevents loss of catalyst clue to diffusion out of the
`pores of the matrix.
`Fusee described the production of L-aspartic acid using
`polyurethane-immobilized cells containing aspartase [37}. Calton er al. reported the
`production of L—aspartic "acid. L-phenylalanine, and t.-alanine using sells entrapped in
`a polyazetidine matrix [38}. Chlbata and co-workers have commercialized processes
`for L—asp:1rtic acid and L-alanine using cells entrapped in carragcenan gels and
`burdened with hexamcthylcnediaminc and glularaldehyde [39}. Calcium alginate gel
`has similarly been used.
`A novel method to overcome the problem of leaching out'of activity was developed
`as a pan of the polyazclidine method by Calton and co-workers. Cells containing
`enzyme are mixed with a polyazetidine polymer and cured by drying or mild heating.
`The polymer chains contain a reactive N-containing ring which opens under
`nucleophilic attack by cellular material or proteins. The result is believed to be a
`combination of entrapment and covalent attachment.
`rendering a more stable
`immobilized enzyme preparation in a hydrophilic environment. which is less prone to
`teaching [40]. Further enhancements to this technique have been developed by Nova
`[41].
`'
`
`13.2.5 Encapsulation or Confinement in a Membrane
`
`Encapsulation is distinguished from entrapment methods by the fact that at solution of
`the enzyme is separated from the bulk solution by a membrane.
`In this approach,
`pioneered by Chang [42, 43], enzymes are encapsulated within membranes that are
`impermeable to the enzymes but permeable to thecnzymc substrate. Such semi-
`permeable microcapsnlcs have been used as artificial cells in which the enzymes,
`cofactors, organelles. and other bioactivc materials are retained [30]. Encapsulation
`offers the opportunity to immobilize larger quantifies of enzyme per unit volume of
`immobilized preparation than any. other procedure [3]. Perhaps the biggest disadvan-
`tage of this immobilization technique is that only relatively small substrate molecules
`
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`

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`{Refs. on p 318]
`J. D. Rozzeli
`316
`_____.___.%_
`can be utilized with the intact membranes. Depending on the type of membrane used,
`encapsulation can also be a relatively expensive way to immobilize enzymes due to
`the high cost of membranes.
`Chang has described the encapsulation of enzymes in a variety of membranes [44]. .
`Two of the membranes that he used most successfully were cellulose nitrate and nylon.
`In another example. the Snamprogetti Company in Italy has entrapped aminoacylase
`and hydantoinase, for the production of amino acids in hollow fibers of cellulose
`acetate [45]. The halt‘~lives were greater than one month under operating conditions.
`Other enzymes have been similarly immobilized for producing a range of different
`products, showing wide applicability of this general method.
`Another membrane system which has shown promising results in biocataiytic
`applications has been developed by Sepracor [46, 47]. This system has been
`particularly effective in facilitating contact between an aqueous phase and a non-water-
`miscible phase. The enzyme is entrapped against the membranes and held in place by
`a slight positive pressure. As long as the stability of the enzyme is high enough. this
`system can operate continuously with little down time.
`In addition, old, deactivated
`enzyme can be flushed out and new enzyme loaded while the membrane cartridge is
`in place.
`
`13.3 CONCLUSIONS
`
`on enzyme immobilization is that, due to the range of methods that has been
`developed, it can be asserted with a high degree of confidence that a successful
`immobilization method can be developed for virtually any enzyme. That does not
`mean, however, that one method will be successful for almost all enzymes.
`In fact,
`the method of choice will likely vary from one case to another. All five methods
`surveyed and described here have been found useful in the past in certain instances.
`Even more importantly, a number of these methods have been proven successful at the
`commercial scale, thus giving the researcher several options to choose from which
`have withstood the "test of practicality" at the manufacturing scale.
`Products manufactured today using immobilized enzymes include high-fructose corn
`syrup, 6—aminopenicillanic acid, certain fatty acids and derivatives. L—malic acid, and
`isomaltulose. Specifically. a number of amino acids are also made using immobilized
`enzyme catalysts, including L-aspartic acid from fumaric acid and ammonia, L-alanine
`from L-aspartic acid, B-4-hydroxyphenylgiycine from the corresponding hydantoin,
`L—omithine and I.-citrulline from L-arginine, L-tryptophan from indole and L-serine._
`Depending on the economics, technology has been developed for other amino acids
`J
`
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`I3 lznmohilized Enzymes: Techniques 3:. Applications
`
`317
`
`t.-tyrosine. 1.-serinc. L-4-phenyl-
`including L—lysine. L-phenylalanine,
`as well,
`2~;uninobt:tanoic acid. L-norvaline, and L-DOPA. Genetic engineering has played a
`key role in ll number of cases in making finzymcs available in quantity at costs that
`previously could not be achieved.
`Fulurc applications will
`likely have an increasing dependence on gcnctic
`engineering as a tool to enhance enzyme-catalyzed processes by allowing improve-
`mcms tn the properties oi the enzymes: modified substrate specificity, improved
`stability. and more suitable tempcrnrure or pH optima. Figure l3.l illustrates in [low
`chart form how this may be done.
`
`NATIVE ENZYME
`
`CLONENG CF GEM
`
`txvncssion IN
`SUlTJ\El.E HOST
`
`°““R"°TE“'Z“T'°“
`
`SELECTION or new
`MOD|F|ED EN2‘rI.‘|E5
`
`PRODLETION AND -
`APPLICATION
`
`SITE-DlRECTED
`MUTAGENESIS
`
`l’-Tgure 13.1. -Using Genetic
`Engineering to Improve the
`Catalytic Properties of_
`Enzymes.
`
`KMKRCMUZMON
`
`Thc field of immobilized hiocatnlysts is rich with technology awaiting exploitation.
`Furthermore, blocntalysis is not standing still. The use of enzymes in nomaqueous
`solutions is rapidly cxpanding'1lte scope of applications for biological catalysts.
`Continuing advances in g¢fl€lit‘.' engineering. as mentioned above, nnd also in reactor
`design. should help to accelerate the realization of the potential
`licttclits of this
`technology.
`
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`
`J. D. Rozzell
`
`13.4 REFERENCES
`
`1.
`2.
`3.
`4.
`5.
`6.
`7.
`8.
`9.
`IO.
`11.
`12.
`I3.
`14.
`15.
`16.
`17.
`18.
`19.
`20.
`21.
`22.
`23.
`24.
`
`_
`
`Chibata, 1. Immobilized Enzymes; John Wiley & Sons, New York, 1978.
`Mosbach, K.. Methods Enzymoi. 44. V-vi (1974).
`Messing, R. A., Methods Enzymol. 44. 148 (1974).
`Chibata. '1'. et al.. Methods Enzymoi. 44. 746 (1974).
`Tosa, T.. Soto. T.. Mori, T.. Matuo. Y.. Chibata. 1.. Biotechol. Bioeng. 15, 69 (1973).
`Schwabe. (3.. Biochemistry 8. 795 (1969).
`Rozzell. J. D.. unpublished results.
`Fukumura, T.. Jpn. Pat. 74-15795 (.1974).
`Goldslein. L., Biochemistry 11. 4072-4084 (1972).
`Axen. R.. Porath. J.. Emback. S.. Nature (London) 214. 1302 (1967).
`Porath, 1.. Axen, R.. Ernback. S.. Nature (London) 215. 1491 (1967).
`Kohn. J.. Wilchek. M.. Enzyme and Microbial Technology 4. 161-163 (1982).
`Kohn, J., Wiichek, M.. Appl. Biochem and Biotechnology 9, 285-305 (1984).
`Rozzell. J. D.. Methods Enzymoi. 136. 479 (1987).
`Messing, R. A.. Weetall, H. H., U.S. Pat. 3,519,538 (1970).
`Weetall. H. 11.. Science 166. 615 (1969).
`Weetall. H. H.. Filbert. A. M.. Methods Enzymol. 34, S9 (1974).
`Messing. R. A.. Yaverbaum. S.. U.S. Pat. 4.071.409 (1978).
`Rohrbach. R. P.. U.S. Pat. 4.525.456 (1985).
`Laqtero. O. J..-U.S. Pat. 4.438.196 (1984).
`Chiang. J. P.. Lantero. 0. 1.. U.S. Pat. 4.713.333 (1987).
`Goldberg. B. S.. U.S. Pat. 4.102.746 (1978).
`Goldberg, B. S.. U.S. Pat. 4,169,014 (1978).
`Crump. S. P.. Meier. J. S.. Rozzell, I. D. In Biocotalysis; Abramowicz. D. A.. Ed.; Van
`Nostrand Reinhold, New York. 1990; pp 115-133.
`. Leuba, I.-L., Ranker. A.. Flaschel, E., U.S. Pat. 4.913.016 (1990).
`Bigwood, M. P.. Naples. J. 0.. U.S. Pat. 4.582.360 (1986).
`Bihari. V.. Buchholz. K.. Biotech. Letters 6, 571-576 (1984).
`Paulsen. P., Enzyme and Microbial Technology 3. 271 (1981).
`Silman. I. 11.. Albu-Weissenberg. M.. Katchalski. E.. Biopolymers 4, 441 (1966).
`Quiocho, F. A.. Richards. F. M.. Proc. Natl. Acad. Sci. USA 52, 833 (1964).
`Quiocho. F. A.. Richards. E M.. Biochemistry 5. 4062 (1986).
`Bemfield, P., Wan. 1., Science 142. 678 (1963).
`Bauman. E. U., Goodson, L. U..Guilbau1t, G. G.. Kramer. D. N., Anal. Chem. 37, 1378
`(1965).
`
`_
`Vieth. W. R.. Venkatsubramanian.K.. Methods Enzymol. 44. 243 (I976).
`Poliack, A., Blumerfeld. H., Baughlin. R. L., Whitesides. G. M.. J. Am. Chem. Soc. 102.
`6324-6336 (1980).
`
`Crans. D. C.. Kaziavskas. R. J.. Hirschbein. B. L.. Wong. C.-1-1., Abril. 0., Whitsides. G.
`M.. Methods Enzymoi. I36. 263 (1987).
`-1.
`'
`Fusee. M. (3.. Methods Enzymol. 136, 463 (1987).
`Calton. G. J.. Wood. L. L., Campbell. M. L., Methods Enzymol. 136. 497 (1987).
`Chibata. 1.. Tosa. T.. Takamatsu. S.. Methods Enzymoi. 136. 472 (1987).
`
`F‘ea°“"e 3”"'a°e3
`
`-
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`

`
`ATTACHMENT D
`
`Petition for Inter Partes Review of U.S. Pat. No. 8,394,618 B2
`
`"Immobilized Aminotransferases for Amino Acid Production": J.
`David Rozzell., "Methods in Enzymology” Volume 136, Pages
`479-497, (1987) Immobilized Enzymes and Cells, Part C,
`ISBN:978-0-12-182036-7.
`
`Reactive Surfaces Ltd., LLP Ex. 1010 - Page 143
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`ATTACHMENT D:
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`
`479
`IMMOBILIZED AMINOTRANSFERASES
`[44]
`lized pH- and glutaraldehyde-treated P. dacunhae cells. The substrate
`solution (adjusted to pH 8.5 with ammonia) consisting of 1.5 M ammo-
`nium fumarate and 1 mM Mg 2+ is applied to the column containing immo-
`bilized E. coli cells at a flow rate of space velocity = 1.0/hr. After addition
`of PLP and pyruvic acid (these concentrations are 0.1 and 1 mM, respec-
`tively) into the effluent, pH of the solution is adjusted to 6.0 by addition of
`acetic acid. The solution is passed through the immobilized P. dacunhae
`closed column at a flow rate of space velocity = 0.06/hr and a pressure of
`about 8 kg/cm 2 achieved by plunger pump.
`Crystallization of L-Alanine from Column Effluent. The effluent of
`appropriate volume is concentrated to about one-fourth of its original
`volume and cooled to 15 °. L-Alanine crystallized is collected by centrifu-
`gation or by filtration and washed with 80% aqueous ethanol. The yield of
`L-alanine from ammonium fumarate is about 90% (theoretical). [~]~ =
`+ 14.8 (c = 10 in 6 N HCI).
`
`Conclusion
`In 1982, Tanabe Seiyaku Co. Ltd. successfully industrialized a contin-
`uous production system of L-alanine from ammonium fumarate, using a
`column reactor containing immobilized pH-treated E. coli cells and a
`closed column reactor containing immobilized pH- and glutaraldehyde-
`treated P. dacunhae cells.
`By this system, L-alanine has been produced at low cost. This is
`considered to be the first industrial application of sequential enzyme reac-
`tions using two immobilized microbial cells.
`
`[44] Immobilized Aminotransferases for
`Amino Acid Production
`By J. DAVID ROZZELL
`
`Aminotransferases
`Background
`transaminases, EC
`(more commonly called
`Aminotransferases
`2.6.1._) are a widely distributed class of enzymes. These enzymes cata-
`lyze the synthesis and breakdown of amino acids in microorganisms,
`
`METHODS IN ENZYMOLOGY, VOL. 136
`
`Copyright © 1987 by Academic Press, Inc.
`All rights of reproduction in any form reserved.
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`480
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`ENZYME ENGINEERING (ENZYME TECHNOLOGY)
`
`[44]
`
`H2N ~'H
`
`+
`
`0
`
`0
`
`+ H 2 N~'%H
`
`SCHEME 1.
`
`plants, and animals by the transfer of an amino group from an a-amino
`acid to a 2-ketoacid as shown in Scheme 1.
`The first evidence for aminotransferases was published by Needham j
`and Szent-Gy6rgyi and co-workers 2 who noticed a relationship between
`the L-glutamic acid, L-aspartic acid, and oxaloacetic acid levels in pigeon
`breast muscle. Banga and Szent-Gy6rgyi 3 demonstrated the reversibility
`of glutamic-pyruvic transaminase (EC 2.6.1.2, alanine aminotransferase)
`by chemically isolating the amino acid products L-glutamate and L-
`alanine. Since that time, a large number of aminotransferases have been
`discovered and characterized. One feature of aminotransferases is the
`requirement for the small molecule, pyridoxal Y-phosphate, for catalytic
`activity, this cofactor being bound through a Schiff base linkage to the e-
`amino group of an active-site lysine. Although the binding of pyridoxal 5'-
`phosphate to the enzyme is reversible, most aminotransferases show
`maximal catalytic activity at cofactor concentrations of 100/~M or less.
`Such low saturating concentrations of pyridoxal phosphate are an impor-
`tant property of aminotransferases; at concentrations of 100/.~M or less,
`the cost of the cofactor in biocatalytic transamination processes is a rela-
`tively minor component of the total cost.
`The mechanism of transamination is well known, and has been re-
`viewed previously.* The reaction catalyzed by aminotransferases occurs
`as the result of two distinct half-reactions: the first involves transfer of the
`amino group of the L-amino acid donor to pyridoxal 5'-phosphate to yield
`a 2-ketoacid product which is released from the enzyme and an enzyme-
`bound pyridoxamine Y-phosphate; the second is the binding of the 2-
`ketoacid to be transaminated to the enzyme and the transfer of the amino
`group from pyridoxamine 5'-phosphate to this 2-ketoacid to produce the
`desired L-amino acid and regenerate the pyridoxal 5'-phosphate. As a
`result, aminotransferases characteristically exhibit Ping-Pong kinetics.
`
`D. M. Needham, Biochem. J. 24, 208 (1930).
`2 E. Annau, I. Banga, A. Blazo, V. Bruckner, K. Laki, F. B. Staub, and A. Szent-Gy6rgyi,
`Z. Physiol. Chem. 224, 105 (1936).
`I. Banga and A. Szent-Gy6rgyi, Z. Physiol. Chem. 245, 118 (1937).
`A. E. Braunstein, "The Enzymes IX" (P. D, Boyer, ed.), Part B, pp. 379-481. Academic
`Press, New York, 1973.
`
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`481
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`IMMOBILIZED AMINOTRANSFERASES
`[44]
`Advantages and Disadvantages for Use in Biocatalysis
`Although aminotransferases have been known for decades, these en-
`zymes have seen little use as biocatalysts until now. However, since the
`normal function of aminotransferases is the biosynthesis and metabolism
`of amino acids, it is natural to look to these enzymes as potentially useful
`catalysts for the production of amino acids. In principle, almost any de-
`sired amino acid can be produced from the appropriate 2-ketoacid using
`an inexpensive amino acid as the amino donor. There are a number of
`advantages to the use of this kind of technology. (1) The aminotransferase
`enzymes catalyze the stereoselective synthesis of only L-amino acids
`from their corresponding 2-ketoacids. No D isomer is produced, and no
`resolution is required. (2) Aminotransferases have uniformly high cata-
`lytic rates, capable of converting up to 400/~mol of substrate/min per
`milligram of protein. (3) Many of the required 2-ketoacid precursors can
`be conveniently prepared by chemical synthes