`
`19
`
`(
`
`Canadian
`) l* Intellectual Property
`Office
`An Agency of
`Industry Canada
`
`0 Office de la Propri,t,
`Intellectuelle
`du Canada
`Un organisme
`d'lndustrie Canada
`
`11
`
`) CA 2 337 884
`(
`(40) 03.02.2000
`43
`03.02.2000
`(
`)
`
`13
`
`(
`
`) A1
`
`
`
`(12)
`
`(21) 2337884
`(22)
`02.07.1999
`
`(51) m 01.7;
`
`C12N 11/02, C12P 41/00,
`0086 18/32
`
`(85)
`
`16.01.2001
`
`(86) PCT/EP99/04590
`
` wow/05354
`
`(30)
`
`(71)
`
`198 32 547.9 DE 21.07.1998
`
`(72)
`
`STURMER, RAINER (DE).
`
`'
`
`BASF AKTIENGESELLSCHAFT,
`ROBIC
`D-67056, LUDWIGSHAFEN, xx (DE).
`
`
`(74)
`
`POLYMERES CONTENANT UNE ENZYME
`ENZYME-CONTAINING POLYMERS
`
`(54)
`(54)
`
`(57)
`
`222The present invention relates to novel enzyme-
`containing polymers,
`having at 2least one
`of
`the
`following structural elements (1 and 2), wherein the
`2abbreviations and symbols have the following meaning:
`E stands for enzyme; X1, 2X2 independently represent
`oxygen, sulfur or NH from a functional group of the
`2enzyme; Q represents hydrogen or group (a); A1, A2,
`A3, A4 independently 2represent oxygen or sulfur; p is
`0 or
`1 with the proviso that A1, A2, A3 and 2A4
`represent oxygen when p = 0; w is 1-4; q is 1-4; v is 1-
`100;
`R1
`represents
`2an optionally substituted or
`functionalized hydrocarbon group with two to five
`zbonds; R2 represents an optionallysubstituted or
`functionalized hydrocarbon 2group with two to five
`bonds; R4 and R5 independently represent hydrogen, a
`C1-2C4-alkyl,
`aryl or alkylaryl group. The invention
`further relates to a method 2for the production of said
`enzyme-containing
`polymers,
`their
`utilization
`as
`2catalysts
`in chemical
`reactions and their use for
`separating enantiomer 2mixtures.2
`
`(1)
`
`(2)
`
`(I)
`
`

`

`OPlC
`OFFICE DE LA pnovxié'ré
`[NTELLECTUELLE m: CANADA
`
`PROPERTY OFHCE
`
`CIPO
`CANADIAN INTELLECTUAL
`
`(72) STURMER, RAINER, DE
`(711BASF AKTIENGESELLSCHAFT, DE
`(51)lnt.Cl.7 ClZN 11/02, COSG 18/32, C12P 41/00
`(30) 1998/07/21 (198 32 547.9) DE
`(54) POLYMERES CONTENANT UNE ENZYME
`(54) ENZYlVIE—CONTAINING POLYMERS
`
`(12) (19) (CA) Demande-Application
`
`(21)(A1) 2,337,884
`
`(86) 1999/07/02
`(871 2000/02/03
`
`(1)
`
`(2)
`
`(a)
`
`ALW%““%FN’KU\‘L@ 1
`
`concerne de nouveaux polymeres
`(57) L'invention
`oontenant une enzyme, component au moins l‘un des
`elements structuraux suivants (1 et 2) dans lesquels les
`abréviations et les symboles ont 1a signification snivante:
`E est une enzyme, X', X2 sont indépendannnent l‘un de
`l'autre oxygene, soufre ou NI-I provenant d‘un groupe
`fonctionnel de l'enzyme, Q est hydrogene 011 le groupe
`(a), A], A2, A3, A4 sont, independaimnent les uns des
`autres. oxygene ou soufre, p vaut 0 ou l a condition que
`A1, A2, A3, A4 représentent oxygene lorsque p = 0, w
`Vaut de l :14, qvaut de l 51,4, v vaut cle 1
`51 100, RI est un
`groupe hydroearbure avec 2 a 5 liaisons, evenluellement
`substitué
`on
`fonctionnalisé, R2
`est
`un
`groupe
`hydrocarbure avec
`2
`a 5
`liaisons,
`e’ventuellement
`substitué
`ou
`fonctionnalise,
`R4
`et
`R5
`sont,
`independamment l'un de l‘autre, hydrogene, un groupe
`alkyle C1-C4, aryle ou alkylaryle. En outre, l’invention
`concerne un procede pour
`la
`fabrication de ces
`polyineres contenant une enzyme,
`leurs utilisations
`comme cataly seurs dans des reactions chimiques et pour
`la separation de mélan ges d'enantiom eres,
`
`I*l Industrie Canada
`
`Industry Canada
`
`invention relates to novel enzyme—
`(57) The present
`oontaining polymers, having at least one of the following
`structural elements (I and 2), wherein the abbreviations
`
`and symbols have the following meaning: 3 stands for
`enzyme; Xl , X2 independently represent oxygen, sulfur
`or NH from a functional group of the enzyme; 0
`represents hydrogen or group (a); A1, A2, A3, A4
`independently represent oxygen or sulfur; p is 0 or 1
`with the proviso that A], A2, A3 and A4 represent
`oxygen when p = 0; w is 1-4; q is 1-4; v is 1-100; Rl
`represents an optionally substituted or functionalized
`hydrocarbon group with two to five bonds; R2 represents
`an optionally substituted or functionalized hydrocarbon
`group with two to five bonds; R4 and R5 independently
`represent hydrogen, a Cl—C4—alkyl, aryl or alkylaryl
`group. The invention further relates to a method for the
`production of said enzy me-containing polymers= their
`utilization as catalysts in chemical reactions and their use
`for separating enantiomer mixtures.
`
`

`

`CA 02337884 2001-01-16
`
`
`
`Intematlonales Bilro
`WEL‘DORGANISATION FOR 081911055 EIGEN'I'UM
`PCT
`INTERNATIONALE ANMELDUNG VEROFFENTLICHT NACH DEM VERTRAG UBER DIE —
`INTERNATIONALE ZUSAMMENARBEIT AUF DEM GEBIET DES PATENTWESENS (PCT)"
`
`
`
`(51) Internationale Patentklassifikation 7 i
`(11) Internationale Ven‘iffmflichungsnummer: WO 00/05354
`
`
`C12N 11/00
`'
`
`
`
`(43) lnternadonaloe
`Verot’fentlichungsdatmn:
`3. Februar 2000 (03.02.00)
`
`
`PCT/EP99/04590
`(81) Bestimmungsstaaten: CA. JP. US. europaisches Patent (AT,
` (21) Internationales Aktenzeichen:
`BE. CH. CY, DE, DK, ES. FI. FR. GB, GR, IE, IT. LU,
`MC, NL, PT, SE).
`(22) Internationale: Anmeldedatum:
`2. Juli 1999 (02.07.99)
`
`
`
`
` Verfifi‘entlichl
`(30) Prioritiitsdaten:
`
`
`21. Juli 1998 (2i 07.98)198 32 547.9 DE
`
`
`Ohne international-en Recherchenbericht and errteut w
`verdfienllichen nach Erhall dea' Ben't‘hls.
`
`(71) Anmelder (fiir alle Besrimmungsslaalen ausser US): RASF AK»
`
`
`
`TIENGESELLSCHAFT [DE/DE]; D—67056 Ludwigshafen
`
`
`(DE).
`
`(72) Erfinder; and
`
`(75) Erfinder/Anmelder
`(nur
`fiir US):
`STURMER, Rainer
`
`(DFJDEJ; Hauptstrasse 153, D-67l27 Rodersheim-Gronau
`
`
`
`(74) Gemeinsamer Vertreter: BASF AK’I‘IENGESELLSCHAFT;
`D—67056 Ludwigsbafen (DE).
`.
`
`(DE).
`
`L__._
`(54) Title: ENZYME—CONTAINING POLYMERS
`
`
`
`(54) Bezeichnung: ENZYMHALTIGE POLYMERE
`
`(57) Abstract
`
`The Present
`
`invention relates to
`
`polymers.
`enzyme—Containing
`novel
`having at
`least one of the following
`structural elements (I and 2), wherein
`the abbreviations and symbols have
`the
`following meanin:
`E stands
`for enzyme; X1.
`)Ggindependently
`represent oxygen. sulfur or NH from
`a functional group of the enzyme; Q
`represents hydrogen or group (a): A‘.
`A1, A3, A4 independently represent
`oxygen or sulfur:
`lp
`i520 03r
`l with
`the proviso that A. A , A and A4
`represent oxygen when p = 0; w
`is
`1—4; q is
`1—4;
`v is
`l—lOO; Rl
`
`1: @‘Xii‘fl
`
`. “Li'fl’ifLXL@
`
`;
`r
`Air
`ace—’05
`’-
`
`H
`
`'
`'g
`, A l
`, 3+" '
`N!
`"7—0
`'
`
`'
`(“1‘1 i
`‘
`
`’
`
`'
`
`I AH)
`n I
`
`n
`
`i”
`
`[2)
`
`(I)
`
`represents an optionally substituted or functionalized hydrocarbon group with two to five bonds; R2 represents an optionally substituted or
`functionalized hydrocarbon group with two to five bonds; R4 and R5 independently represent hydrogen. a C1—C4—alkyl, aryl or alkylaryl
`group. The invention further relates to a method for the production of said enzyme—containing polymers, their utilization as catalysLs in
`chemical reactions and their use for separating enantiomer mixtures.
`‘
`
`(57) Zusamxnenfzmung
`
`Die vorliegende Erfindung berrifft neue enzymhaltige Polymere. enthaltend mindestens eines der Strukturelemente. (I odor 2) wobei
`die Abkilrzungen und Symbole folgende Bedeurung haben: E- Enzym, X1, X2: unabhangig voneinander Sauerstoff, Sehwefel oder NH
`aus einer funktionellen Gruppe des Enzyms, Q: Wasserstoff odor die Gruppe (a). A“. A2, A3. A‘: unabhilngig voneinander Sauerstoff
`Oder Schwefel, p: 0 odor l. mit der MaBgabe, daB A‘. A2. A3, A4 Sauerstol‘f bedeuten. wenn p u D ist, w:
`1—4, q:
`1—4, v:
`l—lOO,
`R‘: eiue 2— bis S-fach gebundene, gegebenenfalls substituierte oder funktionalisierte Kohlcnwasserstoffgmppe, R2: eine 2— bis 5—fach
`gebundene, gegebenenfalls substituierte oder funktionalisierte Kohlenwasserstoffgruppe. R“ und R5: unabhingig voneinander Wasserstoff,
`eine Cl—C4—Alkyl-, Aryl— oder Alkylarylgruppe. Darilber hinaus belrifft die Brfindung ein Verfahren zur Herstellung cler enzymhaltigen
`Polymers, Anwendungen der enzymhaltigen Polymere als Katalysatoren in chemischen Reaktion en und die Verwendung der enzymhaltigen
`Polymere zur Trennung von Enantiomerengemischen.
`_J
`
`
`
`
`
`

`

`CA 02337884 2001-01-16
`
`0050/49212
`
`Enzyme-containing polymers
`
`The present invention relates to novel enzyme—containing polymers
`comprising at least one of
`the following structural elements:
`
`Structural element
`
`1
`
`Structural element 2
`
`A1
`
`IIL
`
`.
`
`10
`
`15
`
`20
`
`25 where the abbreviations and symbols have the following meanings:
`
`E
`
`enzyme
`
`x1, x2
`
`30
`
`independently of one another oxygen, sulfur or NH from
`a functional group of the enzyme
`
`Q
`
`35
`
`hydrogen or the group:
`
`A3
`
`We
`
`40 A1. A2,
`
`A%
`
`A4
`
`independently of one another oxygen or sulfur
`
`p
`
`45
`
`0 or 1, with the proviso that Al,
`When p is 0
`
`A2, A% A4 are oxygen
`
`

`

`0050/49212
`
`CA 02337884 2001-01-16
`
`w
`
`q
`
`v
`
`R1
`
`10
`
`R2
`
`15
`
`2
`
`1—4
`
`1—4
`
`1 — 100
`
`an alkane, alkene, cycloalkane, cycloalkene, arene,
`arylalkane, diaryl ether, diaryl thioether or
`
`diarylamine group which is bonded 2 to 5 times, it
`
`being possible for the aromatic or nonaromatic cyclic
`groups in turn to be substituted by one to four
`
`C1—C4—alkyl, C1—C4—haloalkyl and/or halogen radicals,
`
`an alkane, alkene, cycloalkane, cycloalkene, arene,
`arylalkane, dialkyl ether, dialkyl thioether, diaryl
`ether, diaryl thioether, diarylamine or
`piperazinedialkanediyl group which is bonded 2 to 5
`times, it being possible for the aromatic or
`
`nonaromatic cyclic groups in turn to be substituted by
`one to four C1—C4-alkyl, C1-C4—haloalkyl and/or halogen
`radicals,
`
`20
`
`25
`
`3O
`
`35
`
`40
`
`R4 und R5 independently of one another hydrogen,
`aryl or alkylaryl group.
`
`a C1~C4—alkyl,
`
`invention also relates to the preparation and use of
`The present
`the enzyme-containing polymers.
`
`The enzyme—containing polymers are employed as enzyme catalysts
`in chemical reactions. Compared with the free enzymes,
`the
`
`immobilized enzymes are distinguished by an increased stability
`and useful life when reactions are carried out continuously and
`batchwise, and by easy recovery of the catalytically active
`species in the case of batchwise reactions.
`
`It is known to incorporate enzymes into polymers by covalent
`bonding with retention of
`the activity. It is further known to
`
`use polyurethanes, polyureas and polyamides as polymeric carrier
`
`material. US 5,482,996 describes protein—containing
`polyurethanes, polyureas, polyamides and polyesters.
`
`In this
`
`the proteins are transferred from the aqueous solvent
`case,
`system into the organic solvent system, with retention of the
`
`45
`
`activity, by attaching an amphiphilic spacer (polyalkylene oxide)
`which has the terminal functional group suitable for the type of
`polymerization. The monomers are then reacted with the functional
`
`group of the spacer. This method makes only low coverage possible
`
`

`

`0050/49212
`
`CA 02337884 2001-01-16
`
`3
`
`and is moreover very elaborate and costly because there is no
`
`direct incorporation of the enzymes into the polymer, but
`
`the
`
`attachment of the amphiphilic spacer unit comprises a preceding
`additional step.
`
`US 3,672,955 discloses the preparation of enzymes immobilized in
`polyurethane, where there is initial preparation of amphiphilic
`isocyanate prepolymers bridged with polyether and polyesterpolyol
`units, and these are emulsified and reacted in a water—immiscible
`solvent with the aqueous enzyme solution. The contact with water
`converts the isocyanates which have not reacted with the
`
`10
`
`functional groups of the enzymes into unstable carbamic acid
`
`groups which then decompose into the corresponding amines with
`
`15
`
`elimination of C02. The resulting amino groups react with
`isocyanate groups which are still present,
`to give crosslinking,
`and the C02 produced leads to foaming of the polymer mass.
`
`Us 4,342,834 follows the analogous prepolymer strategy of
`'transferring the polymerization into aqueous systems,
`the
`difference from Us 3,672,955 being that the reaction is
`completely carried out in aqueous solution.
`
`20
`
`The use of the amphiphilic prepolymers Hypol® which is
`analogously bridged with polyalkylene oxide units (produced by
`reacting a polyether— or polyesterpolyol with polyisocyanates in
`the presence of linking reagents) and PU—3® (prepolymer saturated
`with two terminal TDI units and containing polyethylene oxide and
`
`polypropylene oxide units as spacer) for immobilizing a number of
`enzymes from aqueous solution has been described in scientific
`articles.
`
`It was possible to use these amphiphilic prepolymers to
`
`immobilize phosphotriesterases from aqueous solution for breaking
`down neurotoxins (K.E. Lejeune et al., Biotechnology and
`Bioengineering 1997, 54, 105-114). Lipases immobilized from
`aqueous solution with prepolymers have been used for the
`
`acylation (S.F. Dias et al., Biocatalysis 1991, 5, 21-34.)_and
`
`for the enantioselective acylation (S. Koshiro et al., Journal of
`
`Biotechnology 1985, 2, 47—57) of alcohols in organic solution.
`
`In all these processes,
`the polymerization (immobilization of the
`enzyme)
`is carried out in the presence of water with prepolymers.
`This has several disadvantages:
`
`—
`
`The amphiphilic prepolymers must be prepared separately.
`
`25
`
`30
`
`35
`
`40
`
`45
`
`

`

`0050/49212
`
`CA 02337884 2001-01-16
`
`4
`
`~
`
`-
`
`Only short useful lives are achieved with prior art
`enzyme—containing polymers.
`
`Only low loading densities with active enzyme species are
`achieved. The typical loading of a polymer with enzyme in the
`abovementioned studies is a maximum of 1% of the total mass.
`Low space—time yields (STY) result from this.
`
`10
`
`15
`
`It is an object of the present invention to remedy the
`deficiencies described and to provide novel enzyme~containing
`polymers which have been prepared by a simplified process and
`have optimized properties, such as longer useful life and higher
`loading density with catalytically active enzyme species.
`
`We have found that these objects are achieved by the
`enzyme—containing polymers according to the invention described
`at the outset.
`
`20
`
`The structural elements are produced by reacting the functional
`groups (amino, hydroxyl, mercapto)
`located on the enzyme surface
`with monomers having at least bifunctionally reactive groups, and
`subsequently adding at least bifunctional amines.
`
`25
`
`30
`
`35
`
`40
`
`45
`
`The enzymes are to be regarded as at least monofunctional,
`usually polyfunctional, amines, alcohols and/or thiols. They can
`be obtained, for example,
`from organisms such as fungi, bacteria
`(Gram+ or Gram'), yeasts or mammals, and have an enzymatic
`activity in organic solvents.
`
`Examples which may be mentioned are hydrolases such as esterases,
`lipases, amidases, proteases and haloperoxidases,
`aminotransferases, aspartate aminotransferases, pyruvate
`decarboxylases,
`lyases/laccases, benzene dioxygenase, aspartases,
`dehydrogenases,
`fumarases, dehalogenases, amino-acid
`dehydrogenases, oxygenases, aminopeptidases, aminoamidases,
`alkylaminopeptidases and racemases.
`
`Preference is given to lipases from Aspergillus niger,
`Aspergillus oryzae, Candida antarctica, Candida cylindracea,
`Candida lipolytica, Candida utilis, Candida rugosa, Mucor
`javanicum, MUcor miehei, Rhizomucor miehei, Rhizopus arrhizus,
`Rhizopus delemar, Rhizopus niveus, Penicillium acylase,
`Penicillium rogueforti, Thermus aquaticus, Thermus flavus,
`Thermus thermophilus, Chromobacterium Viscosum, Pseudomonas
`putida, Pseudomonas fluorescens, Pseudomonas cepacia,
`
`

`

`0050/49212
`
`CA 02337884 2001-01-16
`
`5
`
`and pig pancreatic lipase (PPL) and wheat germ lipase, esterases
`from Bacillus subtilis, Bacillus stearothermophilus, Bacillus
`thermoglucosidasius, Candida lipolytica, Mucor miehei, equine
`liver, porcine liver, Saccharomyces cerevisiae, Thermoanaerobium
`brockii, Elektrophorus electricus,
`
`thermolysin, subtilisin Carlsberg,
`proteases such as subtilisin,
`nagarse or from Bacillus subtilis, Tritirachium alba, Aspergillus
`oryzae, Aspergillus sp.,
`
`10
`
`benzene dioxygenase from Pseudomonads, Alcaligenes sp.,
`Micrococcus sp., Pseudomonas oleovorans,
`
`dehalogenases from Pseudomonas putida,
`
`oxygenases from Pseudomonas oleovorans, Corynebacterium equi,
`Nocardia carallina, Mycobacter, Xanthobacter,
`
`aminoamidases and alkylaminopeptidases from Mycobacterium,
`
`15
`
`dehydrogenases from Pseudomonas putida
`
`and nitrilases and nitrile hydratases from
`
`20
`
`Aspergillus niger JCM 1925, Fusarium sp. MY-Z, Rhodococcus
`rhodocrus J 1, K22,
`PA 34 and NCIB 11216, Pseudomonas
`
`chlororaphis B 23, Corynebacterium sp. N—774.
`
`The lipase from Burkholderia plantarii is particularly preferred.
`Structural element 1 describes the simplest case in which two
`enzymes are linked by an at least bifunctional monomer unit. X (X1
`or X2) represents the reacted functional group (hydroxyl, mercapto
`or amino group) on the enzyme surface. X can be according to the
`invention oxygen, sulfur or NH.
`
`The case where p = 1 corresponds to linkage of the enzyme to an
`at least bifunctional and at most pentafunctional
`(w a maximum of
`4)
`isocyanate (A = oxygen)
`(A1, A2, A3 or A4),
`isothiocyanate (A =
`sulfur) or mixed isocyanate/isothiocyanate (A = oxygen or sulfur)
`via a urethane (X = oxygen, A = oxygen),
`thiocarbamic acid
`H
`
`NH, A =
`O—ester (thiourethane; X = oxygen, A = sulfur), urea (X
`oxygen),
`thiourea (X = NH, A = sulfur),
`thiocarbamic acid S-ester
`(X = sulfur, A = oxygen) or dithiocarbamic acid diester (X =
`sulfur, A = sulfur) group.
`
`the enzyme to an
`The case where p = 0 corresponds to linkage of
`at least bifunctional and at most pentafunctional
`(w a maximum of
`I!
`4) active ester via an ester (X = oxygen), amide (X
`NH) or
`thiolcarboxylic ester (X = sulfur) group.
`
`25
`
`30
`
`35
`
`40
`
`45
`
`

`

`0050/49212
`
`CA 02337884 2001-01-16
`
`6
`
`R1 represents an alkane, alkene, cycloalkane, cycloalkene, arena,
`arylalkane, diaryl ether, diaryl thioether or diarylamine group
`which carries the functional groups and is bonded 2
`to 5
`times,
`it being possible for the aromatic or nonaromatic cyclic groups
`5 in turn to be substituted by one to four C1-C4-alkyl, such as
`methyl, ethyl, propyl,
`isopropyl, n—butyl,
`isobutyl or t—butyl,
`and/or C1-C4—haloalkyl, such as trichloromethyl,
`trifluoromethyl
`or trifluoroethyl, and/or halogen radicals, such as Cl, Br,
`I or
`F. Preferred R1 radicals are the basic organic moities underlying
`the corresponding polyisocyanate or isothiocyanate monomers known
`from polyurethane chemistry. The following specific structures
`may be listed as particularly preferred,
`the linkage points being
`indicated by a dash:
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`

`

`0050/49212
`
`CA 02337884 2001-01-16
`
`arcs: /
`m = 2—12
`
`CH2
`
`/CH2
`
`/CH2
`\
`
`(EH
`
`3
`
`_$H
`
`j:
`
`"OH
`
`I
`
`CH3
`
`[:::I//
`
`‘
`
`C‘
`
`CH3
`
`::
`
`CH
`
`CH3
`
`x/[:j::I//
`
`0i 0 O 4:?
`
`HaC
`
`H30
`
`GHQ—-
`
`CH3
`
`CH3
`
`H 3
`
`10
`
`15
`
`2O
`
`25
`
`30
`
`35
`
`The enzymes, symbolized by the letter B in a circle, may be
`various enzyme molecules or different binding sites on the same
`enzyme molecule.
`In the particular case where w > 1,
`that is to
`
`40
`
`the enzyme symbols
`say branched polyfunctional monomers are used,
`may also represent various adjacent binding sites on the same
`enzyme molecule.
`'
`
`Structural element
`
`2
`
`takes account of the additional
`
`45
`
`incorporation of the at least bifunctional amines. The linkage of
`
`the at least bifunctional monomers to the enzyme is analogous to
`
`Structural element 1 and has been described there.
`simplest case (v = O), a bifunctional amine (q = 1)
`
`In the
`links two at
`
`

`

`0050/49212
`
`CA 02337884 2001-01—16
`
`8
`
`least bifunctional monomers, which are bonded terminally by the
`appropriate functional group to the enzyme, via a urea (A1 =
`oxygen, p = 1),
`thiourea (A = sulfur, p = 1) or amide (A =
`oxygen, p = 0) group. The unit [at least bifunctional monomer-at
`least bifunctional amine] may be repeated V times to form
`
`polyurea (A = oxygen, p = 1), polythiourea (A = sulfur, p = l) or
`polyamide (A = oxygen. p = O) chains (w = l; q = 1) or crosslinks
`
`(either w > 1 or q > 1 or w and q > 1). The case Q = hydrogen
`covers the termination of a chain without linkage to another
`enzyme molecule.
`
`10
`
`R2 is an alkane, alkene, cycloalkane, cycloalkene, arene,
`arylalkane, dialkyl ether. dialkyl thioether, diaryl ether,
`diaryl thioether, diarylamine or piperazinedialkanediyl group
`which carries the amino groups and is bonded 2 to 5
`times, it
`
`being possible for the aromatic or nonaromatic cyclic groups in
`turn to be substituted by one to four C1—C4-alkyl, C1—C4—haloalkyl
`and/or halogen radicals, e.g. as mentioned above for R1.
`
`Preferred radicals are the basic organic moieties underlying the
`corresponding polyamine monomers known from polyurethane and
`
`polyamide chemistry. The following specific structures may be
`listed as particularly preferred:
`
`H2 H2
`,C—C
`
`]
`HZC‘C—C/
`
`H H
`
`O—(—+—C—}a—
`H2
`2
`
`r=23A
`
`H30
`
`3
`
`HC
`
`3
`
`C—
`
`H2
`
`H+CH2—}7CIZH+CH§—]y—CIDH+CH2-L—H
`
`whae x=O—10
`y=O—1Q
`z=0—10 andx+y+z=O—1O
`
`H*H— CO 67 6/ (5
`
`H
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`

`

`0050/49212
`
`CA 02337884 2001—01-16
`
`9
`
`Depending on the incorporation of primary or secondary at least
`bifunctional amines, R4 and R5 are,
`independently of one another,
`
`hydrogen or a C1~C4—alkyl radical such as methyl, ethyl, propyl,
`isopropyl, n—butyl,
`isobutyl,
`t-butyl or an aryl or alkylaryl
`
`radical, such as phenyl or C1-C4—substituted phenyl such as tolyl.
`
`It is also necessary to take into account with Structural element
`
`2 that the enzymes, symbolized by the letter E in a circle, may
`be various enzyme molecules or different binding sites on the
`same enzyme molecule.
`
`10
`
`The contents of the enzyme by weight in the polymeric carrier are
`
`preferably between 0.01% by weight and 50% by weight,
`
`15
`
`particularly preferably between 1% by weight and 10% by weight,
`based on the total mass. As shown in Example 3,
`the enzyme
`
`activity and the useful life decrease if the loading is less
`
`than 1% does
`than 1%. By contrast, greater loading (5%)
`(0.5%)
`not result in a decrease in the enzyme activity and the useful
`life. The greater loading allows a higher space—time yield (STY)
`
`20
`
`to be achieved in the enzyme-catalyzed reactions described
`hereinafter.
`
`It has additionally been found that the enzyme-containing
`polymers according to the invention are particularly
`
`25
`
`advantageously obtained in a simplified, anhydrous process by
`direct reaction of the enzyme in organic solution with
`crosslinking organic compounds with terminal reactive functional
`
`30
`
`groups. This entails reacting the enzyme in an organic solvent in
`the first step with the at least bifunctional monomer of
`the
`formula I
`
`R1 (x)s(Y) t
`
`I
`
`35
`
`where
`
`R1
`
`4O
`
`and
`
`45
`
`is an alkane, alkene, cycloalkane, cycloalkene, arene,
`arylalkane, diaryl ether, diaryl thioether Cr
`diarylamine group which is bonded 2 to 5
`times, it
`
`being possible for the aromatic or nonaromatic cyclic
`groups in turn to be substituted by one to four
`
`C1—C4-alkyl, Cl-C4-haloalkyl or halogen radicals,
`
`

`

`0050/49212
`
`CA 02337884 2001—01-16
`
`is the isocyanate (-NCO) functional group and
`
`10
`
`is the isothiocyanate (-NCS)
`
`functional group
`
`with s 20 and t 2 O and 5 2 s+t Z 2
`
`x
`
`Y
`
`or
`
`10
`
`X=Y
`
`is the —COR3 functional group
`
`with 5 2 s+t Z 2,
`
`15
`
`in which R3 is a leaving group which can be displaced
`by the amino, hydroxyl or mercapto functionality of the
`enzyme,
`
`and adding in a 2nd step an at least bifunctional amine or a
`mixture of at least bifunctional amines.
`
`20
`
`Organic solvents mean according to the invention aprotic solvents
`Such as aliphatic, aromatic, optionally halogenated hydrocarbons,
`and ethers. Preferred organic solvents are those which are inert
`toward the at least bifunctional monomers, dissolve the at least
`
`25
`
`bifunctional monomers, and do not denature the enzyme.
`Particularly preferred organic solvents are toluene, benzene,
`
`chloroform, methylene chloride, hexane, heptane, diethyl ether
`and methyl t~butyl ether (MTBE), dioxane, THF and halogenated
`aromatic compounds such as chlorobenzene.
`
`30
`
`in a first
`The enzymes are suspended in an organic solvent and,
`step, reacted with an at least bifunctional monomer. The monomer
`
`35
`
`has as functional groups at least two and not more than five,
`particularly preferably two or three,
`isocyanate and/or
`
`isothiocyanate, or at least 2 and a maximum of five, particularly
`preferably two or three, active ester groups of the formula -COR3
`in which R3 is a leaving group which can be displaced by the
`amino, hydroxyl or mercapto functionality of the enzyme. Examples
`which may be mentioned of active ester groups and thus of acyl
`donors are carbonyl chlorides, carboxylic anhydrides, carboxylic
`esters such as N-hydroxysuccinimide esters of carboxylic acids,
`
`phenol esters and halophenol esters such as pentafluorophenol
`esters and trichlorophenol esters. Accordingly, examples which
`
`may be mentioned of the leaving group R3 are halides such as C11
`Br',
`I—, carboxylates such as acetates and propionates or
`alcoholates such as N-succinimidoxide, phenolates, halophenolates
`
`40
`
`45
`
`

`

`0050/49212
`
`CA 02337884 2001-01-16
`
`11
`
`such as pentafluorophenolate,
`
`trichlorophenolates or
`
`1-benzotriazoloxides. The functional groups on the enzyme surface
`(amino, hydrcxyl and/or mercapto groups) react with the terminal
`isocyanate and/or isothiocyanate functionalities of the monomers
`
`thiocarbamic acid O-ester(thiourethane), urea,
`to form urethane,
`thiourea,
`thiocarbamic acid S—ester and/or dithiocarbamic acid
`. diester linkages.
`In analogy to this on use of the polyfunctional
`active ester monomers there is formation with the functional
`
`groups on the enzyme surface (amino, hydroxyl and/or mercapto
`groups) of amide (Jerry March, Advanced Organic Chemistry, 1992,
`4th edition, John Wiley & Sons, New York, pp. 417—425), ester
`(Jerry March, Advanced Organic Chemistry, 1992, 4th edition, John
`Wiley & Sons, New York, pp. 392—398) and/or thiolcarboxylic ester
`linkages (Houben-Weyl, Volume IX, 4th edition, pp.753~756). These_
`linkage types may occur in any combination depending on the
`presence and number of the respective functional groups on the
`enzyme surface and the monomer used.
`
`The diisocyanate, polyisocyanate, diisothiocyanate,
`polyisothiocyanate, mixed polyisocyanates/isothiocyanate or bi—
`to pentafunctional active ester monomers used are alkane, alkene,
`alkyne, cycloalkane, cycloalkene, arene, alkylarene, arylalkane,
`diaryl ether, diaryl thioether or diarylamine compounds, it being
`possible for the aromatic or nonaromatic cyclic groups in these'
`compounds in turn to be substituted by one to four C1—C4-alkyl,
`C1-C4—haloalkyl and/or halogen radicals. Preferred radicals are
`
`the diisocyanates, polyisocyanates, diisothiocyanates,
`polyisothiocyanates and mixed polyisocyanates/polyisothiocyanates
`or polyfunctional carboxylic acid or carboxylic ester derivatives
`
`which are known from polyurethane chemistry and have the basic
`moieties analogous to the polyisocyanates described, and have the
`active ester groups described above as functional groups in place
`of the isocyanate or isothiocyanate groups. Particularly
`preferred monomers are
`
`1,4—diisocyanatobutane.
`
`1,6—diisocyanatohexane,
`
`1,8-diisocyanatooctane,
`
`l,lZ—diisccyanatododecane,
`
`1,S—diisocyanato-2—methylpentane,
`
`1,3—bis—isocyanatomethylbenzene,
`
`trans—l,4-diisocyanatocyclohexane,
`
`1,5—diisocyanatonaphthalene,
`
`4,4'—methylenediphenyl diisocyanate,
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`4O
`
`45
`
`.WM,_ m...
`.m.
`.
`..,,
`
`we»...
`
`.. riflw... . _
`
`__ comm...“ .,
`
`_
`
`. M... ”.m—«uu—wuu -—~.
`
`,
`
`.,
`
`

`

`peso/49212
`
`CA 02337884 2001~01-16
`
`12
`
`1—(p-isocyanat0phenyl)—2~isocyanato—2-methylpropane,
`
`tolylene 2,4-diisocyanate (4»methyl—m—phenylene diisocyanate),
`
`tolylene 2,5~diisocyanate,
`
`tolylene 2,6—diisocyanate,
`
`1,3—diisocyanatobenzene,
`
`1,4—diisocyanatobenzene (p—phenylene diisocyanate),
`
`4,4’-oxydiphenyl diisocyanate,
`
`10
`
`3,3'-bistolyl-4,4’-diisocyanate,
`
`3,3’-dimethyl*4,4'—diisocyanatodiphenylmethane,
`
`2,4—diisocyanato~5~chlor0toluene,
`
`isophorone diisocyanate
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`40
`
`45
`
`and the analogous polyisothiocyanates or analogous mixed
`polyisothiocyanates/isocyanates.
`
`In the case where it is advantageous to obtain the
`
`enzyme—containing polymer as foam (e.g.
`
`to increase the internal
`
`surface area) it is possible simultaneously with the addition of
`
`the monomer to add physical blowing agents such as hydrocarbons,
`in particular methane, ethane, ethylene, propane, propylene,
`pentane, cyclopentane, cyclohexane, or halogenated hydrocarbons,
`in particular trichlbrofluoromethane, chloromethane,
`dichlorodifluoromethane, dichlorofluoromethane,
`
`chlorodifluoromethane, chloroethane, dichlorotetrafluoroethane,
`octafluorocyclobutane, hexafluoropropane,
`l,l-difluoro—2,2—
`dichloroethane, 1,2-difluoro—1,2—dichloroethane,
`
`tetrachloroethane,
`trichloroethane,
`1,1—dichloroethane,
`l—fluoro~1,2,2—trichloroethane, 1—bromoethane or
`
`l,l,2-trifluoro—Z—chloroethane, or to pass inert gases such as
`nitrogen, argon, carbon dioxide or air in during the reaction.
`
`the upper limit is
`The reaction temperature is not critical but
`determined by the sensitivity of the particular enzyme to
`temperature. The preferred temperature range is between -30°C and
`60°C, particularly preferably between —lO°C and 10°C.
`
`In a second step, at least bifunctional amines or mixtures of at
`
`least bifunctional amines are added. The time between step 1 and
`2 is preferably not more than 1 s to 10 min, particularly
`preferably 30 s to 5 min.
`
`..WW" .
`
`.
`
`.
`
`,.. l inn—,wanm 0...;
`
`.
`
`.. .. W~.W__._..l_wwuw
`
`

`

`0050/49212
`
`CA 02337884 2001-01-16
`
`13
`
`The added at least bifunctional amines react with as yet
`unreacted functional groups of the monomers to give crosslinking
`and/or chain extension. The at least bifunctional amines used are
`primary or secondary amines such as amines of alkanes, alkenes,
`alkynes, cycloalkanes, cycloalkenes, arenas, alkylarenes,
`arylalkanes, diaryl ethers, diaryl thioethers, diarylamines,
`dialkylamines or piperazine dialkanes, it being possible for the
`aromatic or nonaromatic cyclic groups in these amines in turn to
`be substituted by one to four C1—C4-alkyl, C1—C4—haloalkyl and/or
`halogen radicals. Preferred radicals are the amine monomers known
`from polyurethane and polyamide chemistry.
`
`Particular preference is given to
`
`bis~3—aminopropyl—l,4—piperazine,
`
`bis-B—aminopropyl ether,
`
`bis-Z—aminoethyl ether.
`
`N—phenylethylenediamine,
`
`N,N'~diethylethylenediamine,
`
`N,N’-diethylpropylenediamine,
`N,N’—diethylbntylenediamine,
`
`N,N’—diethylhexylenediamine,
`
`isophoronediamine,
`
`1,2—diaminocyclohexane (cis and trans),
`
`l,2—diamino~1,2—diphenylethane (cis and trans),
`
`1,8—diaminonaphthalene,
`
`aliphatic amines of the formula II
`
`NH2
`NH2
`H+CHflroH+CHflroH+CHflTH
`
`u
`
`with x = O - 10
`
`y = 0 — 10
`
`z = 0 ~ 10
`
`and 0 S x+y+z S 10,
`
`such as 1,2-diaminobutane,
`
`l,n-diaminoalkanes, by which are meant linear
`C2—C12-diaminoalkanes with terminal amino functionalities
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`

`

`0050/49212
`
`CA 02337884 2001-01-16
`
`(n = 2 to 12; E x = O and z = 0 and y = O
`
`to 10), such as
`
`14
`
`1,2-diaminoethane,
`
`1,3—diaminopropane,
`
`1,4-diaminobutane,
`
`1,5—diaminopentane,
`
`1,6—diaminchexane,
`
`1,7-diaminoheptane,
`
`10
`
`1,8—diaminooctane,
`
`1,9—diaminononane,
`
`l,lO-diaminodecane,
`
`l,ll-diaminoundecane or
`
`15
`
`1,12-diaminododecane,
`
`and in each case the N—monoalkylated or N—monoarylated
`l,n—diaminoalkanes (n = 2
`to 12),
`
`and the N-alkylated and N'—arylated, N,N’—dialkylated or
`N,N’—diarylated l,n—diaminoalkanes (n= 2 to 12).
`
`20
`
`the at least bifunctional monomers and of the at
`The choice of
`least bifunctional amines has a negligible effect on the yield of
`
`25
`
`enzyme-containing polymers. It is therefore possible to use any
`suitable at least bifunctional monomers and any suitable at least
`bifunctional amines, and any suitable combinations.
`
`Particularly good polymer and catalyst properties, such as
`increased useful life and increased conversions, are obtained
`when, as is evident in Example 2,
`the at least bifunctional
`
`30
`
`' monomer
`
`in the first step and at least bifunctional amine in the
`
`second step are preferably used in the following combinations:
`
`35
`
`4O
`
`45
`
`p~phenylene diisocyanate with 1,n-diaminoalkane,
`
`4—methyl~m—phenylene diisocyanate with l,n—diaminoalkane,
`
`4,4’-methylenebisphenyl diisocyanate(MDI) with l,n—diaminoalkane,
`
`p—phenylene diisocyanate with N~phenylethylenediamine,
`
`p-phenylene diisocyanate with 1,6—diaminohexane,
`
`4~methyl—m—phenylene diisocyanate with
`N,N’-diethyl-ethylenediamine or
`
`4-methyl—m-phenylene diisocyanate with 1,4-diaminobutane.
`
`The enzyme-containing polymers are isolated from the reaction
`
`solution by conventional methods for separating solids from fluid
`
`systems, e.g. by filtering off and drying the resulting
`
`

`

`0050/49212
`
`CA 02337884 2001-01-16
`
`15
`
`enzyme-containing polymer. When using solvents which lead to
`
`is preferably added
`t—butyl ether (MTBE)
`tacky polymers, methyl
`in order to obtain free—flowing products.
`before the filtration,
`Without addition of physical blowing agents at the start of the
`reaction,
`the enzyme-containing polymers result as amorphous
`solids. To adapt
`the solid to particular embodiments of reactors,
`the amorphous solid can undergo subsequent processing, for
`example grinding

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