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Efficient Heterogeneous Biocatalysts by Entrapment of Lipases in Hydrophobic SolЦGel Materials.

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Efficient Heterogeneous Biocatalysts
by Entrapment of Lipases
in Hydrophobic Sol- Gel Materials
1 (TMOS)
\/v Si(OCH&
3 (BTMS)
2a R= CH3 (MTMS)
R = C2H5 (ETMS)
C R = n-C& (PTMS)
d R = n-C4Hg (BTMS)
?t R = n-ClaHs7 (ODTMS)
Manfred T, Reetz,* Albin Zonta,
and Jorg Simpelkamp
Lipases are amongst the most widely used enzymes in organic
chemistry.['] I n aqueous emulsions, they catalyze the chemo-,
regio-, and stereospecific hydrolysis of esters, and when suspended in organic solvents"] they catalyze the reverse reaction, effecting selective esterification."] In order to increase the activity and
stability of lipases and at the same time to facilitate their recovery,
many studies on the immobilization of lipases have been carried
We report here on the entrapment of lipases in hydrophobic sol -gel materials, which results in the formation of highly
active. stable and reusable heterogenous biocatalysts.r61
Following the procedures described earlier for the entrapment
of biomolecules in silica gel (SiO,) using the sol-gel process,['. 'I
tetramethoxysilane (TMOS) 1 was hydrolyzed in the presence of
various hpases (e.g. from ps. cepaciu, Amano Ps). Classical solgel processes of this type are acid or base catalyzed; hydrolysis
and condensation of TMOS lead to nanometer-sized sol particles
which then cross-link to form insoluble amorphous SiO, gels.[']
Unfortunately. the immobilized lipases that were produced by
this method displayed extremely low activities. For example, relative activities of only 5 YOwere obtained in the esterification of
n-octanol (B) (0.1 M) in isooctane to
lauric acid ( A ) ( 0 . 0 5 ~with
give octyl laurate (C). In this test reaction that was used in all
of the following studies, the relative activity .Y is defined as
v(immobilized enzyme) / v(commercia1 enzyme), where L' is the
initial rate of the reaction (in pmol(hmg1ipase)- I ) .
4 (PDMS)
catalyst.["] Figure 1 shows the dependence of the activity on the
"methyl content" of the gel. In a mixed gel with 50% MTMS,
the relative activity was still only 30%. Further increases in the
MTMS content caused the relative activity to rise dramatically,
reaching 1300% for a pure MTMS gel.
A 100
Fig. 1. Dependence ofthe activity A [pmol(hmglipase)- '1 of immobilized Ps. i'epuciu lipase in TMOSiMTMS mixed gels on the gel composition. For the definition of
the relative activity .'i see the text.
Taking a constant TMOS/RSi(OCH,), ratio of l / l and varying the R group of the silane, the relative activity sharply rises
in the order CH, <C,H, <n-C,H, <n-C,H,. In contrast, a further increase in the size of the R group and the associated rise
in the lipophilicity by use of n-octyl or n-octadecyl groups causes
only a small additional increase in activity (Fig. 2).
Since lipases are interphase-active enzymes with hydrophobic
domains, they can form ionic as well as hydrophobic interactions with other molecules.[91For this reason, we speculated that
alkyl-modified silica gels["] with hydrophobic character, produced from silanes of the type RSi(OCH,), , might be more suitable host matrices for lipases. In preliminary experiments, we
therefore immobilized the lipase from Ps.cepaciu (Amano PS)
in a sol-gel process using CH,Si(OCH,), (MTMS) (2a) or mixtures of TMOS (1) and MTMS (2a). N a F was used as the
P r d . Dr. M T. Re&. Dip1.-Biochem. A. Zonta. Dr. J. Simpelkamp
Max-Pliinck-lnstitut fur Kohlenforschung
Kaiser-Wilhclm-Platz 1. D-45470 Mulheim an der Ruhr (Germany)
Telefix. Int code + (2010306-2985
~ ' I I ~ w In,
I . Ed. EiiRl. 1995. 34. N o . 3
Fig. 2. Influence of the chain length o f R on the activity of immobilized Ps. cepuciu
lipase in gels of the type TMOS,!RSi(OMe), ( l / l ) . n = number of C atoms in R.
VCH l4dugsgesrll.schufi mhH. D-6Y451 Wcinheiin, 1Y95
US70-0X33;Y5~0303-U3301R lO.OO+ .25 '0
The results summarized in Figures 1 and 2 clearly show that
the silane component is an important parameter for optimizing
the enzymatic activity. We therefore investigated the commercially available silanes 3 and 4, n = 5-9 as gel precursors in
combination with TMOS and several lipases.[121In all cases gel
materials were obtained with excellent activities. U p to 93 Oh of
the protein was immobilized.
The representative results given in Figure 3 indicate that there
is no general recipe for an optimal gel matrix, although in many
cases TMOSjPTMS (1/5) gels turned out to be particularly well
suited. An increase in activity by a factor of 5 and more (relative
to the commercial powders) was achieved with many entrapped
lipases. With the lipase SP 523 (Novo) the factor is even 88.
Fig. 4. Scanning electron micrograph of an immobilized lipase (P.Y.cepacia lipase in
MTMS gel). Right: lox inagnification of the area marked on the left. The length of
the white bar corresponds to 60 pm.
The first attempts at application of the heterogeneous biocatalysts were carried out in the kinetic resolution of l-phenylethan01 (5)[”] using Amano PS lipase in a MTMS gel. The commerwhereas the
cial enzyme preparation resulted in 92%
same amount of entrapped lipase produced the enantiomerically
pure products 6 and 7 ( e r > 9 9 % ) .
Fig. 3. Relative activity s of immobilized lipases in three different gel materials.
Solid: MTMS; outlined: MTMS\PDMS (6;l); shaded: PTMSlTMOS (5;l).
The long-term stability of the entrapped lipases is remarkably
high. The test reaction was performed over 30days in batch
reactions, in which the solid catalyst was filtered off after 22 h,
washed, and then reused.[131The activity decreased typically by
15-20% after the first two to three cycles and then remained
constant at 80-85% of the initial activity, for example, for
Amano PS lipase in pure MTMS or MTMSjPDMS (6/1) gels.
In contrast, the same lipase adsorbed on a pure MTMS gel lost
more than 75% of its initial activity after only eight reaction
cycles. Probably only a small fraction of the lipase (15-20%) is
adsorbed or only weakly stabilized in the sol-gel process and is
hence more easily deactivated or lost.
The morphology of the sol-gel immobilizates was examined by
scanning electron microscopy (SEM)
Figure 4 shows a typical scanning electron micrograph of a lipase-containing MTMS
gel containing amorphous regions as well as spherical particles.
What are the reasons for the increased relative enzyme activities? Since the comparisons are based on suspensions in organic
solvents, the primary cause is probably the fine distribution and
therefore increased accessibility of the enzyme. It can also be
speculated that the hydrophobic groups R in the sol-gel matrix
are involved in stabilizing and activating interactions with the
hydrophobic domains of the lipases. The observed “alkyl effect”
could also be intrinsic during the formation of the gel, possibly
by a smaller damaging effect to the enzyme by the hydrophobic
silane monomers. An increase in activity caused by a local rise in
substrate concentration in the hydrophobic matrix is also con~eivable.[~]
A favorable influence of possible hydrophobic interactions in the immobilization of lipases o n other supports, albeit
with much smaller increases in activity, has been described
VCH Verlagsgesellschaf~mbH. 0-69451 Weinhrrm. 1992
92% ee
92% ee
in MTMS gel
99.6% ee
99.6% ee
The method described herein leads to immobilized lipases
with excellent activity and stability. The resulting biocatalysts
are easy to
and should also be suitable for application
in continuous processes. A particular advantage is that by varying a range of parameters[”] it is possible to generate tailormade gels for each lipase.[61
Received: July 14. 1994
Revised version: September 17, 1994 [271251E]
German version: Angew. Clrem. 1995, 107. 373
Keywords: enzymatic catalysis . immobilization . lipases . sol gel processes
11) K. Faber, Bi~~truan.r/orninfin.s
itz Organic Clzmiistry. Springer, Berlin, 1992;
En:ymes a s Caralwts in Organic Sjxthesir (Ed.: M. P. Schneider), Reidel,
Dordrecht, 1986; W. Boland. C . Frossl, M. Lorenz, Synthesis 1991, 1049.
[2] A. Zaks. A . M . Kiibanov, Pruc. Narl. Acad. Sci. LISA 1985. 81. 3192; A. M.
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131 E. Guibe-Jampel, G . Rousseau, Tetrahcrirori Lett. 1990.31. 132; F. X. Malcata,
H. R . Reyes. H. S. Garcia, C. G . Hill. Jr., C. H. Amundson. J. A m . Oil Chrrn.
Soc. 1990, 67, 890; M. Arroyo. J. M. Moreno. J. V. Sinisterra, J. Mol. Catal.
1993, 83, 261: see also R . V. Parthasarathy. C. R. Martin, Nature 1994, 369,
[4] J. A. Bosley. J. C. Clayton, Bioterhnol. Bioeng. 1994, 43, 934; M. Norin. J.
Boutelje, E. Holniberg. K. Hult. Appl. Microhiol. Biotechnnl. 1988. 28. 527.
[5] Y. Kimurd, A. Tanaka, K. Sonomoto. T. Nihira, S. Fukui. Appl. Microhiol.
Bioteclinol. 1983. f7,107.
[6] ,.lmmohili.siivtc L i p u ~ ~in
i z li~drophobenSol-Ci,l-Murcriulien”: M. T. Reetz, A.
Zonta, J. Simpelkamp (Max-Planck-Institut fur Kohlenforschung), DE-A
4408152.9. 1994.
S 10.00+ .2S/O
An,qw. Cllrm. Int. Ed. En,?/. 1995. 34,
lengths of the molecules achieved so far in the series of oligo(tetraethynylethene)s,['] up to 50 A, oligo(thienylenethynylene)s,[21
up to 100 A, and oligo(phenylenethynylene)s.[31up to 128 A,
have reached the size of the smallest achievable structural dimensions of lithographic n a n o s t r u ~ t u r e s . [Although
of the first series can be charged, preferdbly by reduction. and
are suitable as molecular conductors owing to delocalization,
these properties have not yet been established for oligomers
linked by alkyne units.
a-Conjugated oligothiophenes are defined model compounds
for electrically conducting polythiophenes. Because of their
easily oxidized n-electron systems, they exhibit conductivities of
up to 20 Scm- I , values comparable to those of the correspond17. ?Y
ing polymers.[5i The correlation between the chain length of
F Schwertleger. W. Glaubitt. U. Schubert. J. !Von-Crw/. S d i d ~1992. 145, 85;
oligothiophenes and their electronic properties have already
Y. Haruvy. A Heller, S. E. Weber in Suprunioleculur A r ~ h i l e ~ r i i r r - S ~ n l l ~ i , / i c
been investigated.L6] The properties of an organic semiconducC'iin/ro/ i i i 7 ' h i i F i l n n and Solirly (Ed.: T. Bein) (ACS Symp. Sw. 1992, 499).
p. 405.
tor are also influenced. however, by the spatial arrangement of
Thc Iipase was dissolved in water or buffer. centrifuged to remove undissolved
the molecules in the solid material.l'] Garnier et al.[7.81and
solid\. and mixed with aqueous solutions of polyvinyl alcohol and sodium
Ostoja et aLi9I demonstrated that by improving the molecular
fluoride. The ailanes were then added in the order of increasing reactivity. The
order in thin layers of sexithiophene derivatives on silicon, they
reaction mixture was thoroughly mixed and shaken until gelation occurred (ca.
1 m i i i ) . The gela thus obtained were allowed to stand in sealed vessels for 24 h.
were able to increase the conductivity and simultaneously imtlricd foi- 3 d;iys at 37'C. washed with water, acetone. and pentane. dried and
prove the mobility of the charge carrier improved. For the transground into il powder.
fer of charge from chain to chain (interchain hopping), not only
We thank Novo Nordisk AIS (Denmark) for a sample of SP 523 (NOVO) and
the distance separating the conjugated n-electron systems of the
Amano En/yme Europe Ltd. (England) for PS lipase.
Whereas thc commercial enzyme powder combined with the water produced
molecules but also their spatial overlap appear to be decisive.[71
l'rmi tlic rcnction and formed a viscous residue which was difficult to separate,
The images of poly(3-alkylthiophene)~obtained by high-resoluthe sol gel immobilized lipase could be recycled without any difficulty.
tion scanning tunneling microscopy (STM) and atomic force
We thank Dr. B. Tesche (Fritz Haber Institut. Berlin) for the scanning electron
microscopy (AFM) have not been convincing up to now. As
D. Bianchi. P Cesti, E. Battistel. J. Org. Chrm 1988. 53, 5531.
Chang and Bard have shown,["] details in the images recorded
.4ccording IO C'esri [15] the reaction resulted in 95% er. However. it is
by Lacaze et al.["l of the polymers on graphite cannot readily
known th;it the enantioselectivity of enzyme-catalyzed reactions can vary for
be distinguished from defects in the graphite structure and asdifferent batchesm of enzyme.
signed to clearly defined molecules. Kamrava et a1.1'21used
The influence of other parameters. for example the use of additives, the stoichiometrq water:silane. the amount of catalyst, and the use of other silane
STM to record images of an electrochemically prepared polymonoinerc :ire presently tinder investigation in our group.
thiophene film, partially oxidized with FeCI, ; however, they
obtained a resolution of only 5-10 nm. Similarly. AFM investigations did not yield images with molecular resolution." 31
We report here on the synthesis of a homologous series of
isomerically pure a-linked oligo(a1kylthiophene)s 1-4. According to calculations, sedecithiophene 4, the longest known and
Oligothiophenes-Yet Longer? Synthesis,
clearly characterized oligothiophene, should be 64 8, long when
Characterization, and Scanning Tunneling
Previously, oligo(alky1thiophene)s up to a dodecamer
Microscopy Images of Homologous, Isomerically
were known,"41 and it is only recently that a pentadecamer has
Pure Oligo(alkylthiophene)s**
been described."'] Because of their improved solubility in organic solvents, the new oligothiophenes 1-4 can be successfully
Peter Bauerle,* Thomas Fischer, Bernd Bidlingmeier,
purified and their physical properties, even those of the longer
Andreas Stabel, and Jiirgen P. Rabe*
homologues, were determined in solution. In this way the structure -property relationships can be extended to chain lengths
The synthesis of structurally defined conjugated oligomers with
that were previously unobtainable. In addition it was possible to
dimensions on the nanometer scale have recently awakened inobtain the first STM images of physisorbed two-dimensional
terest not only because of the possibility of investigating trans(2D) crystalline layers of homologues 1-4 with submolecular
port behavior along the "molecular wire". Interestingly, the
[*I Prof. Dr. P Bauerlc. Dipl.-Chem. T. Fischer. B. Bidlingmeier
The synthesis of the new oligo(alky1thiophene)s 1-4 is shown
Imtitut fiir Organische Chemie der Universitit
in Scheme 1. 3,3"'-Didodecylquaterthiophene 1 was obtained in
Am Hubland. D-97074 Wurzbtirg (Germany)
75 % yield by the nickel-catalyzed coupling of two equivalents
Telefitx Ink. code + (931)888-4606
Prof. Dr. J. P. Rabe
of the Grignard reagent prepared from 2-bromododecylthioInstitut fur Physikalischc Chemie der Universitit
phene['61with 5,5'-dibromo-2,2'-bithiophene.
After the analogJakob-Wclder-Weg 11. D-55099 Mainz (Germany)
ous Grignard coupling of an appropriate monobrominated
Tclefax. I n t . code + (6131)39-3768
quaterthiophene failed, the conversion of 1 to higher homoDipl.-Cheni. A. Stahel
logues was carried out by oxidative dimerization of the lithiated
Max-Plank-lnstitut fur Polymerforschung, Mainz (Germany)
compound with CUCI,.["~ It had to be taken into account
[**I We would like to thank the Bundesministerium fur Forschung und Technologie
that because of exchange reactions the lithiation of oligothio("Tr;insport-Phinomene in Polymer-Substrat-Grenzflichen" and "Muster
se1bstorg;inisierender Molekule") and ESPRIT Basic Research Project 7282
phenes generally leads to a mixture of mono- and dilithiated
(TOPFIT) foi- financial support. A. S. thanks the Fonds der Chemischen
product^,["^^ and that the yields drop rapidly as the size of
lndustrie for il Kekule fellowship. We would like to thank DipLPhys. U .
the coupling components increases.[''' Thus. the reaction of 1
Segelbachcr (2. Phys. Inst. Universitit Stuttgart) for the temperature-dependent spectroelectrochemical measurements.
with equimolar amounts of n-butyllithium and CuCI, yielded as
J ,4m. Chriii. S I I ~1955.
59, 695. P. Johnson. T. L. Whateley. ./.
1971.557: L. M. Ellerby. C. R. Nishida, t Nishida, S. A.
Y;iitiannka. B. Dunn. J. Selverstone Valentine, J. J. Zink. Scirnce 1992. 255.
1 1 13: Y , Tatsu. K. Yamashita. M. Yamaguchi. S. Yamamura. H. Yamamoto. S.
\'o\hikawa. Chrw?.M I . 1992. 1615: S. Braun. S. Rappoport. S. Shtelrer. R.
Ztisman. S. 1)ruckmann. D Avnir. M. Ottolenghi in Biotivhnolog~:BritlXing
H N Y I I - C ~m i ( / .A/ip/;cu~;(in(Eds. ' D. Kaniely, A. Shakrabarty. S. E. Kornguth).
Kluwer. Boston. 1991, p. 205; H. Weetall. B. Robertson. D. Cullin. J. Brown.
M W;ilch. U i o d i m t . B i o p h ~ ! Acro
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.So/-Gr,I-Srwni I r/lcP/ r i n d Cheniis 1ri' of Sol-Ge/-Pror.ersinp. Academic
Prcss. Boston. 1990.
A . M. Br/o/ouski. U . Derewenda. Z. S. Derewenda, G. G. Dodson. D. W.
Lawson. J P Turkenburg. F. Bjorkling, B. Huge-Jensen, S. A. Patkar. L.
Thim. . Y o / i i r e iLontLin) 1991. . I S / . 491; K.-E. Jaeger, S. Ransac. B. W.
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i ~ r Sci.
Anger!. Chon. In/ Ed. Engl. 1995. 34. No. 3
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biocatalysts, efficiency, heterogeneous, lipase, material, hydrophobic, entrapment, solцgel
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